Innervation of the Gastrointestinal Tract (Autonomic Nervous System)

INNERVATION OF THE GASTROINTESTINAL TRACT

The Autonomic Nervous System A series of books discussing all aspects of the autonomic nervous system. Edited by Geoffrey Burnstock, Autonomic Neuroscience Institute, Royal Free Hospital School of Medicine, London, UK. Volume 1 Autonomic Neuroeffector Mechanisms edited by G. Burnstock and C.H.V. Hoyle

Volume 8 Nervous Control of Blood Vessels edited by T. Bennett and S.M. Gardiner

Volume 2 Development, Regeneration and Plasticity of the Autonomic Nervous System edited by I.A. Hendry and C.E. Hill

Volume 9 Nervous Control of the Heart edited by J.T. Shepherd and S.F. Vatner

Volume 3 Nervous Control of the Urogenital Systsem edited by C.A. Maggi

Volume 10 Autonomic – Endocrine Interactions edited by K. Unsicker

Volume 4 Comparative Physiology and Evolution of the Autonomic Nervous System edited by S. Nilsson and S. Holmgren

Volume 11 Central Nervous Control of Autonomic Function edited by D. Jordan

Volume 5 Disorders of the Autonomic Nervous System edited by D. Robertson and I. Biaggioni

Volume 12 Autonomic Innervation of the skin edited by J.L. Morris and I.L. Gibbins

Volume 6 Autonomic Ganglia edited by E.M. McLachlan

Volume 13 Nervous Control of the Eye edited by G. Burnstock and A.M. Sillito

Volume 7 Autonomic Control of the Respiratory System edited by P.J. Barnes

Volume 14 Innervation of the Gastrointestinal Tract edited by S. Brookes and M. Costa

This book is part of a series. The publisher will accept continuation orders which may be cancelled at any time and which provide for automatic billing and shipping of each title in the series upon publication. Please write for details.

INNERVATION OF THE GASTROINTESTINAL TRACT

Edited by

Simon Brookes and Marcello Costa

London and New York

First published 2002 by Taylor & Francis 11 New Fetter Lane, London EC4P 4EE Simultaneously published in the USA and Canada by Taylor & Francis Inc, 29 West 35th Street, New York, NY 10001 Taylor & Francis is an imprint of the Taylor & Francis Group

This edition published in the Taylor & Francis e-Library, 2003. © 2002 Taylor & Francis All rights reserved. No part of this book may be reprinted or reproduced or utilised in any form or by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying and recording, or in any information storage or retrieval system, without permission in writing from the publishers. Every effort has been made to ensure that the advice and information in this book is true and accurate at the time of going to press. However, neither the publisher nor the authors can accept any legal responsibility or liability for any errors or omissions that may be made. In the case of drug administration, any medical procedure or the use of technical equipment mentioned within this book, you are strongly advised to consult the manufacturer’s guidelines.

British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging in Publication Data A catalog record for this book has been requested

ISBN 0-203-21701-2 Master e-book ISBN

ISBN 0-203-27305-2 (Adobe eReader Format) ISBN 0–415–28377–9 (Print Edition)

Contents Preface to the Series — Historical and Conceptual Perspective of the Autonomic Nervous System Book Series

vi

Preface

xii

Contributors

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1 Enteric Reflexes that Influence Motility J.C. Bornstein, J.B. Furness, W.A.A. Kunze and P.P. Bertrand 2 Motor Control of the Stomach David Grundy and Michael Schemann

1 57

3 Control of Gastric Functions by Extrinsic Sensory Neurons Peter Holzer

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4 Neural Control of the Large Intestine Kalina Venkova, Beverley Greenwood-Van Meerveld and Jacob Krier

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5 Neurons of the Gallbladder and Sphincter of Oddi Gary M. Mawe, Erin K. Talmage, Lee J. Jennings, Kirk Hillsley and Audra L. Kennedy

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6 Pharmacology of the Enteric Nervous System Marcello Tonini, Fabrizio De Ponti, Gianmario Frigo and Francesca Crema

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7 Neuroeffector Transmission in the Intestine Charles H.V. Hoyle, Pam Milner and Geoffrey Burnstock

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8 Neural Control of Intestinal Vessels Neela Kotecha

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9 Enteric Neuro-Immunophysiology Jackie D. Wood

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10 Cellular Organisation of the Mammalian Enteric Nervous System Simon J.H. Brookes and Marcello Costa

393

11 Development of the Enteric Nervous System Michael D. Gershon

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Index

527

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Preface to the Series — Historical and Conceptual Perspective of the Autonomic Nervous System Book Series The pioneering studies of Gaskell (1886), Bayliss and Starling (1899), and Langley and Anderson (see Langley, 1921) formed the basis of the earlier and, to a large extent, current concepts of the structure and function of the autonomic nervous system; the major division of the autonomic nervous system into sympathetic, parasympathetic and enteric subdivisions still holds. The pharmacology of autonomic neuroeffector transmission was dominated by the brilliant studies of Elliott (1905), Loewi (1921), von Euler and Gaddum (1931), and Dale (1935), and for over 50 years the idea of antagonistic parasympathetic cholinergic and sympathetic adrenergic control of most organs in visceral and cardiovascular systems formed the working basis of all studies. However, major advances have been made since the early 1960s that make it necessary to revise our thinking about the mechanisms of autonomic transmission, and that have significant implications for our understanding of diseases involving the autonomic nervous system and their treatment. These advances include: (1) Recognition that the autonomic neuromuscular junction is not a “synapse” in the usual sense of the term where there is a fixed junction with both pre- and post-junctional specialization, but rather the transmitter is released from mobile varicosities in extensive terminal branching fibres at variable distances from effector cells or bundles of smooth muscle cells which are in electrical contact with each other and which have a diffuse distribution of receptors (see Hillarp, 1959; Burnstock, 1986a). (2) The discovery of non-adrenergic, non-cholinergic nerves and the later recognition of a multiplicity of neurotransmitter substances in autonomic nerves, including monoamines, purines, amino acids, a variety of different peptides and nitric oxide (Burnstock et al., 1964, 1986b, 1997; Rand, 1992; Milner and Burnstock, 1995; Lincoln et al., 1995; Zhang and Snyder, 1995; Burnstock and Milner, 1999). (3) The concept of neuromodulation, where locally released agents can alter neurotransmission either by prejunctional modulation of the amount of transmitter released or by postjunctional modulation of the time-course or intensity of action of the transmitter (Marrazzi, 1939; Brown and Gillespie, 1957; Vizi, 1979; Fuder and Muscholl, 1995; MacDermott et al., 1999). vi

PREFACE TO THE SERIES

G. Burnstock — Editor of The Autonomic Nervous System Book Series

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(4) The concept of cotransmission that proposes that most, if not all, nerves release more than one transmitter (Burnstock, 1976; Hökfelt, Fuxe and Pernow, 1986; Burnstock, 1990a; Burnstock and Ralevic, 1996) and the important follow-up of this concept, termed “chemical coding”, in which the combinations of neurotransmitters contained in individual neurones are established, and whose projections and central connections are identified (Furness and Costa, 1987). (5) Recognition of the importance of “sensory-motor” nerve regulation of activity in many organs, including gut, lungs, heart and ganglia, as well as in many blood vessels (Maggi, 1991; Burnstock, 1993), although the concept of antidromic impulses in sensory nerve collaterals forming part of “axon reflex” vasodilatation of skin vessels was described many years ago (Lewis, 1927). (6) Recognition that many intrinsic ganglia (e.g., those in the heart, airways and bladder) contain interactive circuits that are capable of sustaining and modulating sophisticated local activities (Saffrey et al., 1992; Ardell, 1994). Although the ability of the enteric nervous system to sustain local reflex activity independent of the central nervous system has been recognized for many years (Kosterlitz, 1968), it has been generally assumed that the intrinsic ganglia in peripheral organs consist of parasympathetic neurones that provided simple nicotinic relay stations. (7) The major subclasses of receptors to acetylcholine and noradrenaline have been recognized for many years (Dale, 1914; Ahlquist, 1948), but in recent years it has become evident that there is an astonishing variety of receptor subtypes for autonomic transmitters (see Pharmacol. Rev., 46, 1994). Their molecular properties and transduction mechanisms have been characterised (see IUPHAR Compendium of Receptor Characterisation and Classification 2000). These advances offer the possibility of more selective drug therapy. (8) Recognition of the plasticity of the autonomic nervous system, not only in the changes that occur during development and ageing, but also in the changes in expression of transmitter and receptors that occur in fully mature adults under the influence of hormones and growth factors following trauma and surgery, and in a variety of disease situations (Burnstock, 1990b; Saffrey and Burnstock, 1994; Milner et al., 1999). (9) Advances in the understanding of “vasomotor” centres in the central nervous system. For example, the traditional concept of control being exerted by discrete centres such as the vasomotor centre (Bayliss, 1923) has been supplanted by the belief that control involves the action of longitudinally arranged parallel pathways involving the forebrain, brain stem and spinal cord (Loewy and Spyer, 1990; Jänig and Häbler, 1995). In addition to these major new concepts concerning autonomic function, the discovery by Furchgott that substances released from endothelial cells play an important role in addition to autonomic nerves, in local control of blood flow, has made a significant impact on our analysis and understanding of cardiovascular function (Furchgott and Zawadski, 1980; Burnstock and Ralevic, 1994). The later identification of nitric oxide as the major endothelium-derived relaxing factor (Palmer et al., 1988; see Moncada et al., 1991) (confirming the independent suggestion by Ignarro and by Furchgott) and endothelin as an endothelium-derived constricting factor (Yanagisawa et al., 1988; see Rubanyi and Polokoff, 1994) have also had a major impact in this area.

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In broad terms, these new concepts shift the earlier emphasis on central control mechanisms towards greater consideration of the sophisticated local peripheral control mechanisms. Although these new concepts should have a profound influence on our considerations of the autonomic control of cardiovascular, urogenital, gastrointestinal and reproductive systems and other organs like the skin and eye in both normal and disease situations, few of the current textbooks take them into account. This is largely because revision of our understanding of all these different specialised areas in one volume by one author is a near impossibility. Thus, this Book Series of 14 volumes is designed to try to overcome this dilemma by dealing in depth with each major area in separate volumes and by calling upon the knowledge and expertise of leading figures in the field. Volume I, deals with the basic mechanisms of Autonomic Neuroeffector Mechanisms which sets the stage for later volumes devoted to autonomic nervous control of particular organ systems, including Heart, Blood Vessels, Respiratory System, Urogenital Organs, Gastrointestinal Tract, Eye Function, Autonomic Ganglia, Autonomic-Endocrine Interactions, Development, Regeneration and Plasticity and Comparative Physiology and Evolution of the Autonomic Nervous System. Abnormal as well as normal mechanisms will be covered to a variable extent in all these volumes depending on the topic and the particular wishes of the Volume Editor, but one volume edited by Robertson and Biaggioni, 1995, has been specifically devoted to Disorders of the Autonomic Nervous System (see also Mathias and Bannister, 1999). A general philosophy followed in the design of this book series has been to encourage individual expression by Volume Editors and Chapter Contributors in the presentation of the separate topics within the general framework of the series. This was demanded by the different ways that the various fields have developed historically and the differing styles of the individuals who have made the most impact in each area. Hopefully, this deliberate lack of uniformity will add to, rather than detract from, the appeal of these books. G. Burnstock Series Editor

REFERENCES Ahlquist, R.P. (1948). A study of the adrenotropic receptors. Am. J. Physiol., 153, 586–600. Ardell, J.L. (1994). Structure and function of mammalian intrinsic cardiac neurons. In Neurocardiology, edited by J.A. Armour and J.L. Ardell, pp. 95–114. Oxford: Oxford University Press. Bayliss, W.B. (1923). The Vasomotor System. Longman: London. Bayliss, W.M. and Starling, E.H. (1899). The movements and innervation of the small intestine. J. Physiol. (Lond.), 24, 99–143. Brown, G.L. and Gillespie, J.S. (1957). The output of sympathetic transmitter from the spleen of a cat. J. Physiol. (Lond.), 138, 81–102. Burnstock, G. (1976). Do some nerve cells release more than one transmitter? Neuroscience, 1, 239–248. Burnstock, G. (1986a). Autonomic neuromuscular junctions: Current developments and future directions. J. Anat., 146, 1–30. Burnstock, G. (1986b). The non-adrenergic non-cholinergic nervous system. Arch. Int. Pharmacodyn. Ther., 280(suppl.), 1–15. Burnstock, G. (1990a). Co-Transmission. The Fifth Heymans Lecture – Ghent, February 17, 1990. Arch. Int. Pharmacodyn. Ther., 304, 7–33.

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Burnstock, G. (1990b). Changes in expression of autonomic nerves in aging and disease. J. Auton. Nerv. Syst., 30, 525–534. Burnstock, G. (1993). Introduction: Changing face of autonomic and sensory nerves in the circulation. In Vascular Innervation and Receptor Mechanisms: New Perspectives, edited by L. Edvinsson and R. Uddman, pp. 1–22. San Diego: Academic Press Inc. Burnstock, G. (1997). The past present and future of purine nucleotides as signalling molecules. Neuropharmacology, 36, 1127–1139. Burnstock, G., Campbell, G., Bennett, M. and Holman, M.E. (1964). Innervation of the guinea-pig taenia coli: Are there intrinsic inhibitory nerves which are distinct from sympathetic nerves? Int. J. Neuropharmacol., 3, 163–166. Burnstock, G. and Milner, P. (1999). Structural and Chemical Organisation of the autonomic nervous system with special reference to non-adrenergic, non-cholinergic transmission. In Autonomic Failure: A Textbook of Clinical Disorders of the Autonomic Nervous System. 4th edn, edited by C.J. Mathias and R. Bannister, Oxford: Oxford University Press, pp. 63–71. Burnstock, G. and Ralevic, V. (1994). New insights into the local regulation of blood flow by perivascular nerves and endothelium. Br. J. Plast. Surg., 47, 527–543. Burnstock, G. and Ralevic, V. (1996). Cotransmission. In The Pharmacology of Smooth Muscle, edited by C.J. Garland and J. Angus, Oxford: Oxford University Press. Dale, H. (1914). The action of certain esters and ethers of choline and their reaction to muscarine. J. Pharmacol. Exp. Ther., 6, 147–190. Dale, H. (1935). Pharmacology and nerve endings. Proc. Roy. Soc. Med., 28, 319–332. Elliott, T.R. (1905). The action of adrenalin. J. Physiol. (Lond.), 32, 401–467. Fuder, H. and Muscholl, E. (1995). Heteroceptor-mediated modulation of noradrenaline and acetylcholine release from peripheral nerves. Rev. Physiol. Biochem. Physiol., 126, 265–412. Furchgott, R.F. and Zawadski, J.V. (1980). The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature, 288, 373–376. Furness, J.B. and Costa, M. (1987). The Enteric Nervous System. Edinburgh: Churchill Livingstone. Gaskell, W.H. (1886). On the structure, distribution and function of the nerves which innervate the visceral and vascular systems. J. Physiol. (Lond.), 7, 1–80. Hillarp, N.-Å. (1959). The construction and functional organisation of the autonomic innervation apparatus. Acta Physiol. Scand., 46 (suppl. 157), 1–38. Hökfelt, T., Fuxe, K. and Pernow, B. (Eds.) (1986). Coexistence of neuronal messengers: A new principle in chemical transmission. In Progress in Brain Research, Vol. 68, Amsterdam: Elsevier. IUPHAR Compendium of Receptor Characterisation and Classification 2000, IUPHAR Media Ltd (London), UK. Jänig, W. and Häbler, H.-J. (1995). Visceral-Autonomic Integration. In Visceral Pain, Progress in Pain Research and Management, edited by G.F. Gebhart, Vol. 5, pp. 311–348. Seattle: IASP Press. Kosterlitz, H.W. (1968). The alimentary canal. In Handbook of Physiology, edited by C.F. Code, Vol. IV, pp. 2147–2172. Washington, DC: American Physiological Society. Langley, J.N. (1921). The Autonomic Nervous System, part 1. Cambridge: W. Heffer. Lewis, T. (1927). The Blood Vessels of the Human Skin and Their Responses. Shaw & Sons: London. Lincoln, J., Hoyle, C.H.V. and Burnstock, G. (1995). Transmission: Nitric oxide. In The Autonomic Nervous System, Vol. 1 (reprinted): Autonomic Neuroeffector Mechanisms, edited by G. Burnstock and C.H.V. Hoyle, pp. 509–539. The Netherlands: Harwood Academic Publishers. Loewi, O. (1921). Über humorale Übertrangbarkeit der Herznervenwirkung. XI. Mitteilung. Pflügers Arch. Gesamte Physiol., 189, 239–242. Loewy, A.D. and Spyer, K.M. (1990). Central Regulations of Autonomic Functions. New York: Oxford University Press. MacDermott, A.B., Role, L.W. and Siegelbaum, S.A. (1999). Presynaptic iootropic receptors and the control of transmitter release. Ann. Rev. Neurosci., 22, 443–485. Maggi, C.A. (1991). The pharmacology of the efferent function on sensory nerves. J. Auton. Pharmacol., 11, 173–208. Marrazzi, A.S. (1939). Electrical studies on the pharmacology of autonomic synapses. II. The action of a sympathomimetic drug (epinephrine) on sympathetic ganglia. J. Pharmacol. Exp. Ther., 65, 395–404. Mathias, C.J. and Bannister, R. (eds.) (1999). Autonomic Failure. 4th edn, Oxford: Oxford University Press. Milner, P. and Burnstock, G. (1995). Neurotransmitters in the autonomic nervous system. In Handbook of Autonomic Nervous Dysfunction, edited by A.D. Korczyn, pp. 5–32. New York: Marcel Dekker. Milner, P., Lincoln, J. and Burnstock, G. (1999). The neurochemical organisation of the autonomic nervous system. In Handbook of Clinical Neurology Vol 74(30): The autonomic nervous system – Part 1 – Normal Functions, edited by O. Appezeller, Amsterdam: Elsevier Science, pp. 87–134.

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Moncada, S., Palmer, R.M.J. and Higgs, E.A. (1991). Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev., 43, 109–142. Palmer, R.M.J., Rees, D.D., Ashton, D.S. and Moncada, S. (1988). Arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation. Biochem. Biophys. Res. Commun., 153, 1251–1256. Rand, M.J. (1992). Nitrergic transmission: nitric oxide as a mediator of non-adrenergic, non-cholinergic neuroeffector transmission. Clin. Exp. Pharmacol. Physiol., 19, 147–169. Rubanyi, G.M. and Polokoff, M.A. (1994). Endothelins: molecular biology, biochemistry, pharmacology, physiology, and pathophysiology. Pharmacol. Rev., 46, 328–415. Saffrey, M.J. and Burnstock, G. (1994). Growth factors and the development and plasticity of the enteric nervous system. J. Auton. Nerv. Syst., 49, 183–196. Saffrey, M.J., Hassall, C.J.S., Allen, T.G.J. and Burnstock, G. (1992). Ganglia within the gut, heart, urinary bladder and airways: studies in tissue culture. Int. Rev. Cytol., 136, 93–144. Vizi, E.S. (1979). Prejunctional modulation of neurochemical transmission. Prog. Neurobiol., 12, 181–290. von Euler, U.S. and Gaddum, J.H. (1931). An unidentified depressor substance in certain tissue extracts. J. Physiol., 72, 74–87. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K. and Masaki, T. (1988). A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature, 332, 411–415. Zhang, J. and Snyder, S.H. (1995). Nitric oxide in the nervous system. Annu. Rev. Pharmacol. Toxicol., 35, 213–233.

Preface Eating is an extremely hazardous way to make a living, biologically speaking. The strategy of ingesting foreign organisms, breaking them down and reusing the spare parts is fraught with risks. For one thing, the organism concerned is likely to resist being broken down for scrap and may, in fact, have designs on the body parts of the diner. For another, the chemicals required to digest food are equally capable of breaking down the structure of the gut wall and the rest of the consumer’s body as well. It seems that minimising these risks, while meeting the imperative to absorb nutrients, has influenced many of the design principles of the gastrointestinal tract. This long tube is composed of a variety of tissue types and is the largest internal organ of the body. Its main function is to digest food and absorb the released nutrients. It is subdivided into functionally distinct regions, so that ingested material is processed serially in a way that serves to minimise the risks of self-digestion and attack by ingested pathogens. After rapid propulsion through the thorax via the oesophagus, food is subjected to mechanical and chemical breakdown in the harshly acidic environment of the stomach. This tends to reduce the viability of (inadvertently) swallowed micro-organisms. If toxic material is detected, propulsion can be reversed and the ingested material can be rapidly expelled by vomiting. During its sojourn in the stomach, sphincteric muscles at either end of the organ prevent spillage of the corrosive acidic content into the susceptible neighbouring regions of the duodenum and oesophagus. Gastric contents are then gradually aliquoted into the duodenum at a carefully controlled rate, adapted to the calorific and nutrient content of the meal. In the duodenum, the acidity of the contents is neutralised and they are mixed with digestive enzymes, bile and watery secretions, to allow the complex chemistry of digestion to get to work. The resulting chyme is mixed, moved to and fro and gradually propelled along the small intestine, at a rate adapted to the requirements for absorption of nutrients. A few hours after the meal, the small intestine is basically empty, the residual matter having been expelled into the colon. Here it may reside for several days, as water and ions are reabsorbed, playing a potentially vital role in the water balance of the organism. Eventually, the near-solid material is expelled from the body. In mechanical terms, this is a decidedly non-trivial event. The prolonged retention of contents in the colon makes it inevitable that large numbers of bacteria will be present, posing an enormous potential danger to the organism. If large numbers of micro-organisms colonise the small intestine, the barrier between the vasculature and the outside world is rapidly compromised and xii

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massive septicaemia results. Parasites in food may survive the digestive process and then attack the wall of the gut. The consuming organism defends itself against these perils with specialised motor patterns, which are triggered to remove such noxious threats with great rapidity from the body. The multiplicity of functions required of the digestive tract makes it a fascinating subject for biological research by a range of disciplines, including immunology, microbiology, biochemistry, oncology etc. However it provides an especially appealing subject for neuroscientists. Neuronal circuitry is intimately involved in controlling many of the functions of the gut. It controls the activity of the smooth muscle in the gut wall, which mixes and propels the contents. Between meals, specialised patterns of motor activity periodically empty sloughed-off epithelial cells and accumulated mucous, thus preventing the build-up of a culture medium for the ever-threatening bacteria. Other highly propulsive motor patterns can be triggered by toxic chemicals, bacterial infection, parasitic infestation or identified allergens. While we consider the symptoms associated with these motor patterns (diarrhoea and pain) to be unpleasant, there is little doubt that these specialised mechanisms are of profound adaptive significance to our survival. The feature of the innervation of the gastrointestinal tract that is most appealing to neuroscientists is the presence of extensive networks of neural circuits embedded in its walls – the enteric nervous system. Containing approximately the same number of nerve cells as the spinal cord, it is connected with the central nervous system via visceral afferents and sympathetic and parasympathetic efferents of the autonomic nervous system. The presence of an entire, specialised nervous system within this peripheral organ system provides an opportunity for detailed study, without having to contend with the central nervous system. Since the studies of Bayliss and Starling (1899), Langley and Magnus (1905) and Trendelenburg (1917), it has been apparent that the enteric nervous system is capable of displaying a great deal of autonomy in controlling gastrointestinal function. Thus the enteric nervous system is studied by gastroenterologists seeking to understand how gut function is controlled, and also by neuroscientists endeavouring to understand how a relatively simple, autonomous mammalian neural circuit is organised. Obviously these two motivations are not mutually exclusive and many students of the enteric nervous system have foot in both camps. The complexity of the enteric nervous system began to be recognised with the discovery of the two major neural plexuses, the submucous plexus by Meissner (1857) and the myenteric plexus by Auerbach (1862). Langley (1921) placed the intrinsic enteric innervation in a class of its own after he recognised that many of the neural functions depend on the circuits entirely intrinsic to the digestive tract, rather than being solely determined by extrinsic neural inputs. Despite this early insight, it took many more years before the enteric nervous system was recognised by gastroenterologists as a relatively independent component of the autonomic nervous system. Combined studies by neuroanatomists, pharmacologists and physiologists eventually demonstrated that the study of the enteric nervous system was not only relevant to gastroenterology, but essential for its progress. During much of the twentieth century, the rate of progress was relatively slow in this field, which seemed to have fallen into a gap between neuroscience and gastroenterology. During this period, much emphasis was placed on the myogenic control of gastrointestinal motility, with little direct investigation of the roles of enteric neurons. It is only recently

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that the cellular basis of the spontaneous electrical activity of gut smooth muscle has begun to be understood in detail. It has been discovered that the mysterious cells, first described by Cajal as “interstitial cells” are involved both in generating myogenic activity and in mediating the input of enteric motor neurons to the smooth muscle (Sander, 1996; Huizinga et al., 1997). This breakthrough promises eventually to bridge the historical divide between the two schools of thought (myogenic versus neurogenic) about the control of gut motility. In this volume of the series on the autonomic nervous system, the innervation of the gut by the enteric nervous system, and its interface with the extrinsic innervation, is examined from a number of different perspectives. It will become apparent that all of these different aspects of nervous control can be related directly to how gut function is adapted to minimise risk, while maximising digestive efficiency. For example, the detailed analysis of enteric neural circuits controlling reflex motor activity of the intestine is elegantly summarised in the chapter by Joel Bornstein and colleagues. They provide an insightful synthesis of data gathered over many years by their own laboratory and by others around the world, as to how simple circuits may propel the gut contents, preventing excessive distension and perhaps contribute to the expulsion of pathogens. As described above, the stomach plays a very different role in the process of dealing with food, as it is not involved in digestion or absorption but is rather adapted for storage, mechanical breakdown and aliquoting of contents. Not surprisingly, the neuronal control of gastric motility has many different features from those of the small intestine. This is summarised in the extensive review of David Grundy and Michael Schemann who have contributed widely to the study of both the extrinsic and intrinsic innervation of the stomach. A particularly important role for extrinsic sensory nerves in protecting the stomach from a range of damaging agents has been identified in the last decade. Peter Holzer has been at the centre of this field and has written an authoritative review, summarising the role and mechanisms of action of these nerves in gastric protection. At the other end of the gastrointestinal tract, the colon also plays a significant role as an organ of storage, but it has to deal with very different material. In particular, much of the activity of the colon is modulated by extrinsic neuronal inputs from both the sympathetic and parasympathetic divisions of the autonomic nervous system. For many years the contributions of the extrinsic innervation have been rather over-simplified. This is corrected in the chapter of Kalina Venkova, Beverely Greenwood-Van Meerveld and the late Jack Krier, who carried out so many foundation studies on the extrinsic innervation of the distal gut. The last specialised region to receive attention in this volume is the gallbladder and sphincter of Oddi. Gary Mawe’s group have established themselves as leading investigators of this often overlooked accessory to the gut. Given the amount of trouble that it causes clinically, the biliary system and its motility are medically important subjects for study. The major differences between the organisation of the enteric nervous system of the gallbladder and sphincter of Oddi and that of the extensively studied small intestine are highlighted in this chapter. Since the pioneering study of Paton (1955), the small intestine, particularly of the guinea-pig, has been a favoured preparation for pharmacologists around the world. A vast literature has developed about the effects of drugs acting on a huge range of receptors found in the enteric nervous system and smooth muscle of this preparation. Marcello

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Tonini and his colleagues have summarised an enormous body of work to provide an accessible, yet comprehensive summary of this field. It should be pointed out that the future ability of gastroenterologists to provide treatment for disorders of the gut will rely on the development of new drugs. Understanding the pharmacology of the enteric nervous system is a crucial step in this undertaking. This analysis is greatly extended by the review of Charles Hoyle, Pam Milner and Geoffrey Burnstock on the nature of neuroeffector transmission in the gut. A wide-ranging summary of the transmitters, receptors and pharmacology of neuromuscular transmission is provided in their contribution. Frequently, enteric neurobiologists forget about the secretory and absorptive functions of the gastrointestinal tract, which are so central to its role in digestion. Charles Hoyle and colleagues also give us a wonderful account of the major pathways, largely arising from the submucous plexus, which control epithelial function. Of course, the secretory and absorptive capability of the epithelium is closely coupled with maintenance of an adequate blood supply, as are the requirements for mucosal protection described in Peter Holzer’s review. Neela Kotecha’s chapter summarises what is known about the enteric and extrinsic control of the gut vasculature, providing a valuable foundation for appreciating how different aspects of neuronal control are integrated. Lay people and funding agencies are often particularly interested in what scientists can tell them about the nature of medical disorders. This is as true for the innervation of the gut as it is for any other organ system. Many of the disorders of the gastrointestinal tract are related to the interactions between the immune system and the nerve cells and fibres in the gut wall. Jackie Wood and his many colleagues, over a long period, have been pioneers in this field. In his chapter, Professor Wood provides a compelling account of the central role of mast cells and mediators from leukocytes in modifying and sometimes determining the motor patterns and secretion of the small and large intestine during pathogen challenge. In the chapter by Simon Brookes and Marcello Costa, the cellular organisation of the enteric nervous system and some of the changes associated with gut disorders have been summarised. This raises the question of how the enteric nervous system develops to the normal adult form. This field has made enormous progress in the last decade and at the forefront of the advance has been the New York-based group of Michael Gershon. In a scholarly review, Professor Gershon describes some of the major influences on the colonisation and phenotypic development of enteric neurones. He discusses evidence for the involvement of a number of powerful growth factors and their receptors. In addition, he examines how defects in them may contribute to the aganglionosis of the distal colon, which is one of the most common developmental disorders of the enteric nervous system. The investigations summarised in this volume are beginning to reveal a large repertoire of neural mechanisms present in the digestive tract. It is not possible for a single book to encompass all knowledge about such a large and rapidly growing field. However, the chapters presented here give a good indication of the state of current knowledge. By reading between the lines, the astute reader will also be able to identify some of the current gaps in our knowledge. For example, it should not be surprising that there have been only few attempts to link cellular mechanisms to the functions of the entire organ, either in vitro or in vivo. In only a few cases have investigators been able to establish the circumstances under which a particular neural mechanism actually operates in normal living

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conditions. The gap between cellular physiology and organ physiology is still very wide. It should be clear from the reviews presented here that we now know a lot about what the neural system in the gut can do, but we know much less about how it actually operates under normal conditions. We know even less about how it changes, at the cellular level, to deal with different diets, with pathogens and in disease states. This represents a major challenge for the next generation of investigators. It can be expected that addressing these questions will be invaluable in the rational design of new therapies for disorders of the gastrointestinal tract. Simon Brookes and Marcello Costa

REFERENCES Auerbach, L. (1862). Über einen plexus myentericus, einen bisher unbekannten ganglionervösen Apparat im Darmkanal der Wirbelthiere. Breslau, Morgenstern, E. p. 13. Bayliss, W.M. and Starling, E.H. (1899). The movements and innervation of the small intestine. Journal of Physiology (London), 24, 99–143. Huizinga, J.D., Thuneberg, L., Vanderwinden, J.M. and Rumessen, J.J. (1997). Interstitial cells of Cajal as targets for pharmacological intervention in gastrointestinal motor disorders. Trends in Pharmacological Sciences, 18, 393–403. Langley, J.N. and Magnus, R. (1905). Some observations of the movements of the intestine before and after degenerative section of the mesenteric nerves. Journal of Physiology (London), 33, 34–51. Meissner, G. (1857). Über die Nerven der Darmwand. Zeitschrift für Ration Medezin, 8, 364–366. Paton, W.D.M. (1955). The response of the guinea-pig ileum to electrical stimulation by coaxial electrodes. Journal of Physiology (London), 127, 40P–41P. Sanders, K.M. (1996). A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract. Gastroenterology, 111, 492–515. Trendelenburg, P. (1917). Physiologische und Pharmakologische Versuche über die Dunndarmperistaltik. Naunyn Schmiedebergs Archiv für Experimentelle Pathologie und Pharmakologie, 81, 55–129.

Marcello Costa and Simon Brookes — Editors of this volume

Contributors P.P. Bertrand Department of Physiology University of Melbourne Victoria 3010 Australia J.C. Bornstein Department of Physiology University of Melbourne Victoria 3010 Australia Simon J.H. Brookes Department of Human Physiology and Centre for Neuroscience Flinders University GPO Box 2100, Adelaide South Australia 5001 Geoffrey Burnstock Autonomic Neuroscience Institute Royal Free and University College Medical School Royal Free Campus Rowland Hill Street London NW3 2PF UK Marcello Costa Department of Human Physiology and Centre for Neuroscience

Flinders University GPO Box 2100, Adelaide South Australia 5001 Francesca Crema Department of Internal Medicine and Therapeutics Section of Clinical and Experimental Pharmacology University of Pavia Piazza Botta 10 I-27100 Pavia Italy Fabrizio De Ponti Department of Internal Medicine and Therapeutics Section of Clinical and Experimental Pharmacology University of Pavia Piazza Botta 10 I-27100 Pavia Italy Gianmario Frigo Department of Internal Medicine and Therapeutics Section of Clinical and Experimental Pharmacology University of Pavia Piazza Botta 10 I-27100 Pavia Italy xviii

CONTRIBUTORS

J.B. Furness Department of Anatomy and Cell Biology University of Melbourne Victoria 3010 Australia Michael D. Gershon Department of Anatomy and Cell Biology Columbia University College of Physicians and Surgeons 630 W 168th Street New York NY 10032 USA Beverley Greenwood-Van Meerveld Oklahoma Foundation for Digestive Research Basic Science Labs V.A. Medical Center Oklahoma City OK 73104 USA David Grundy Department of Biomedical Science The University of Sheffield The Alfred Denny Building Western Bank Sheffield S10 2TN UK Kirk Hillsley Department of Anatomy and Neurobiology C423 Health Science Complex The University of Vermont Burlington VT 05405 USA Peter Holzer Department of Experimental and Clinical Pharmacology University of Graz, Universitätsplatz 4 A-8010 Graz Austria

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Charles H.V. Hoyle Department of Anatomy and Developmental Biology University College London Gower Street London WC1E 6BT UK Lee J. Jennings Department of Anatomy and Neurobiology C423 Health Science Complex The University of Vermont Burlington VT 05405 USA Audra L. Kennedy Department of Anatomy and Neurobiology C423 Health Science Complex The University of Vermont Burlington VT 05405 USA Neela Kotecha Department of Physiology Monash University Clayton Victoria-3800 Australia Jacob Krier Oklahoma Foundation for Digestive Research Basic Science Labs V.A. Medical Center Oklahoma City OK 73104 USA Wolfgang A.A. Kunze Department of Anatomy and Cell Biology University of Melbourne Victoria 3010 Australia

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Gary M. Mawe Department of Anatomy and Neurobiology C423 Health Science Complex The University of Vermont Burlington VT 05405 USA Pam Milner Autonomic Neuroscience Institute Royal Free and University College Medical School Royal Free Campus Rowland Hill Street London NW3 2PF UK Michael Schemann Department of Physiology School of Veterinary Medicine Bischofsholer Damm 15/102 D-30173 Hannover Germany Erin K. Talmage Department of Anatomy and Neurobiology C423 Health Science Complex The University of Vermont Burlington VT 05405 USA

Marcello Tonini Department of Internal Medicine and Therapeutics Section of Clinical and Experimental Pharmacology University of Pavia Piazza Botta 10 I-27100 Pavia Italy Kalina Venkova Oklahoma Foundation for Digestive Research Basic Science Labs V.A. Medical Center Oklahoma City OK 73104 USA Jackie D. Wood Department of Physiology College of Medicine The Ohio State University 300 Hamilton Hall 1645 Neil Avenue Columbus Ohio 43210 USA

1 Enteric Reflexes that Influence Motility J.C. Bornstein1, J.B. Furness2, W.A.A. Kunze2 and P.P. Bertrand1 1

Department of Physiology and Department of Anatomy and Cell Biology, University of Melbourne, Victoria 3010, Australia

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The gut exhibits a variety of movements that depend on the region studied and the timing and composition of the last meal. Primary control of these movements is exerted by the enteric nervous system. Except in the oesophagus and the sphincters, external inputs modulate enteric neural activity, rather than directly controlling muscle movements. In isolated intestine, localised mechanical and chemical stimuli excite enteric neurons to produce stereotypic reflexes in the smooth muscle. The reflex circuits form the basis of the circuitry responsible for more complex motor patterns evoked by more generalised stimuli. The enteric neural circuitry responsible for stereotyped reflexes in the guinea-pig ileum has been studied in detail and the neurons involved have been identified. These include at least three types of intrinsic primary afferent neurons (IPANs), a population of ascending interneurons, three populations of descending interneurons, excitatory and inhibitory motor neurons innervating the circular muscle and longitudinal muscle motor neurons. Synaptic transmission in the ascending excitatory reflex pathway is primarily via fast excitatory synaptic potentials (EPSPs) mediated by acetylcholine acting at nicotinic receptors; slow EPSPs mediated via NK3 tachykinin receptors may also be involved. Acetylcholine is less important in descending pathways. Transmission from interneurons to inhibitory motor neurons depends on ATP acting at P2X receptors and tachykinins play a significant, but not exclusive, role in transmission from IPANs to descending interneurons. Transmitters at other synapses in this pathway remain to be identified. These properties of simple reflexes will be important for understanding more complex behaviour. KEY WORDS: myenteric plexus; intestinal reflexes; synaptic transmission; intrinsic primary afferent neurons; enteric neural circuits; neurotransmitters.

INTRODUCTION The gastrointestinal (GI) tract breaks down food into absorbable nutrients by mechanisms that are almost entirely without conscious control. While chewing, swallowing and defaecation are consciously initiated; processes such as digestion, secretion and absorption, and the motility of the GI tract are essentially automatic. Both motility and secretion are under the direct control of the nervous system. This is manifested at several different levels. The enteric nervous system (ENS) is contained 1

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entirely within the wall of the GI tract and can mediate many gastrointestinal functions without input from the central nervous system. Nevertheless, under normal circumstances several levels of external control can be identified. Emotions such as fear have well known gastrointestinal effects without the involvement of a visceral stimulus. In addition, many visceral stimuli alter gastrointestinal behaviour via pathways that include neurons of the central nervous system (e.g. vago-vagal reflexes). However, some stimuli applied to the gut modify gastrointestinal functions via neural pathways that bypass the central nervous system, but include the prevertebral sympathetic ganglia. Furthermore, a variety of hormonal mechanisms can also regulate intestinal function. For example, the interdigestive motor patterns of the stomach and both acid secretion and gastric emptying after a meal are modified by the chemical composition of the chyme in the small intestine (Lin et al., 1990a,b; Spencer et al., 1990; Orloff et al., 1992). The latter two effects are depressed when all neural pathways into the stomach are interrupted, but they can still be observed. This implies that both the extrinsic nerves and hormonal influences are involved in the control of gastric function by the contents of the small intestine. Behaviours evoked by stimulation of the GI tract, that are independent of the endocrine system, fall into the general class of entero-enteric reflexes. Many, notably the vago-vagal reflexes referred to above (for review see Grundy and Scratcherd, 1989), depend on the integrity of connections between the GI tract and the central nervous system. Others depend only on intact connections between the GI tract and the prevertebral sympathetic ganglia (for review see Szurszewski and King, 1989). A characteristic feature of the extrinsic nervous supply to the GI tract is that efferent nerves provide terminals largely to the intrinsic ganglionated plexuses rather than directly to the muscle, except in the sphincters and in the striated muscle of the oesophagus (Furness et al., 1999). Thus, the extrinsic reflex pathways coordinate the activities of different regions of the GI tract largely by modifying the activity of enteric neurons, rather than via direct effects upon the muscle, the enteric neurons representing a final common level of control of gastrointestinal function. This chapter focuses on the control of motility. In particular, we discuss recent data about the neural circuitry responsible for motility reflexes and the way this circuitry is organised to produce the motor programs responsible for complex patterns of movement. MOTILITY REFLEXES AND MOTOR PROGRAMS The neural circuits that control real motor behaviours anywhere in the body are complex and are generally studied by the use of reduced preparations in which the number of factors affecting a motor response is kept to a minimum. In the extreme case, such reduced preparations give rise to simple reflexes in which each element of the circuit can be readily identified. At the next level, the neural circuits that produce more complex behaviours can be considered to act as motor pattern generators, producing coordinated and often complex interactions between different muscles to give rise to stereotyped behaviour. To take an example from another system, in the simple tendon jerk reflex, primary afferent neurons from muscle spindles directly excite motor neurons. However, when the spinal cord is isolated from central input, appropriate sensory stimuli trigger the entire suite of coordinated movements required for normal walking. The difference between the simple reflexes of reduced preparations and the more complex, but still stereotyped, motor

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patterns is that the simple reflexes always have the same form when initiated by a specific stimulus, while a single organ can generate several distinct motor patterns. In systems where pattern generators have been studied, including the GI tract, the neurons in simpler reflex circuits are an integral part of the pattern generator circuits. GI tract reflexes vary in complexity as they do in all other parts of the nervous system. Even if those reflexes involving the central nervous system or the prevertebral ganglia are excluded, enteric reflexes range from local constrictions resulting from pinches applied to the serosal surface to coordinated waves of activity sweeping along the intestine. The former may be analogous to the spinal monosynaptic reflex, while the latter are due to activation of complex motor patterns controlled by circuitry analogous to the motor pattern generators of the central nervous system. This chapter explores the circuitry underlying simple reflexes and the reflex motor patterns seen in the gut, with an emphasis on the ileum of the guinea-pig, about which we have the most detailed information.

PHYSIOLOGICAL STUDIES OF ENTERIC REFLEXES The first studies of intestinal reflexes were performed towards the end of the nineteenth century when it was shown that various stimuli applied to the intestine trigger movement of intestinal content or secretion across the intestinal wall (Nothnagel, 1882; Mall, 1896; Bayliss and Starling, 1899, 1900a,b). By 1899, it was clear that a substantial part of the mechanisms that control propulsion of intestinal contents must be confined to the intestinal wall. Bayliss and Starling (1899) showed that a bolus inserted into a region of intestine triggered a complex motor pattern which caused the bolus to pass anally along the intestine even when all connections to the central nervous system had been severed. This study identified two reflexes which appear to underlie much of the normal propulsive behaviour of the intestine; the ascending excitatory reflex, in which the circular muscle contracts oral to the point of stimulation, and the descending inhibitory reflex, in which the circular muscle relaxes anal (or aboral) to it. Bayliss and Starling believed that any stimulus that evoked one of these reflexes would evoke the other as well and they termed this the “Law of the Intestine”. Other workers, including Langley and Magnus (1905), quickly confirmed that propulsion of material along the intestine did not depend on the central nervous system. However, the motility patterns of the intestine in the presence, and absence, of food in vivo are more complex than the propulsive response to an inserted stimulus in vitro. STUDIES OF MOTOR PATTERNS IN VIVO Using radiometric methods, Cannon identified several distinct types of propulsive behaviour in the presence of food in vivo (Cannon, 1902, 1906, 1912). These can be broadly described as segmentation, peristalsis and peristaltic rushes. Segmentation seems to be a mixing behaviour and involves rhythmic stationary contractions that repeatedly divide and redivide a mass of intestinal content. In contrast, peristalsis and peristaltic rushes each propel the intestinal content in an anal direction, with the difference between them being a matter of degree, duration and velocity. He also identified propulsion of content in an oral direction, antiperistalsis.

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Radiometric methods require the presence of a material opaque to X-rays in the lumen of the GI tract and have been extended by the use of radioactive labels within the food so that its movement can be monitored with scintigraphy. Such studies, in combination with the multisite recording methods discussed below, have provided valuable information about the effects of varying the composition of a meal on the motility of the intestine. An important observation is that the rate of movement of a meal through the stomach and along the intestine is critically dependent on its composition. Chemically inert meals move along the intestine more rapidly than meals with significant nutritive content, with the relative proportions of fat, carbohydrate and protein being significant determining factors (Eeckhout et al., 1984; Schemann and Ehrlein, 1986c; Buhner et al., 1988; Buhner and Ehrlein, 1989; Siegle, Schmid and Ehrlein, 1990; Schmid and Ehrlein, 1993). Even the pattern of contractions seen within the intestine varies according to the chemical composition of the meal. This is illustrated by a study by Schemann and Ehrlein (1986c) who found that meals containing nutrients induced a larger proportion of stationary contractions, attributed to segmentation, than did non-nutritive meals. The rate of propagation of propulsive contractions (peristalsis) was unchanged, but the length over which they propagated was substantially reduced by increased nutrient density. Thus, segmentation predominates when nutrient is present in lumen, while peristalsis predominates when the chyme is nutrient free (Schemann and Ehrlein, 1982, 1986b,c; Siegle and Ehrlein, 1988; Buhner and Ehrlein, 1989; Schmid and Ehrlein, 1993; Huge, Weber and Ehrlein, 1995). In many species, different patterns of activity occur in fed and fasted states. Studies using a variety of widely spaced recording devices – strain gauges, extracellular electrodes, multichannel pressure sensing catheters – have shown that when an animal is fasted a complex wave of activity propagates repeatedly, but slowly, along the intestine from the stomach to the caecum. The behaviour is variously known as the migrating myoelectric complex (MMC), the migrating motor complex or the interdigestive cycle (for a recent review of this, and other motility patterns in vivo, see Hasler (1999)). The MMC essentially consists of an anally propagating wave of strong excitation of the intestinal muscle that alternates with a longer period of very low activity or possibly inhibition of the muscle (Figure 1.1). These two phases (Phase III and Phase I, respectively) are separated by periods of irregular muscular activity (Phases II and IV). Interestingly, while MMCs in the small intestine have never been convincingly recorded in isolated tissue, they are seen in extrinsically denervated GI tract in vivo. Thus, the neural circuit generating MMCs must be contained within the intestinal wall, i.e. part of the enteric nervous system (Sarna et al., 1981). In many animals, including humans, MMCs disappear immediately after the animal eats and the activity of the intestine changes to the fed, or post-prandial state described above. MMCs are widely identified with the gastric antrum, the duodenum, jejunum and ileum. However, migrating motor patterns, which have been termed MMCs, have also been observed in the colon, but not in the caecum (Sarna, Prasad and Lang, 1988). The colonic MMCs are independent of those in the small bowel. Another motor pattern seen predominantly in the fasted state is the giant migrating contraction (GMC): a large contraction that is much briefer than phase III of the MMC, propagates rapidly and continues for a substantial distance (Sarna, Prasad and Lang, 1988; Sarna, 1991a,b; Otterson and Sarna, 1994; Sethi and Sarna, 1995). GMCs are seen in the ileum, the caecum and the

ENTERIC REFLEXES THAT INFLUENCE MOTILITY

J1 J2 J3 J4 J5 J6 J7 J8 J9 J10 J11 J12

III

I

II

5

III

24 min

80 mmHg

Figure 1.1 Multichannel manometric recordings taken from 12 sites (J1–J12) within the jejunum of a fasting human. Each recording site was separated from its neighbours by 2 cm. The different phases of the migrating motor complex are indicated for the upper trace as Phase I (I), Phase II (II) and Phase III (III), in this set of recordings Phase IV was not readily discernable. Recordings provided by Prof. Sushil Sarna.

colon and are clearly distinct from MMCs, but are similar to the peristaltic rushes seen in the fed state. In the fed state, the distal ileum has a characteristic pattern of motility not seen elsewhere in the GI tract (Fich et al., 1990). When a meal arrives in the terminal ileum of the dog, a series of high frequency contractions of the circular muscle is initiated. These contractions occur more frequently than the slow waves that are usually thought to control the frequency of contractions within the intestinal muscle. They are seen only when the material entering the terminal ileum contains nutrient; i.e. they are not seen during an isotonic saline infusion. As yet their function is unknown. Thus, in vivo studies have demonstrated the presence of several distinct motor patterns whose presence and persistence depends on the region being studied, the content of that region and of other regions. The relationship between some of these behaviours and the intestinal microcircuitry mediating simple reflexes will be discussed in the last section of this chapter. REFLEXES AND MOTOR PATTERNS IN ISOLATED SYSTEMS The work of Mall (1896) and then Bayliss and Starling (1899; 1900a,b) may fairly be described as the first substantial studies of motor patterns in isolated segments of intestine and hence in reduced systems. The results were essentially similar to those seen over the next 2 decades by other workers in studies performed on segments of intestine in vitro (Magnus, 1904; Langley and Magnus, 1905; Trendelenburg, 1917). Stimulation of the intestinal segment by irritating the mucosa or by distension with either a solid or liquid bolus evoked a stereotyped propulsive movement of the intestine that tended to move the stimulus in an oral to anal direction. The movement involved a shortening of the longitudinal muscle, followed by a contraction of the circular muscle oral to the stimulus and,

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in some cases, a relaxation of the circular muscle anal to the stimulus. This last component of the response was not seen by all subsequent workers (e.g. Bolzer, 1949b), perhaps because the smooth muscle must first generate some active tension for a relaxation to be detected. Bolzer (1949a,b) recognised that peristalsis evoked by stroking the mucosa or by distension was a more complex phenomenon than a simple reflex. He found that contractions evoked oral to a solid bolus propagate anally along the intestine at a rate faster than the movement of the bolus itself. The contraction passed over the bolus implying that movement of the stimulus is not the sole mechanism leading to anal propagation of the contractions in the circular muscle. He speculated that the consistency of the bolus may be an important determinant of the final pattern of the propulsive behaviour. The idea that differing stimuli evoke different patterns of contractile behaviour has been supported by studies of intestinal segments isolated from the central nervous system in vivo and in vitro by Hukuhara and colleagues in the late 1950s and throughout the 1960s (Hukuhara, Yamagami and Nakayama, 1958; Hukuhara and Miyake, 1959; Hukuhara, Nakayama and Sumi, 1959; Hukuhara et al., 1959; Hukuhara, Nakayama and Nanba, 1960a,b, 1961; Hukuhara, Sumi and Kotani, 1961; Hukuhara and Fukuda, 1965; Hukuhara, Neya and Tsuchiya, 1969). In these studies, stretching the intestinal wall inhibited ongoing contractions oral to, and anal to, the point of stimulation in the dog and the rabbit small intestine, but mechanical or acid stimulation of the mucosa evoked contraction on the oral side and inhibition on the anal side. The response to a specific stimulus also depended on the species tested, with guinea-pigs showing only oral excitation as a result of chemical or mechanical stimulation of the mucosa. In this species, stretch evoked contractions on both the oral and the anal side, unlike the responses seen in dogs and rabbits. Anal excitation evoked by stretch has been seen only intermittently in subsequent studies of guinea-pig intestine (see below). Hukuhara’s studies demonstrated several important points about the motor behaviour of the intestine. First, removal of the myenteric plexus interrupted transmission of reflex activity along the intestine, but disruption of the submucous plexus did not. Thus, the myenteric plexus probably contains the interneurons and the cell bodies of motor neurons mediating these reflexes. Second, reflex responses in the circular muscle to chemical and mechanical stimulation of the mucosa were essentially identical. Third, mucosal stimulation does not always evoke responses similar to those evoked by stretch. Thus, the nature of the motor program activated by a stimulus may depend on the overall state of the tissue at the time the stimulus is applied. Finally, they showed that chemical stimuli (acetylcholine, histamine, pilocarpine or BaCl2) applied to the serosa could evoke stereotyped motor responses in the intestine. The variety of stimuli employed by Hukuhara and his colleagues, raises the issue of the modes of stimulation appropriate for studies of motor reflexes. Recently, three distinct sensory modalities have been shown to excite stereotyped intestinal reflexes consisting of excitation oral and inhibition anal to the point of stimulation in isolated segments of small intestine or distal colon in vitro. Distension, mechanical stimulation of the mucosa and acid applied to the mucosa, all evoke such reflexes (Smith and Furness, 1988; Smith, Bornstein and Furness, 1990, 1991, 1992; Smith et al., 1992; Yuan et al., 1991; Grider, 1994; Grider and Jin, 1994; Foxx-Orenstein, Kuemmerle and Grider, 1996; Smith and McCarron, 1998).

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Despite the dependence of propulsion and local motor patterns on the chemical composition of the chyme (see above), mechanical stimuli are nearly always used in studies of enteric motility reflexes, perhaps because the onset and offset of the stimulus can be well controlled by the investigator. Distension, or a localised stretch applied to the intestinal wall, has been the stimulus of choice in the vast majority of studies, either in vivo or in vitro. This does not affect chemoreceptors and so distension is a relatively pure stimulus. However, exclusive use of any stimulus modality to analyse propulsive behaviours carries the inherent assumption that the neural circuitry activated by that stimulus includes all the elements that might be excited by other stimuli. Detailed electrophysiological analysis of the functional classes of neurons excited by different sensory modalities in the guinea-pig small intestine indicates that these different stimuli excite common populations of circular muscle motor neurons (Smith, Bornstein and Furness, 1992). However, as discussed below, many of the primary afferent neurons excited by the different sensory modalities that trigger intestinal reflexes are functionally distinct, indicating that this assumption does not hold. The other underlying assumption of exclusive use of distension to initiate reflexes is that the motor program excited by this stimulus provides the basic pattern underlying all motility in the GI tract. Although the basic reflexes excited by the three distinct sensory modalities appear similar, the results outlined earlier in this chapter indicate that chemical stimuli may initiate motor patterns distinctly different from those evoked by distension. Thus, as “physiological stimuli” used in most studies of enteric reflexes are constrained to a single sensory modality or a mix of mechanical stimuli (see below), they may excite only a proportion of the behaviours of which the stimulated region is capable. When an isolated segment of intestine is distended by a static increase in the pressure of isotonic saline contained within the lumen, it exhibits rhythmic contractions of the circular muscle (e.g. Trendelenburg, 1917; Tonini et al., 1981; Buchheit and Buhl, 1991). These contractions propagate anally, propelling the saline in the same direction and can empty the entire segment. Prior to each circular contraction, the longitudinal muscle contracts in what is known as the “preparatory phase” of peristalsis. In a variant of this method, a length of intestine is steadily distended with increasing amounts of physiological saline until a propulsive reflex is generated (Bülbring and Lin, 1958; Kottegoda, 1969; Waterman, Costa and Tonini, 1992, 1994a). The behaviour evoked consists of a ring of contraction of the circular muscle, which typically starts at the oral end of the intestinal segment and propagates anally (Hennig et al., 1999; SchulzeDelrieu, 1999). The contraction usually occludes the lumen and is preceded by a relaxation of the circular muscle, so that the saline within the segment is propelled anally and expelled from its anal end. This method has the advantage of allowing a threshold for the initiation of the behaviour to be determined along with both the pressure changes and the transport of liquid through the intestinal segment. Such studies have shown how complex the propulsion of material along the intestine can be. For example, intraluminal acid reduces the threshold pressure needed to trigger propulsion, but leads to a reduced flow at any given stimulus pressure once threshold is exceeded (Hukuhara, Nakayama and Sumi, 1959). Similarly, blockade of inhibitory motor neurons reduces the threshold pressure for initiation of the contraction, but can lead to a complete cessation of propulsion (Waterman and Costa, 1994, 1994b). Moreover, while these methods produce responses that are similar to the behaviours seen in vivo, there are several possible differences. For example,

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the normal content of the intestine is not as chemically inert as physiological saline and does not steadily, or evenly, distend long segments of intestine. A simpler behaviour can also be evoked by gradual filling of a segment of the GI tract. This is reflex accommodation which involves a relaxation of the circular muscle at the site of distension when the pressure inside the stomach (Azpiroz and Malagelada, 1990; Hennig et al., 1997), the small intestine (Waterman, Costa and Tonini, 1994a) or the large intestine (Davison and Pearson, 1979; Ciccocioppo et al., 1994) exceeds a threshold value. This threshold is typically below that required to evoke a propulsive reflex (Waterman, Costa and Tonini, 1994a). Thus, reflex accommodation may be part of the preparatory phase of the propulsive behaviour evoked in the intestine by a fluid distension, as well as a separate behaviour in its own right. Saline distensions have also been used to study motor behaviours in the stomach (Hennig et al., 1997) and colon (Ciccocioppo et al., 1994; Smith and Robertson, 1998). In the latter, however, artificial faecal pellets, which represent a more physiologically realistic stimulus, are often used (Costa and Furness, 1976; Foxx-Orenstein and Grider, 1996; Kadowaki, Wade and Gershon, 1996; Foxx-Orenstein, Jin and Grider, 1998). The pellet is inserted into the oral end of an isolated segment of colon and the time taken to expel it from the anal end is measured. This time is consistent between trials and provides a reliable measure of the propulsive behaviour of the colon. The propulsive mechanism is very similar to that in the small intestine with pellets being propelled anally by waves of contraction within the circular muscle that are preceded by relaxation of the same muscle layer. An elegant refinement of this method has been the use of a mobile balloon attached to a device for measuring rate of transit as the artificial pellet (Crema, Frigo and Lecchini, 1970; Frigo and Lecchini, 1970; Tonini et al., 1989; Ciccocioppo et al., 1994; Onori et al., 2000). This allows the size of the balloon to be varied between trials and the pressure generated to be determined along with the speed of propulsion. These studies have shown that, in the rabbit colon, the dependence of propulsion on inhibitory neuromuscular transmission is greater for larger pellets. Another type of experiment uses a confined stimulus, which is held stationary, to evoke reflexes within the intestine. Responses are measured as changes in the contractility of the smooth muscle layers (Holzer, 1989; Tonini and Costa, 1990; Holzer, Schluet and Maggi, 1993; Maggi et al., 1994; Spencer, Walsh and Smith, 1999, 2000), as electrical responses in the muscle (Hirst, Holman and McKirdy, 1975; Smith and Furness, 1988; Smith, Bornstein and Furness 1990, 1991; Smith et al., 1992; Yuan et al., 1991) or as changes in the activity of enteric neurons measured either extracellularly or intracellularly (Hirst and McKirdy, 1974; Hirst, Holman and McKirdy, 1975; Bornstein et al., 1991a; Smith, Bornstein and Furness, 1992). Local distension with a balloon evokes an excitatory reflex oral to the point of stimulation (Holzer, 1989; Smith, Bornstein and Furness, 1990, 1992; Tonini and Costa, 1990; Holzer, Schluet and Maggi, 1993; Maggi et al., 1994), but this reflex typically propagates a few centimetres orally rather than anally (Smith, Bornstein and Furness, 1990, 1992). Such stimuli (and also mechanical and chemical stimuli applied to the mucosa) may excite local reflexes which do not propagate at all. However, recent studies by Spencer, Walsh and Smith (1999, 2000) found that both radial distension and mechanical stimulation of the mucosa evoked anally propagating contractions of both longitudinal and circular muscle in the guinea-pig ileum.

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In contrast, electrophysiological studies have rarely shown any evidence that the excitatory reflex contractions evoked by stationary distensions propagate anally in the small intestine. Similarly, stimulation of the mucosa either mechanically or with acid evokes an orally propagating excitatory reflex (Smith and Furness, 1988; Smith, Bornstein and Furness, 1991), which has yet to be shown to propagate anally. Thus, the anal propagation of contraction seen in the propulsion experiments cannot be explained by a simple combination of reflexes evoked by stationary stimuli applied to a confined region of intestine. This will be discussed in the last section of this chapter.

NEURAL CIRCUITS MEDIATING REFLEXES EVOKED BY STATIONARY STIMULI IN VITRO The basic circuit excited by a stationary stimulus is now clearly established for the small intestine of the guinea-pig (Figure 1.2). This is based on the responses of enteric neurons and the intestinal muscle and correlated electrophysiological, immunohistochemical and morphological studies of individual enteric neurons. In combination, these experiments

Figure 1.2 Diagram showing the neural circuitry underlying simple reflexes within the guinea-pig small intestine. This circuit, which contains seven types of neurons, has been inferred from many experimental sources. At each point along the intestine there is a network of IPANs with outputs to all other neuronal subtypes other than SOM descending interneurons. The interneurons form chains along the intestine and also make specific connections to motor neurons (ascending interneurons to excitatory motor neurons, descending interneurons to inhibitory motor neurons), which also receive input from local IPANs. The identities of specific neuronal subtypes are shown below the main circuit and are used in subsequent figures.

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have allowed virtually all component neurons of the reflex pathways to be identified both structurally and physiologically. A stationary stimulus excites one or more populations of intrinsic primary afferent neurons (IPANs), which project circumferentially to excite local motor neurons and separate populations of orally and anally directed interneurons. Orally directed (ascending) interneurons activate both excitatory motor neurons and other ascending interneurons, while the anally directed (descending) interneurons excite other descending interneurons and inhibitory motor neurons. A significant subset of the IPANs project anally to contact inhibitory motor neurons (Johnson et al., 1996; Johnson, Bornstein and Boucher, 1998; Bian, Bertrand and Bornstein, 2000). These studies are discussed in detail in the remainder of this section. CLASSIFICATION OF ENTERIC NEURONS There have been four primary methods of classifying enteric neurons prior to identification of their functions: morphological, neurochemical, pharmacological and electrophysiological. Morphological studies date back to the work of Dogiel (1899) in the nineteenth century. This work provided the basic description of the major morphological types of cell bodies seen in the many subsequent studies (e.g. Stach, 1979, 1989; Scheuermann and Stach, 1983; Brehmer and Stach, 1998; Brehmer et al., 1998). In the small intestine of the guinea-pig, two broad classes of neurons can be identified using morphological techniques: large multipolar neurons are usually called Dogiel type II neurons, and several poulations of monoaxonal neurons (Figure 1.3) (Furness, Bornstein and Trussell, 1988).

Figure 1.3 Representative silhouettes of myenteric neurons from the guinea-pig duodenum that had been injected with neurobiotin during electrophysiological recordings (Clerc et al., 1998a). These shapes are typical of the shapes of myenteric neurons in the duodenum, ileum, proximal colon and distal colon.

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A subset of the monaxonal neurons has short lamellar dendrites; these are Dogiel type I neurons. Other subsets of the monoaxonal neurons include a population with long filamentous dendrites and a population of small neurons (Furness, Bornstein and Trussell, 1988). Small neurons with short filamentous dendrites have been identified in the guineapig duodenum (Clerc et al., 1998a,b) and in the proximal colon (Messenger, Bornstein and Furness, 1994) and distal colon of this species (Lomax et al., 1999). These small filamentous neurons have similar projection patterns to the small neurons of the ileum and may represent the same functional class. The immunohistochemical studies that have led to classification of enteric neurons according to their neurochemistry and projections are described in detail elsewhere in this volume (see Chapter 11 of this volume). Electrophysiological identification of enteric neurons began in the early 1970s with studies using extracellular recording methods (for review see Wood, 1975). These were rapidly overtaken by classification schemes based on intracellular recordings that began a little later. The first substantial intracellular study of enteric neurons published was that of Nishi and North (1973) who divided neurons into type 1 and type 2 on the basis of their firing properties, specifically whether they adapted rapidly or slowly to an imposed depolarization. In work published a few months later, Hirst, Holman and Spence (1974) divided myenteric neurons of the duodenum into two groups on the basis of their synaptic inputs and the after-potentials following their action potentials. S-neurons exhibited prominent fast excitatory synaptic potentials (EPSPs) in response to electrical stimulation of synaptic inputs and had only a brief (<100 ms) hyperpolarization following an action potential. The second population, AH-neurons, appeared to lack fast EPSPs and their action potentials were followed by prolonged after-hyperpolarizing potentials (AHPs). The AH/S classification scheme has proved highly successful in the small intestine of the guinea-pig; in part, because intracellular dye injection has shown that all AH-neurons have Dogiel type II morphology, while all S-neurons are monoaxonal (for review see Bornstein, Furness and Kunze, 1994). An important extension has been the recognition that all AH-neurons have inflections on the falling phases of their action potentials (Iyer et al., 1988; Schutte, Kroese and Akkermans, 1995) and that these persist even when the AHP is suppressed by synaptic input (Kunze et al., 1998). In the duodenum and distal colon, some neurons classified as Dogiel type II neurons by shape lack a prolonged AHP, but still have an inflection on the falling phase of the action potential (Clerc et al., 1998a; Lomax et al., 1999). Although intracellular injection of dye allows prediction of whether a neuron belongs to the AH- or S-category from its morphology, the experimental conditions are crucial for determining some electrophysiological properties of enteric neurons. For example, the characteristic AHP of an AH-neuron is not prominent in Dogiel type II neurons in stretched preparations when the muscle is free to contract (Kunze et al., 1998), but is seen in all such neurons when muscle contraction is blocked with L-type calcium channel blockers (Iyer et al., 1988). Similarly, three recent reports differ in their description of the firing of ascending interneurons during prolonged depolarizations and in whether they exhibit slow EPSPs. One found that such neurons are rapidly accommodating under all conditions tested and exhibited slow EPSPs when synaptic inputs were stimulated electrically (Kunze et al., 1997), while another (Brookes et al., 1997) reported that they are slowly accommodating and lacked slow EPSPs. These studies differ in that the former was

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carried out in freshly isolated tissue, while the latter was performed with neurons that had been in organ culture for about 3 days. In the third study, ascending interneurons were found to be slowly accommodating, but also to have slow EPSP input (Smith, Burke and Shuttleworth, 1999). Where possible in the discussion below, neurons are categorized on the basis of both their electrophysiology and their morphology determined by intracellular dye injection from the recording electrode. REFLEXES EVOKED BY LOCAL DISTENSIONS In the small intestine (Costa et al., 1985; Grider, 1989; Holzer, 1989; Tonini and Costa, 1990; Holzera, Schluet and Maggi, 1993; Tonini et al., 1996), and in the colon (Costa and Furness, 1976; Grider and Makhlouf, 1992; Grider, 1994; Grider and Jin, 1994; FoxxOrenstein, Kuemmerle and Grider, 1996), distension of the intestinal wall with a stationary balloon or a mechanical stretching device evokes a contraction that extends several centimetres orally from the stimulus. In the colon, such stimuli also evoke relaxations on the anal side of the stimulus site (Costa and Furness, 1976; Grider and Makhlouf, 1992; Grider, 1994; Grider and Jin, 1994; Foxx-Orenstein, Kuemmerle and Grider, 1996; Spencer, McCarron and Smith, 1999). The first electrophysiological studies of reflexes evoked by balloon distension focussed on anally directed reflex pathways and showed that inflation of a balloon inserted into the lumen of a segment of intestine evoked inhibitory junction potentials (IJPs) in the circular muscle 2–3 cm anal to the balloon (Hirst and McKirdy, 1974; Hirst, Holman and McKirdy, 1975). Essentially identical results have been obtained in subsequent electrophysiological studies (Figure 1.4B) (Smith, Bornstein and Furness, 1990; Yuan et al., 1991; Yuan, Furness and Bornstein, 1992). A second finding of the early electrophysiological studies of distension evoked anally directed reflexes was that IJPs evoked by distension were often followed by excitatory junction potentials (EJPs), although the EJPs were much more labile (Hirst, Holman and McKirdy, 1975). This descending excitation may be the electrophysiological equivalent of the anally propagating contractions that pass over a stimulating bolus as described by Bolzer (1949b) and others. A similar descending excitation has also been observed in both the ileum (Spencer, Walsh and Smith, 1999, 2000) and the distal colon (Smith et al., 1992). However, electrophysiological studies of the guinea-pig ileum have failed to consistently observe a descending excitation. The reason(s) for this remain unclear. Electrophysiological analysis of orally directed reflex pathways in the small intestine excited by stationary distensions has confirmed that the ascending reflex contractions of the circular muscle are accompanied by EJPs in individual muscle cells (Figure 1.4A) (Smith, Bornstein and Furness, 1990; Yuan et al., 1991; Yuan, Furness and Bornstein, 1992). They have also shown that an ascending inhibitory pathway is activated by distension (Smith et al., 1990), although this has not been seen in contractility studies, possibly because of the lack of background tension discussed above. Both ascending excitatory and ascending inhibitory reflexes have been seen in the distal colon (Smith et al., 1992). Stationary balloon distension also evokes electrophysiologically detectable reflexes in various regions of the stomach (Yuan, Brookes and Costa, 1997). However, the predominant

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Figure 1.4 Intracellular recordings (upper traces each panel) of (A) an excitatory junction potential (EJP) evoked in a circular muscle cell by a distension with a balloon located 15 mm anal to the impaled muscle cell and (B) an inhibitory junction potential (IJP) evoked in a circular muscle cell by a distension applied 15 mm oral. The lower traces in each panel show when the distension was applied and removed; the small fluctuations in these traces reflect a time marker operating every 2 s. In both cases, contractions were blocked by addition of nicardipine to the bathing solution. No EJP was recorded on the anal side of the distension. Note, prominent slow waves were present in each preparation.

response appears to be inhibition of the circular muscle both oral and anal to the distending stimulus. REFLEXES EVOKED BY MECHANICAL OR CHEMICAL STIMULATION OF THE MUCOSA Electrophysiological and contractility studies in the guinea-pig small intestine (Smith and Furness, 1988; Yuan et al., 1991; Yuan, Furness and Bornstein, 1992; Spencer, Walsh and Smith, 1999, 2000), contractility studies in the guinea-pig colon (Spencer, McCarron and Smith, 1999) and contractility studies in the rat colon and human jejunum in vitro indicate that mechanical stimulation of the mucosa evokes reflexes similar to those evoked by distension of the same preparations (Grider, 1994; Grider and Jin, 1994; Foxx-Orenstein, Kuemmerle and Grider, 1996; Smith and McCarron, 1998). In each preparation, disturbing the mucosa either by brushing the mucosal surface or, in the small intestine, by compressing the villi evokes excitation oral (Figure 1.5A) and inhibition anal to the region stimulated (Figure 1.5B). The properties of these reflexes are discussed below. Studies of chemical stimuli applied to the mucosa in vitro are rare. Dilute acid applied to the mucosa of the distal ileum of the guinea-pig evoked ascending excitatory and descending inhibitory reflexes similar to those evoked by mechanical stimulation of the mucosa (Smith, Bornstein and Furness, 1991). The responses were transient and when stimuli were repeated within a refractory period of about 2 min, no further response was

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Figure 1.5 Intracellular recordings of (A) an EJP evoked by mucosal compression applied 15 mm anally and (B) an IJP evoked by mucosal compression applied 15 mm orally (arrows show duration of the compression). Contractions were blocked in each preparation with nicardipine. No EJP was recorded on the anal side of the compression. Slow waves were recorded in (A), but not in (B).

evoked. As yet other potential chemical stimulants have not been shown to excite reflexes in vitro, although certain fatty acids can trigger peristalsis in vivo (Thomas and Baldwin, 1971; Baldwin and Thomas, 1975; Stewart and Bass, 1976a,b) and fatty acids and other nutrients alter intestinal transit of a meal (Schemann and Ehrlein, 1986a,c; Ehrlein et al., 1987; Richardson et al., 1991; Huge, Weber and Ehrlein, 1995). INTERACTIONS BETWEEN REFLEXES EVOKED BY DIFFERENT STIMULI Interactions between reflexes evoked by different stimuli have been studied using a preparation in which distension was applied from the serosal surface and mechanical stimulation was applied to the mucosa and responses were recorded electrophysiologically from the circular muscle. Both distension evoked responses and responses to mucosal stimuli are transient even when the stimulus is maintained (Smith and Furness, 1988; Smith, Bornstein and Furness, 1990; Yuan et al., 1991). This is modality specific (Smith, Bornstein and Furness, 1991; Yuan et al., 1991); when the membrane potential change in the muscle evoked by distension has completely disappeared, mucosal stimulation still evokes a reflex, and vice versa. Indeed, maintained mucosal stimulation markedly enhances responses to distension even when any response to the former has vanished (Smith, Bornstein and Furness, 1991). These results indicate that the two types of stimulus activate reflex pathways that are at least partially separate. The relationship between the reflex pathways excited by acid pH and those excited by distension is similar. Prior treatment with dilute acid enhances distension evoked reflexes

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when the response to acid has disappeared (Smith, Bornstein and Furness, 1991). However, as the final concentration of acid at the level of the mucosa is unknown, it is difficult to determine the physiological significance of this observation. There are also interactions between reflexes evoked by similar stimuli at different sites. For example, if the intestine is distended at two distinct sites, then the response to the more distant distension is depressed if applied within 2 min of the nearer distension (Yuan, Furness and Bornstein, 1992). The depression is seen in both ascending and descending pathways and may be due to interference with transmission within the pathways, rather than an interaction at the level of the smooth muscle. REFLEX RESPONSES RECORDED FROM NEURONS Many results of studies of reflexes evoked in the circular muscle by either distension or mechanical stimulation of the mucosa are paralleled by observations of the activity of myenteric neurons following similar stimuli. Hirst and coworkers (Hirst and McKirdy, 1974; Hirst, Holman and McKirdy, 1975) found that luminal distension evoked bursts of fast EPSPs in virtually all myenteric S-neurons lying 2–5 cm anal to the stimulating balloon. AH-neurons did not respond to this stimulus. Many S-neurons responded only transiently to distension; the latencies of responses in these neurons were relatively short. Other S-neurons exhibited prolonged responses with much longer latencies even when distension was brief. Both mucosal deformation and distension from the serosal surface evoked bursts of fast EPSPs in S-neurons lying either oral (Figure 1.6A) or anal to the region stimulated (Figure 1.6B) (Bornstein et al., 1991a; Smith, Bornstein and Furness, 1992). The responses to distension were transient with maintained distension (Figure 1.6) and the number of fast EPSPs evoked declined markedly when distensions were repeated within 2 min of each other. When the mucosa was deformed, some neurons on the anal side exhibited very prolonged responses, but most responses in either orally or anally located neurons were transient (Figure 1.6) and repeated stimuli led to marked declines in the number of fast EPSPs evoked. There has been no similar analysis of the responses to chemical stimulation of the mucosa, although chemical stimulation of the mucosa evokes both fast and slow EPSPs in S-neurons lying circumferential to the site of stimulation (Kunze, Bornstein and Furness, 1995; Bertrand et al., 1997, 1998). Smith, Bornstein and Furness (1992) also showed that the reflex pathways excited by distension and deformation of the mucosa converge onto the same S-neurons, which were identified as motor neurons by their projections (see below). This study confirmed that stimulation via one modality evokes a normal reflex even when the response to the other stimulus at the same site had completely disappeared (see above). These results imply that separate populations of IPANs mediate the two reflexes, but that the motor neurons are the final common pathway for all the reflex circuits. While convergence of the responses to the different sensory modalities was not unexpected, a second type of convergence was more surprising. A significant subset of the S-neurons received synaptic input from both orally and anally directed reflex pathways (Smith, Bornstein and Furness, 1992). Most of these neurons were identified morphologically as longitudinal muscle motor neurons, others may have been circular muscle motor neurons or even interneurons.

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Figure 1.6 Responses in two myenteric neurons to distension and brushing the mucosa at the same site. The neuron illustrated in (A) lay oral to the stimuli and thus was in the ascending reflex pathway. The neuron illustrated in (B) lay anal to the stimuli and was in the descending reflex pathway. In each case, the responses consisted of a transient burst of fast EPSPs. Both neurons were identified from their projections as circular muscle motor neurons, see Smith, Bornstein and Furness (1992). In both (A) and (B), the onset of the mucosal stimulation is indicated by the arrow.

IDENTITIES OF NEURONS IN THE REFLEX PATHWAYS INTRINSIC PRIMARY AFFERENT NEURONS It has recently been proved that some intestinal reflexes are mediated by primary afferent neurons with cell bodies in the enteric nervous system. Studies in rat colon and guinea-pig small intestine have shown that, after degeneration of all extrinsic nerve terminals in the gut wall, mechanical stimulation of the mucosa evokes ascending and descending reflexes indistinguishable from those seen when the extrinsic nerve supply is intact (Grider and Jin, 1994; Furness et al., 1995). While the cell type that detects this mechanical stimulus has not been identified, these results indicate unequivocally that some primary afferent neurons have their cell bodies within the intestinal wall. That is, there must be IPANs that are either directly sensitive to mechanical distortion of the mucosa or sensitive to some mediator released by non-neuronal cells within the mucosa.

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The same studies were less consistent when it came to responses to distension. While extrinsic denervation did not affect responses to distension in the guinea-pig small intestine (Furness et al., 1995), it abolished such responses in the rat colon (Grider and Jin, 1994). Thus, it appears that IPANs mediate distension evoked reflexes in the guinea-pig small intestine, while similar reflexes in rat colon may be mediated by extrinsic primary afferent neurons whose terminals remain functional when acutely isolated from their cell bodies. In this case, the outputs of the distension sensitive extrinsic primary afferent neurons may be via axon collaterals or synapses of passage in the myenteric plexus. Speculation about the identity of the IPANs precedes proof of their existence and dates back at least to Dogiel (1899), who suggested that they correspond to his type II neurons. He based this on the projections of neurons with this morphology, as they send processes both to the mucosa and to other myenteric ganglia, the ideal projection pattern for an IPAN mediating reflexes evoked by mucosal stimuli. The basic observation that type II neurons in the myenteric plexus project to the mucosa and make synapses with other myenteric ganglion cells has been abundantly confirmed (Furness, Bornstein and Trussell, 1988; Pompolo and Furness, 1988, 1995; Pompolo et al., 1989; Furness et al., 1990; Song, Brookes and Costa, 1991, 1994; Bornstein et al., 1991b; Brookes et al., 1995). A second population of Dogiel type II neurons has been identified in the submucous plexus (Bornstein, Furness and Costa, 1989) and these too project to both the mucosa (Costa et al., 1981; Keast, Furness and Costa, 1984) and the myenteric plexus (Kirchgessner and Gershon, 1988; Kirchgessner, Tamir and Gershon, 1992). Initial electrophysiological studies suggested AH-neurons might be IPANs, because fast synaptic inputs were not observed and so they were unlikely to be anything other than the first neuron in a reflex pathway (Hirst, Holman and Spence, 1974). This suggestion converged with that of Dogiel when intracellular injection of neuronal markers showed that myenteric AH-neurons had the morphology of Dogiel type II cells in the guinea-pig small intestine (Bornstein et al., 1984a, 1991b; Katayama, Lees and Pearson, 1986; Iyer et al., 1988). A similar observation was made for submucous AH-neurons (Bornstein, Furness and Costa, 1989). However, while fast synaptic input to AH-neurons is rare, these neurons exhibit prominent and powerful slow EPSPs in response to electrical stimulation of enteric nerve trunks (Katayama and North, 1978; Wood and Mayer, 1978b, 1979; Grafe, Mayer and Wood, 1979, 1980; Katayama, North and Williams, 1979; Johnson, Katayama and North, 1980; Johnson et al., 1981). Thus, the projections of AH/Dogiel type II neurons are consistent with them being IPANs, but their electrophysiological properties indicate that they may also act as interneurons. This is not unique to the IPANs of the intestine as primary afferent neurons with cell bodies in the mesencephalic trigeminal nucleus also have prominent synaptic inputs (Copray et al., 1990; Pedroarena et al., 1999). Confirmation that myenteric AH-neurons are IPANs has come from recent electrophysiological studies that directly measured the responses of these neurons to chemical and mechanical stimulation of the mucosa (Kunze et al., 1995; Bertrand et al., 1997, 1998). These studies show that application of acid (pH 1–5), base (pH 9–11) or the volatile fatty acid, acetate (pH 7.0), to the mucosa evokes bursts of action potentials in myenteric AH-neurons lying within 2 mm circumferential to the site of application (Figure 1.7). This occurs even when synaptic transmission is blocked with a low Ca2+, high Mg2+ solution, indicating that the action potentials are due to a sensory rather than a synaptic process.

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Figure 1.7 Responses of three different AH/Dogiel type II neurons to application of (A) acetate (buffered to pH 7.2), (B) acid saline (pH 3) and (C) 5-HT to a patch of intact mucosa lying 1–3 mm circumferential to the impaled neurons. Each stimulus evoked a burst of action potentials in the impaled neuron. Figure modified from Bertrand et al. (1997).

Most AH/Dogiel type II neurons that respond to chemical stimulation of the mucosa do not respond to mild mechanical stimulation of the same region, but some do (Bertrand et al., 1998). Thus, many myenteric AH/Dogiel type II neurons appear to be chemosensitive IPANs. IPANs that are muscle mechanoreceptors The fact that distension evokes reflexes indicates that many primary afferent neurons are sensitive to mechanical changes in the intestinal smooth muscle. In the guinea-pig ileum, these mechanosensitive primary afferent neurons are intrinsic to the enteric nervous system (Furness et al., 1995). Furthermore, both ascending and descending reflexes can be evoked by distension when the mucosa and submucosa have been removed (Hirst, Holman and McKirdy, 1975; Smith, Bornstein and Furness, 1990; Yuan, Bornstein and Furness, 1994), indicating that IPANs sensitive to mechanical changes in the muscle have cell bodies and processes in the myenteric plexus. A variety of studies indicate that many IPANs are sensitive to an increase in the circumference of the intestinal tube, i.e. to the length of the circular muscle. For example, placing a sleeve around a segment of intestine to prevent an increase in intraluminal pressure stretching the intestinal wall, also prevents the initiation of the normal peristaltic motor pattern (Kosterlitz and Robinson, 1959). Similarly, slow stretch applied to an entire segment of intestine first excites reflex accommodation which relaxes circular muscle (Waterman, Costa and Tonini, 1994a) and then, as the length of the muscle continues to

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increase, the peristaltic motor pattern (Waterman, Costa and Tonini, 1992, 1994a,b; Waterman and Costa, 1994; Brookes et al., 1999a). However, while it is clear that the increase in length excites IPANs, whether this is due to an increase in tension within the muscle or to the actual change in length is unknown. Recent electrophysiological studies indicate that some myenteric IPANs are sensitive to tension in the outer smooth muscle layers (Kunze et al., 1998). Indeed, these neurons, which fire spontaneously in stretched preparations when the muscle is free to contract, appear to respond to the active generation of tension in the muscle as their firing disappears when contractions of the muscle are blocked (Figure 1.8). However, the change in firing between low and high levels of stretch may be slow, as only 3 of 10 neurons whose firing was followed for one second after the application of a rapid stretch increased their firing during this period. The effect of stretching the muscle can be mimicked by increasing spontaneous contractions of the muscle with the Ca2+ ionophore Bay-K 8864 and prevented by treatment with dispase, which breaks the connective tissue cross-bridges between the muscle and the myenteric plexus (Kunze et al., 1999). This indicates that these IPANs respond to the tension within the preparation rather than its macroscopic length. The spontaneous firing arises in the processes of the tension sensitive IPANs, because not all action potentials fully invade the soma and hyperpolarization of the soma often does not alter the rate of firing in the processes. However, there are discrepancies between the properties of the tension-sensitive IPANs directly identified by Kunze et al. (1998) and those that can be inferred for the IPANs that initiate reflexes evoked by distension. The compound EJPs evoked in circular muscle and bursts of fast EPSPs evoked in myenteric neurons oral to a stimulus by distension are transient and decay to baseline within 2–3 s even when the distension is maintained for up to 30 s (Smith, Bornstein and Furness, 1990, 1991, 1992; Smith et al., 1992; Yuan, Furness and Bornstein, 1992). The distensions used to evoke reflexes typically reach their peak in 150–500 ms and the latencies of the responses evoked in either neurons or the circular muscle are of the same order. This implies that

Figure 1.8 Intracellular recordings from a Dogiel type II neuron taken when the preparation was stretched to (A) 120% of its resting circumference, (B) 140% of its resting circumference and (C) 140% of its resting circumference in the presence of isoprenaline to relax the muscle. By contrast to Figures 1.4–1.7, nicardipine was not present in this experiment and the preparation was free to contract in (A) and (B). Modified from Kunze et al. (1998).

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rapid increases in tension or muscle length excite some IPANs with very short delays for sensory transduction. Furthermore, these rapid distensions evoke reflex responses in neurons and circular muscle when the muscle is prevented from contracting by L-type Ca2+ channel blockers. By contrast, the directly identified tension-sensitive IPANs fire throughout a maintained stretch and depend on muscle contraction to produce their full response during maintained stretch (Kunze et al., 1998). These differences suggest that mechanosensitive myenteric IPANs may have two different responses to changes in the state of the intestinal muscle. Most, perhaps all, respond to a maintained increase in the active tension within the muscle with ongoing firing of action potentials in their sensory processes. These mechanoreceptors depend on contraction of the muscle and cease firing when the muscle relaxes. Some neurons, which may be a subset of the tension receptors, respond to rapid stretch with a short latency increase in firing that does not depend on muscle contraction. This type of response appears to adapt rapidly, declining to the static type response when the distension is maintained or disappearing entirely. Macroscopically, rapidly adapting mechanoreceptive IPANs would respond to the dynamic component of a tension increase (or perhaps to a length increase), while slowly adapting IPANs would signal the static component of the tension. Whether the mechanoreceptive IPANs are also chemosensitive remains to be determined. However, the slowly adapting IPANs have been unequivocally identified as AH/Dogiel type II neurons (Kunze et al., 1998), and neurons of this morphological type all project to the mucosa (Song, Brookes and Costa, 1994), so the projections of these neurons are consistent with this possibility. Furthermore, 50–80% of AH/Dogiel type II neurons respond to stretch, while more than 30% of these neurons have been found to respond to chemical stimuli applied to the mucosa. Thus, some overlap might be expected. However, although reflexes evoked in the circular muscle by acid stimulation of the mucosa are transient and depressed by prior stimulation with acid, such stimuli enhance subsequent responses to distension for several minutes even in the absence of a direct response to the acid (Smith, Bornstein and Furness, 1991). This suggests that reflexes evoked by acid stimuli and by rapid distension may be mediated by separate IPANs. Mucosal mechanoreceptors There is strong evidence that the IPANs that respond to mechanical stimulation of the mucosa are distinctly different from those that respond to distension. Studies using various activity dependent markers for neurons (Kirchgessner, Tamir and Gershon, 1992; Kirchgessner, Liu and Gershon, 1996) indicate that the cell bodies of the former are located in the submucous plexus. When nicotinic synaptic transmission was blocked, the only neurons that responded to mechanical stimulation of the mucosa, either by expression of Fos protein or by taking up FM1–43, had cell bodies in the submucous plexus. These neurons were immunoreactive for substance P, which is a characteristic feature of the submucous AH/Dogiel type II neurons in the guinea-pig small intestine (Bornstein, Furness and Costa, 1989). Retrograde and anterograde tracing studies indicate that these neurons project to the myenteric plexus (Kirchgessner and Gershon, 1988; Kirchgessner, Tamir and Gershon, 1992), as predicted for neurons mediating the reflexes evoked by mechanical stimulation of the mucosa.

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The projections of the IPANs responding to different sensory modalities provide further evidence that they represent distinct populations. Injection of intracellular markers into myenteric AH/Dogiel type II neurons has shown that most, if not all, have predominantly circumferential projections (Bornstein et al., 1991b). Some project more than half way around the intestinal wall (Stebbing and Bornstein, 1996). However, retrograde tracing studies indicate that some of these neurons also have long anal projections (Brookes et al., 1995). Analysis of the reflex pathways excited by stationary stimuli also indicates that some IPANs have long anal projections and that these are preferentially sensitive to distension rather than mechanical deformation of the mucosa (Johnson et al., 1996; Johnson, Bornstein and Boucher, 1998). This is consistent with the observation that the mechanosensitive IPANs in the submucosa have predominantly circumferential projections within the myenteric plexus (Kirchgessner and Gershon, 1988; Kirchgessner, Tamir and Gershon, 1992). A further distinction is that transmission from the IPANs sensitive to mucosal deformation appears to include a component mediated by NK3 tachykinin receptors, while transmission from the distension-sensitive IPANs does not (Johnson, Bornstein and Boucher, 1998). Thus, at least three distinct classes of IPANs can be identified in the guinea-pig ileum (Figure 1.9). These are a population of mechanoreceptive Dogiel type II neurons in the myenteric plexus, a population of myenteric Dogiel type II neurons that are sensitive to the chemical environment of the mucosa and a population of submucous Dogiel type II neurons that respond to mucosal deformation. Reflexes evoked by rapid stretch indicate that some mechanoreceptive myenteric IPANs respond preferentially to either rapid increases in tension or changes in length of the intestinal wall. Whether these functionally distinguishable IPANs are completely separate populations of neurons or whether they overlap in the sensory modalities they prefer remains to be determined.

Figure 1.9 Diagram showing the three different classes of intrinsic primary afferent neurons (IPANs) that have been unequivocally identified in physiological experiments on the guinea-pig myenteric plexus, all are AH/Dogiel type II neurons. The interconnections of these neurons are also shown. Chemosensitive IPANs (cross hatch) have outputs to, and receive input from, tension sensitive IPANs (solid) in the myenteric plexus. Mechanosensitive IPANs (checks) in the submucous plexus respond to mechanical deformation of the mucosa and have outputs to tension sensitive myenteric IPANs, which may in turn have outputs to the submucous IPANs (? indicates that this is less certain). MP, myenteric plexus; SMP, submucosal plexus.

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INTERNEURONS Interneurons play a major role in the transmission of reflex activity along the intestine. Direct evidence for this has been obtained in a number of structural and functional studies, most notably in the guinea-pig ileum. Interneurons have been identified immunohistochemically and via direct injection of intracellular markers during electrophysiological experiments (see below). That they have a significant role in reflexes is illustrated by the observation that mucosal deformation evokes EJPs in the circular muscle 35 mm oral to the point of stimulation (Smith and Furness, 1988), but IPANs project no more than 1 mm orally and the excitatory motor neurons project less than 10 mm in the same direction. There is only one population of myenteric neurons that could bridge this gap, ascending interneurons that have been identified as S-neurons, have orally projecting axons and are immunoreactive for calretinin (Brookes et al., 1997). The axons of two neurochemically distinct subsets of anally projecting S-neurons, one immunoreactive for somatostatin and the other immunoreactive for nitric oxide synthase (NOS), provide collateral branches and synaptic terminals to other myenteric ganglia (Pompolo and Furness, 1995; Portbury et al., 1995; Stebbing and Bornstein, 1996). Another population of neurons, those immunoreactive for 5-hydroxytryptamine (5-HT), have similar projections within the myenteric plexus (Furness and Costa, 1982), but have not been identified electrophysiologically, although they have shapes characteristic of S-neurons. Each electrophysiologically identified class of descending interneurons makes ultrastructurally identifiable synaptic connections with inhibitory motor neurons (Pompolo and Furness, 1995; Portbury et al., 1995; Mann et al., 1997). The descending interneurons immunoreactive for 5-HT do not make significant numbers of synaptic connections with inhibitory motor neurons (Young and Furness, 1995). Descending interneurons are immunoreactive for choline acetyltransferase (ChAT) (Steele, Brookes and Costa, 1991; Costa et al., 1996; Li and Furness, 1998). Thus, there are at least three classes of cholinergic descending interneurons and one class of cholinergic ascending interneurons.

MOTOR NEURONS Electrophysiological studies of the projections of the excitatory and inhibitory motor neurons supplying the circular muscle indicate that each functional population can be subdivided on the basis of the lengths of their axons (Bornstein et al., 1986; Smith et al., 1988). Similar conclusions have been drawn from retrograde tracing studies, which also identified the neurochemistries of the different populations (for review see Costa et al., 1996, and Chapter 10 of this volume). In the guinea-pig small intestine, the inhibitory motor neurons are all immunoreactive for both NOS and vasoactive intestinal peptide (VIP), and the excitatory motor neurons are immunoreactive for ChAT and tachykinins. The short inhibitory motor neurons can be distinguished because they are immunoreactive for neuropeptide Y (NPY) (Uemura, Pompolo and Furness, 1995) and γ-aminobutyric acid (GABA) (Williamson, Pompolo and Furness, 1996), while the long neurons are immunoreactive for gastrin releasing peptide (GRP) (Uemura, Pompolo and Furness, 1995). Correlated electrophysiological, morphological and immunohistochemical studies indicate that all the

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circular muscle motor neurons are monoaxonal S-neurons (Bornstein et al., 1984a, 1991a; Katayama, Lees and Pearson, 1986; Stebbing and Bornstein, 1996; Kunze et al., 1997). The motor neurons that innervate the longitudinal muscle are also monoaxonal S-neurons (Bornstein et al., 1991a; Smith, Bornstein and Furness, 1992). Most are cholinergic and excitatory (Brookes, Steele and Costa, 1991; Brookes et al., 1992; Steele, Brookes and Costa, 1991). However, a small proportion contains GABA, NOS and/or VIP and may be inhibitory (Williamson, Pompolo and Furness, 1996). This is consistent with the results of some studies that indicate the presence of an inhibitory innervation (Kosterlitz and Lydon, 1968; Osthaus and Galligan, 1992), although electrophysiological studies of the longitudinal muscle usually only record EJPs (Hirst, Holman and McKirdy, 1975; Bauer and Kuriyama, 1982; Bywater and Taylor, 1983; Neil, Bywater and Taylor, 1983; Bornstein et al., 1986; Niel et al., 1986). FUNCTIONAL POPULATIONS OF NEURONS IN OTHER PREPARATIONS As yet there has been no complete study of the reflex pathways in other tissues comparable to those of the reflex pathways in the guinea-pig ileum, however, some features are common to all species and regions. For example, the circular muscle receives both excitatory and inhibitory innervation in every preparation studied. Nevertheless, immunohistochemical and electrophysiological studies of neurons elsewhere in the guinea-pig GI tract or in other species indicate that, despite the substantial similarities, there are differences between preparations. For example, in the guinea-pig, NOS is not found in the terminals of interneurons within the myenteric plexus of the duodenum (Clerc et al., 1998b) and somatostatin-immunoreactive interneurons project orally, rather than anally, in the distal colon (Messenger and Furness, 1990). Similarly, neurons electrophysiologically identical to the IPANs of the guinea-pig ileum have been identified in the guinea-pig duodenum (Hirst, Holman and Spence, 1974; Clerc et al., 1998a), proximal colon (Messenger, Bornstein and Furness, 1994), distal colon (Wade and Wood, 1988; Lomax et al., 1999) and rectum (Tamura and Wood, 1989; Tamura, 1992) as well as the mouse distal colon (Furukawa, Taylor and Bywater, 1986), rat duodenum (Brookes, Ewart and Wingate, 1988) and human colon (Brookes, Ewart and Wingate, 1987). However, such neurons are absent from the corpus of the guinea-pig stomach (Schemann and Wood, 1989), are rare in the antrum of the guinea-pig (Tack et al., 1992; Tack and Wood, 1992) and possibly in the pig ileum (Cornelissen et al., 1996). Thus, there are substantial differences between functionally equivalent neurons in the stomach and the rest of the GI tract, with less significant differences between similar neurons in other intestinal regions.

SYNAPTIC INTERACTIONS BETWEEN FUNCTIONAL CLASSES OF ENTERIC NEURONS TYPES OF SYNAPTIC POTENTIALS Electrical stimulation of enteric nerve trunks has revealed two forms of excitatory transmission in the myenteric plexus: fast (Figure 1.6) and slow EPSPs (Figure 1.10).

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Figure 1.10 Slow excitatory synaptic potentials (EPSPs) in (A) an AH/Dogiel type II neuron and (B) and (C) two different S-neurons. In (A) and (B) the upper traces show hyperpolarizing current pulses injected through the recording electrodes to monitor resistance changes during the slow EPSPs. In both cases, there was an increase in input resistance of the neuron during the early part of the slow EPSP, although the relative magnitude of this fall was much greater in the AH/Dogiel type II neuron than in the S-neuron. (C) shows the effect of activation of a slow EPSP on the firing induced in an S-neuron by three successive 500 ms depolarizing current pulses of equal magnitude (1, 2, 3). A train of 10 stimuli at 20 Hz was delivered to an internodal strand between depolarizations 1 and 2 and evoked fast EPSPs in the S-neuron. These were insufficient to evoke an action potential during depolarization 2. Depolarization 1 evoked only a single action potential. However, during the rising component of the slow EPSP, which began at the end of depolarization 2, depolarization 3 triggered a train of action potentials that lasted throughout the current injection. i.e. the neuronal firing pattern was converted from rapidly adapting to slowly adapting. (C) was modified from Kunze et al. (1996).

Inhibitory synaptic potentials have also been seen in myenteric neurons (Wood and Mayer, 1978a; Johnson, Katayama and North, 1980; Johnson et al., 1981). However, the stimulus regime needed to evoke such inhibitory synaptic potentials is identical to that which evokes slow EPSPs, so that the excitatory responses usually occlude the inhibitory responses unless the slow EPSPs are blocked in some way (Christofi and Wood, 1993b). As a result, the inhibitory synaptic potentials have yet to be characterized. Similarly, while various different presynaptic inhibitory mechanisms have been described or inferred to exist in pharmacological studies (e.g. Morita, North and Tokimasa, 1982b), these have also not been well studied and this chapter will focus on the excitatory events. Three pharmacologically distinct types of fast EPSPs have been identified in the myenteric plexus. All involve similar conductance increases to Na+ and K+ and reverse at about 0 mV (Zhou and Galligan, 1996, 1998, 1999). It has long been known that many fast EPSPs are blocked by nicotinic antagonists (Nishi and North, 1973; Hirst, Holman and Spence, 1974). However, recent studies have shown that, although nicotinic blockade almost abolishes EPSPs in about a third of S-neurons, it is less effective in the remainder. P2X-receptor antagonists, suramin and PPADS, substantially depress the hexamethonium-insensitive fast EPSPs in most of these S-neurons (Galligan and Bertrand, 1994; LePard, Messori and Galligan, 1997). In around 10% of S-neurons, hexamethonium-insensitive fast EPSPs are blocked by the 5-HT3 receptor antagonist ondansetron (Zhou and Galligan, 1999).

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The exclusively nicotinic fast EPSPs appear to be in neurons of the ascending reflex pathways, while the fast EPSPs mediated via P2X receptors are in neurons of the descending pathways and may be confined to inhibitory motor neurons (LePard and Galligan, 1999; Johnson et al., 1999; Bian, Bertrand and Bornstein, 2000). The identity of the S-neurons exhibiting 5-HT3 receptor mediated fast EPSPs is unknown. Blockade of such receptors depresses reflexes in the ascending excitatory pathway (Yuan, Bornstein and Furness, 1994), but the only 5-HT immunoreactive neurons are descending interneurons (Costa et al., 1996). It has been suggested that glutamate may also mediate fast EPSPs in myenteric neurons (Liu et al., 1997). However, detailed pharmacological analysis of the effects of glutamate on enteric neurons indicates that these neurons express type 1 metabotropic glutamate receptors and not ionotropic glutamate receptors (Ren et al., 2000). Slow EPSPs last from 10 s to several tens of minutes depending on whether they are evoked in S- or AH-neurons and on the stimulus regime used to excite them (Katayama and North, 1978; Wood and Mayer, 1978b; Grafe, Mayer and Wood, 1980; Johnson, Katayama and North, 1980; Bornstein et al., 1984b; Clerc et al., 1999). In some S-neurons, a single focal stimulus applied to a circumferentially directed internodal strand evokes a slow EPSP (Bornstein et al., 1984b); but in AH-neurons, it is more common to employ a train of stimuli lasting 1–3 s at frequencies of 10–20 Hz. Prolonged trains of stimuli, up to 10 min, at a relatively low frequency (1 Hz) also evoke large slow EPSPs in AH-neurons (Clerc et al., 1999). The slow EPSPs in S-neurons are markedly shorter and have shorter latencies than those in AH-neurons for any given stimulus regime (Bornstein et al., 1984b). All slow EPSPs appear to involve a reduction in the K+ conductance of the postsynaptic membrane; in AH-neurons, this is often associated with an increase in the Cl − conductance (Bertrand and Galligan, 1994); whether a similar increase occurs in S-neurons is unknown. In both AH- and S-neurons, slow EPSPs apparently involve closure of a resting, voltage independent K+ conductance (Galligan, North and Tokimasa, 1989). In AH-neurons, however, closure of a calcium-dependent K+ channel also makes a major contribution (Grafe, Mayer and Wood, 1980; Galligan, North and Tokimasa, 1989; Bertrand and Galligan, 1994, 1995). Although calcium-dependent K+ channels have been identified in S-neurons (Tokimasa, Cherubini and North, 1983), these do not appear to be open at rest so they are not involved in the slow EPSPs of these neurons. The calciumdependent K+ channel in AH-neurons is blocked by BK channel specific toxins, iberiotoxin and charybdotoxin, but differs from a conventional BK channel in that it is insensitive to tetraethylammonium (TEA) and is not rapidly activated by calcium ions entering during an action potential (Kunze et al., 1994). The calcium-dependent K+ conductance underlies the slow AHPs of AH-neurons, so that slow EPSPs in these neurons depress their AHPs and enhance their firing during imposed depolarizations. The AHPs in AH-neurons normally limit their firing to the start of a prolonged depolarization, but during slow EPSPs the neurons fire throughout (see for example, Johnson, Katayama and North, 1980; Grafe, Mayer and Wood, 1980; Tokimasa, Cherubini and North, 1983; Kunze et al., 1994). That is, the AH-neuron is converted from a rapidly accommodating state to a slowly accommodating state. Slow EPSPs also cause a change in firing state from rapidly accommodating to slowly accommodating in most S-neurons (Figure 1.10C) (Kunze et al., 1997). In many S-neurons,

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the change in state is due to blockade of an early onset voltage activated outward current that is blocked by TEA, but not by 4-aminopyridine (Kunze et al., 1997). A similar change in firing state is also seen under conditions in which IPANs are spontaneously active (Kunze et al., 1997, 1998). The identity of the neurotransmitters that mediate the slow EPSPs has been debated since they were first identified. It is now clear that the slow EPSPs are produced by a combination of different transmitters, and that this combination varies between different functional classes of neurons. Acetylcholine (ACh) acting at muscarinic receptors, 5-HT and tachykinins may all play a role in mediating these events. Muscarinic antagonists block an early component of the slow EPSPs in 25% of S-neurons and 10% of AH-neurons; this appears to be due to an action at M1 muscarinic receptors (North and Tokimasa, 1982; Morita, North and Tokimasa, 1982a; North and Tokimasa, 1983; North, Slack and Surprenant, 1985; Galligan, North and Tokimasa, 1989). This muscarinic component has a physiological role because pharmacological studies of reflexes indicate that muscarinic receptors play a role in transmission from IPANs to interneurons (Tonini and Costa, 1990; Johnson et al., 1996). However, most of the slow EPSPs seen in enteric neurons are not cholinergic. There is a body of pharmacological evidence indicating that many of the slow EPSPs in AH-neurons are mediated by 5-HT acting at a receptor subtype that has been identified as 5-HT1P (Branchek, Mawe and Gershon, 1988). The most powerful evidence comes from blockade of electrically evoked slow EPSPs by various antagonists and by anti-idiotypic antibodies, each of which also blocks the slow depolarizing effects of 5-HT on AH-neurons (Takaki et al., 1985; Mawe et al., 1989; Wade et al., 1994). However, slow EPSPs can still be evoked in both AH- and S-neurons after lesions that cause degeneration of all 5-HT containing nerve terminals (Bornstein et al., 1984b). Indeed, such lesions do not alter the slow EPSPs in S-neurons and those in AH-neurons show only very subtle changes. Furthermore, it has been shown using electron-microscopic immunocytochemistry that 5-HT neurons rarely make synapses with AH/Dogiel type II neurons (Young and Furness, 1995). Thus, 5-HT is unlikely to mediate slow EPSPs in S-neurons and may play only a minor role in AH-neurons. Recent data derived from experiments using specific antagonists against NK1 and NK3 tachykinin receptors confirm the conclusions of many less direct studies that tachykinins mediate many of the slow EPSPs in myenteric neurons. In particular, slow EPSPs evoked in AH-neurons by stimulating circumferentially directed internodal strands are blocked by either the specific NK3 receptor antagonist SR 142801 (Figure 1.11) or the specific NK1 receptor antagonist SR 140333 (Figure 1.12), or both (Johnson and Bornstein, 2002). Other pharmacological data indicating a role for tachykinins includes the fact that all the major types of mammalian tachykinins depolarize these neurons via a mechanism that mimics the slow EPSPs (Katayama and North, 1978; Katayama, North and Williams, 1979; Morita, North and Katayama, 1980; Johnson et al., 1981; Galligan, Tokimasa and North, 1987; Morita and Katayama, 1992; Bertrand and Galligan, 1994, 1995). Furthermore, slow EPSPs can be depressed by peptidases and by desensitization with substance P (Morita, North and Katayama, 1980). Tachykinin mediated slow EPSPs have also been identified in gall bladder neurons by blockade of NK3 receptors (Mawe, 1995) and AH-neurons of the guinea-pig proximal colon (Neunlist, Dobreva and Schemann, 1999).

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Figure 1.11 Effect of the NK3 tachykinin receptor antagonist SR 142801 on a slow EPSP evoked in an AH/Dogiel type II neuron by ten stimuli delivered at 10 Hz to a circumferentially directed internodal strand (star). (A) shows the control slow EPSP, while (B) shows the effect of the same stimulus after the preparation had been exposed to SR 142801 (100 nM) for 45 min. The slow EPSP is abolished. Upper traces show the membrane potential, while the lower traces show hyperpolarizing current pulses injected via the recording electrode to monitor resistance changes associated with the slow EPSP.

NK3 receptors have also been implicated in slow EPSPs in myenteric neurons of the guinea-pig stomach (Schemann and Kayser, 1991). Pharmacological evidence that other neurotransmitters mediate slow EPSPs of AH-neurons has been put forward recently. For example, specific antagonists of the actions of cholecystokinin (CCK) depress slow EPSPs in some myenteric AH-neurons (Schutte et al., 1997), although the relationship of these slow EPSPs to those mediated by tachykinins is unknown. While tachykinins depolarize both AH- and S-neurons in the guinea-pig ileum, direct pharmacological evidence for a role for these peptides in mediating slow EPSPs in the latter is very sketchy. There is indirect evidence that NK3 receptors play a role in transmission from IPANs to interneurons in both the ascending and descending reflex pathways (Johnson et al., 1996; Johnson, Bornstein and Boucher, 1998). Furthermore, studies using NK1 receptor internalization as a surrogate measure of receptor activation indicate that many S-neurons respond to tachykinins released when the mucosa is stimulated mechanically (Southwell et al., 1998). Nevertheless, in a study of myenteric neurons in the guinea-pig ileum, the slow EPSPs in 6 S-neurons were not blocked by an NK3 receptor antagonist and an NK1 antagonist blocked these responses in only 1 of 3 S-neurons (Johnson unpublished). As these experiments were performed in the presence of a muscarinic antagonist and 5-HT is unlikely to mediate slow EPSPs in S-neurons (see above), this suggests

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Figure 1.12 Effect of the NK1 tachykinin receptor antagonist SR 140333 on a slow EPSP evoked in an AH/ Dogiel type II neuron by 20 focal pulses delivered at 10 Hz to a circumferentially directed internodal strand (arrow). (A) shows the control slow EPSP, (B) shows the effect of the same stimulus after the preparation had been exposed to SR 140333 (100 nM) for 40 min. Note, the slow EPSP in this experiment was faster in time course than that in Figure 1.11.

that a neurotransmitter other than a tachykinin, ACh or 5-HT mediates slow EPSPs in many S-neurons. Several other candidates are present in myenteric nerve terminals, all of which depolarize myenteric neurons. These include VIP (Williams and North, 1979), somatostatin (Katayama and North, 1980) and pituitary adenyl cyclase activating peptide (Christofi and Wood, 1993a). However, no direct evidence is available about these or other possible candidates as yet. TRANSMISSION BETWEEN IPANs AND MOTOR NEURONS (Figure 1.13) There is abundant structural and electrophysiological evidence for monosynaptic transmission between IPANs and excitatory and inhibitory motor neurons supplying each muscle layer. Pompolo and Furness (1988) used the fact that 80–90% of all IPANs in the myenteric plexus of the ileum are immunoreactive for calbindin, but no other neurons are, to study the synaptic connections of IPANs at an ultrastructural level. They found that calbindin-immunoreactive terminals contacted all myenteric neurons and those neurons

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Figure 1.13 Diagram showing the connections from intrinsic primary afferent neurons (IPANs) to motor neurons and the transmitters that can be inferred to act at the specific synapses. Acetylcholine (ACh) is likely to play a major role in transmission to local motor neurons, but is unlikely to be involved in transmission from the long anally projecting axons of some myenteric IPANs. Whether tachykinins (TK) have a role is unclear. In this and later Figures “?” indicates that the transmitter or receptor subtype has not been established unequivocally.

that were not IPANs had large numbers of such synapses. More recently, similar methods provided direct evidence for synapses between the IPANs and longitudinal muscle motor neurons and a subset of circular muscle motor neurons (Pompolo and Furness, 1990, 1993). Simultaneous recordings made from IPANs and structurally identified motor neurons showed that myenteric IPANs can cause slow EPSPs in many motor neurons (Kunze et al., 1993). Electrical stimulation of long circumferentially directed pathways within the myenteric plexus evokes fast EPSPs in motor neurons (Stebbing and Bornstein, 1996). As the only neurons in the myenteric plexus with long circumferential projections appear to be IPANs, this suggests that these neurons transmit to motor neurons via fast, as well as slow, EPSPs. Furthermore, chemical stimulation of the mucosa evokes volleys of fast EPSPs in nearby S-neurons with latencies similar to those of the action potentials evoked in IPANs by the same stimuli (Kunze et al., 1995; Bertrand et al., 1997, 1998). This

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indicates that the fast EPSPs are not secondary to a slow EPSP in an interneuron as the rise time of a slow EPSP would add several hundred milliseconds to the latency of any secondary response. Analysis of transmission at these synapses indicates that both tachykinins and ACh play a role (Figure 1.13), but also implies that at least one other transmitter contributes to this process. The mechanoreceptive mucosal IPANs contain both substance P and ChAT (Bornstein and Furness, 1988; Kirchgessner, Tamir and Gershon, 1992). Nicotinic blockade substantially reduces the number of enteric neurons that express Fos or take up activity dependent markers in response to mechanical stimulation of the mucosa indicating an involvement of ACh at nicotinic receptors somewhere in this pathway (Kirchgessner, Tamir and Gershon, 1992; Kirchgessner, Liu and Gershon, 1996). Similar results with distension suggest that ACh may also have a role in transmission from distension-sensitive IPANs (Ritter, Costa and Brookes, 1997). This is consistent with the observations that all myenteric IPANs are immunoreactive for ChAT (Li and Furness, 1998) and that hexamethonium blocks fast EPSPs evoked in S-neurons by electrical stimulation of long circumferential pathways (Stebbing, 1996). Studies of descending inhibitory reflexes strongly suggest involvement of a transmitter other than ACh, ATP or a tachykinin in transmission from IPANs to inhibitory motor neurons. Divided organ bath studies indicate that synaptic transmission from the long anally projecting axons of distension-sensitive IPANs to inhibitory motor neurons was unaffected by blockade of nicotinic and muscarinic ACh receptors, P2X ATP receptors or NK1 and NK3 tachykinin receptors in the recording chamber (Johnson et al., 1996; Johnson, Bornstein and Burcher, 1998; Bian, Bertrand and Bornstein, 2000). Despite the failure of specific tachykinin antagonists to modify reflex transmission from IPANs to inhibitory motor neurons, slow EPSPs clearly have a physiological role in transmission between these two classes of neurons. When IPANs are spontaneously active, many morphologically and immunohistochemically identified inhibitory motor neurons change their firing state from rapidly adapting to slowly adapting (Kunze et al., 1997, 1998). TRANSMISSION FROM IPANs TO INTERNEURONS (Figure 1.14) Electrophysiological analyses indicate that ascending interneurons receive fast EPSPs (Stebbing and Bornstein, 1996; Brookes et al., 1997) and slow EPSPs (Kunze et al., 1993) from myenteric IPANs. However, slow EPSPs were not recorded in ascending interneurons that had been in organ culture for 3 days (Brookes et al., 1997). A direct analysis of the synaptic inputs from submucous IPANs to the ascending interneurons has not yet been undertaken. Nevertheless, substantial conclusions can be drawn from studies using divided organ baths and similar preparations. Blockade of nicotinic receptors in the stimulation chamber depresses ascending reflexes evoked in the circular muscle in a more oral recording chamber by either distension or mucosal distortion by 40–60% (Tonini and Costa, 1990; Johnson et al., 1996). These responses are further depressed to less than 20% of the control by desensitization of NK3 tachykinin receptors (Johnson et al., 1996). The response to mucosal distortion, but not that to distension, is also depressed by an NK3 antagonist in the presence of hexamethonium (Johnson, Bornstein and Burcher, 1998).

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Figure 1.14 Sites of transmission from intrinsic primary afferent neurons (IPANs) to ascending and descending interneurons and the transmitters/receptors that can be inferred to act at those sites. Transmission to ascending interneurons is likely to be primarily due to acetylcholine (ACh), but there is evidence for a lesser role for tachykinins acting at NK3 receptors. Tachykinins acting at NK3 receptors are involved in transmission to descending interneurons, presumably the nitric oxide synthase (NOS) interneurons, but the major transmitter at this site is unknown (indicated by + ?). Note, no connection between IPANs and SOM-immunoreactive descending interneurons has been identified. See Figure 1.2 for identities of specific neuronal subtypes.

Hyoscine also depresses the non-nicotinic component of each reflex when applied to the stimulation chamber and virtually abolishes reflexes evoked by mucosal distortion when added together with hexamethonium and an NK3 antagonist (Tonini and Costa, 1990; Johnson et al., 1996; Johnson, Bornstein and Burcher, 1998). Thus, these pharmacological studies indicate that distension-sensitive IPANs transmit to ascending interneurons via ACh acting on nicotinic and muscarinic receptors, which would produce fast and slow EPSPs, respectively. Submucous mechanosensitive IPANs employ both of these receptors and a tachykinin acting via NK3 receptors, which also implies transmission via fast and slow EPSPs. Ascending interneurons make their first synapses 3–4 mm oral to their cell bodies (Brookes et al., 1997), so some synapses affected by drugs in the stimulation chamber would be between interneurons and these probably involve exclusively cholinergic fast EPSPs (see next section). Thus, the possibility that all synaptic transmission between IPANs and ascending interneurons is via slow EPSPs cannot be excluded. Computer simulation of the enteric circuitry mediating ascending reflexes indicates that the time courses of the electrophysiologically recorded responses are more compatible with transmission at these synapses being exclusively via slow EPSPs than via fast EPSPs (Bornstein et al., 1997, 1998; Thomas, Bertrand and Bornstein, 1999). The nature of transmission from IPANs to descending interneurons is more difficult to infer. Pharmacological studies of the descending pathways indicate that nicotinic transmission plays only a minor role in transmission from either the distension-sensitive IPANs or the mucosal mechanoreceptors. Hexamethonium has virtually no effect on descending inhibitory reflexes over distances of up to 2 cm from the stimulus site (Smith, Bornstein and Furness, 1990; Johnson et al., 1996; Smith and Robertson, 1998; Bian, Bertrand and Bornstein, 2000). Furthermore, although descending interneurons that contain NOS receive fast EPSP input from what are presumably myenteric IPANs, the descending

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interneurons that contain somatostain (SOM) rarely do (Stebbing and Bornstein, 1996). The SOM-immunoreactive descending interneurons do not appear to exhibit prominent slow EPSPs (Song et al., 1997), so it is possible that these neurons do not receive significant synaptic input from local myenteric or submucous IPANs. Indeed, quantitative immunocytochemical analysis of the neurochemistry of the inputs to the SOM-immunoreactive descending interneurons indicates that they receive very few structurally identifiable inputs from IPANs (Mann et al., 1997) (Pompolo and Furness, 1998). This suggests that the SOM-immunoreactive interneurons are not involved in the initial phase of transmission of descending reflexes. By contrast, slow EPSPs are common in NOS-immunoreactive descending interneurons, so it is possible that these slower non-cholinergic synaptic potentials mediate transmission from IPANs into the descending reflex pathways. This is consistent with studies showing that interference with NK3 tachykinin receptors in the stimulus region depresses descending inhibitory reflexes evoked by either mucosal distortion or distension, with the former being much more susceptible (Johnson et al., 1996; Johnson, Bornstein and Burcher, 1998). However, there has been no direct demonstration that NK3 receptors mediate slow EPSPs in the NOS-immunoreactive descending interneurons and other explanations for the effects of NK3 antagonists are possible (see below). There is no evidence available about transmission from IPANs to the 5-HT-immunoreactive class of descending interneurons. The results of divided organ bath experiments indicate that a neurotransmitter or receptor subtype, other than those already discussed, must be involved in transmission from IPANs to descending interneurons. Even when nicotinic receptors, suramin-sensitive receptors, NK1 and NK3 receptors are blocked in the region of the sensory stimulus transmission to the interneurons during reflexes still occurs. The nature of this extra transmitter remains unknown, as does the type of synaptic potential it produces. TRANSMISSION BETWEEN ASCENDING INTERNEURONS (Figure 1.15A) Only a single population of ascending interneurons, immunoreactive for ChAT, substance P, enkephalin and calretinin (Brookes, Steele and Costa, 1991; Costa et al., 1992; Brookes et al., 1997) has been identified immunohistochemically. These interneurons form chains with each receiving synapses from more anally located interneurons (Pompolo and Furness, 1993; Brookes et al., 1997). Most project 4–8 mm orally, although much longer neurons have been identified in retrograde tracing studies (Brookes et al., 1997). Both electrophysiological and pharmacological evidence indicates that transmission between ascending interneurons is via nicotinic fast EPSPs (Tonini and Costa, 1990; Bornstein et al., 1991a; Johnson et al., 1996; Brookes et al., 1997). For example, hexamethonium completely abolishes transmission of ascending reflexes through the central chamber of a three-chambered organ bath (Johnson et al., 1996). As the only synapses likely to be active in this chamber are those between ascending interneurons, this is strong evidence that transmission at such synapses is via nicotinic receptors (Figure 1.15A). Furthermore, local stimulation of ascending interneurons excites more oral ascending interneurons via fast EPSPs (Brookes et al., 1997). Despite the presence of tachykinin-immunoreactivity in ascending interneurons, three-chambered organ bath experiments indicate that tachykinins have no role in transmission between ascending interneurons (Johnson et al., 1996).

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Figure 1.15 Transmitters employed at synapses between (A) ascending interneurons and other ascending interneurons (exclusively ACh) and ascending interneurons and excitatory circular muscle motor neurons (predominantly ACh, with a minor role for tachykinins acting at NK3 receptors) and (B) descending interneurons and other descending interneurons and descending interneurons and inhibitory circular muscle motor neurons. The cell symbols are as shown in Figure 1.2. In B, synapses between NOS-immunoreactive descending interneurons and SOM-immunoreactive descending interneurons are shown as having no known transmitter, while synapses from the former to inhibitory motor neurons appear to use ATP acting at P2X receptors to produce fast EPSPs. The role of ACh in transmission between descending interneurons is uncertain, although these neurons are immunoreactive for ChAT and the vesicular acetylcholine transporter.

TRANSMISSION FROM ASCENDING INTERNEURONS TO MOTOR NEURONS (Figure 1.15A) Intracellular recordings from morphologically identified circular muscle motor neurons during ascending excitatory reflexes evoked by distension or mucosal distortion indicate that ascending interneurons transmit to motor neurons via fast EPSPs (Figure 1.6A) (Bornstein et al., 1991a; Smith, Bornstein and Furness, 1992). The divided organ bath studies of such reflexes indicate that this transmission is virtually abolished by nicotinic antagonists (Tonini and Costa, 1990; Johnson et al., 1996). Thus, it is probable that the fast EPSPs are mediated by ACh. However, the effects of blockade of NK3 receptors in the recording chamber of a divided organ bath suggest that these receptors have a minor role in transmission from ascending interneurons to the circular muscle motor neurons (Johnson, Bornstein and Boucher, 1998). Interestingly, when transmission along the ascending reflex pathway is blocked by hexamethonium for long periods, the reflex response recovers from 10% to about 30% of its control value and this may be due to increased activity of the tachykinins (Barthó, Holzer and Lembeck, 1989; Holzer, 1989). Transmission to the longitudinal muscle motor neurons from ascending interneurons is via fast EPSPs. Recordings made from such neurons identified by intracellular injection of

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biocytin indicate that, like circular muscle motor neurons, they respond to activation of ascending reflex pathways with a burst of fast EPSPs (Smith, Bornstein and Furness, 1992). These are likely to be cholinergic as the ascending interneurons are immunoreactive for ChAT (Brookes, Steele and Costa, 1991; Steele, Brookes and Costa, 1991; Brookes et al., 1997). As yet no evidence for a role for slow transmission or a role for tachykinins has been identified. TRANSMISSION BETWEEN DESCENDING INTERNEURONS (Figure 1.15B) Morphologically identified descending interneurons (2–25 mm anal to the stimulus) respond to activation of descending reflex pathways by mucosal distortion with a burst of fast EPSPs (Bornstein et al., 1991a). The mucosal mechanoreceptive IPANs do not appear to have prominent anal projections (Johnson et al., 1996; Johnson, Bornstein and Boucher, 1998), so these EPSPs are probably from descending interneurons. However, these fast EPSPs may not be predominantly mediated via nicotinic receptors, as hexamethonium has little effect on these reflexes or on distension evoked reflexes over these distances (Johnson et al., 1996). Divided organ bath studies indicate that antagonists acting at 5-HT3 or 5-HT4 receptors do not modify either type of descending reflex (Yuan, Bornstein and Furness, 1994). Similar studies indicate that blockade of NOS activity actually enhances transmission from IPANs and has no effect on transmission between interneurons (Yuan, Bornstein and Furness, 1995). Another possible candidate is a purine acting through P2X receptors, but suramin does not depress descending reflexes in divided organ bath experiments, when added to a chamber lying between the stimulus chamber and the recording chamber (Johnson et al., 1999). Many of the synapses active in this chamber would have been expected to be between interneurons. Thus, no clear candidate has emerged as the likely neurotransmitter mediating fast transmission between the descending interneurons involved in descending inhibitory reflexes. Whether there is a role for slow excitatory transmission between descending interneurons is an open question. In two studies of the effects of mucosal distortion and/or distension on neurons in the descending reflex pathways, only one convincing slow EPSP has been recorded and this was in a neuron that was not identified morphologically (Bornstein et al., 1991a; Smith, Bornstein and Furness, 1992). By contrast, desensitization of NK3 tachykinin receptors in the intermediate chamber of a divided organ bath markedly depressed descending inhibitory reflexes evoked either by distension or mucosal deformation (Johnson et al., 1996). However, the NK3 antagonist SR 142801 in the same chamber had no effect on the reflexes (Johnson, Bornstein and Burcher, 1998). Furthermore, tachykinins are not found in descending interneurons, while they may be in the long anal projections of some IPANs. Thus, if tachykinins were involved in transmission within the intermediate chamber, it may have been at synapses between IPANs and one or more populations of descending interneurons. Muscarinic receptors are unlikely to be involved in transmission between the descending interneurons, because hyoscine in the intermediate chamber had little effect on descending reflexes (Johnson et al., 1996). The neurochemistries of the descending interneurons provide some clues as to the transmitters involved at interneuron to interneuron synapses. Interneurons immunoreactive for SOM and interneurons immunoreactive for NOS are each likely to be involved in motility

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reflexes because they contact inhibitory motor neurons (Mann et al., 1997) and each contains substances that excite myenteric neurons. Both classes of interneurons contain ACh, together with either SOM or VIP, pituitary adenylate cyclase-activating peptide and GRP (Costa et al., 1996), all of which excite myenteric neurons (Williams and North, 1979; Katayama and North, 1980; Zafirov et al., 1985; Christofi and Wood, 1993a). Furthermore, each makes chains of neurochemically identical interneurons (Portbury et al., 1995; Young, Furness and Povey, 1995; Mann et al., 1997) and has cross-connections to neurons of the other neurochemical class (Mann et al., 1997). Thus, it is unlikely that a single substance mediates transmission at all synapses between descending interneurons. TRANSMISSION BETWEEN DESCENDING INTERNEURONS AND MOTOR NEURONS (Figure 1.15B) Activation of descending reflex pathways by either distension or distortion of the mucosa evokes bursts of fast EPSPs in many morphologically identified inhibitory motor neurons supplying the circular muscle (Bornstein et al., 1991a; Smith, Bornstein and Furness 1992). The resistance of descending inhibition to hexamethonium discussed above indicates that these fast EPSPs are unlikely to be solely mediated by ACh acting at nicotinic receptors. They are also unlikely to be due to 5-HT acting at 5-HT3 receptors as granisetron (a 5-HT3 receptor antagonist) has no effect on descending reflexes when applied in the recording chamber of a divided organ bath (Johnson et al. unpublished observation). The transmitter mediating the major portion of these fast EPSPs is probably a purine acting at P2X receptors. Such receptors are prominent on inhibitory motor neurons and rare on excitatory motor neurons (Johnson et al., 1999) and purinergic fast EPSPs are preferentially evoked by electrical stimulation of descending pathways (LePard and Galligan, 1999). Furthermore, P2X antagonists in the recording chamber depress descending inhibitory reflexes recorded in the circular muscle to a substantially greater extent than they depress IJPs evoked by local electrical stimulation (Bian, Bertrand and Bornstein, 2000). There is no direct evidence for slow excitatory transmission between descending interneurons and inhibitory motor neurons. Blockade of tachykinin NK1 receptors in the recording chamber of a divided organ bath depresses descending reflexes and the effect appears to be selective for transmission from descending interneurons (Johnson, Bornstein and Boucher, 1998). However, these neurons do not contain tachykinins (Costa et al., 1996), so this effect is unlikely to be simply due to blockade of transmission between interneurons and the motor neurons. Rather it may be due to blockade of tachykinins released from spontaneously active IPANs in the vicinity of the inhibitory motor neurons thereby depressing the excitability of the latter (see below for a more complete discussion). Descending reflex input to longitudinal muscle motor neurons is also via fast EPSPs (Bornstein et al., 1991a; Smith, Bornstein and Furness, 1992). Whether these inputs arise from one or more populations of descending interneurons or from the anally directed axons of IPANs is unknown. There is structural evidence that the NOS/VIP/GRP interneurons make synaptic connections with longitudinal muscle motor neurons and with ascending interneurons, although the significance of the latter is unknown (Pompolo and Furness, 1995). The longitudinal muscle motor neurons also receive ultrastructurally defined inputs

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from myenteric IPANs (Pompolo and Furness, 1995), although these may come from circumferentially directed pathways rather than descending pathways. While there is no definitive evidence as to the identity of the transmitter mediating the fast EPSPs evoked in these neurons by descending reflexes, there is no shortage of possible candidates. ACh, 5-HT, ATP and glutamate could all be involved. RETROGRADE TRANSMISSION FROM INTERNEURONS TO IPANs While nitric oxide (NO) has no role in transmission between descending interneurons, it does modulate the excitability of the descending reflex pathways. When NO synthesis is blocked in the stimulation chamber of a divided organ bath, the descending inhibitory response evoked either by distension or mucosal deformation is significantly enhanced (Yuan, Bornstein and Furness, 1995). This effect is not seen with ascending reflexes. This suggests that NO released from the cell bodies of descending interneurons depresses synaptic transmission between IPANs and descending interneurons (presumably those containing NOS). This is consistent with the suggestion that NOS-immunoreactive descending interneurons carry the excitatory signal from the IPANs into the descending reflex pathway. The effect of inhibition of NO synthesis on transmission between enteric neurons has not been directly studied. However, NO donors depress slow EPSPs evoked by electrical stimulation in IPANs (Tamura et al., 1993). Such slow EPSPs may result from activation of other IPANs (see next section). By contrast, NO donors did not modify fast EPSPs in the small number of neurons studied to date (Tamura et al., 1993). TRANSMISSION BETWEEN IPANs (Figure 1.16) IPANs are relatively unusual amongst primary afferent neurons in general because they make significant synaptic connections with each other. Ultrastructural studies indicate that calbindin neurons make synaptic connections with virtually all other myenteric neurons including Dogiel type II neurons (Pompolo and Furness, 1988). Furthermore, intracellular stimulation of an individual myenteric IPAN can evoke slow EPSPs in a second myenteric IPAN, if the stimulated neuron has an axon contacting the other neuron (Kunze et al., 1993). Repeated mucosal distortion enhances distension reflexes, even when the direct reflex response to mucosal stimulation has disappeared, which suggests an excitatory interaction between the submucosal and myenteric IPANs (Smith, Bornstein and Furness, 1991). This phenomenon persists for as long as the mucosal stimulation and for a considerable time afterwards suggesting that the decline in the direct response is not due to cessation of firing in the submucosal IPANs. The simplest explanation is that prolonged firing of mechanoreceptive submucosal IPANs evokes slow EPSPs in distension-sensitive myenteric IPANs thereby increasing their excitability. This implies differences in the transmission process between synapses from IPANs to interneurons and from IPANs to myenteric IPANs. As discussed above, there is strong evidence that the mechanosensitive submucosal IPANs transmit to ascending interneurons via fast EPSPs mediated by ACh acting on nicotinic receptors and slow EPSPs mediated via NK3 tachykinin receptors. The same

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Figure 1.16 Transmitters employed at synapses between IPANs. No distinction is made between different classes of IPANs in this diagram, because as yet pharmacological studies have not allowed such distinctions to be drawn. Both tachykinins acting at NK3 and NK1 receptors and ACh acting at muscarinic receptors appear to be involved. The predominant role of NK3 receptors is indicated by the heavier type.

neurons appear to transmit to descending interneurons via slow EPSPs mediated by a tachykinin acting on NK3 receptors and via another, unidentified, transmitter system. These conclusions are consistent with the immunohistochemical findings that submucosal IPANs are immunoreactive for both tachykinins and ChAT (Bornstein and Furness, 1988; Kirchgessner and Gershon, 1988; Kirchgessner, Tamir and Gershon, 1992; Kirchgessner, Liu and Gershon, 1996). Maintained distension does not enhance responses to mucosal distortion in the same region (Smith, Bornstein and Furness, 1991). This suggests that distension-sensitive myenteric IPANs do not provide synaptic input to submucosal IPANs. However, myenteric IPANs provide terminals to submucous ganglia (Furness et al., 1990) and there is indirect evidence that some slow EPSPs recorded in submucosal IPANs are due to activation of those terminals (Galligan et al., 1988). Other evidence that IPANs directly excite IPANs comes from analysis of the responses to chemical stimuli applied to the mucosa just circumferential to an impaled AH/Dogiel type II neuron. Although most neurons that responded did so by firing a burst of action potentials, some also exhibited a slow EPSP-like response and others only showed this slow EPSP-like response to the stimulus (Bertrand et al., 1997, 1998). Consideration of the known anatomy suggests that the slow EPSPs arose from connections between chemosensitive IPANs and other IPANs. This is consistent with the finding that chemical stimulation of the mucosa enhances reflexes evoked by distension (Smith, Bornstein and Furness, 1991). Thus, a variety of evidence indicates that mechanoreceptive IPANs with cell bodies in the submucosa and myenteric chemosensitive IPANs each modulate the activity of distension-sensitive IPANs via slow EPSPs. The question then arises whether mechanoreceptive myenteric IPANs have similar outputs to other myenteric IPANs, whether

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mechano- or chemosensitive. Many myenteric IPANs in freely contracting stretched preparations of guinea-pig ileum are more excitable than in relaxed preparations (Kunze et al., 1998). The prolonged AHPs, characteristic of IPANs in relaxed preparations, are greatly diminished in stretched contracting preparations and the firing of IPANs accommodates more slowly in the latter condition. These are all features characteristic of the behaviour of IPANs during slow EPSPs, which typically reduce the amplitudes and durations of the AHPs in IPANs (Grafe, Mayer and Wood, 1980; Morita and North, 1985). This suggests that the firing of the myenteric IPANs sensitive to maintained tension leads to maintained slow EPSPs in virtually all other myenteric IPANs. These are not detected as individual synaptic events, but rather are seen as an overall increase in the excitability of the target neurons. Myenteric IPANs also exhibit slow EPSPs when pathways running longitudinally along the myenteric plexus are stimulated electrically, even when the stimulus is applied up to 1 cm away (Hodgkiss and Lees, 1984). The source of these slow EPSPs is unknown, but they may be due to activation of the long anally directed axons of distension-sensitive IPANs.

SENSORY TRANSDUCTION STRETCH As discussed above, the mechanosensitive myenteric IPANs can be inferred to have up to three distinct responses to stretch. Many of these neurons have been shown in electrophysiological recordings to respond to sustained stretch when the muscle is free to contract (Kunze et al., 1998). Others, or a subset of these, respond to the onset of stretch independently of muscle contraction (see above), while some appear to also respond to the passive length of the muscle (see above). In each case, the actual mechanisms that transduce changes in the muscle coat into neuronal firing have not been determined. They presumably all involve the presence of mechanosensitive ion channels in the neuronal membranes with the exact nature of the stimulus being determined by the nature of the coupling of these channels to the extracellular matrix or the intestinal muscle. Indeed, the IPANs that respond to sustained stretch in contracting preparations lose their ability to respond to such stretch when the collagen coupling them to the muscle is enzymatically removed (Kunze et al., 1999).

MUCOSAL MECHANORECEPTORS The cell bodies of the IPANs mediating responses to mechanical distortion of the mucosa have been localised to the submucosa (see above). There is a large body of evidence indicating that sensory stimuli act on the mucosa to release 5-HT from the enterochromaffin cells, and that this excites IPAN terminals in the mucosa. 5-HT applied to the lumen enhances peristaltic reflexes (Bülbring and Crema, 1958; Bülbring and Lin, 1958; Craig and Clarke, 1991; Costall, Naylor and Tuladhar, 1993; Tuladar, Kaisar and Naylor, 1997). Specific antagonists for 5-HT3 and 5-HT4 receptors either by themselves or in combination

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block reflexes evoked by mucosal distortion in the dog ileum (Neya, Mizutani and Yamasato, 1993), the human jejunum (Foxx-Orenstein, Kuemmerle and Grider, 1996), the rat colon (Grider, Kuemmerle and Jin, 1996) and the guinea-pig colon (Foxx-Orenstein, Kuemmerle and Grider, 1996). Studies in the guinea-pig small intestine have been less compelling with 5-HT3 and 5-HT4 antagonists having no apparent effect on the initiation of reflexes by mucosal deformation when applied to the region of the IPANs (Yuan, Bornstein and Furness, 1994). However, blockade of 5-HT1P receptors prevents the expression of Fos protein normally evoked in submucous AH/Dogiel type II neurons by mechanical deformation of the mucosa (Kirchgessner, Tamir and Gershon, 1992). Thus, 5-HT may act as a sensory intermediate between mechanical deformation of the mucosa and the IPANs that mediate the subsequent reflexes. Release of 5-HT from mucosal epithelial cells by mechanical deformation must be at least partially independent of extracellular Ca2+. Indeed, the mechanisms transducing chemical and distension stimuli must also be partially Ca2+-independent. Low Ca2+, high Mg2+ solutions in the region of the stimulated IPANs do not block descending reflexes evoked by mechanical deformation of the mucosa or by distension (Johnson et al., 1996; Johnson, Bornstein and Burcher, 1998) nor do they alter responses of chemosensitive IPANs to either acid or neutral acetate in the guinea-pig small intestine (Kunze et al., 1995; Bertrand et al., 1997). Calcitonin gene-related peptide (CGRP) has also been implicated in the sensory transduction process in studies of rat colon, human jejunum and guinea-pig colon (Grider, 1994; Foxx-Orenstein, Kuemmerle and Grider, 1996; Grider, Kuemmerle and Jin, 1996). However, immunohistochemical localization of CGRP in the intestinal mucosa to date do not provide a clear picture of where this peptide acts within the reflex pathways. In particular, most of the CGRP in mucosa of the guinea-pig ileum seems to be contained either within the terminals of extrinsic primary afferent neurons or in a population of secretomotor neurons (Furness, Costa and Keast, 1984; Gibbins et al., 1985). CHEMORECEPTORS The mechanisms of transduction of chemical stimuli applied to the mucosa are less clear. The mucosal epithelium represents a barrier between the stimulant and the sensory terminals of chemosensitive IPANs and whether the stimulants cross the mucosa or lead to release of a sensory mediator from epithelial cells is unknown. Electrical stimulation of the mucosa can evoke delayed bursts of action potentials in myenteric chemosensitive IPANs and these have been attributed to release of a chemical mediator from the mucosal epithelium (Bertrand et al., 1997, 1998). It is tempting to suggest that it is this unidentified mediator that transduces chemical stimuli applied to the mucosa into neural activity. The mediator is unlikely to be 5-HT, because 5-HT applied to the mucosa stimulates the chemosensitive myenteric IPANs of the guinea-pig ileum via 5-HT3 receptors and the delayed bursts of action potentials are unaffected by 5-HT3 receptor antagonists (Bertrand et al., 2000). Thus, other chemicals found within mucosal endocrine cells mediate responses to chemical stimuli. For example, CCK is found within these cells (Rehfeld, 1978; Noyer et al., 1980; Williams, 1982), enhances intestinal motility (Stewart and Bass, 1976a; Barthó et al., 1982; Scheurer et al., 1983; Chang, Lotti and Chen, 1984; Rodriguez-Membrilla et al.,

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1995) and is a powerful excitant of enteric neurons. Indeed, myenteric IPANs are specifically excited by this peptide when it is applied to their cell bodies (Schutte et al., 1997). However, whether CCK or another peptide mediates chemical stimuli remains to be investigated.

RELATIONSHIPS BETWEEN SIMPLE REFLEXES, MOTOR PATTERNS AND MORE COMPLEX BEHAVIOUR Most of the discussion thus far has been about the simple reflexes evoked by stationary stimuli, usually of a single sensory modality, but the real situation is much more complex. When food is present in the intestine, the behaviour of any region will be determined by the interactions between simple reflexes evoked at many locations and via several different sensory modalities at each location, together with any neural mechanisms that may be activated solely by mixed stimuli. This might suggest that the overall outcome would be impossible to predict. However, some clear and consistent behaviours are seen in these circumstances and these appear to result from activation of motor patterns more complex than those responsible for the simple reflexes. Peristalsis, or the peristaltic reflex, is the most intensively studied of the motor patterns, largely because it can be repeatedly activated in a stereotyped fashion in vitro (see above). Other motor patterns seen in vivo such as the MMC and segmentation have been less easy to analyse. Once activated, the peristaltic pattern evoked by fluid distension of the small intestine involves a contraction of the circular muscle oral to a distended region of intestine containing much of the original fluid stimulus (a bolus) and a relaxation anal to the contracted region (Hennig et al., 1999). Both the contraction and the relaxation progress anally pushing the contents in an anal direction. This coordinated behaviour usually propagates for almost the full length of the intestinal segment in vitro, but in vivo the contraction typically dies away after progressing for varying distances along the gut. There have been many studies of peristaltic motor patterns and it has been assumed that they arise because the simple ascending excitatory and descending inhibitory reflexes are excited by the distension at the level of the fluid bolus. The anal propagation of the bolus is then due to movement of the bolus into a new segment of intestine, thereby stimulating a new set of reflexes. This idea is attractive in its simplicity, however, there are several observations, both recent and old, that cast doubt on the idea that the peristaltic motor pattern is a moving version of the stationary reflexes. This can be illustrated by three recent in vitro studies. In the first, it was found that the ascending excitatory reflex is evoked at a lower threshold distension than the peristaltic motor pattern (Tonini et al., 1996). Thus, activation of the ascending excitatory reflex is not sufficient to initiate propulsion of fluid along the intestine. In the second, it was found that circumferential stretch of an opened segment of intestine evoked a contraction of the circular muscle that progressed anally even though the stimulus could not move (Brookes et al., 1999a,b). In a third, an anally propagating peristaltic wave was initiated in an isolated segment of guinea-pig ileum by a localized mechanical stimulus even though the segment contained no content and so the stimulus could not move (Spencer, Walsh and Smith, 1999). These studies indicate that the peristaltic motor pattern involves activation of neural elements additional to those excited by

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simple reflexes and the second and third studies imply the existence of an anally directed excitatory pathway. Further evidence that the simple reflexes do not fully account for the peristaltic motor pattern comes from observations of the behaviour of the intestine or colon when a solid bolus is used to stimulate peristalsis. In both regions, it is common to observe a wave of circular muscle contraction passing over the bolus (Bolzer, 1949b), rather than remaining on the oral side of the bolus as would be expected if the contractions depended only on ascending excitation. Any model of the neural mechanisms underlying propulsive motor patterns needs to account for the basic properties and interconnections of the various functional classes of neurons identified above. Two features appear to be critical: the fact that virtually all neurons receive synaptic inputs from local IPANs and the fact that many of these inputs are via slow EPSPs lasting from several seconds to minutes. Figure 1.9 illustrates the basic circuit that can be deduced for the interconnections of the IPANs with each other, ignoring the possibility that there is a population of length sensitive neurons. Slowly adapting tensionsensitive IPANs contact most other myenteric IPANs including other neurons of the same class, chemosensitive IPANs and rapidly adapting distension-sensitive myenteric IPANs. The last population may also receive input from submucosal mechanosensitive IPANs. Chemosensitive neurons also contact the rapidly adapting distension-sensitive IPANs. All of these connections operate via slow EPSPs, so that changes in neuronal excitability can be expected to last much longer than the stimulus. POSSIBLE FEEDBACK EFFECTS WHEN THE MUSCLE TENSION CHANGES IPANs that are activated by muscle tension are in a peculiar situation, because the consequence of their activation, a change in muscle tension, will in turn change their excitability. We can speculate on the consequences of this feedback. Many of these IPANs provide inputs via slow EPSPs to neighbouring IPANs, interneurons and motor neurons (Kunze et al., 1993, 1997, 1998) (Figure 1.6B). Thus, changes in muscle tension would alter the firing of the slowly adapting tension-sensitive IPANs and, over time, modify the excitability of all the other myenteric IPANs and indeed all the other myenteric neurons in the region. This slow modulation of excitability would not play a role in the simple reflexes recorded electrophysiologically that were described above. Those reflexes were evoked under conditions where muscle contractions were prevented by blockade of L-type Ca2+ channels, thereby precluding ongoing activity in the slowly adapting tension-sensitive IPANs. By contrast, the descending excitatory reflex identified by Spencer, Walsh and Smith (1999, 2000) may depend on the ability of the muscle to contract thereby loading the slowly adapting receptors. This may account for the ability of P2 antagonists in the intermediate chamber of a three chambered organ bath to depress this reflex, even though they have no effect on descending inhibitory reflexes. However, the modulation of IPAN firing by activity in other IPANs can be expected to play a significantly greater role in responses to fluid distension in vitro or to the presence of food in the lumen in vivo. A slow fluid distension would increase firing in some slowly adapting tension-sensitive IPANs, which would then excite local motor neurons and also increase the excitability of other slowly adapting and rapidly adapting stretch-sensitive

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IPANs. Because the local motor neurons include both excitatory and inhibitory motor neurons, the tension change in the muscle would depend on the relative strength of the input from local IPANs, on their local projections and the interactions of their outputs within the muscle. The excitatory motor neurons project either directly to the circular muscle or orally, but the inhibitory motor neurons project anally. Thus, it might be predicted that the effect of a slow stretch in a given region would be to increase the tension in the circular muscle at the oral edge of the region by comparison to that at the anal edge. This would tend to load the more oral slowly adapting tension-sensitive IPANs, while unloading the equivalent neurons lying more anally. Thus, firing would increase in the oral tension-sensitive IPANs and the rapidly adapting distension-sensitive IPANs would become more excitable, while more anal neurons would remain less excitable. The muscle at the oral end of the region originally distended would provide a positive feedback into the motor circuit. The effect of this in an isolated segment would be for a slow rise in excitation in the most oral part of the segment with only a small increase in tension until a threshold is passed and all the local neurons would fire together. Such a slow increase in tension leading to a threshold has recently been demonstrated in a slowly stretched opened segment of intestine which appears to mimic many of the normal properties of the peristaltic motor pattern (Brookes et al., 1999a). A key feature of this mechanism is that the peristaltic motor pattern is initiated at a site where descending inhibitory pathways are not active either because there is no more oral stimulus or because the reflex pathways have been interrupted. The latter has been observed in isolated intestinal segments in which the myenteric plexus has been lesioned (Waterman, Costa and Tonini, 1994b). In the intact intestine, antiperistalis resulting from the excitatory feedback would be prevented by the presence of food in more oral segments exciting descending inhibitory reflexes. Chemical stimulants within the lumen may modulate the positive feedback IPAN circuit in at least two ways. Firstly, chemical stimulants excite descending inhibitory pathways, so that slow distensions anal to a local increase in the concentration of a stimulant would be less likely to excite slowly adapting tension-sensitive IPANs and peristalsis would be more difficult to initiate. In essence, this would reduce the ability of a peristaltic motor pattern to enter the relaxed region until the chemical either dispersed or was absorbed. This would provide a mechanism for ensuring that digesta remains in a specific region to allow further digestion and absorption. Secondly, a raised concentration of a chemical at a particular location would tend to set the oral edge of a functional segment, because activity in chemosensitive IPANs would have a similar effect to that of slowly adapting tension-sensitive IPANs. This would feed into the same positive feedback loop excited by slow distension, thereby exaggerating any effects of distension and localising them to the region containing the chemicals. The chemical stimulant would be expected to reduce the threshold for initiation of the peristaltic motor pattern in this region. Widely distributed chemicals in the intestinal lumen enhance intestinal propulsion both in vivo (Thomas and Baldwin, 1971; Richardson et al., 1991) and in vitro (Bülbring and Crema, 1958; Bülbring and Lin, 1958; Craig and Clarke, 1991; Costall, Naylor and Tuladhar, 1993; Tuladar, Kaisar and Naylor, 1997). The pattern would, however, not propagate very far until the local concentration of the chemical stimulant falls to a level that no longer evokes descending inhibition to act as a brake on the effect of distension. Removal of the chemical stimulus might be expected to lead to

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a more rapid decline in the excitability of the inhibitory motor neurons than that of the local IPANs, because the slow EPSPs in S-neurons are much briefer than those in AH/Dogiel type II neurons (Bornstein et al., 1984b). Thus, the inhibitory input to the muscle may decline while the excitability of mechanoreceptive myenteric IPANs is still elevated. Accordingly, once a particular component of the digesta has been absorbed, the peristaltic motor pattern would be released and progress for some distance until descending inhibition again unloaded slowly adapting mechanoreceptive IPANs in front of it. The idea that a descending inhibition brakes the peristaltic motor pattern by unloading tension-sensitive neurons is also consistent with the long anal projections of the rapidly adapting stretch-sensitive IPANs (Brookes et al., 1995; Johnson et al., 1996; Johnson, Bornstein and Burcher, 1998). These neurons excite the inhibitory motor neurons (Johnson, Bornstein and Burcher, 1998) and would relax the circular muscle and unload slowly adapting tension-sensitive neurons. Thus, a component of the simple descending inhibitory reflexes would be expected to increase the overall threshold for the peristaltic motor program anal to the point of maximum activity. This would tend to slow the propagation of the peristaltic wave, if not prevent its propagation. Evidence for the existence of a nutrient driven brake on the peristaltic motor pattern has come from studies of intestinal motor patterns in vivo, where glucose infusion has been shown to produce a marked inhibition of jejunal contractions (Schmid and Ehrlein, 1993). These examples of possible interactions between IPANs, the excitatory and inhibitory motor neurons and the muscle show how several different motor patterns may be generated within a common, relatively simple, reflex circuit. The actual behaviour will depend on the temporal and spatial balance of activation of the different IPANs. This will depend on the chemical state of the lumen, the texture of the luminal content, the contractile state of the muscle, and the rate of filling and emptying of the segment. Because the muscle normally has its own rhythmic pacemakers, the slow waves, in any final output one would also need to consider the intrinsic rhythm that would be imposed by these upon the activity of slowly adapting mechanosensitive IPANs. These interactions may also affect the results of pharmacological experiments on simple reflexes evoked by stationary stimuli. For example, blockade of NK1 tachykinin receptors in the recording chamber of a divided organ bath depresses descending inhibitory reflexes recorded in the circular muscle (Johnson, Bornstein and Burcher, 1998). While this effect appears specific for transmission from descending interneurons to inhibitory motor neurons, it may not be due to direct interference with transmission between these two classes of neurons. Many IPANs are spontaneously active when the mucosa is intact (Kunze et al., 1997; Bertrand et al., 1997, 1998), so that the inhibitory motor neurons would be expected to be receiving a background slow EPSP input from local IPANs (Kunze et al., 1997). The NK1 antagonist may block the background activity, thereby reducing the excitability of the inhibitory motor neurons. If the safety factor for transmission from descending interneurons is low relative to the descending axons of IPANs, then the apparent effect would be a specific reduction in transmission from the former to the inhibitory motor neurons. The predominant motor pattern in an animal fed an inert meal appears to be propulsive, i.e. propagating waves of circular muscle contraction that are very similar to the peristaltic motor pattern seen in vitro (Schemann and Ehrlein, 1986c; Buhner and Ehrlein, 1989).

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By constrast, when the meal contains nutrients, the predominant pattern consists of local contractions that spread only short distances along the intestine (Schemann and Ehrlein, 1986c; Buhner and Ehrlein, 1989). This appears to the analogue of the segmentation originally described by Cannon. Whether this is a neurally mediated motor programme is unclear. It has been argued that segmentation, which has not been recognised in vitro, is actually a myogenic phenomenon (Hasler, 1991). However, as can be seen from the discussion above stationary or slowly propagating rings of contraction may be produced by simple enteric reflex circuits by varying the level of excitability of the different neural elements. It is tempting to speculate that it is the release of the chemical (nutrient) brake postulated above which switches the motor activity of a segment of intestine from mixing to propulsion.

A FINAL COMMENT Over the last few years, methods for study of enteric reflexes and reflexly activated motor programs have grown in power and sophistication. However, many questions remain to be answered because the neural architecture is complex, there is a wide range of different neural interactions and several different stimuli may be considered to be physiological. The motor programmes and complex behaviour that govern intestinal function will probably prove to be elaborations of the neuronal mechanisms responsible for the simple reflexes described here. However, these represent only the base on which many other influences, like circulating hormones and the extrinsic nerves, impinge.

ACKNOWLEDGEMENTS This work was supported by a Program Grant (963213) of the NH&MRC (Australia).

REFERENCES Azpiroz, F. and Malagelada, J.-R. (1990). Perception and reflex relaxation of the stomach in response to gut distention. Gastroenterology, 98, 1193–1198. Baldwin, M.V. and Thomas, J.E. (1975). The intestinal intrinsic mucosal reflex; a possible mechanism of propulsive motility. In Functions of the stomach and intestine, edited by M.H.F. Friedman, pp. 75–91. Baltimore: University Park Press. Barthó, L., Holzer, P., Donnerer, J. and Lembeck, F. (1982). Effects of substance P, cholecystokinin octapeptide, bombesin, and neurotensin on the peristaltic reflex of the guinea-pig ileum in the absence and in the presence of atropine. Naunyn-Schmiedeberg’s Archives of Pharmacology, 321, 321–328. Barthó, L., Holzer, P. and Lembeck, F. (1989). Evidence for an involvement of substance P, but not cholecystokininlike peptides, in hexamethonium-resistant intestinal peristalsis. Neuroscience, 28, 211–217. Bauer, V. and Kuriyama, H. (1982). Evidence for non-cholinergic, non-adrenergic transmission in the guinea-pig ileum. Journal of Physiology, 330, 95–110. Bayliss, W.M. and Starling, E.H. (1899). The movements and innervation of the small intestine. Journal of Physiology, 24, 99–143. Bayliss, W.M. and Starling, E.H. (1900a). The movements and innervation of the small intestine. Journal of Physiology, 26, 125–138.

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2 Motor Control of the Stomach David Grundy1 and Michael Schemann2 1

Department of Biomedical Science, The University of Sheffield, The Alfred Denny Building, Western Bank, Sheffield S10 2TN, UK 2 Department of Physiology, School of Veterinary Medicine, Bischofsholer Damm 15/102, D-30173 Hannover, Germany The complex repertoire of gastric motor function is controlled and coordinated by reflexes that match gastric tone and peristaltic activity to the digestive needs of the individual. These reflexes operate both at the level of the enteric nervous system and through autonomic connections to the brain and spinal cord, with excitatory and inhibitory enteric motor neurones providing the final pathway for both intrinsic and extrinsic reflexes. The electrophysiological properties, neurochemical coding and projection patterns of these enteric neurones have been well characterised enabling the detailed circuit diagrams for contraction and relaxation of the corpus and antrum to be described. Extrinsic influences, from the vagus and splanchnic nerves, have a widespread action on enteric neural transmission, despite the relative paucity of extrinsic to intrinsic neurones. The stomach has a rich sensory innervation and the properties of vagal and spinal afferents have been well characterised. In contrast, the nature of the intrinsic primary afferent neurone has not been resolved despite abundant functional data for their existence. A concept for permissive control is presented, whereby autonomic inputs to enteric circuits set the “gain” for enteric reflexes. Plasticity in these circuits accounts for the adaptation of gastric function that occurs when the extrinsic innervations is compromised by surgery or disease. KEY WORDS: enteric nervous system; vagus; splanchnic nerves; stomach; neurochemistry; smooth muscle; gastric reflexes.

INTRODUCTION THE GASTRIC REFLEX REPERTOIRE Neural reflexes, together with endocrine and paracrine controls, serve to coordinate and control gastric motor and secretory function. Gastric motor function can be divided into two distinct activities which can be differentiated on a regional basis. The proximal stomach consisting of corpus and fundus serves as an important reservoir and has a remarkable capacity to accommodate large meals for subsequent delivery to the digestive and absorptive machinery of the gastrointestinal tract. The smooth muscle cells in this region generate myogenic tone and as such can be actively inhibited to bring about gastric 57

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relaxation (see Szurszewski, 1987). This occurs during deglutition by a process known as receptive relaxation (Cannon and Lieb, 1911). Gastric relaxation also occurs during gastric filling and was recognised by Kelling (1903) as a means of adapting gastric capacity to increasing contents by maintaining the pressure within very narrow limits, hence the term adaptive relaxation. Relaxation also occurs as a prelude to vomiting when the stomach dilates to receive chyme being returned from the intestines in readiness for expulsion (see Grundy and Reid, 1994). In all three cases the relaxation is mediated by the parasympathetic innervation via the vagus nerve and, in particular for the adaptive relaxation, by the enteric nervous system (ENS). Gastric tone is also influenced by the sympathetic innervation especially during the widespread inhibition of gut motor function that occurs during “adynamic ileus” following surgical manipulation of the gut. However, the vagus nerve may also play a role in this response illustrating the importance of reflex interactions between vagal and splanchnic pathways to the gut (Abrahamsson, Glise and Glise, 1979). The antrum, in contrast, functions as a peristaltic pump to mix, grind and propel gastric contents through the pylorus into the duodenum. These phasic, rather than tonic, contractions originate in a pacemaker region in the middle or orad corpus with the dominant site of origin on the greater curve (Alvarez and Mahoney, 1922; Kelly and Code, 1971). The contractions propagate aborally towards the pylorus and in the fasting state periodically serve to clear the stomach of accumulated secretions, cell debris and undigested residue as part of the migrating motor complex (Code and Martlett, 1975). However, during gastric emptying of a meal, the corpus and antrum, together with the pylorus and duodenum, must act in a coordinated fashion to optimise delivery of chyme to the small intestine. Reflexes between corpus and antrum are responsible for the fine tuning of this coordinated activity and, when disrupted, the rate of gastric emptying can be excessively fast (post-vagotomy dumping syndrome) or slow (diabetic gastroparesis) leading to a range of symptoms including bloating, nausea and pain (see Meyer, 1994). However, under normal circumstances gastric reflexes serve to coordinate the corpus and antrum, while feedback from the small intestine concerning nutrient delivery, osmolarity and pH tend to inhibit the gastric drive to emptying and so can match the rate of gastric emptying to the capacity of the intestine to digest and assimilate nutrients. THE NEURAL BASIS OF REFLEX CONTROL The motor programmes for controlling gastric tone and peristaltic contractions rely on feedback from sensory receptors positioned both within and outside the gastric wall. These monitor the prevailing conditions and trigger the appropriate response in the motor supply to the gastric smooth muscle. This reflex activity is organised at a number of different levels, all of which interact in an integrative way (Figure 2.1). At the local level is the enteric nervous system which Langley (1921) recognised as containing all the elements of an independent nervous system. Reflexes are triggered by local sensory inputs with the integrative ability of the ENS allowing the assimilation of information from the muscle and mucosa to determine the appropriate motor response (Wood, 1994b). The ENS in turn is influenced by the extrinsic elements of the autonomic nervous system through the parasympathetic and sympathetic nerves, which modulate activity in the ENS according to the digestive needs of the individual as determined by both somatic and autonomic demands.

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Figure 2.1

59

Schematic representation of the hierarchy of neural controls regulating gastric motor function.

Motor neurones in the ENS, be they excitatory or inhibitory, therefore provide the final pathway for both intrinsic and extrinsic reflexes. In this review the organisation of the neural control mechanisms which are responsible for this wide repertoire of gastric motor activity will be discussed.

THE ENTERIC NERVOUS SYSTEM The enteric nervous system in the stomach differs from other regions of the gut in that it consist of only one ganglionated plexus. The intrinsic innervation of the stomach is primarily located in the myenteric plexus since a ganglionated submucosal plexus is either very sparsely present or completely absent (Müller, 1920; Radke, Stach and Weiss, 1980; Christensen and Rick, 1985; Keast, Furness and Costa, 1985; Furness et al., 1991). Therefore, one must conclude that the myenteric plexus contains neurones involved in the regulation of both muscle and mucosal functions. Myenteric ganglia throughout the stomach do not run parallel to the circular muscle as they do in the distal small intestine, but rather they form a honeycombed-like meshwork similar to that seen in the proximal small intestine and the colon. The density and size of myenteric ganglia increases from the fundus to the antrum and there is also a decreasing gradient from lesser to greater curvature.

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A subserosal plexus located along the lesser curvature has been described in the rat and opossum (Skad-Nielsen, Poulsen and Holst, 1987; King and Szurszewski, 1984). There is evidence that these parafascicular ganglion cells receive input from extrinsic vagal fibres and it has been suggested that they serve as relay stations to transmit vagal signals to the periphery (King and Szurszewski, 1984).

ELECTROPHYSIOLOGY AND NEUROPHARMACOLOGY OF GASTRIC MYENTERIC NEURONES Electrophysiological properties of gastric myenteric neurones Different classes of enteric neurones with specific electrical and synaptic properties have been described in different regions of the gut (for review see Wood, 1994a; Bornstein, Furness and Kunze, 1994; Chapters 1 and 10 of this volume). Their electrical properties range from inexcitable cells to highly excitable cells with spontaneous activity. In addition, the full repertoire of synaptic transmission, i.e. fast and slow excitatory and inhibitory potentials, has been described in the enteric nervous system and these may be modulated by presynaptic inhibitory or excitatory mechanisms. All neurones in the enteric nervous system appear to receive synaptic input although any one neurone does not usually receive the entire complement of synaptic input (see Wood, 1994b). Although the issue of terminology has not been finally resolved there is a general agreement on the existence of two major classes of cells which were first described in the small intestine (Nishi and North, 1973; Hirst, Holman and Spence, 1974, see also Chapters 1 and 10 in this volume). One class of cell has phasic or tonic spike discharge during the injection of depolarising current pulses and usually exhibits fast excitatory post-synaptic potentials (fEPSP) after stimulation of synaptic input. These cells have been termed S-cells or Type 1 neurones. In contrast AH-cells, also described as Type 2 neurones, usually fire fewer action potentials than S-cells and are characterised by a prominent Ca-hump during the repolarising phase of the action potential and by a prolonged after-hyperpolarisation following their action potentials, hence the term AH. While action potentials in S-cells are carried mainly by sodium currents, calcium currents contribute to action potentials in AH-cells. AH-cells very rarely receive fast synaptic input but slow EPSPs can be readily induced in these cells. S-cells may be activated by fast and slow excitatory input. In addition to S and AH cells, a number of other cell types with intermediate electrophysiological behaviour have been described throughout the gut (for review see Wood, 1994a; Bornstein, Furness and Kunze, 1994). AH-cells have received particular attention over the last years because a sub-population of these are likely to function as intrinsic sensory neurones (Kunze, Bornstein and Furness, 1995). The electrical and synaptic properties of gastric myenteric neurones differ from those in the small and large intestine in a number of ways. The most striking difference is the total lack of AH cells in the corpus region (Schemann and Wood, 1989a). A few neurones with properties similar to AH cells have been described in one study on antral myenteric neurones (Tack and Wood, 1992a). The majority of gastric myenteric neurones behave like phasic or tonic S cells and they have been termed Gastric I and Gastric II neurones, respectively (Schemann and Wood, 1989a). A small population of neurones (less than 5%)

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which received fast synaptic input but were otherwise inexcitable were called Gastric III neurones (Schemann and Wood, 1989a). In the small intestine, initially inexcitable cells have been described which in time develop characteristics of AH cells (Wood, 1994b). However, it is unlikely that the gastric III neurones are AH cells since no spike generation occurs even after hours of impalement. In addition, unlike their counterparts in the intestine, inexcitable neurones in the gastric corpus can not be activated by forskolin. Unlike in the small intestine (see Chapters 1 and 10 of this volume) correlations between the neuronal types, i.e. Gastric I, II or III, with their morphological characteristics, their function as motor- or interneurones, and their pharmacology were not as obvious. The only relation found so far was that descending inhibitory neurones mostly exhibit Gastric I properties whereas Gastric I and II neurones were equally distributed in the population of ascending excitatory neurones. However, it still remains open at the moment whether the different neuronal types represent one cell class exhibiting different excitability levels, or whether their behaviour reflects functional specialisations. Synaptic properties of gastric myenteric neurones In addition to differences in electrical behaviour, there are also differences in the synaptic properties of gastric and intestinal enteric neurones. Synaptic transmission to gastric myenteric neurones, whether originating from intrinsic myenteric or extrinsic vagal fibres, appears to rely mainly on fEPSPs. In contrast to intestinal enteric neurones, all gastric myenteric neurones receive fEPSPs (Schemann and Wood, 1989b; Tack and Wood, 1991). The transmitter for these fEPSPs is acetylcholine which activates post-synaptic neurones via nicotinic receptors. The fEPSPs in Gastric I neurones usually reach threshold for spike discharge whereas the fEPSPs in Gastric II neurones are often subthreshold and of smaller amplitude. The small amplitude fEPSPs in Gastric III neurones never reach threshold for spike discharge. This may reflect differences in the number of cholinergic terminals synapsing onto the different cell types or may be simply related to different excitability level and passive properties of the membrane. Stimulation of either vagal or intrinsic fibres produced only occasionally slow EPSPs which are very likely mediated by acetylcholine or tachykinins (Schemann and Wood, 1989b; Tack and Wood, 1991; Schemann and Grundy, 1992). The relative paucity of slow excitatory and inhibitory synaptic events in gastric compared to intestinal enteric neurones is difficult to explain, in particular since the putative mediators of these two events are present in the gastric myenteric plexus or in extrinsic fibres supplying the myenteric plexus (Mawe et al., 1989; Schemann, Schaaf and Mäder, 1995) and moreover, exogenous application of mediators, including serotonin (Schemann, 1991a; Tack et al., 1992), substance P (Schemann and Kayser, 1991), noradrenaline (Schemann, 1991b; Tack and Wood, 1992b) and motilin (Tack, 1995) mimic slow EPSP-like responses in many neurones. Noradrenaline also has to be included in the list of putative mediators for slow EPSPs because it excites a small population of gastric myenteric neurones via α1 receptors (Schemann, 1991b; Tack et al., 1992). The functional relevance of the noradrenergic excitatory response remains unclear. It could be involved in excitatory and inhibitory responses observed after stimulation of intrinsic and vagal fibres because activation of α1 receptors decreases the amplitude of the rebound contractions while simultaneously

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increasing the contractile on-response and the relaxation in guinea pig stomach (Hillsley, Schemann and Grundy, 1992). It is notable that many mediators which enhance or decrease excitability in intestinal myenteric neurones do not affect gastric myenteric neurones (Schemann, 1990, 1992). These include substances like vasoactive intestinal peptide (VIP), calcitonin gene related peptide (CGRP), gastrin releasing peptide (GRP) and galanin. Antidromic stimulation of extrinsic sensory nerves to cause the peripheral release of tachykinins might also contribute to the long lasting activation of myenteric neurones (Delbro et al., 1983). The concept of axon reflexes is supported by data showing that nearly all neurones in the gastric myenteric plexus are excited by the sensory neurotoxin capsaicin. The excitatory effect of capsaicin can be abolished by desensitising the neurones to substance P or to [MePhe7]NKB which would suggest that neuronal NK3 receptors are involved in the effects of capsaicin-sensitive extrinsic sensory neurones on the gastric myenteric plexus. Although slow EPSPs are only rarely induced, a long lasting excitation of gastric neurones may be achieved by prolonged stimulation of cholinergic synapses. This is possible because the fEPSPs in the stomach do not show the run-down phenomenon that suppresses fEPSP discharge in myenteric neurones of the small intestine. Recurrent activation and positive feedback regulation involving release of acetylcholine are also mechanisms to raise the excitation level over extended periods of time (Schemann and Wood, 1989b). Auto-inhibition of acetylcholine release via presynaptic muscarinic receptors does not appear to play a significant role in the stomach, which could explain the high discharge rate of stimulus evoked fEPSPs (Schemann and Wood, 1989b). Spontaneous fEPSPs in a minority of gastric myenteric neurones is evidence for ongoing release of acetylcholine which coincides with basal cholinergic tone present in isolated stomach preparations (Beani, Bianchi and Crema, 1971; Hennig, Brookes and Costa, 1997). Neither slow nor fast inhibitory post-synaptic potentials have been described in myenteric neurones of the stomach. Inhibitory mechanisms, however, do operate in the stomach and presynaptic inhibition is one mechanism by which fEPSP discharge is modulated. However, a substantial number of fEPSPs appear to be resistant to presynaptic modulation, which is not surprising in view of the dominant role of fEPSPs for synaptic transmission. The most effective substances which presynaptically inhibit acetylcholine release are neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (Schemann and Tamura, 1992). In addition, noradrenaline and serotonin act to presynaptically inhibit cholinergic fEPSPs and non-cholinergic slow EPSPs (Schemann, 1990, 1991a,b; Tack and Wood, 1992). Vago-enteric interactions The majority of gastric myenteric neurones receive vagal input as demonstrated directly by electrophysiological recordings (Schemann and Grundy, 1992). Electrical stimulation of the vagus evokes cholinergic fEPSPs mediated via nicotinic receptors. Thus both intrinsic and vagal extrinsic transmission is dependent on cholinergic nicotinic synapses. Cholinergic and nitrergic motor, as well as non-motorneurones (presumably interneurones), are synaptically activated by vagal fibres. Occasionally, high frequency stimulation of the

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63

vagus was seen to produce a small amplitude slow EPSP, however, the mediator of the slow EPSPs has not been identified. Presynaptic inhibition is not restricted to fEPSPs evoked by intrinsic myenteric nerve fibres, but the same substances can also suppress vagal input to myenteric neurones. Indeed, some selectivity for vagal rather than intrinsic fEPSPs has been described for noradrenaline acting on α2 receptors to mediate presynaptic inhibition (Schemann, 1991b). This might represent a way in which vagal input to gastric myenteric neurones is selectively removed in order to favour, under certain conditions, the activation of intrinsic reflex circuits. Functional studies would support this view since a noradrenaline induced inhibition on gastric motility requires vagal cholinergic tone (Muren, 1957). The α2 receptor-mediated inhibition of transmitter release, mainly affecting acetylcholine release, is involved in the inhibition of electrically evoked contractile response in the stomach (Hillsley, Schemann and Grundy, 1992). Serotonin and NPY also have presynaptic inhibitory effects on some vagal synapses. NPY is released upon stimulation of nicotinic receptors and its release is blocked by hexamethonium (McIntosh, Dadgar and Kwok, 1992). PYY, which would presynaptically block both vagal and intrinsic fEPSPs has been shown to inhibit both basal gastric muscle contractions and those evoked by nerve stimulation by inhibiting cholinergic neurotransmission in the guinea-pig stomach (Wiley, Lu and Owyang, 1991). NPY-immunoreactivity in the myenteric plexus is not significantly affected by chemical sympathectomy (Mawe et al., 1989). Therefore, it seems possible that the presynaptic inhibitory effect on vagal inputs by NPY reflects the ability of myenteric NPY-positive neurones to actively shut down the vagal preganglionic input. This however is not a selective inhibition because in the same neurones intrinsic fEPSPs are also NPY-sensitive. Both electrophysiological and morphological studies indicated convergence as well as divergence of vagal inputs to myenteric ganglia (Schemann and Grundy, 1992; Berthoud, Jedrzejewska and Powley, 1990; Powley et al., 1994). These studies clearly demonstrated that the extent of vagal influences on gastric myenteric neurones had been previously underestimated. The concept that extrinsic fibres, in particular vagal fibres, innervate only a small number of myenteric neurones which would serve as command neurones has to be abandoned in favour of a more widespread influence of vagal fibres on the activity of gastric myenteric neurones (see below). NEUROCHEMICAL CODING OF ENTERIC NERVES IN THE STOMACH Plurichemical neurotransmission is one of the key features in the enteric nervous system and recognition of the way certain putative neurotransmitters or neuronal markers co-exist has been an important development in our ability to identify functionally distinct populations of neurones. The presence of a variety of transmitters in gastric myenteric neurones has been demonstrated in a number of different species (Gabriel et al., 1992; Kyosola, Veijola and Rechardt, 1975; Furness et al., 1991; Sternini, DeGiorgio and Furness, 1992; Van Aswegen et al., 1994; Jensen and Holmgren, 1991; Junquera et al., 1986; Schemann, Schaaf and Mäder, 1995; Schultzberg et al., 1980; Scheuermann et al., 1991; Wathuta, 1986; Groenewald, 1994; Yamamoto et al., 1994; Sann et al., 1998). Although the detailed chemical code of myenteric neurones varies between different regions of the gut and also between different species, it is notable that certain populations and their code appear to be

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highly conserved. This applies especially for neurones which contain nitric oxide synthase (NOS) and choline acetyltransferase (ChAT) which, except for minor overlap, belong to separate populations. These two enzymes are markers for cholinergic and nitrergic neurons respectively (Schemann, Schaaf and Mäder, 1995) which appear to be the primary transmitters for enteric reflexes. The most detailed information on the neurochemical coding of myenteric neurones in the stomach has been obtained for the guinea-pig and the description below is derived mainly from this species (Table 2.1). The proportion of cholinergic versus nitrergic neurones is about 70–30% both in the fundus and corpus. However, the cholinergic and nitrergic populations may be further subdivided according to the additional presence of other antigens and in this respect more than ten neuronal subpopulations have been described in the guinea-pig stomach (Table 2.1). Cholinergic subpopulations (at least 8) outnumber the nitrergic subpopulations (at least 2). ChAT-positive cholinergic cell bodies are the predominant neuronal class in the gastric myenteric plexus. A substantial number of these do not appear to contain any other markers from a long list so far examined (Schemann, Schaaf and Mäder, 1995).

TABLE 2.1 Neurochemical coding of guinea-pig gastric myenteric neurones, their relative proportions and putative functions in the corpus and fundus. Chemical code

Population size (Corpus/Fundus)

Main projections

Putative functions

NOS/VIP/NPY/±ENK

28/16

NOS/VIP/NPY/GRP/ ±ENK NOS/(GRP)/(ENK) NOS/–

/4

descending to muscle (only/+ENK) descending to mucosa (only/–ENK) ?

inhibitory muscle motor neurones secreto and/or vasomotor neurones ?

/3 5/9

? descending to muscle

? inhibitory muscle motor neurones secreto-and/or vasomotor neurones interneurones

descending to mucosa

ChAT/VIP/NPY/GRP/ ±DBH ChAT/SP/ENK/±GRP ChAT/SP/ENK/CALRET/ ±GRP ChAT/SP/SOM/±GRP ChAT/±ENK/±GRP

11/12

descending intraganglionic ascending to mucosa

21/15

ascending to muscle

6/6

ascending to muscle

6/2 21/37

? ascending to muscle ascending to mucosa

5-HT/±SP/±VIP/±ChAT

2/2

intraganglionic intraganglionic

secreto-and/or vasomotor neurones excitatory muscle motor neurones excitatory muscle motor neurones ? excitatory muscle motor neurones secreto-and/or vasomotor neurones interneurones interneurones

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However, substance-P is exclusively localised in one subpopulation of cholinergic neurones while some substance-P-positive as well as substance-P-negative cholinergic neurones also contain calretinin, enkephalins (ENK), GRP, 5-hydroxytryptamine (5-HT) or somatostatin (SOM). It is noteworthy that the number of SP-ergic myenteric neurones is twice as high in the corpus compared to the fundus region. Most nitrergic neurons in the guinea-pig are also VIP- and NPY-immunoreactive (Table 2.1). NO or a related compound, VIP and NPY are all candidates for inhibitory transmitters to gastric smooth muscle. A subpopulation of NOS, VIP and NPY containing neurones show also ENK immunoreactivity. Retrograde tracing studies would indicate that these neurones primarily innervate the muscle layers, in particular the circular muscle. Some nitrergic neurones did not show any neuropeptide-immunoreactivity (at least for the neuropeptides tested, see Table 2.1). They were NADPH-diaphorase positive and might be purely nitrergic. A proportion of these neurones were also found to be circular muscle motor neurones. ATP is also a prime candidate according to the purinergic hypothesis (Burnstock, 1972; Hoyle and Burnstock, 1989) and the coexistence of ATP and nitric oxide (NO) has been demonstrated in a subpopulation of myenteric neurones in the small and large intestine (Belai and Burnstock, 1994). However, it is unknown whether ATP is colocalised in these neurones in the stomach. NOS, VIP and NPY are also colocalised in ferret myenteric neurones but in this species one can visualise VIP-immunoreactive cell bodies without the need to pre-treat with colchicine to block axonal transport (Sann et al., 1998). This may mean that expression of VIP is higher in the ferret and could possibly relate to the eating habits of this carnivore which devour small prey whole and might therefore require a more pronounced and powerful relaxation of the stomach. In contrast NO appears to be the major mediator of relaxation in rodents. VIP and NPY are not exclusive to nitrergic neurones but are also present in a population of cholinergic neurones which in addition express DBH-immunoreactivity (Table 2.1). The DBH-cells do not show TH-immunoreactivity and it is unlikely that they synthesise noradrenaline (Mawe et al., 1989; Gabriel, Fekete and Halasy, 1989). Furthermore, this population is unlikely to be involved in the regulation of muscle function because they mainly project to the mucosa (Reiche, Pfannkuche and Schemann, 1997). A few TH-positive but DBH-negative, very likely dopaminergic cells, are however present in the ferret stomach (Sann et al., 1998). PNMT (phenylethanolamine-N-methyltransferase), the enzyme catalyzing the formation of adrenaline is not present in intrinsic neurones in the stomach (Back et al., 1995) but noradrenergic, TH/DBH-positive, and dopaminergic TH-positive and DBH negative nerve terminals can be visualised in sympathetic fibres surrounding myenteric ganglion cells (Mawe et al., 1989; Back et al., 1995). Some of the myenteric VIP neurones might project to the celiac ganglion, but their detailed coding is not known (Lee et al., 1986). There are significant differences in the chemical coding between the stomach and intestine which might be of functional significance. The relative high proportion of the putatively “pure” cholinergic neurones as well as the pronounced colocalisation of NPY in cholinergic neurones is striking in the stomach. CGRP is not present in intrinsic neurones in the stomach but is present in extrinsic sensory fibres where it is often colocalised with substance-P (see below). However, probably the most striking difference is the total lack of the calcium binding protein, calbindin, in myenteric neurones in the corpus. Very few

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calbindin-positive neurones have been reported in the antrum region (Furness et al., 1988) and the fundus (less than 2%). This is of functional significance because one population of calbindin positive neurones are strong candidates for intrinsic sensory neurones (see below). Either the stomach contains only very few intrinsic sensory neurones, which is unlikely, or some other cell type serves this role. Moreover, the presence or absence of calbindin immunoreactivity should not be taken, per se, as evidence in favour of a particular function. The chemical coding as well as the morphology of calbindin-immunoreactive enteric neurones is different between species and various regions in the gut (Hodgkiss and Lees, 1983; Scheuermann et al., 1991; Furness et al., 1988) and hence they very likely subserve a variety of functions. Other putative transmitters classically involved in neural regulation within the central nervous system have been identified in the gastric myenteric plexus but their detailed chemical coding is unknown. GABAergic myenteric cell bodies are present in the stomach with fibres innervating the circular muscle (Krantis and Webb, 1989; Gilon Tappaz and Remacle, 1991; Tsai, Tsai and Wu, 1993). Glutamatergic nerve cell bodies and fibres in the circular muscle have also been found (Tsai, Tsai and Wu, 1994; Liu et al., 1997). Galanin and thyrotropin-releasing hormone are found in myenteric cell bodies and fibres supplying all layers of the stomach (Ekblad et al., 1985; Wang et al., 1995; Tsuruo et al., 1988). Neurochemical coding of vagal fibres Nerves within the vagus have either motor (efferent fibres) or sensory (afferent fibres) function. The vast majority of vagal motor fibres use acetylcholine as the transmitter and are therefore ChAT-positive. There is evidence for additional transmitters in vagal fibres but it is not known whether they are colocalised with acetylcholine or represent separate populations. Thus, galanin, CGRP, NOS, and substance P have been immunohistochemically localised in vagal fibres (Kirchgessner and Gershon, 1989; Forster and Southam, 1993). Only a minor proportion of vagal afferents innervating the stomach contained CGRP (Sternini and Anderson, 1992). Using different strategies and approaches there is evidence that cholinergic neurones exist in the vagal afferent system (Tsubomura et al., 1987; Ternaux et al., 1989; Falempin et al., 1989). However, their function has not been unequivocally demonstrated. Glutamate is the major transmitter of vagal afferent inputs to the brainstem (Sykes, Spyer and Izzo, 1997). Neurochemical coding related to motor activity in the isolated stomach It is noteworthy that both in the fundus and corpus the cholinergic neurones by far outnumber the nitrergic neurones despite the primary task of the proximal stomach to accommodate volume and hence to initiate inhibition of muscle activity. This reservoir function is therefore not paralleled by a higher number of inhibitory muscle motor neurones. This might point to an important role of disinhibition of cholinergic neurones in the mediation of relaxation (see below). Nevertheless, the chemical coding of gastric myenteric neurones agrees with the differential motor responses evoked by electrical stimulation of the isolated intact stomach or of gastric muscle strip preparations (Paton

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and Vane, 1962; Armitage and Dean, 1966). The response to field stimulation is generally biphasic with an initial transient excitation followed by a longer lasting relaxation of the muscle. In some cases, an excitatory off (or rebound) response and a late excitation may occur while the relaxation may also have multiple components. The contraction/relaxation response is dependent on the stimulus frequencies with contractile responses dominating at lower frequencies (<1 Hz) and relaxation following the contractile response at higher frequencies (1–10 Hz) (Costall, Naylor and Tan, 1983). The initial tone of the preparation is also important in determining the balance of excitatory or inhibitory responses. Low tone favours an excitatory, high tone an inhibitory response (McSwiney and Wadge, 1928). The major mediator of the contractile response at the neuroeffector junction is acetylcholine acting at muscarinic receptors. The acetylcholine is probably released from more than one population of cholinergic myenteric neurones along with other transmitters including substance-P. The relaxation is mediated by non-adrenergic non-cholinergic (NANC) nerves. Several NANC transmitters including purines (Burnstock, 1972), VIP (Fahrenkrug et al., 1978), PACAP (Katsoulis et al., 1996) or NO (Bult et al., 1990) have been proposed and shown to be released upon field stimulation (Belai, Lefebvre and Burnstock, 1991). NPY is another candidate for inhibitory transmission to the muscle since immunoneutralisation to NPY caused a loss in vagal-induced inhibitory drive to the muscle (Grundy, Schemann and Hutson, 1993). At least in muscle strips of guinea-pig fundus, GRP which is present in a small population of nitrergic neurones evokes a relaxation. There has been an explosion of interest in the transmitter(s) released from these NANC inhibitory neurones because of the recent evidence suggesting NO may be involved. The novelty of a transmitter which is neither stored in pre-synaptic vesicles but synthesised de novo in response to an action potential, nor acts on post-synaptic receptor, but interacts intracellularly with guanylate cyclase to cause relaxation, has aroused considerable interest. Since NO is synthesised from L-arginine and analogues of L-arginine which act as inhibitors of NO synthesis are readily available, a role for NO in relaxations throughout the GI tract, including the stomach, has rapidly become established. It is not possible to determine which one of this list of substances is the primary transmitter because their contribution to the relaxation might vary with the species and/or the experimental conditions. Moreover, the coordinated release of two or more substances is likely to be involved in the relaxation response which can often reveal multiple components. A brief burst of low frequency stimulation evokes the short lasting relaxation which is followed by a sustained relaxation as stimulus frequencies and duration is increased. It is generally accepted that NO or a NO-releasing substance is involved in the initial, rapid relaxation, while the additional longer lasting relaxation is mediated by VIP (De Beurme and Lefebvre, 1988; D’Amato, Curro and Montuschi, 1992; Grundy, Gharib-Naseri and Hutson, 1993b; Meulemans, Eelen and Schuurkes, 1995). This conclusion is consistent with release studies demonstrating that low frequency stimulation is sufficient for maximal NO release whereas higher stimulation rates are required to release VIP (Boeckxstaens et al., 1992; Li and Rand, 1990; Takahashi and Owyang, 1995). Muscle cGMP levels increase before cAMP levels during intramural nerve stimulation supporting the view that NO and VIP act sequentially (Ito et al., 1990). Based on the finding that NO and VIP are colocalised in inhibitory myenteric neurones it seems reasonable to assume that these substances are frequency-dependently released from one specific population of inhibitory muscle motor

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neurones. The effects of NO released from inhibitory motor neurones are considered to be more complex than that of VIP and not restricted to a direct effect on muscle activity but might involve an intermediary action on ICC (Daniels and Berezin, 1992; Ward et al., 1997). NO may also be able to amplify VIP effects on muscle by enhancing presynaptically the release of VIP (Grider et al., 1992). There is also evidence for VIP induced release of NO from smooth muscle (Murthy et al., 1993). Blockade of NO release also enhances neurally-induced cholinergic responses evoked by electrical field stimulation and ganglionic stimulation by nicotine or dimethylphenylpiperazinium (DMPP), but not muscular contractions evoked directly by acetylcholine or methacholine (Lefebvre, deVries and Smits, 1992; Baccari, Bertini and Calamai, 1993). This suggests that NO might exert an additional inhibitory influence on acetylcholine release at a prejunctional level. This would be in agreement with the putative role of a subset of NOS-positive myenteric gastric neurones as interneurones (Schemann, Schaaf and Mäder, 1995). However, we have been unable to demonstrate any presynaptic inhibitory effects of the NO-donor sodium nitroprusside on cholinergic fEPSP in the small intestine (Tamura, Schemann and Wood, 1993) or the stomach (own unpublished results). If this holds true, the inhibition of acetylcholine release by NO must occur at the neuromuscular junction rather than at the level of the interneuronal synapses. VIP and ATP also depress neurally induced cholinergic contractions but, in contrast to NO, both ATP and more significantly VIP, depress the muscular contractions evoked by exogenous acetylcholine which might indicate that ATP and VIP have postjunctional effects (Baccari, Calamai and Staderini, 1994). Alterations in bowel motility, such as ileus, might result from excessive concentrations of NO generated during endotoxicosis and inflammatory bowel disease (Salzmann, 1995). However, a significant role of nNOS is supported by a study in animals with gastric hypomotility. Dairy cows suffering from abomasal displacement show signs of upregulation of nitrergic pathways which has been suggested to be involved in the occurrence of a dilated abomasum (Geishauser, Reiche and Schemann, 1998). Electrical stimulation of the vagus nerve in vivo mediates essentially identical responses to those described above for intramural field stimulation, i.e. cholinergic excitation followed by NANC-mediated inhibition (Klee, 1912) with both NO and VIP contributing largely to the latter (Grundy, Gharib-Naseri and Hutson, 1993b). Both the vagal excitatory and inhibitory responses are hexamethonium sensitive, mimicked by the nicotinic agonist DMPP and therefore dependent upon a predominantly cholinergic nicotinic transmission (Greef and Holtz, 1956a,b; Greef, Kasperat and Osswald, 1962; Paton and Vane, 1962; Armitage and Dean, 1966; Yano et al., 1983). This conclusion is supported by the electrophysiological demonstration of vagally evoked nicotinic fEPSPs in gastric myenteric neurones (Schemann and Grundy, 1992) and the observation that vagal stimulation-induced NO and VIP release is mediated via nicotinic receptors (Ito et al., 1988; Takahashi and Owyang, 1995). Transmural stimulation is reported to evoke stronger contractions, more vigorous peristalsis and a more pronounced relaxation than electrical stimulation of the vagus alone (Paton and Vane, 1963; Armitage and Dean, 1966) while DMPP evokes a stronger relaxation than that evoked by stimulation of the vagus (Yano et al., 1983). It appears, therefore, that transmural stimulation activates nerves inaccessible to the vagus, which may be truly “autonomic” (Paton and Vane, 1963). This has been confirmed by electrophysiological studies demonstrating

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that not all gastric myenteric neurones receive vagal input (Schemann and Grundy, 1992). PROJECTIONS AND CIRCUITS WITHIN THE MYENTERIC PLEXUS OF THE STOMACH Recent studies have demonstrated that the projection of cholinergic and nitrergic pathways within the myenteric plexus are polarised into ascending and descending projections in all regions including corpus, fundus (Brookes, Schemann and Hennig, 1994; Schemann and Schaaf, 1995; Pfannkuche et al., 1998; Brookes, Hennig and Schemann, 1998) and antrum (own unpublished results). The intracellular injection of myenteric neurones with neurobiotin allows the cell bodies of electrophysiologically characterised neurones to be identified and the terminals traced into the circular muscle, the longitudinal muscle or to other myenteric neurones (Schemann and Schaaf, 1995). From these morphological study it can be concluded that the gastric myenteric plexus contains motor neurones which project to the muscle. Other neurones project to myenteric cell bodies which we refer to as non-motor neurones, which because of their varicose endings in close proximity to other myenteric neurones, are likely to function as interneurones. The heterogeneous appearance of the terminals of these non-motor neurones, ranging from heavily branching basket-like endings to more simple varicose endings, might be taken as a preliminary indication of functional specialisation. The vast majority of myenteric neurones in the stomach were uniaxonal with paddle-shaped or filamentous dendrites but a small proportion of uniaxonal gastric neurones have axon collaterals supplying a number of different targets. One process always remains within the plexus, whereas the other branches project to the muscle, the mucosa or to both. Whether some specialised multitargeted neurones with intraganglionic endings or terminals in the muscle function as sensory neurones or have a specialised function as integrative neurones remains to be investigated. Motor neurones make up about two third of the gastric myenteric neurones; only a small population of less than 5% are multitargeted neurones; the remaining neurones belong to the class of non-motorneurones, which includes interneurones and the yet unidentified sensory neurones. Motor, non-motor and multitargeted neurones had no distinguishable electrophysiological characteristics. In the stomach, as in the small intestine, there is a projection preference of cholinergic and nitrergic myenteric neurones (Figure 2.2) (Schemann and Schaaf, 1995; Brookes, Hennig and Schemann, 1998; Pfannkuche et al., 1998). Intracellular labelling of myenteric neurones, sampled at random, indicates that the vast majority of cholinergic motor neurones project in an ascending direction. In contrast, the vast majority of nitrergic motor neurones are descending (Schemann and Schaaf, 1995). This projection pattern applies also for interneurones. Thus cholinergic interneurones have primarily ascending and nitrergric interneurones primarily descending projections. The fibres typically run for 0.5–20 mm in the longitudinal direction with 90% of the fibres projecting within 0.5–5 mm. Long projections over several centimetres are extremely rare in all regions of the stomach. An alternative labelling technique using the retrograde tracer DiI (1′-didodecyl3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate) placed on a small area of circular

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Figure 2.2 Ascending muscle motor neurones are cholinergic whereas descending muscle motor neurones are nitrergic. A small 50 µm bead of a retrograde tracer (DiI) has been placed onto the circular muscle. The tissue has been kept in organotypic culture and processed for immunohistochemistry for ChAT and NOS. On the left side (A–C) chemical code of ascending neurones are illustrated. The DiI filled cells are shown in B. The same field shows that the DiI cells are ChAT-positive but NOS-negative. On the right side (D–F) chemical coding of descending neurones are illustrated. The DiI filled cells (shown in E) are NOS-positive (D) but ChATnegative (F).

muscle (50 µm) has confirmed the projection polarity for myenteric neurones (Figure 2.3) (Brookes, Hennig and Schemann, 1998; Pfannkuche et al., 1998). In addition, using this technique, we have determined the detailed chemical coding of circular muscle motor neurones (Table 2.1). Ascending cholinergic neurones to this layer are both substance P-positive and substance P-negative and can be further divided by the additional presence or absence of GRP. Both populations of descending nitrergic neurones, those colocalised with VIP and NPY and those lacking the two peptides, project to the circular muscle. Functionally, this projection preference of cholinergic and nitrergic motor and interneurones would indicate the presence of a hardwired circuit which upon stimulation could initiate ascending excitation and descending inhibition of the circular muscle (Figure 2.4). It has to be stressed that this projection pattern of cholinergic and nitrergic myenteric neurones appears to be a general principle throughout the gut. The polarised circuits in the stomach may be differentially activated during gastric emptying in order to adjust excitatory and inhibitory tone to an appropriate level (Table 2.2 and Figure 2.4). This differential control has been recently demonstrated for the action of serotonin (Michel et al., 1997). Both muscle motor and interneurones are activated by serotonin. A fast transient excitatory response is mediated by 5-HT3 receptors and a slow long lasting activation is mediated by 5-HT1p receptors. It has been shown by intracellular dye injection in combination with the neurochemical coding that a proportion of the nitrergic neurones (30%) are activated by 5-HT1p receptors. In contrast, the 5-HT3 mediated activation is mainly restricted to cholinergic substance P-ergic neurones with 70% of the

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Figure 2.3 Distribution of DiI filled myenteric circular muscle motorneurones. The retrograde tracer DiI was applied to the circular muscle. The spatial distribution of DiI filled cells is shown. In addition the figure demonstrates that both in the fundus and corpus NOS-positive neurones are mainly located oral to the application site (descending neurones) whereas ChAT-positive neurones are primarily located aboral to the application site (ascending neurones). The broken lines indicate the border between fundus, corpus and antrum. The dotted lines indicate the course of the circular muscle.

cholinergic neurones responding. The functional implication is apparent from the observation that the 5-HT1p receptor agonist 5-hydroxyindalpine induces muscle relaxation and inhibits the response to cholinergic transmural stimulation (Michel et al., 1997). In contrast the 5-HT3 receptor agonist 2-methyl-5-hydroxytryptamine induces an atropine sensitive increase in muscle tone and potentiate the cholinergic transmural stimulation response (Desai, Warner and Vane, 1994; Michel et al., 1997). In agreement with these observations, gastric emptying is increased by a 5-HT1p antagonist (Mawe, Branchek and Gershon, 1989; Coulie et al., 1997) whereas gastric emptying of a protein rich meal is delayed by a 5-HT3 antagonist (Forster and Dockray, 1990). In the isolated stomach preparations it has been shown that 5-HT evoked relaxation is linked to NO (Meulemans, Helsen and Schuurkes, 1993a). Comparable differential control may be postulated for other transmitters (Table 2.2), like noradrenaline and tachykinins which are known to affect only a subpopulation of myenteric neurones in the stomach (Schemann, 1991;

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Cholinergic (nicotinic) enteric activation Cholinergic (nicotinic) vagal activation Presynaptic inhibition (NPYergic?) Presynaptic sympathetic inhibition (alpha-2)

Ascending excitatory cholinergic motorneurones

Descending non-cholinergic interneurone

Vagus

Sympathetics

5-HT1p mediated activation

NK-3 mediated activation

Descending inhibitory NANC motorneurones

5-HT3 mediated activation

Ascending cholinergic interneurone

Figure 2.4 This figure illustrates a wiring diagram based on results of neuropharmacological and neurochemical coding studies. The hardwired circuits may be activated through different receptors leading to graded and opposite changes in muscle activity. The sensory elements which might activate the circuits are unknown. Ascending cholinergic and descending nitrergic neurones might form the hardwired basis for a putative peristaltic-like reflex in the stomach.

Tack et al., 1991; Schemann and Kayser, 1991). In addition to the differential activation through post-synaptic receptors there also appears to be differential presynaptic inhibition. Thus, noradrenaline appears to predominantly inhibit cholinergic input to cholinergic motor neurones. There is no differential activation through cholinergic nicotinic receptors because all gastric myenteric neurones can be excited by exogenous acetylcholine and receive cholinergic fEPSPs independent of their function or neurochemical code (Schemann and Wood, 1989; Schemann and Schaaf, 1995). In the small intestine there is strong evidence that the projection polarity of cholinergic and nitrergic neurones in the myenteric plexus is the hardwired basis for the peristaltic reflex (see Chapters 1 and 10). This reflex is responsible for aboral transport of luminal

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TABLE 2.2 Neuropharmacology of gastric myenteric neurones. Neurotransmitter/ Neuromodulator

Response

Receptor

Neuronal populations responding

Acetylcholine

post-synaptic excitatory: fast, transient post-synaptic excitatory: slow, long lasting post-synaptic inhibitory: slow, long lasting post-synaptic excitatory: fast, transient

~100% ~10% ~1% ~10%

Motilin Neuropeptide Y Noradrenaline

post-synaptic excitatory: slow, long lasting presynaptic inhibitory: block of fEPSP post-synaptic excitatory: slow, long lasting post-synaptic inhibitory: slow, long lasting resynaptic inhibitory: block of fEPSP

nicotinic muscarinic muscarinic strychnine sensitive ? ? α1 α2 α2

5-hydroxytryptamine

post-synaptic excitatory: fast, transient

5-HT3

post-synaptic excitatory: slow, long lasting

5-HT1p

post-synaptic inhibitory: long lasting presynaptic inhibitory: block of fEPSP post-synaptic excitatory: long lasting

5-HT1a 5-HT1a NK3

Glycine

Substance P

? ~80% ~17% ~7% ~30% (primarily cholinergic) ~70% of ascending cholinergic (primarily SPergic) neurones ~30% of descending nitrergic neurones ? ? ~80% (cholinergic and nitrergic)

content and mediates relaxation below and contraction above a stimulus (Bayliss and Starling, 1899; Langley and Magnus, 1905; Magnus, 1908). The coordinated behaviour of muscle activity might also be relevant for peristalsis in the stomach. The degree of relaxation and the level of contractile activity or tone at adjacent regions is a major determinant of the propulsive force of the peristaltic wave and thereby influences gastric emptying (Keinke, Schemann and Ehrlein, 1984). A functional descending inhibition is indicated by myotomy experiments designed to sever the intramural pathways between the corpus and antrum (Holle, 1992). As a result of this projection the contractile activity aboral to the myotomy increased and led to a constricted antrum. Increase in basal tone of isolated stomach preparations after blocking neural activity with tetrodotoxin would support an important role of an inhibitory tone (Meulemans, Helsen and Schuurkes, 1993b). Increased tone of the isolated stomach treated with the L-arginine analogue (L-NAME) would indicate that NO is responsible, although not solely, for maintenance of inhibitory tone (Hennig, Brookes and Costa, 1997). Irrespective of the possible existence of a local peristaltic reflex in the stomach, other nerve mediated reflexes have been demonstrated. In isolated guinea-pig stomach, gradual filling of the stomach results in reproducible adaptive responses consisting of a relaxation of the fundus together with a substantial increase in intra-gastric volume. This response is independent of extrinsic innervation and is NANC-mediated (Carlson, 1913; Paton and Vane, 1963; Desai, Sessa and Vane, 1991; Hennig, Brookes and Costa, 1997). Distension of the LES caused gastric relaxation in the isolated stomach preparation, a reflex which depends on intramural NANC mechanisms (Ohta, Nakazato and Ohga, 1985). Both

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functional and morphological studies indicate that intrinsic gastric nerves may also modulate LES tone (Schulze-Delrieu et al., 1989; Brookes et al., 1996). Involvement of intrinsic neurones in distension evoked relaxation of the rat stomach is strongly suggested by a recent demonstration of c-fos expression in gastric myenteric neurones after refeeding fasting rats or distension of the rat stomach (Dimaline et al., 1995). While refeeding increased c-fos mRNA, physiological distension induced expression of c-fos, c-jun and c-myc in myenteric neurones. In this context it should be remembered that the initial increase in gastric pressure which occurs during distension is absent during feeding (when the stomach fills naturally) (Sick and Tedesco, 1908; Hennig, Brookes and Costa, 1997). INTRINSIC CONTROL OF GASTRIC MOTOR ACTIVITY Studies based on X-Ray examination of the stomach indicate the importance of muscle activity and tone just oral and aboral to a peristaltic wave (Keinke, Schemann and Ehrlein, 1984). The degree of relaxation and the level of contractile activity above and below a peristaltic contraction determines the propulsive force of the peristaltic wave and thereby the net transport of gastric content towards the pylorus. The spatial specificity and local modulation of neural activity determines the occurrence and timing of lumen occlusion relative to adjacent segments. Peristaltic activity in the stomach can be observed in the isolated stomach (Goltz, 1872; Paton and Vane, 1963; Armitage and Dean, 1966). Therefore, it must be concluded that intact extrinsic reflexes are not required for peristalsis, however, local axon reflexes might still be operative (see below). Moreover, even a muscle preparation where the myenteric plexus has been removed will respond to stretch with contractions indicating that myogenic mechanism may also be involved (Sick and Tedesco, 1908). Myogenic properties of gastric muscle are therefore involved in the intrinsic adaptations of gastric muscle tension to stretch and might be highly relevant for gastric compliance (Schulze-Delrieu, 1983; Hennig, Brookes and Costa, 1997). Around the time when Bayliss and Starling (1899) were describing the peristaltic reflex in the small intestine a number of investigators were attempting to investigate similar reflex responses in the stomach. However, it appears that the classical peristaltic reflex consisting of a contraction oral and a relaxation aboral to the stimulus (Bayliss and Starling, 1899) is not readily reproducible in the stomach. Yet there is extensive evidence for intrinsic reflex activity in the isolated stomach of a variety of species starting from the pioneering work of the end of the last century which demonstrated responses to mechanical (touching the serosal surface), chemical (crystals of sodium chloride) and electrical stimuli which sometimes induced peristaltic waves and produced variable motor responses in the stomach at the site of the stimulus as well as at oral and aboral sites (Lüderitz, 1891; Schultz, 1897a,b; Winkler, 1898; Auer, 1907; Sick and Tedesco, 1908, Schulze-Delrieu, 1990; Hennig, Brookes and Costa, 1997). In most cases, atropine sensitive local contractions and contractions oral to the stimulus were observed. Mechanical or chemical stimuli can also evoke an aboral contraction which sometimes obscured the small relaxations (Sick and Tedesco, 1911). These cholinergic influences are readily demonstrable in isolated muscle strips (Kuroda, 1924; Beani, Bianchi and Crema, 1971) where phasic activity will cease after removing the myenteric plexus (Sick and Tedesco, 1908).

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The existence of ascending excitatory cholinergic and descending inhibitory nitrergic myenteric neurones in the stomach might represent the hardwired circuit for the stimulus evoked oral contraction and aboral relaxation (Figure 2.4), despite the inconsistency of this particular reflex pattern. The dilemma and a possible explanation for the high variability in reflex activity reported for the stomach is that in most studies reflexes occurring close to the site of stimulus have not been carefully distinguished from reflexes occurring at a distance. This is relevant as the projection of ascending and descending neurones in the gastric myenteric plexus is only a few millimetres (Schemann and Schaaf, 1995; Brookes, Hennig and Schemann, 1998; Pfannkuche et al., 1998). For initiation of phasic contractions and for the propagation of peristaltic contractions continuity within the myenteric plexus has to be preserved (Sick and Tedesco, 1908; Poos, 1923; Holle, Steinbach and Forth, 1994; Cannon, 1911; Chaplin et al., 1987; Chaplin and Duke, 1990). It is likely that a chain of cholinergic neurones is involved in the longitudinal spread of the peristaltic wave because the ganglionic blocker hexamethonium is able to prevent peristalsis in response to transmural stimulation in an isolated stomach preparation (Bennet Bucknell and Dean, 1966; Armitage and Dean, 1966). The neural control mechanisms in the stomach can obviously initiate a variety of different reflex patterns depending on whether adjustment more locally or at more distant sites are required. Recent studies in the isolated stomach indicate complex reflex responses coordinating motor activities in functionally distinct gastric regions like fundus, corpus and antrum (Yuan, Brookes and Costa, 1997). These reflex pathways have been deduced from intracellular muscle recordings in response to distension. Descending inhibitory reflex pathways existed between the fundus and corpus, ascending inhibitory and excitatory reflex pathways between the antrum and corpus and between corpus and fundus (Yuan, Brookes and Costa, 1997). It is not known whether there are specific innervation patterns for the circular and longitudinal muscle. In the stomach circular and longitudinal muscle layer contract at a given locus simultaneously suggesting that they are activated in a complementary fashion whereas in the intestine the two muscle layers are activated in a reciprocal fashion (Sarna, 1993). Gastric reflexes exist that coordinate antral, corporal and fundal activity. These reflexes are usually opposite to the peristaltic reflex and are significantly reduced but not totally abolished after extrinsic denervation (Andrews and Bingham, 1990). The remaining long descending reflexes are hexamethonium sensitive and hence depend on intramural polysynaptic pathways (Andrews, Grundy and Scratchard, 1980a). THE QUESTION OF INTRINSIC SENSORY NEURONS From the beginning of this century it has become apparent that the isolated stomach can orchestrate complex contractile responses to mechanical stimulation of the muscle or mucosa despite the absence of connections to the brain and spinal cord. Distension of the isolated stomach evokes or enhances peristaltic activity (Hofmeister and Schütz, 1886; Schütz, 1886). In an excised stomach it is possible to evoke peristaltic activity after mucosal stimulation (Bickel, 1925). Muscle strips with mucosa attached produce coordinated peristaltic activity which changed into a more irregular activity when the mucosa has been removed (Poos, 1923). In the isolated stomach preparation instillation of acidic solutions initiated a much more vigorous peristalsis than water or NaCl solutions (Sick

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and Tedesco, 1908). Sensory nerves in the mucosa might transmit information to the myenteric plexus or directly to the muscle as indicated by the observation that lidocaine slows down propagation of peristaltic contractions (Ichikawa, 1989). A significant proportion of gastric myenteric cell bodies send their processes to the mucosa (Reiche, Pfannkuche and Schemann, 1997; Reiche and Schemann, 1998) and might be involved in mucosa/ muscle reflexes. However, neither in electrophysiological nor in neurochemical studies has the population of intrinsic sensory neurones in the stomach been identified to date. Such reflexes are organised entirely within the gut wall indicating that there must either be enteric sensory neurones or that collaterals of primary afferents, with cell bodies outside the gut wall, possess axon collaterals which project to elements of the enteric nervous system. While there is little information about the nature of the intrinsic sensory system in the stomach there is strong support for this concept as a basis for local reflexes in the small and large intestine. Dogiel (1899) recognised the heterogenous nature of enteric neurones and speculated that the Dogiel type-II neurons were sensory. Recent immunocytochemical studies demonstrating Dogiel type-II myenteric neurones with calbindin immunoreactivity and terminals extending to the mucosa provide support for this hypothesis (Furness et al., 1995). Intracellular recordings from these cells have been made during the application of mechanical and chemical stimuli to the mucosa and have confirmed that the peripheral terminals serve a sensory function and transmit information into the enteric circuits to modulate the excitability of S-neurones (Kunze, Bornstein and Furness, 1995). In contrast, Kirchgessner, Tamir and Gershon (1992), using the immuno-detection of fos as a marker for enteric neurones activated by mucosal stimulation, have suggested that the first-order sensory neurones have their cell bodies in the submucosal rather than myenteric plexus and that the extensive fos-immunoreactivity in the myenteric plexus arises from a cholinergic input from submucosal sensory neurones. However, the pattern of intrinsic afferent activation may be stimulus specific since Smith, Bornstein and Furness (1992) showed that distension and deformation of the mucosa activate different sensory neurones but a common final motor pathway. Numerous studies have demonstrated gastrointestinal reflexes in vitro which are abolished by tetrodotoxin and therefore are of intrinsic origin. Using elegant variations of the basic whole mount preparations for intracellular electrophysiology, several studies have characterised the synaptic responses in the ascending and descending enteric pathways that are an essential element of the peristaltic reflex (Hirst, Holman and McKirdy, 1975; Smith, Bornstein and Furness, 1990). The convergence of inputs following stimulation of mucosal endings and mechanoreceptors in the muscle points to a common motor programme for propulsion independent of the mechanism of activation. This programme could be triggered by intrinsic sensory neurones or axon collaterals of primary afferents and using capsaicin as a neurotoxin selective for the latter, there does indeed appear to be a deficit in reflex behaviour when primary afferents are eliminated in the colon (Grider and Jin, 1994). Distension localised to different regions of the isolated stomach also evokes a complex pattern of reflex excitation and inhibition revealed by intracellular recordings from the guinea-pig circular muscle (Yuan, Brookes and Costa, 1997). Both ascending excitation and descending inhibition were evident over relatively long distances between corpus and antrum. Thus, distension of the antrum evoked inhibition of the

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corpus while distension of the corpus evoked inhibition followed by excitation of the fundus. When these inhibitory junction potentials were attenuated pharmacologically with L-NOARG and apamin, distension was shown to evoke ascending excitatory reflexes. No reflex activity was found in the antrum following stimulation of the corpus. The authors conclude that these reflexes could arise from intrinsic sensory neurones or from collaterals of extrinsic vagal or spinal afferents but offer no evidence either way. From experiments in which the movement of colonic segments were examined after degenerative section of the extrinsic innervation, Grider and Jin (1994), suggested that responses to muscle stretch were mediated by axon reflexes while reflexes from the mucosa were unaffected. In the small intestine, however, both muscle and mucosal reflex responses were maintained in chronically denervated segments (Furness et al., 1995). Thus there may be different patterns of sensory innervation to mucosa and muscle in colon and small intestine. Relating these observations to the stomach is difficult because in this region there is no ganglionated sub-mucosal plexus and neurones with Dogiel type II morphology, calbindin immunoreactivity or AH electrophysiology in the myenteric plexus are rare.

THE EXTRINSIC INNERVATION EXTRINSIC AFFERENTS The upper gastrointestinal tract has an extensive afferent innervation which conveys sensory information to the brainstem and spinal cord via vagal and splanchnic afferents respectively. Under normal circumstances the information conveyed in these afferents does not reach consciousness but is involved in reflexes which control gastric tone and motility. However, these afferents can mediate sensations which can range from vague feelings of fullness and satiety to discomfort, pain and nausea. According to the summation theory of nociception, pain is elicited when receptors are excessively stimulated rather than by specific nociceptors. In this respect splanchnic afferents can respond to subnoxious levels of stimulation which mediate reflex inhibition of gastrointestinal motor function and might mediate pain when the level of stimulation becomes noxious. Some afferents respond only at noxious levels and may be specific nociceptors. These may become sensitised during inflammation while other “silent” nociceptors may be recruited as a consequence of a range of inflammatory mediators (see Sengupta and Gebhart, 1994). In organs like the stomach (although the observations have been made in the bladder and colon) which can generate a range of sensations from mild fullness to intense pain, it is likely that the relative activation of low and high threshold afferent fibres is a determining factor (Cervero and Jänig, 1992). The splanchnic afferents have been likened to a trip-wire alarm system because the relatively small number of afferent fibres (<7% of DRG cells innervate the gastrointestinal viscera) have widespread connections in the spinal cord. There is little capacity for discrimination in the system but once it is activated it can have massive effector responses (Cervero and Tattersall, 1986). There is little evidence that vagal afferents mediate sensations but instead convey moment-to-moment information on gastrointestinal function to the brainstem. At the level of the diaphragm vagal afferent fibres outnumber efferent fibres by about 10 to 1

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providing an enormous volume of afferent traffic that plays an important role in reflex control of gastrointestinal function and behavioural responses such as satiety (see Grundy, 1988). Vagal afferents have their cell bodies in the nodose ganglia and provide peripheral and central projections to the gut and the brain respectively. The terminals of primary afferents within the stomach wall have been visualised by a variety of fibre-tracing and immunocytochemical approaches. Vagal afferents have been shown to terminate in both mucosa and muscle where the endings may subserve a sensory function while other terminations in the myenteric plexus are more consistent with a role in mediating axon reflexes (Berthoud and Powley, 1992; Berthoud et al., 1997). However, the possibility that these intraganglionic endings may sense the neurochemical environment or electrical activity within the ganglia, as suggested by Kolossow and Milochin (1963), can not be discounted, especially in the absence of good evidence for functional synaptic inputs from vagal afferents to gastric myenteric neurones (Schemann and Grundy, 1992; Zheng et al., 1997). Using confocal microscopy, the terminal arborisations of individual vagal afferents have been identified in both the muscle and mucosal layers of the stomach and duodenum (Berthoud et al., 1997; Berthoud and Powley, 1992; Powley et al., 1994). The projections into the myenteric plexus have been termed intraganglionic laminar endings (IGLE) and have a high density in the stomach with about 30 to 50% of ganglia receiving at least one IGLE. A parent axon typically branches to form a highly arborised network on the surface of an individual myenteric ganglia immediately beneath the basal lamina and interdigitating with glial processes. This has been suggested to provide a mechanical link to the connective tissue matrix around the ganglia and is consistent with a mechanosensitive role detecting sheer forces generated by contraction of the overlying or underlying muscle. However, since the same fibres project into the muscle layer and endings here are better positioned to detect muscle tension, the role of IGLEs remains speculative. An “efferent” role for vagal afferent terminals in the myenteric plexus is inferred from the presence of a variety of peptides including substance P, cholecystokinin (CCK), SOM, VIP, GABA and CGRP in the cell bodies of these neurones in the nodose ganglia and which accumulate on the oral side of the ligated vagus indicating transport towards the periphery (Dockray and Sharkey, 1986; Varro et al., 1988; Szabat et al., 1992). Large numbers of nodose ganglion cells contain NADPH-diaphorase (Ichikawa and Helke, 1996). However, there is little information on the function of any of these agents derived from vagal afferents on gastric motor function although axon reflexes are inferred from responses to vagal stimulation that are hexamethonium resistant (Delbro and Lisander, 1982; Delbro et al., 1983). The terminations of splanchnic afferents are also extensively distributed within the gut wall and also project into the prevertebral ganglia where they can influence the activity of post-ganglionic sympathetic neurones. A major feature of the splanchnic afferent fibres in the stomach is that they are immunoreactive to CGRP and, to a variable extent in different species, also contain substance P (Green and Dockray, 1988). They supply the muscle layers, myenteric plexus and submucosal blood vessels and disappear after treatment with capsaicin or coeliac ganglionectomy. Retrogradely labelled gastric sensory neurones in the DRG were predominantly (85%) CGRP-positive and the majority of these neurones colocalised substance P (Green and Dockray, 1988). Other peptides and neurochemical

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markers present in DRG neurones include ChAT, NOS, galanin, SOM, CCK, VIP, GRP, oxytocin but the functional significance of these is unknown (See Szurszewski and Miller, 1994). Peripheral release of CGRP from spinal afferents following noxious stimulation is implicated in hyperaemia that has a cytoprotective role following mucosal damage (Holzer 1994; Chapter 3 of this volume) and appears to play a role in the inhibition of gastric emptying seen after surgery (Plourde et al., 1993). The gastric emptying of acid and hypertonic saline is increased following immunoneutralisation of CGRP which implies that axon reflexes may also have a more physiological role (Forster and Dockray, 1991). CGRP nerve terminals are also distributed around gastric myenteric ganglia although an influence on synaptic transmission at this level has not been demonstrated. Substance P, in contrast, has a potent depolarising action on gastric myenteric neurones via the NK-3 receptor, but because substance P can also be of intrinsic origin it is not clear if this represents a potential action of afferent axon collaterals on myenteric reflex circuits (Schemann and Kayser, 1991). Splanchnic afferents also have collateral branches supplying the prevertebral ganglia where they may modulate transmission through the sympathetic pathway (see Szurszewski and Miller, 1994). Immunoreactive CGRP and substance P terminals form pericellular nests around principle ganglion cells in a number of species. Application of substance P mimics the noncholinergic sEPSP that follows electrical stimulation of the inputs to the prevertebral ganglia and both are reduced by desensitisation to substance P and after capsaicin treatment in vitro. Thus both morphological and electrophysiological data support the view that splanchnic afferents participate in peripheral reflexes and at the same time convey nociceptive information to the spinal cord. The role of CGRP in the pre-vertebral ganglia is not yet known. Another mechanism for reflexes through the prevertebral ganglia stems from a population of VIP containing myenteric neurones that project from the stomach and intestine to the coeliac and other prevertebral ganglia (Lee et al., 1986). These may also be ChAT positive (Mann et al., 1995). Postganglionic sympathetic neurones have been classified according to their transmitter phenotype into those that contain noradrenaline alone and others which co-localise either NPY or somatostatin (Macrae, Furness and Costa, 1986). These innervate different functional targets in the small intestine with those containing NPY supplying mainly blood vessels and, in the small intestine, those containing SOM supplying the submucosal plexus. Those containing only noradrenaline have the myenteric plexus as their target. All receive input from preganglionic sympathetic neurones but it is predominantly the latter and rarely the former neurones which receive inputs from spinal afferent and viscerofugal fibres suggesting that activation of these pathways will lead to inhibition of motor function rather than influencing gastric blood flow (Mann et al., 1995). This observation is consistent with in vitro studies that have demonstrated the loss of intestino-gastric inhibitory reflexes after the connection to the coeliac ganglion is severed (Kreulen, Muir and Szurszewski, 1983). An inhibitory reflex from stomach to intestine via the coeliac ganglia has also been described. This mechanism appears to utilise NO in ganglionic transmission, which is consistent with the presence of NADPH-diaphorase stained endings in the coeliac ganglia of rabbit and guinea-pig (Mazet et al., 1993; Mann et al., 1995) although in the latter study this may derive from fibres projecting from the

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large intestine. Some preganglionic sympathetic neurones in the spinal cord may also be NADPH diaphorase positive (Anderson et al., 1993). The nature of the afferent inputs to the pre-vertebral ganglia have been assessed by recordings from principle ganglion cells during stimulation of the viscera. The synaptic input to the ganglion cell from these myenteric afferents appears to arise from mechanoreceptive elements that (in the colon) are “in-parallel” with the circular muscle. Thus, as the viscus fills there is tangential lengthening of the circular muscle which activates mechanosensitive afferents associated with it. Contractions which cause the viscus to empty are associated with off-loading of the mechanoreceptor despite the increase in wall tension and intraluminal pressure. Thus the viscerofugal myenteric neurone may be activated by stretch and not tension as is the case for primary vagal afferents (see below). A number of mediators are implicated in the distension evoked depolarisation of post-ganglionic sympathetic neurones including ACh, VIP, GABA and CCK with both pre-synaptic and post-synaptic mechanisms of action (see Szurszewski and Miller, 1994). There may also be inhibitory influences which allow the preganglionic inputs to gate the inputs from the periphery and as such the functional importance of these two inputs may vary under certain physiological conditions (Miolan and Neil, 1996). Afferent inputs to the CNS While sensory information is available to the local reflex circuits it is only in the CNS that a global picture of gastrointestinal events can be constructed from the convergent inputs from along the entire length of the gastrointestinal tract and from different receptor types signalling different information. These extrinsic afferents are therefore important to allow the stomach to function within the wider context of the entire gastrointestinal tract. There is a considerable literature on the discharge characteristics of gastrointestinal primary afferents, recorded directly either from the axon en route to the CNS or from the cell body in the nodose or dorsal root ganglia. A particular focus in these studies has been the functional characteristic of afferent fibres as determined by the stimulus-response characteristics of their receptive ending. In this way, the frequency of action potentials encodes the stimulus either as a consequence of a direct action on a generator region on the nerve terminal or following the stimulation of a secondary sense cell which encodes stimulus intensity by the release of a neuroactive substance which in turn causes afferent firing. The properties of afferents have also been inferred from recordings made from second order neurones in the pre-vertebral ganglia, spinal cord and brainstem (Raybould, Gayton and Dockray, 1988; Barber, Yuan and Burks, 1989; Anthony and Kreulen, 1990). However, these indirect recordings are influenced by the convergence of incoming information which might be a problem in the brainstem where information from different receptor types along almost the entire GI tract is channelled. Gastric mechanoreceptors Vagal mechanosensitive afferents in the stomach were first described by Paintal (1954). In 1957, Iggo coined the term “in-series tension receptors” because of the ability of these afferents to respond to both passive stretch and active contraction, although this may relate

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to a functional arrangement rather than to an anatomical one (Berthoud and Powley, 1992; Grundy, 1988). Although the properties of muscle mechanoreceptors are homogeneous (all respond to stretch and contraction), they do show a range of spontaneous activity and threshold for activation (Cottrell and Iggo, 1984). Furthermore, receptors in different parts of the stomach behave quite differently because of the different functions of the proximal and distal stomach (Takeshima, 1971; Andrews, Grundy and Scratcherd, 1980b). The properties of muscle mechanoreceptors in the corpus reflect the reservoir function of this region. With the stomach empty they generate a low frequency irregular spontaneous activity which increases dramatically when the volume of the stomach is increased (Andrews, Grundy and Scratcherd, 1980b; Blackshaw, Grundy and Scratcherd, 1987). They therefore effectively monitor the degree of gastric filling. However, being tension receptors, they can also signal increases and decreases in muscle tone, the latter occurring during receptive relaxation. In contrast, the properties of receptors in the antrum reflect the function of this region as a muscular pump. Being less distensible than the corpus, with much of a meal being accommodated in the latter, antral mechanoreceptors do not readily respond to increases in gastric volume, but instead generate a burst of impulses as each wave of contraction passes over the receptive field. The amplitude and waveform of the contraction is reflected in the rate and duration of discharge in the mechanoreceptors, while the velocity and direction of propagation of the contraction will be encoded in the discharge from adjacent receptors. The response of gastric mechanoreceptors to stretch has recently been suggested to be influenced by CCK released when nutrients enter the duodenum. Gastric afferent activity is stimulated by CCK at high doses (Davison and Clarke, 1988) while at low doses CCK can sensitise the afferent’s response to stretch (Schwartz, McHugh and Moran, 1993). This sensitivity is suggested to underlie the satiety effect of CCK with gastric afferents signalling both the quantity of ingested material through distension and its quality in terms of nutrient density following its delivery to the duodenum. However, in the gastric corpus which undergoes pronounced relaxation in response to CCK, the reverse occurs and vagal mechanoreceptors are off-loaded as the stomach relaxes (Grundy, Bagaev and Hillsley, 1995). This observation is consistent with data which suggests that inhibition of food intake by gastric distension and intestinal nutrients is mediated by independent mechanisms (Cheng et al., 1993) and that the action of endogenous CCK on satiety is not via an endocrine action but through local paracrine mechanisms. A more likely target for CCK action is on the terminations of intestinal mucosal afferents which lie in close proximity to the enteroendocrine cells which release CCK in response to luminal nutrients, and which might be implicated in afferent signal transduction (Berthoud and Patterson, 1996). Splanchnic afferents are also mechanosensitive and will respond to stretch and during contraction (Morrison, 1977). However, these afferents are associated with the serosa and its mesenteric connections, where a single afferent terminates in multiple receptive fields which can cover a wide area and extend over more than one visceral target. As such, the relationship between wall tension and afferent activity is less precise than is the case with vagal afferents and these afferents are commonly referred to as “in parallel” receptors. The threshold for activation of splanchnic afferents is generally higher than for vagal afferents which is consistent with their role in visceral sensation. However, because they are active at physiological levels of stimulation these afferents may also play a role in reflex control

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as well as the generalised suppression of gastrointestinal activity which accompanies pathophysiological levels of distension. In addition, splanchnic afferents, at least in more distal regions, can be sensitised by inflammatory agents such as bradykinin and other agents that can be released during ischaemia (Blumberg et al., 1983; Pan and Longhurst, 1996).

Mucosal afferents A diverse population of mucosal receptors have been described on the basis of experiments in which vagal afferent discharge is recorded in response to a range of stimuli applied to the mucosal epithelium (Grundy and Scratcherd, 1989). The endings are ideally positioned to signal information on the physical and chemical environment of the gut lumen and a complex picture of mucosal sensitivity has emerged (Mei, 1985). Much of the literature on mucosal afferents is concerned with those supplying the small intestine and especially the duodenum where chemosensitivity underlies the feedback mechanisms for gastric emptying. However, it is clear that mucosal sensitivity exists also in the stomach. These endings have a multi-modal pattern of activation (Iggo, 1957; Davison, 1972a; Clarke and Davison, 1978; Blackshaw and Grundy, 1990b) and respond whenever the luminal contents differ from iso-osmotic or neutral pH. The relevance of the latter for an organ that secretes acid is unclear but could be related to the normal cytoprotective role of gastric mucous and blood flow. Only when the normal cytoprotective mechanisms breakdown would acid back diffuse to the afferent endings in the lamina propria. Vagal mucosal afferents are also highly sensitive to deformation of the mucosa, generating a burst of impulses when the mucosa is stroked or touched. In this respect, these endings will detect the particulate nature of gastric contents and may play a role in trituration of solids (Davison, 1972b). Spinal afferents may also be sensitive to luminal acid but these have not been characterised electrophysiologically. However, it is clear that noxious agents applied to the mucosa (including acid, especially when the protective mucous has been eliminated with alcohol) activate capsaicin sensitive spinal afferents which mediate reflex vasodilatation via the local release of CGRP and protect the mucosa against pending damage (Holzer, 1994). These spinal afferents are suggested to be the “first line of defence” against mucosal damage while at the same time can activate autonomic reflexes to initiate more widespread reactions.

Intestinal afferents and feedback mechanism Intestinal and especially duodenal afferents play an important feedback role in the control of gastric emptying. Thus the rate of gastric emptying is proportional to the nutrient content of the meal to the extent that calories, whether from fats, carbohydrates or proteins, are delivered to the duodenum at a relatively constant rate (Hunt and Stubbs, 1975). However, while it might appear that the duodenum monitors calories, in reality the sensitivity to different nutrients is tuned to produce a comparable end-organ response. The concept of nutrient receptors has been readily accepted in the literature on reflex control of gut function but direct electrophysiological evidence is, in fact, quite sparse. The group of Mei and his colleagues are the main advocates of specific nutrient receptor sensitivity with separate

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publications describing afferent responses to fats (Melone and Mei, 1991), amino acids (Jeanningros and Mei, 1980) and carbohydrates (Mei, 1978). The mechanisms underlying this nutrient sensitivity have not been elucidated but recent studies have focussed on the role of enteroendocrine cells in afferent signal transduction both for intrinsic and extrinsic afferents. The principle is that enteroendocrine cells are secondary sense cells in the gastrointestinal mucosa which are strategically positioned to monitor luminal composition (Fujita et al., 1979). These cells have a apical tuft of microvilli which is proposed to “taste” the luminal contents and in response to an appropriate stimulus release the contents of storage granules across the baso-lateral membrane to stimulate afferent terminals in close proximity. Good evidence is available that CCK acts in such a fashion. Thus, it has been shown that a subset of vagal mucosal afferents are exquisitely sensitive to CCK acting on the CCKa receptor (Richards et al., 1996). Functional data has demonstrated that many of the actions of CCK including the feedback regulation of gastric emptying (Holzer et al., 1994) and the inhibition of feeding behaviour (Smith et al., 1981) are mediated by this vagal action, and, moreover that endogenous CCK released by luminal exposure to protein acts in the same way (Reidelberger, 1992). Data suggesting a similar role for 5-HT are also available in the context of its role in nausea and vomiting and the gastrointestinal consequence of these behaviours (Andrews et al., 1990). Certainly a subpopulation of afferents are exquisitely sensitive to 5-HT (Blackshaw and Grundy 1990a,b; Hillsley, Kirkup and Grundy, 1998). For the latter it appears that the 5-HT3 receptor on vagal mucosal afferents is an important target for 5-HT released from EC cells and this has led to the development of a new class of potent anti-emetics (Andrews et al., 1990). For intrinsic afferents in the small intestine, however, there is good evidence that 5-HT1p receptors may be more important and that 5-HT release is an obligatory step in the transduction of both mechanical and chemical luminal stimuli (Kirchgessner, Tamir and Gershon, 1992). For extrinsic intestinal afferents, however, neither 5-HT- nor CCK-sensitive vagal afferents depend entirely on enteroendocrine cell mediators for their afferent sensitivity since both mechanosensitivity and sensitivity to luminal acid persist after the receptors for these agents have been blocked. It appears therefore that there are multiple transduction pathways for mucosal afferents (Richards et al., 1996).

CENTRAL REFLEX PATHWAYS SPINAL REFLEXES From the dorsal root ganglion the central processes of splanchnic afferents enter the spinal cord and project rostro-caudally in Lissauer’s tract, with collaterals passing into lamina I laterally and medially around the dorsal horn (de Groat, 1986). The main terminal field is in lamina V with some afferent projections to the dorsal grey commissure, to the region around the central canal and to laminae I and V on the contralateral side of the spinal cord. Medial projections of splanchnic afferents extend into lamina I and also into the ipsilateral dorsal column. The substantia gelatinosa (lamina II), which is the terminal site for somatic afferents, does not receive a strong visceral afferent projection, indicating a distinct pattern of processing of visceral and somatic afferent information (Cervero, 1985). This

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pattern of innervation is reflected in the distribution of fos immunoreactivity in the thoracic segments of the spinal cord following gastric distension (Traub, Sengupta and Gebhart, 1996). Second order neurones ascend the spinal cord in the same region as the homologous somatic afferents through pathways such as the spinothalamic tract, spinoreticular tract and dorsal columns (Downman and Evans, 1957; Kuo and De Groat, 1985). Projections into lamina VII may make connections with dendrites of preganglionic sympathetic neurones located in the IML nucleus or near the central canal with ascending and descending pathways projecting to preganglionic neurones in adjacent segments of the spinal cord. Preganglionic fibres to the prevertebral ganglia emerge from the thoraco-lumbar spinal cord in the ventral roots. Projections to the coeliac ganglion arise between T5 and T12 in the guinea-pig and are probably organised in the same way as those to the pelvic organs with visceral vasoconstrictor neurones receiving different inputs from those involved in motility regulation (Jänig, 1988). In most species section of the splanchnics has minimal effects on normal gastric motor function whereas laparotomy and surgical manipulation of the viscera provokes widespread inhibition of gastric tone and motility that depends upon an intact sympathetic innervation (see Furness and Costa, 1987). However, in the dog, section of the splanchnic nerves leads to an increase in gastric motility and gastric emptying suggesting a physiological role (Carlson, 1913; McCrea, 1926). In the ferret, this sympathetic influence on gastric tone is only apparent after prior section of the vagus nerve or after pretreatment with atropine suggesting that there may be functional interactions between vagal and splanchnic influences in the periphery (Andrews, Grundy and Lawes, 1981). Furthermore, this interaction may be bidirectional (McSwiney and Robson, 1931; Mamber and Gershon, 1979). Somatic inputs converge onto the same spinal neurones that receive a splanchnic input. These somatic inputs are generally nociceptive, which provides the basis for the convergence-projection theory of referred pain. However, their role in the control of gastrointestinal function cannot be dismissed, especially since there is functional evidence for splanchnic projections to the dorsal motor vagal nucleus where they can modulate the vagal outflow to the gastrointestinal tract (Grundy, Salih and Scratcherd, 1981; Barber and Yuan, 1993; Renehan et al., 1995; Traub, Sengupta and Gebhart, 1996). BRAINSTEM REFLEXES Pertinent to this discussion is the fact that the full repertoire of vago-vagal reflexes to the entire gastrointestinal tract is achieved by just a few thousand pre-ganglionic efferent fibres, the vagus being a predominantly afferent nerve (Pretchl and Powley, 1990). An added complication arises because there are at least two distinct functional populations of pre-ganglionic vagal motor neurone that supply the stomach (ignoring the sub-classification into motor and secretomotor): those that when stimulated cause contraction and others that cause gastric relaxation. The contractile and relaxatory responses to vagal stimulation are both mediated by C-fibre inputs to gastric myenteric neurones via predominantly nicotonic synapses. However, the contractile response is largely cholinergic and blocked by atropine, and relaxation mediated by the so-called non-adrenergic, non-cholinergic (NANC) pathway that involves NO and VIP (see earlier discussion). One talks of excitatory and

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inhibitory vagal pathways but this is in fact short-hand for two pathways to enteric circuits which influence the release of excitatory or inhibitory transmitters at the neuro-muscular junction. Vagal excitatory or inhibitory reflexes therefore arise from the selective activation or suppression of brainstem neurones projecting to different myenteric circuits for contraction or relaxation. These pathways through the brainstem are probably hardwired for specific peripheral responses in much the same way as those described above for the polarised enteric circuits. The processing of afferent information in the brainstem in relation to vago-vagal reflex control of gastrointestinal function has been investigated in neurophysiological studies in which the efferent discharge in pre-ganglionic vagal fibres was recorded during the application of stimuli designed to activate different afferent sub-populations in different regions of the gastrointestinal tract. Earlier work in the field utilised extracellular single fibre recording techniques to obtain efferent signals on route to the periphery (Davison and Grundy, 1978; Grundy, Salih and Scratcherd, 1981). This approach treats the brainstem as a “black-box” and while providing valuable information on overall central processing could not detail the mechanisms involved. To overcome this problem there has been a move towards direct recordings from the cell bodies of nucleus tractus solitarius (NTS) and vagal pre-ganglionic motor neurones within the brainstem using extracellular and intracellular electrode techniques (Appia et al., 1986; Barber and Burks, 1983; Ewart and Wingate, 1984; McCann and Rogers, 1992; Renehan et al., 1995; Zhang, Fogel and Renehan, 1992, 1995; Fogel, Zhang and Renehan, 1996; Chu et al., 1993). The intracellular approach offers an additional advantage in that the injection of markers such as neurobiotin through the recording electrode enables the morphology of the neurone to be correlated with its electrophysiology. However, in the absence of specific neurochemical or anatomical “markers” for the excitatory and inhibitory vagal motorneurones, investigators have been forced to interpret functions from the behaviour of individual neuronal responses to afferent stimulation which give rise to “specific” end-organ responses. Spontaneous activity If one focuses initially on the pre-ganglionic vagal outflow then a striking feature of these neurones is that more than 90% of them generate a spontaneous discharge in the absence of any intentional stimulation. In most cases this spontaneous firing is irregular and of low frequency, less than 2 imps–1, while in others the discharge is modulated in phase with on-going contractile activity in the stomach. That this discharge is generated centrally is evident from experiments in which the vagal afferent input is severed during maintained efferent recording. In some cases discharge increased after vagotomy demonstrating a tonic inhibitory vagal afferent input, while in other cases the reverse was observed (Davison and Grundy, 1978; Grundy, Salih and Scratcherd, 1981). The presence of spontaneous activity enables gastrointestinal reflex responses to be generated by both increases and decreases in efferent discharge (see below). This spontaneous activity may be generated by neurones of the dorsal motor nucleus of the vagus (DMNV) themselves or may result from synaptic influences elsewhere in the brainstem or higher centres which contribute the diversity of transmitters which exist in the dense synaptic neuropil around these neurones. Brain regions with a prominent input to this region of the brainstem

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include the medullary raphe nuclei, the paraventricular nucleus of the hypothalamus and the central nucleus of the amygdala. These connections provide the basis for emotional and behavioural effects, and in particular the effect of stress on gastrointestinal function through a number of mediators including thyrotropin-releasing hormone, corticotropinreleasing factor and CCK (Tache, Maeda-Hagiwara and Turkelson, 1987; Diop et al., 1991; Bueno, 1993, 1994; Martinez et al., 1997). Central corticotropin-releasing factor may also be involved in the post-surgical inhibition of gastric emptying (Barquist et al., 1996). Reflex modulation of the vagal outflow The modulatory influence of vagal afferent inputs to brainstem circuits is dramatically illustrated when these afferents are electrically stimulated. Firing is suspended for several seconds in some efferent neurones after a single afferent shock, while in others there are reflex-evoked action potentials. Thus reflex circuits include both inhibitory and excitatory mechanisms and in some neurones a complex picture of both inhibition and excitation occurs. In the case of inhibition, it is not possible to determine if this arises as a direct consequence of inhibitory vagal afferent synapses onto vagal pre-ganglionic neurones or through a relay in the NTS. The latter is more likely since the majority of gastrointestinal vagal afferents project to the NTS (Kalia and Mesulam, 1980; Altschuler et al., 1989). Moreover, in a number of electrophysiological studies the predominant vagal input to the NTS is excitatory while DMNV neurones show inhibition in response to the same stimulus (Rogers, McTigue and Hermann, 1995). However, a direct vagal afferent projection to the DMNV has been described while in addition the dendrites of pre-ganglionic vagal motorneurones extend into the NTS where mono-synaptic contacts have been observed electron microscopically (Rinaman et al., 1989). Functional evidence for mono-synaptic vagal reflexes comes from the observation that some vagal efferent fibres generate a single action potential at fixed latency following a single electrical stimulus to vagal afferent fibres in both the contralateral and ipsilateral branches of the cervical vagus nerve. The limited latency “jitter” and the security of the response in terms of the probability of firing are in keeping with a mono-synaptic connection and are in marked contrast to the less secure responses seen in the majority of efferent neurones (Blackshaw and Grundy, 1988). This observation emphasises the variable extent to which the incoming afferent information is processed within the vago-vagal pathway. The function of the mono-synaptic pathway and the modality of afferent information processed in this way is not known but by analogy with the mono-synaptic somatic reflex may be the basis on which other modulating influences are exerted. Of the various subnuclei that make up the NTS, the subnucleus gelatinosus, medial and commissural nuclei are the principal targets for gastric afferents, although an afferent projection to the area postrema has also been noted (Sawchenko, 1983). The projection to area postrema may be associated with emesis triggered from gastrointestinal afferents (Leslie, 1986). From the NTS, neurones project to other nuclei within the brainstem which subserve the vago-vagal reflexes controlling gastric function. Other pathways ascend through the mid-brain and reticular nuclei to higher centres. While these pathways are well established anatomically, the functional implications for gastrointestinal control are unknown, although some undoubtedly have a role in the control of food intake.

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Convergent vagal inputs Neurones in the NTS that receive a gastric vagal input possess a striking horizontal orientation of their dendrites extending over a 600 µm from medial NTS to solitary tract and as such pass through the terminal zones of all gastrointestinal afferent inputs providing a morphological basis for the dramatic convergence seen in the vago-vagal reflex pathway (Rogers and McCann, 1993; Rogers, McTigue and Hermann, 1995). An individual efferent neurone can be modulated by afferent fibres supplying different regions of the gastrointestinal tract, supplying different types of sensory modality and follow different pathways to the brainstem (Blackshaw and Grundy, 1989). The relative proportion of vagal efferent neurone that are excited or inhibited by a given stimulus varies with the region of gut being stimulated and the level of stimulation. In the rat and ferret the ratio of efferent fibres showing excitation and inhibition following gastric distension was about 50:50. However, when progressive more distal regions of the gastrointestinal tract were stimulated, the inhibitory influences became dominant (Grundy, Salih and Scratcherd, 1981). In some cases the inhibition was complete and all ongoing efferent firing was suspended for the duration of the distension. In other cases the inhibition was only partial. Similarly variations in the degree of excitation were also observed which again reflects the extent to which afferent information is processed. The observation that some efferent fibres were excited by the same stimulus, and at the same time as others were inhibited, viewed in the light of inhibitory and excitatory motor pathways, led to the hypothesis that these two motor pathways were reciprocally controlled (Davison and Grundy, 1978; Miolan and Roman, 1974). Thus, during gastric relaxation there would be activation of the vagal inhibitory pathway and concomitant inhibition of the excitatory pathway, an effect which would sharpen the response of either alone. In a number of more recent studies on brainstem mechanisms, reflex suppression of the vagal excitatory drive to the stomach is suggested to be the major means of gastric relaxation. For example, when the afferent stimulus was localised to the antrum by distending a balloon inserted through the pylorus, over 90% of DMNV responses were inhibitory (McCann and Rogers, 1992). A similar preference towards inhibitory responses in the DMNV was described by Zhang, Fogel and Renehan (1992) and Renehan et al. (1995) following intestinal distension. Since, both antral and intestinal distension gives rise to a vagally mediated gastric relaxation, the conclusion from both studies was that reflex events were brought about by a decrease in ongoing vagal cholinergic tone to the gastrointestinal tract. In contrast, functional studies of adaptive gastric relaxation favour a dominant role for enteric inhibitory pathways (Grundy, Gharib-Naseri and Hutson, 1993a; De Ponti, Azpiroz and Malagelada, 1989; Desai, Sessa and Vane, 1991). However, antrally mediated relaxation may have a disinhibition of cholinergic mechanisms as a contributing factor (Krowicki and Harby, 1996). An interpretational difficulty for all of these electrophysiological studies is that the target organ of the neurones under investigation is unknown. One assumes that they supply predominantly the stomach because this region receives the densest vagal input. Both morphological and electrophysiological data confirm that most myenteric ganglia in the stomach are innervated by the vagus while many less are innervated in the small intestine (Schemann and Grundy, 1992; Berthoud Jedrzejewska and Powley, 1990). The

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discrepancy between data from vagal recording and brainstem studies may arise from the ability to target the gastric motor innervation in the brainstem since different gastrointestinal regions are organotopically organised within the DMNV with the gastric projecting neurones located medially. In contrast, recording from the vagus inevitably means a more random selection of fibres. The convergence of afferent inputs onto individual vagal motor fibres is striking. For example, single efferent fibres have been shown to respond to distension of the stomach, duodenum and colon in the ferret (Grundy, Salih and Scratcherd, 1981). Following truncal vagotomy, the gastric input was greatly attenuated while that from the duodenum and colon persisted, indicating that afferents from these more distal regions reached the brainstem via a non-vagal pathway. Convergent inputs from muscle mechanoreceptors and multimodal mucosal chemoreceptors have also been shown (Blackshaw and Grundy, 1989). In general, the vagal input is dominant and mechanoreceptive inputs more powerful than chemoreceptive ones. Nevertheless, the outcome from this considerable afferent convergence is that a particular population of preganglionic fibres provides the efferent limb of a variety of reflexes that require the same end organ response. Gastric corpus relaxation, for example, occurs under a variety of circumstances including receptive and adaptive relaxation to accommodate a meal, following gastric emptying into the duodenum, and during emetic episodes. A single efferent pathway may mediate all these responses and could account for vagal control through a relatively small number of efferent fibres. Vagal motor fibres may also contribute to pathological conditions such as paralytic ileus (Abrahamsson, Glise and Glise, 1979). There is strong evidence that splanchnic afferents project to the dorsal vagal complex and can therefore influence transmission through vago-vagal reflex pathways. Vagal efferent fibres received a non-vagal input following distension of the small and large intestine which was generally inhibitory (Grundy, Salih and Scratcherd, 1981). In contrast an excitatory input to the DMNV was shown following intestinal distension which appeared to bypass the predominantly inhibitory route via the NTS (Renehan et al., 1995). The authors concluded that spinal projections may be direct to neurones of the DMNV an interpretation that might explain the way gastric responses to low threshold stimulation is often reversed when the intensity of stimulation increases. These opposing actions may therefore have their basis in differential inputs from vagal and splanchnic afferents. COMMAND NEURONES Enteric neurones provide the final pathway for both intrinsic and extrinsic reflexes regulating gastric motor function. The command neurone hypothesis was proposed to explain the widespread influence on GI function of just a few thousand preganglionic vagal fibres. This hypothesis suggests that only a few myenteric neurones are synaptically driven by the vagus and these innervated neurones are suggested to play a key role by activating specific microcircuits for contraction or relaxation. Morphological data has produced conflicting data with respect to the extent of the preganglionic input to gastric myenteric neurones. On the one hand this innervation is suggested to be sparse with few enteric neurones receiving direct extrinsic input, an observation in keeping with the command

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hypothesis (Kirchgessner and Gershon, 1989) while on the other hand, vagal efferent terminals were widely distributed within the gastric myenteric plexus (Berthoud et al., 1990). In an extension to this study, Berthoud estimated that in the rat fundus vagal terminals are in close apposition to between 25% and 50% of gastric myenteric neurones although synaptic contacts are impossible to verify with this approach. However, c-fos expression in large numbers of myenteric neurones following vagal stimulation is consistent with an extensive direct synaptic input (Berthoud, 1997) or via polysynaptic pathways within the ENS. Approximately one third of “innervated” neurones were NADPH-diaphorase positive and therefore likely to be inhibitory neurones to the muscle (Berthoud, 1995). The two third NADPH-diaphorase negative neurones that were also vagally innervated would include cholinergic motor neurones providing a morphological basis for the dual vagal motor pathways to the gastric smooth muscle. This proportion of innervated neurones matches the proportion of NOS and ChAT positive cells in the guinea-pig myenteric plexus and suggests that there is no preferential target for the vagal input to the enteric nervous system, a conclusion similar to that of an earlier study in which VIP, GRP and ENK neurones were examined (Berthoud, 1996). Electrophysiological studies in the guinea-pig corpus would tend to support the view that vagal terminals make both divergent and convergent contacts with myenteric neurones (Schemann and Grundy, 1992). Rather than there being few, specialised myenteric neurones receiving synaptic input from vagal preganglionic fibres it was found that the majority of neurones responded to electrical vagal stimulation with nicotinic fEPSPs of which most led to the generation of action potentials. This is suggestive of a highly divergent vagal input with widespread modulatory influence over gastric myenteric neurotransmission. These vagally innervated myenteric neurones also received nicotinic inputs from other myenteric neurones. The ENS is therefore capable of autonomous integrative activity in order to generate reflexes which are dependent upon intrinsic circuits but which are modulated by extrinsic inputs from the vagus. A variety of synaptic responses could be distinguished in response to vagal stimulation. In most cases increasing the stimulus strength above threshold produced a graded EPSP at a fixed latency (<1 ms) until finally firing an action potential. This observation suggests individual neurones receive multiple vagal inputs recruited as the stimulus strength increases and since most cells in a particular ganglion had similar latency this further suggests a monosynaptic input to many neurones. A minority of neurones generated a single fEPSP triggered action potential in an all-or-none fashion when stimulus strength was increased while a third type of response consisted of multiple EPSP at different latencies. A small number of neurones generated a slow EPSP following a train of stimuli delivered to the vagus but inhibitory post-synaptic potentials were never observed. The prominent influence of vagal fibres on myenteric neurones in the stomach might explain the quite dramatic effect of acute vagotomy on gastric functions. Although the circuits responsible for initiating the reflexes reside within the myenteric plexus, these circuits appear to depend on acetylcholine provided by the extrinsic nervous system. Support for this view is the finding that coordinated reflex activity in the stomach may be restored after acute vagotomy by raising the level of neural activity by a background infusion of acetylcholine or carbachol (Grundy, Hutson and Scratcherd, 1986; Grundy, 1989; Papasova and Atanassova, 1984). The intramural nervous system gradually adapts after

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vagotomy so that it can perform as an independent organiser and modulator of motor function. Therefore, changes in gastric motility observed after acute vagotomy are usually fully reversible with time (Atanassova, Kortezova and Papasova, 1986). Based on these results we propose a permissive action of the vagus on stomach motility. PERMISSIVE CONTROL OF ENTERIC REFLEX CIRCUITS The electrophysiological data above suggests that rather than “command neurones” in the stomach which, once activated, produced a stereotyped excitatory or inhibitory response, there is a divergent vagal input to the majority of gastric myenteric neurones. This type of innervation suggests that the vagus may play a more permissive role in regulating enteric reflexes. Consistent with this view is the convergence of intrinsic and vagal inputs to individual neurones. The sympathetic input also fits easily into this concept since noradrenaline acts presynaptically to suppress cholinergic fEPSPs evoked by both the vagus and intrinsic fibres (Schemann, 1991b). Since the vagal input is tonically active, one might predict that after vagotomy one would lose a facilitatory input to the enteric circuits leading to a deficit in reflex control. In addition, sympathetic presynaptic inhibition primarily shuts down cholinergic tone because α2 receptor-mediated inhibition of fEPSPs mostly affects cholinergic neurones (Table 2.2). Certainly, acute vagotomy has dramatic effects on gastric motor function causing a suppression of fed motor patterns in the conscious dog (Hall, El-Sharkawy and Diamant, 1986), an increase in corpus tone (Grundy, GharibNaseri and Hutson, 1993) and disorganised antral peristaltic activity leading to delayed gastric emptying (Malbert, Mathis and Laplace, 1995). These deficits in motor function may not merely reflect the loss of specific reflex pathways but also this loss of a facilitatory input to the enteric reflex circuits. In this respect restoring this vagal tone by electrical stimulation has been shown to restore the corpus relaxatory response to local distension necessary for gastric accommodation (Grundy, 1993) and the antral contractile response to distension (Grundy, Hutson and Scratcherd, 1986). After chronic vagotomy, the deficit in gastric reservoir function appears to be temporary indicating that the enteric nervous system can adapt to the loss of the potentiating effect of the vagus, possibly by up-regulation of the nitrergic innervation (Andrews and Bingham, 1990; Takahashi and Owyang, 1997). It is not clear how changes in the intramural nerves compensates for the loss of vagal input. The degree of up-or-down regulation, reorganisation or plasticity after loss of central inputs is not known. Although the gross morphological integrity of the intramural elements is preserved after vagotomy, degeneration of vagal fibres is accompanied by regressive transneural changes that are rapid in onset (1–3 days after vagotomy) are short lasting (5–7 days) and presumably fully reversible (Vaithilingham, Wong and Ling, 1986). There has also been described a dramatic decrease in the number of synapses after vagotomy (Filogama and Gabella, 1970). Upregulation of muscarinic receptors on corpus muscle has also been reported after vagotomy (Keshavarzian et al., 1990) although the mechanisms contributing to restored reservoir function appear not to be muscarinic in the ferret (Grundy, Gharib-Nasari and Hutson, 1993a). What is lost after vagotomy or splanchnectomy are the reflex pathways that link distant regions of gastrointestinal tract and which normally ensure coordination of gut function and reinforce local reflex circuits. The corpus and antrum, for example, are

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linked by reflex pathways that can be activated in vitro (Yuan, Brookes and Costa, 1997). However, there a bidirectional reflexes through the vagus which provides a facilitatory input to the antrum and may couple the amplitude of antral peristaltic contractions to the level of corpus distension and inhibitory mechanism from the antrum to the corpus which may play a role in retropulsion which helps triturate solid material prior to emptying (Rudge et al., 1990). The polarisation of these reflexes are such that excitatory influences occur in a descending direction while ascending reflexes are inhibitory, in other words in the opposite direction to the way the enteric circuits are wired. Inhibition of both antrum and corpus by chyme in the duodenum has both a vagal and splanchnic component (see Meyer, 1994). Nevertheless, the decentralised stomach is still capable of coordinated activity and non-neural mechanisms are undoubtedly necessary to complete the picture of gastric motor control (Spencer et al., 1990; Van Lier Ribbink, Sarr and Tanaka, 1989).

CONCLUSION The fundamental mechanisms for gastric relaxation and peristalsis exists within the isolated stomach with the basic myogenic properties of smooth muscle being regulated by enteric reflex circuits activated either by intrinsic sensory neurones, the identity of which has still to be determined, or by axon collaterals of primary sensory neurones. However, in the intact state there is the potential for parasympathetic and sympathetic modulation which may influence the extent to which these local circuits give rise to the effector responses. The important contribution of enteric circuits for gastric reflex activity is supported by studies demonstrating a permissive role for the vagus, a concept that recognises the complex interactions between extrinsic nerves and intrinsic circuits. Thus, a basal preganglionic parasympathetic tone, and possible tone in local axon reflexes, is required to keep intrinsic circuits that are responsible for the behaviour of gastric motor function operational.

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Abdominal vagotomy blocks the satiety effect of cholecystokinin in the rat. Science, 213, 1036–1037. Smith, T.K. and Furness, J.B. (1988). Reflex changes in circular muscle activity elicited by stroking the mucosa: an electrophysiological analysis in the isolated guinea-pig ileum. Journal of the Autonomic Nervous System, 25, 205–218. Smith, T.K., Bornstein, J.C. and Furness, J.B. (1990). Distension-evoked ascending and descending reflexes in the circular muscle of guinea-pig ileum: an intracellular study. Journal of the Autonomic Nervous System, 29, 203–217.

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Smith, T.K., Bornstein, J.C. and Furness, J.B. (1992). Convergence of reflex pathways excited by distension and mechanical stimulation of the mucosa onto the same myenteric neurons of the guinea pig small intestine. Journal of Neuroscience, 12, 1502–1510. Spencer, M.P., Sarr, J.G., Soper, N.J. and Hakim, N.S. (1990). Jejunal regulation of gastric motility patterns: effect of extrinsic neural continuity to stomach. American Journal of Physiology, 258, G32–G37. Sternini, C. and Anderson, K. (1992). Calcitonin gene-related peptide containing neurones supplying the rat digestive system: differential distribution and expression pattern. Somatosensory and Motor Research, 9, 45–49. Sternini, C., DeGiorgio, R. and Furness, J.B. (1992). Calcitonin gene-related peptide neurons innervating the canine digestive system. Regulatory Peptides, 42, 15–26. Sykes, R.M., Spyer, K.M. and Izzo, P.M. (1997). 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3 Control of Gastric Functions by Extrinsic Sensory Neurons* Peter Holzer Department of Experimental and Clinical Pharmacology, University of Graz, Universitätsplatz 4, A-8010 Graz, Austria The maintenance of gastric mucosal integrity depends on the rapid alarm of protective mechanisms in the face of pending injury. Afferent neurons of extrinsic origin constitute an emergency system that is called into operation when the gastric mucosa is endangered by noxious chemicals. The function of these chemoceptive afferents can selectively be manipulated and explored with the excitotoxin capsaicin. Since most of the homeostatic actions of capsaicin-sensitive afferents are brought about by peptides released from their peripheral endings in the gastric wall, the roles of these neurons are described as “sensory-efferent”, “sensory-motor”, “local effector” or “noceffector” to emphasise the functional similarity with the roles of autonomic efferents. When stimulated, chemoceptive afferents enhance gastric blood flow and activate hyperaemia-dependent and hyperaemia-independent mechanisms of protection and repair. Furthermore, they can influence the gastric secretion of acid, bicarbonate and mucus and modulate gastric motility and emptying. In the rodent stomach, the noceffector roles of sensory neurons are mediated by calcitonin gene-related peptide acting via CGRP1 receptors and neurokinin A acting via NK2 receptors, both peptides using nitric oxide as their second messenger. In addition, capsaicin-sensitive neurons form the afferent arc of autonomic reflexes that control secretory and motor functions of the stomach. The pathophysiological potential of the neural emergency system is best portrayed by the gastric hyperaemic response to acid backdiffusion, which is signalled by afferent nerve fibres. This mechanism limits damage to the surface of the mucosa and creates favourable conditions for rapid restitution and healing of the wounded mucosa. KEY WORDS: stomach; afferent neurons; capsaicin; calcitonin gene-related peptide; tachykinins; substance P; neurokinin A; nitric oxide; gastric blood flow; gastric secretion; gastric motor activity; peptic ulcer disease; ileus.

INTRODUCTION Nowhere else in the digestive system is the mucosa more endangered than in the gastroduodenal region, the threats being primarily of a chemical nature and of both endogenous and exogenous origin. Most tissues would rapidly disintegrate if exposed to the concentrations of hydrochloric acid that bathe but do not harm the gastric surface epithelium.

*Article completed on 3 February 1997. 103

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This is because a multitude of physical, chemical and physiological factors, collectively forming the gastric mucosal barrier, prevent hydrogen ions from entering the tissue at quantities that would produce cell injury (Allen et al., 1993; Pabst, Wachter and Holzer, 1996). Although autonomic neurons have long been recognised to influence the capacity of the gastric mucosa to defend itself against damage, the possibility that neurons constitute an important element in gastric mucosal protection from injury was hardly thought of. This neglect of neurons may have been related to the understanding that the vagus nerve is a permissive factor in gastric lesion formation, given that subdiaphragmatic vagotomy can be beneficial in peptic ulcer disease. However, central stimulation of the vagus nerve can also protect the gastric mucosa from damage (Kato, Yang and Taché, 1995), which attests to the complexities in the autonomic regulation of gastric mucosal homeostasis. In his initial conception, Langley (1903) considered afferent neurons (“autonomic afferents”) to be a third subdivision of the autonomic nervous system. Although this concept was later abandoned, it remains convenient to label afferent neurons running in the vagus and pelvic nerves as “parasympathetic afferents” and those running in the splanchnic nerves as “sympathetic afferents” to denote their projections in the respective autonomic efferent nerves (Sengupta and Gebhart, 1994). It was only recently that “autonomic afferents” were discovered to constitute an important neural emergency system in the digestive tract. These neurons have been found to participate in the neural control of blood flow, secretory processes and motor activity and to regulate a variety of functions that can be viewed as increasing the resistance of the tissue to injury and facilitating the repair of damaged tissue. Since most of these actions are brought about by release of transmitter substances from the peripheral endings of afferent nerve fibres, the function of these neurons has been portrayed as “sensory-efferent” (Szolcsányi, 1984), “sensory-motor” (Burnstock, 1990), “local effector” (Holzer, 1988) or “noceffector” (Kruger, 1988) to emphasise the operational similarity with the physiological roles of autonomic efferent neurons. Many actions exerted by afferent neurons in the stomach, notably those on blood flow, resemble sensory nerve-mediated “neurogenic inflammatory reactions” seen in other tissues (Geppetti and Holzer, 1996). The discovery of these sensory neuron functions was

Figure 3.1 Use of capsaicin as a selective pharmacological tool to examine the functional implications of primary afferent neurons with fine (C- and Aδ-) nerve fibres.

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made possible by the availability of capsaicin, a pharmacological tool with which the activity of primary afferent neurons with unmyelinated or thinly myelinated nerve fibres can selectively be manipulated (Holzer, 1991; Wood, 1993). Capsaicin is an excitotoxin which acutely stimulates a group of fine afferent nerve fibres and, depending on the dose and route of administration, may cause a long-lasting defunctionalisation of, and depletion of transmitter substances from, these neurons (Figure 3.1). In contrast, afferent neurons with thick myelin sheaths are not sensitive to capsaicin (Holzer, 1991). The selectivity of capsaicin’s excitotoxic action on afferent neurons derives from the fact that only these neurons express receptor binding sites (“vanilloid receptors”) for capsaicin and structurally related ligands (Szallasi, 1994). By taking use of the neurotoxic action of capsaicin it is possible to ablate afferent neurons without severing efferent autonomic neurons which unavoidably are cut by nerve transection. The exploitation of capsaicin’s neuropharmacological potential has brought unprecedented insights into the pathophysiological roles of primary afferent nerve fibres in the gastrointestinal tract, and it is the aim of this review to give an account of the experimental findings that have led to the elucidation of this neural emergency system in the stomach and to consider its physiological and pathophysiological implications.

EXTRINSIC AFFERENT NERVE FIBRES IN THE STOMACH The extrinsic primary afferent neurons supplying the stomach arise from two different sources (Figure 3.2). The spinal sensory neurons originate from cell bodies in the dorsal root ganglia and reach the stomach via the splanchnic and mesenteric nerves, while the sensory nerve fibres running in the vagus nerves have their cell bodies in the nodose and jugular ganglia. In small rodents (rat, mouse, guinea-pig) most of the spinal afferent neurons contain a variety of bioactive peptides including calcitonin gene-related peptide (CGRP) and the tachykinins substance P (SP) and neurokinin (NKA). CGRP and SP show a considerable degree of coexistence in the same afferent neurons but, while CGRP is

Figure 3.2 Schematic diagram showing the innervation of the mammalian stomach by primary afferent neurons.

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contained in both small and large somata, SP occurs in small and medium-sized cell bodies only (Molander, Ygge and Dalsgaard, 1987; Su et al., 1987; Green and Dockray, 1988; McCarthy and Lawson, 1989, 1990; Kashiba et al., 1991). It is important to realise that the chemical coding of extrinsic afferents, i.e., the combination of peptides coexpressed in individual neurons, varies considerably across species. Thus, very little CGRP seems to be expressed in neurons of the human stomach (Tsutsumi and Hara, 1989; Sundler, Ekblad and Håkanson, 1991). The peptidergic innervation of the gastrointestinal tract is complicated by the fact that there are three separate populations of peptide-containing neurons, extrinsic primary afferent, extrinsic autonomic, and intrinsic enteric neurons, which have many peptide transmitters in common (Ekblad et al., 1985; Costa, Furness and Gibbins, 1986; Kirchgessner, Dodd and Gershon, 1988). However, extrinsic afferents differ from the other neuron populations in their chemical coding and sensitivity to capsaicin, let alone their physiological implications. CGRP and SP are coexpressed only in extrinsic afferents (Costa, Furness and Gibbins, 1986) and most of the CGRP present in afferent neurons appears to be CGRP-α, whereas the only form of CGRP in enteric neurons is CGRP-β (Mulderry et al., 1988; Sternini, 1992). Furthermore, only primary afferent neurons are sensitive to the excitotoxic action of capsaicin while autonomic (Holzer, 1991) and enteric (Barthó and Holzer, 1985; Green and Dockray, 1988) neurons are not. Table 3.1 summarises some of the neurochemical and biochemical changes that result from capsaicin-induced defunctionalisation of afferent neurons in the rat stomach and duodenum. These studies have revealed that the CGRP-containing nerve fibres in the rat stomach arise from extrinsic afferent neurons whereas the SP-immunoreactive fibres are of both extrinsic and intrinsic origin (Green and Dockray, 1988; Varro et al., 1988; Sternini, 1992). The extrinsic axons disappear from the wall of the gastrointestinal tract when the gut is surgically severed from its extrinsic nerve supply or the experimental animals are treated with a neurotoxic dose of capsaicin (Furness et al., 1982; Hayashi et al., 1982; Barja, Mathison and Huggel, 1983; Minagawa et al., 1984; Gibbins et al., 1985; Lee et al., 1987b; Sternini, Reeve and Brecha, 1987; Su et al., 1987; Green and Dockray, 1988; Kashiba et al., 1990; McGregor and Conlon, 1991). TABLE 3.1 Effect of capsaicin-induced defunctionalisation of afferent neurons on neurochemical and biochemical parameters in the rat stomach and duodenum. Capsaicin Parameter administration Systemic

Effect of nerve defunctionalisation

References

Alanine aminopeptidase activity No change (duodenum) McGregor and Conlon, 1991 Alkaline phosphatase activity No change (duodenum) McGregor and Conlon, 1991 Calcitonin gene-related peptide Loss Sternini, Reeve and Brecha, 1987; Su et al., 1987; Geppetti et al., 1988; Green and Dockray, 1988; Varro et al., 1988; Evangelista et al., 1991; Inui et al., 1991;

CONTROL OF GASTRIC FUNCTIONS BY EXTRINSIC SENSORY NEURONS

DNA synthesis Galanin Gastrin messenger RNA Glucagon HDCa mRNA Histamine content 5-Hydroxytryptamine content Leucine enkephalin content Methionine enkephalin content Neurokinin A Neuropeptide Y Nitric oxide synthase Prostaglandin formation Somatostatin Somatostatin messenger RNA

Substance P content

Substance P in muscle/ myenteric plexus Substance P in perivascular nerves Substance P in mucosa/ submucosa Vasoactive intestinal polypeptide

McGregor and Conlon, 1991; Renzi et al., 1991; Holzer, Lippe and Amann, 1992; Gray et al., 1994 No change Takeuchi et al., 1994 No change Su et al., 1987; Evangelista et al., 1992 No change (antrum) Sandvik et al., 1993 No change (duodenum) McGregor and Conlon, 1991 No change (corpus) Sandvik et al., 1993 No change Holzer et al., 1981 No change Holzer et al., 1981; McGregor and Conlon, 1991 No change Varro et al., 1988 No change Varro et al., 1988 No change

Renzi et al., 1991; Evangelista et al., 1992 No change Su et al., 1987; Evangelista et al., 1992 No change Forster and Southam, 1993 No change Holzer and Sametz, 1986 No change Inui et al., 1991 Reduction (corpus) Sandvik et al., 1993; Dimaline et al., 1994 No change (antrum) Sandvik et al., 1993; Dimaline et al., 1994 No change Holzer, Gamse and Lembeck, 1980; Geppetti et al., 1991; McGregor and Conlon, 1991; Renzi et al., 1991 Reduction (duodenum) Geppetti et al., 1988 No change Su et al., 1987; Green and Dockray, 1988 Loss Su et al., 1987; Green and Dockray, 1988 Reduction Su et al., 1987; Green and Dockray, 1988 No change Su et al., 1987; Evangelista et al., 1991, 1992; McGregor and Conlon, 1991; Tramontana et al., 1994a,b

Perivagal

Calcitonin gene-related peptide

No change

Raybould et al., 1992

Pericoeliacb

Calcitonin gene-related peptide

Depletion

Raybould et al., 1992

a

107

HDC, histidine decarboxylase; b administration of capsaicin locally to the coeliac ganglion.

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Most of the extrinsic nerve fibres expressing CGRP and/or SP in the rodent stomach are spinal, rather than vagal, afferents which on their way from the dorsal root ganglia to the gut pass through the prevertebral ganglia (Figure 3.2) where they give off collaterals to form axodendritic and axosomatic synapses with the sympathetic ganglion cells (Hayashi et al., 1982; Matthews and Cuello, 1984; Lee et al., 1987a,b; Su et al., 1987; Green and Dockray, 1988; Kondo and Yamamoto, 1988; Lindh, Hökfelt and Elfvin, 1988). However, vagal afferent nerve fibres immunoreactive for SP and CGRP have been found to supply the oxyntic part of the feline and rat stomach (Hayashi et al., 1982; Bäck et al., 1994) while other studies hold that vagal afferents contribute little to the SP and CGRP content of the rat gastric corpus (Lee et al., 1987b; Green and Dockray, 1988). This controversy may be related to regional and species differences in the gastric projections of peptidergic vagal afferents. It seems as if the density of these vagal nerve fibres decreases from the proximal to the distal stomach as the gastric corpus of the cat is innervated by vagal SP-containing fibres (Hayashi et al., 1982) whereas the extrinsic supply of SP fibres in the antrum, pylorus and duodenum of the cat and guinea-pig is derived from spinal afferents (Hayashi et al., 1982; Lindh, Hökfelt and Elfvin, 1988). Within the rodent gastric wall it is particularly the arterial and arteriolar system that receives a dense supply by spinal afferent nerve fibres expressing CGRP, SP and NKA (Figure 3.2), whereas the venous system is rather sparsely innervated (Furness et al., 1982; Barja, Mathison and Huggel, 1983; Sharkey, Williams and Dockray, 1984; Uddman et al., 1986; Sternini, Reeve and Brecha, 1987; Su et al., 1987; Green and Dockray, 1988; Sternini, 1992). The peptide-containing axons run primarily in the connective tissue (tunica adventitia) surrounding the vessels and at the border between adventitia and muscle (media). In addition, some peptide-containing afferent nerve fibres also supply the myenteric plexus, the circular muscle layer and the mucosa of the gut (Furness et al., 1982; Minagawa et al., 1984; Sharkey, Williams and Dockray, 1984; Gibbins et al., 1985; Rodrigo et al., 1985; Uddman et al., 1986; Lee et al., 1987b; Sternini, Reeve and Brecha, 1987; Su et al., 1987; Green and Dockray, 1988; Kashiba et al., 1990; Sundler, Ekblad and Håkanson, 1991; Sternini, 1992; Jakab et al., 1993; Bäck et al., 1994; Schmidt et al., 1996).

PEPTIDERGIC AFFERENT NERVE FIBRES AND GASTRIC BLOOD FLOW VASODILATATION CAUSED BY AFFERENT NERVE STIMULATION The possibility that perivascular afferent nerve fibres control vascular functions in the stomach went unrecognised for a long time, as these axons were considered to subserve only sensory but not effector functions. However, with the advent of capsaicin it became feasible to prove that sensory neurons containing CGRP participate in the regulation of the rat gastric circulation. Stimulation of afferent nerve fibres by intragastric administration of capsaicin or its ultrapotent analogue resiniferatoxin leads to a marked increase in gastric mucosal blood flow (GMBF; Table 3.2) and blood flow through the left gastric artery (Figure 3.3). This effect is brought about by dilatation of submucosal arterioles but not venules (Chen et al., 1992; Chen and Guth, 1995). Other regions of the digestive tract,

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Figure 3.3 Recording of the hyperaemic and dilator effect of intragastric (i.g.) capsaicin (160 µM) on the left gastric artery of an urethane-anaesthetised rat. The traces from the top to the bottom show heart rate (HR), mean arterial blood pressure (MAP), blood flow in the left gastric artery (BF/LGA) and vascular conductance in the left gastric artery (VC/LGA). Data from Wachter et al. (1995).

TABLE 3.2 Effect of capsaicin/resiniferatoxin-evoked stimulation of afferent neurons on motor, secretory and vascular functions of the oesophagus, stomach and duodenum. Species/region

Function

Rabbit oesophagus Blood flow Sphincter pressure Canine LOSa Canine stomach Acid backdiffusion Acid output 2-DODGb-evoked acid output Histamine-evoked acid output Mucosal blood flow TMPDc Plasma gastrin level Rat stomach

Acid backdiffusion Acid output

Effect of capsaicin

References

Increase Increase No change No change Reduction

Bass et al., 1991 Sandler et al., 1993 Sullivan et al., 1992 Soldani et al., 1992; Sullivan et al., 1992 Soldani et al., 1992

No change

Soldani et al., 1992

Increase No change No change

Sullivan et al., 1992 Sullivan et al., 1992 Soldani et al., 1992

No change No change

Lippe, Pabst and Holzer, 1989 Lippe, Pabst and Holzer, 1989; Matsumoto, Takeuchi and Okabe, 1991; Narita et al., 1995; Kang, Teng and Chen, 1996 Abdel Salam, Szolcsányi and Mózsik, 1994, 1995; Brzozowski et al., 1996b Brzozowski et al., 1996b

Decrease Pepsin output

Decrease

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INNERVATION OF THE GASTROINTESTINAL TRACT

TABLE 3.2 Continued Species/region

Function

Effect of capsaicin

References

Rat stomach (Continued)

Bicarbonate output Emptying Motor activity in vivo

Increase Inhibition Increase (low dose) Reduction (high dose) Contraction/ relaxation Relaxation

Takeuchi et al., 1992b,c Kang et al., 1993; Hatakeyama et al., 1995 Holzer et al., 1991

Increase

Lippe, Pabst and Holzer, 1989; Holzer et al., 1990a, 1991; Li et al., 1991; Matsumoto, Takeuchi and Okabe, 1991; Takeuchi et al., 1991b; Chen et al., 1992; Leung, 1992a; Wallace, McKnight and Befus, 1992; Whittle, Lopez-Belmonte and Moncada, 1992; Brzozowski et al., 1993; Grønbech and Lacy, 1995; Merchant et al., 1994; Podolsky et al., 1994; Abdel Salam et al., 1996; Kang, Teng and Chen, 1996; Miyake et al., 1996 Holzer and Lippe, 1988; Lippe, Pabst and Holzer, 1989; Matsumoto, Takeuchi and Okabe, 1991; Takeuchi et al., 1991b Holzer, Pabst and Lippe, 1989 Holzer and Lippe, 1988

Motor activity in vitro (corpus) Motor activity in vitro (fundus) Mucosal blood flow

Takeuchi et al., 1991b Holzer-Petsche, Seitz and Lembeck, 1989 Lefebvre, De Beurme and Sas, 1991

TMPD

No change

TMPD fall due to ASAd TMPD fall due to ethanol Mucus output Vascular permeability

Attenuation No change Increase No change Increase

Kang et al., 1995a Holzer and Lippe, 1988; Hatakeyama et al., 1995; Sann et al., 1996 Lördal et al., 1996

Human stomach

Emptying Mucosal blood flow

Inhibitione Increasee

Horowitz et al., 1992 Graham et al., 1988

Rat duodenum

Bicarbonate output Mucosal blood flow Mucosal tissue integrity Vascular permeability

Increase Increase No change Slight increase

Takeuchi et al., 1991a, 1992b Leung, 1993b; Seno et al., 1996 Takeuchi et al., 1992a; Leung, 1993b Maggi et al., 1987a; Sann et al., 1996

a d

LOS, lower oesophageal sphincter; b 2-DODG, 2-deoxy-D-glucose; c TMPD, transmucosal potential difference; ASA, acetylsalicylic acid; e Effect of capsaicin contained in Jalapeno peppers or chilli powder.

in which capsaicin-evoked stimulation of sensory neurons augments blood flow, include the rabbit oesophagus (Bass et al., 1991), rat duodenum (Leung, 1993b; Seno et al., 1996), rat jejunum (Abdel Salam et al., 1996), rat colon (Leung, 1992b) and the mesenteric arteries of the rat and dog (Holzer, 1992; Jacobson et al., 1994).

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111

The vasodilator effect of capsaicin and resiniferatoxin in the rat stomach, duodenum (Table 3.3), jejunum (Mathison and Davison, 1995) and colon (Leung, 1992b) depends on the integrity of the extrinsic afferent innervation of these organs, because defunctionalisation of afferent neurons by a neurotoxic dose of capsaicin abolishes the hyperaemic response to sensory nerve stimulation. Nerve-selective ablation of afferent nerve fibres has shown that only spinal afferent neurons passing through the coeliac ganglion participate in the capsaicin-evoked increment of GMBF (Li et al., 1991). However, the vagus nerves of the rat also contain afferent nerve fibres that give rise to gastric mucosal vasodilatation following electrical stimulation (Thiefin et al., 1990) but these vagal nerve fibres do not seem to contribute to the hyperaemia caused by intragastric capsaicin (Li et al., 1991) although they are ablated by perineural treatment with a neurotoxic dose of capsaicin (Thiefin et al., 1990).

TABLE 3.3 Neurons and mediators involved in the functional responses to capsaicin/resiniferatoxin-evoked stimulation of afferent neurons in the oesophagus, stomach and duodenum. Effect of capsaicin/ resiniferatoxin

Neurons/mediators

References

Increased acid elimination Increased bicarbonate output

Afferent neurons involved

Lippe, Pabst and Holzer, 1989

Afferent neurons involved

Takeuchi et al., 1991a, 1992b

Indomethacin-sensitive mechanisms involved Muscarinic and nicotinic ACha receptors involved

Takeuchi et al., 1991a, 1992b

Increased motor activity

Tachykinins involved

Holzer-Petsche, Seitz and Lembeck, 1989; Sandler et al., 1993

Decreased motor activity

Extrinsic afferent nerves involved Indomethacin-sensitive mechanisms involved CGRPb, SPc and VIPd ruled out

Lefebvre, De Beurme and Sas, 1991; Takeuchi et al., 1991b Takeuchi et al., 1991b

Afferent neurons involved

Holzer and Lippe, 1988; Holzer et al., 1991; Takeuchi et al., 1991b, 1994; Uchida, Yano and Watanabe, 1991b; Gray et al., 1994 Holzer and Lippe, 1988 Holzer and Lippe, 1988

Protection from injury

Sympathetic neurons ruled out Parasympathetic neurons ruled out Muscarinic ACh receptors involved Muscarinic ACh receptors ruled out

Takeuchi et al., 1991a

Holzer-Petsche, Seitz, and Lembeck, 1989; Lefebvre, De Beurme and Sas, 1991; Takeuchi et al., 1991b

Abdel Salam et al., 1995a Holzer and Lippe, 1988

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INNERVATION OF THE GASTROINTESTINAL TRACT

TABLE 3.3 Continued Effect of capsaicin/ resiniferatoxin

Neurons/mediators

References

Protection from injury (Continued)

CGRP involved

Lambrecht et al., 1993a; Peskar et al., 1993; Kato, Yang and Taché, 1994; Merchant et al., 1994 Peskar et al., 1991; Brzozowski et al., 1993; Lambrecht et al., 1993a; Podolsky et al., 1994 Takeuchi et al., 1991b

Nitric oxide involved SP ruled out Neurokinin A involved Prostaglandins and leukotriene C4 ruled out Indomethacin-sensitive mechanisms ruled out Indomethacin-sensitive mechanisms involved

Vasodilatation

Afferent neurons involved

CGRP involved

Nitric oxide involved

Tachykinins ruled out

Indomethacin-sensitive mechanisms involved Muscarinic ACh receptors ruled out Nicotinic ACh receptors involved Noradrenaline α- and β-adrenoceptors ruled out Mast cell mediators ruled out

Stroff et al., 1996 Holzer et al., 1990a Holzer et al., 1990a Takeuchi et al., 1991b; Uchida, Yano and Watanabe, 1991b; Brzozowski et al., 1993; Mercer, Ritchie and Dempsey, 1994; Hatakeyama et al., 1995 Lippe, Pabst and Holzer, 1989; Holzer et al., 1991; Li et al., 1991; Matsumoto, Takeuchi and Okabe, 1991; Takeuchi et al., 1991b, 1994; Chen et al., 1992; Leung, 1992a; Wallace, McKnight and Befus, 1992; Abdel Salam et al., 1996 Li et al., 1991; Chen et al., 1992; McKie et al., 1994; Merchant et al., 1994; Chen and Guth, 1995 Whittle, Lopez-Belmonte and Moncada, 1992; Brzozowski et al., 1993; Podolsky et al., 1994; Chen and Guth, 1995; Merchant et al., 1995 Matsumoto, Takeuchi and Okabe, 1991; Grønbech and Lacy, 1994; McKie et al., 1994; Stroff et al., 1996 Matsumoto, Takeuchi and Okabe, 1991; Takeuchi et al., 1991b; Brzozowski et al., 1993 Lippe, Pabst and Holzer, 1989; Matsumoto, Takeuchi and Okabe, 1991; Abdel Salam et al., 1996 Leung, 1993a Lippe, Pabst and Holzer, 1989 Matsumoto, Takeuchi and Okabe, 1991; Wallace, McKnight and Befus, 1992; Grønbech and Lacy, 1994

a ACh, acetylcholine; b CGRP, calcitonin gene-related peptide; c SP, substance P; d VIP, vasoactive intestinal polypeptide.

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PATHWAYS AND MEDIATORS OF NEUROGENIC VASODILATATION Calcitonin gene-related peptide (CGRP) and related peptides The neural pathways responsible for the capsaicin-evoked rise of GMBF in the rat stomach, which involve ganglionic transmission via nicotinic acetylcholine receptors (Leung, 1993a), have not conclusively been defined. Since noradrenergic neurons and cholinergic vasodilator neurons have been ruled out (Table 3.3) it would appear that the vasodilator response to capsaicin is due to a local mechanism within the gastric wall and brought about by release of vasodilator peptides from perivascular afferent nerve fibres (Holzer et al., 1990a, 1991). While the vasodilator mediators of vagal afferent nerve fibres have not yet been elucidated, there is conclusive evidence that CGRP plays an important mediator role in the rise of rat GMBF evoked by stimulation of spinal afferent nerve fibres, since the CGRP1 receptor antagonist CGRP8–37 prevents the gastric vasodilatation caused by intragastric and intravascular capsaicin (Table 3.3). It need be added, though, that the data obtained with CGRP8–37 prove the implication of CGRP1 receptors but not necessarily of CGRP itself. This is because peptides with some structural homology to CGRP, such as adrenomedullin formed in endothelial cells (Sugo et al., 1994) and amylin expressed in afferent neurons (Mulder et al., 1995) and endocrine cells of the gastric mucosa (Mulder et al., 1994a), are also capable of activating CGRP1 receptors, and vasodilator responses to both amylin (Gardiner et al., 1991) and adrenomedullin (Nuki

TABLE 3.4 Release of calcitonin gene-related peptide (CGRP), substance P (SP) and neurokinin A (NKA) from presumably afferent nerve fibres in the stomach and duodenum. Species/region

Peptide

Release stimulus

References

Rat stomach

CGRP

Acid (lowering of pH)

Ethanol Acid Capsaicin Capsaicin

Geppetti et al., 1991; Manela et al., 1995; Ren et al., 1995 Holzer et al., 1990b; Chiba et al., 1991; Geppetti et al., 1991; Inui et al., 1991; Renzi et al., 1991; Ren et al., 1993; Gray et al., 1994; Narita et al., 1995; Inaba et al., 1996 Inui et al., 1989 Manela et al., 1992 Inui et al., 1989 Kwok and McIntosh, 1990; Renzi et al., 1991; Katori, Ohno and Nishiyama, 1993; Hayashi et al., 1996 Katori, Ohno and Nishiyama, 1993; Hayashi et al., 1996 Smedfors, Theodorsson and Johansson, 1994 Fujimiya and Kwok, 1995 Renzi et al., 1988

Acidified 2 M NaCl Capsaicin Acidification

Gislason et al., 1995a Schmidt et al., 1996 Mueller et al., 1991

Capsaicin

SP/NKA

Rat duodenum

SP/NKA

Guinea-pig SP/NKA stomach Feline stomach CGRP Porcine stomach SP/NKA Human stomach SP a

Dibutyryl cAMPa Peptone Theophylline Capsaicin

cAMP, adenosine 3′,5′-cyclic monophosphate.

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INNERVATION OF THE GASTROINTESTINAL TRACT

et al., 1993; Entzeroth et al., 1994) are antagonised by CGRP8–37. Whether amylin and adrenomedullin, which are less active than CGRP in causing vasodilatation (Gardiner et al., 1991; Nuki et al., 1993; Entzeroth et al., 1994), play a role in afferent nervemediated hyperaemia has not yet been tested. An implication that CGRP is involved is consistent with the ability of capsaicin and other stimuli to release this peptide from extrinsic afferent nerve fibres in the rat, guineapig and feline stomach (Table 3.4) and with the vascular pharmacology of the peptide. CGRP fails to affect gastric venules but potently dilates submucosal arterioles in the rat stomach (Chen et al., 1992; Chen and Guth, 1995) and enhances blood flow through the oesophagus and stomach of rat and rabbit (Bauerfeind et al., 1989; Lippe, Lorbach and Holzer, 1989; Holzer and Guth, 1991; Li et al., 1991; Holzer et al., 1993; Lopez-Belmonte and Whittle, 1993; Király et al., 1994a; McKie et al., 1994). The vasodilator action of the peptide is in keeping with the abundant presence of CGRP receptors on the muscle and endothelium of arteries and arterioles in the rat and canine stomach (Gates et al., 1989; Sternini, 1992), although one laboratory has failed to observe a hyperaemic response to CGRP in the canine stomach (Nakamura et al., 1996). The ability of CGRP8–37 to block the hyperaemia that in the rat stomach is elicited by both capsaicin and CGRP (Li et al., 1991; Chen et al., 1992; Holzer et al., 1994; Király et al., 1994a; Chen and Guth, 1995) indicates that the peptide dilates gastric blood vessels via an action on CGRP1 receptors. This is also true for the CGRP-evoked rise of mucosal blood flow in the rabbit oesophagus (McKie et al., 1994). Nitric oxide (NO) The vasodilator action of CGRP released by capsaicin in the rat stomach involves the formation of NO, since NO synthase inhibitors suppress the gastric hyperaemic reaction to both capsaicin (Table 3.3) and CGRP (Whittle, Lopez-Belmonte and Moncada, 1992; Holzer et al., 1993; Chen and Guth, 1995). NO is unlikely to be released from the CGRPcontaining axons, because ablation of capsaicin-sensitive afferent nerve fibres does not alter the activity of NO synthase in the rat stomach (Forster and Southam, 1993). The localisation of CGRP receptors on endothelial cells and the observation that the relaxant action of CGRP on precontracted arteries from the human stomach depends on the presence of the endothelium (Thom et al., 1987) suggest that CGRP increases the formation of NO in endothelial cells (Figure 3.4). However, the gastric vasodilator response to CGRP is only partly NO-dependent (Holzer et al., 1993) and less susceptible to inhibition by a NO synthase blocker than the vasodilator response to capsaicin (Chen and Guth, 1995). These observations indicate that part of the vasodilator action of CGRP in the stomach may be due to a direct action of the peptide on the vascular smooth muscle (Figure 3.4) and that the cellular sources of NO formed in response to capsaicin and CGRP are not fully identical. It need be considered in this context that, in addition to being the final vasodilator mediator of CGRP, NO can also facilitate the release of CGRP from afferent vasodilator axons (Wei et al., 1992; Holzer and Jocic, 1994; Hughes and Brain, 1994). While such a role of NO is conceivable in the in vivo stomach (Chen and Guth, 1995), there is little direct evidence that NO activates CGRP-releasing afferent nerve fibres in the vasculature of the rat stomach in vitro (Seyed Ebrahim, Stroff and Peskar, 1996).

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Figure 3.4 Flow chart of the vasodilator and protective mechanisms initiated by stimulation of afferent neurons and subsequent release of peptide transmitters in the stomach. CGRP1-R, CGRP1 receptor; NK1-R, tachykinin NK1 receptor; NK2-R, tachykinin NK2 receptor.

Tachykinins SP and NKA are two other peptides that are released from afferent nerve fibres in the stomach and duodenum in response to capsaicin and other stimuli (Table 3.4). While SP/ NKA and CGRP comediate the capsaicin-evoked dilatation of submucosal arterioles in the guinea-pig ileum (Vanner, 1994), there is no evidence that SP and NKA acting via tachykinin NK1 or NK2 receptors play a role in the afferent nerve-mediated rise of GMBF in the oesophagus and stomach (Table 3.3). Thus, SP, NKA and NK2 receptor-selective agonists fail to dilate submucosal arterioles (Katori, Ohno and Nishiyama, 1993) and to augment blood flow in the rat stomach (Yokotani and Fujiwara, 1985; Holzer and Guth, 1991; Grønbech and Lacy, 1994; Holzer et al., 1994; Heinemann et al., 1996a; Stroff et al., 1996), and tachykinin NK1 or NK2 receptor antagonists do not alter the hyperaemic reaction to afferent nerve stimulation by intragastric capsaicin (Matsumoto, Takeuchi and Okabe, 1991; Stroff et al., 1996). To the contrary, SP has in fact been found to constrict rat gastric submucosal venules in a leukotriene C4-independent manner (Katori, Ohno and Nishiyama, 1993), which is consistent with the ability of NKA and NK2 receptor-selective analogues to decrease blood flow through the rat stomach (Heinemann et al., 1996a; Stroff et al., 1996; Lippe, Wachter and Holzer, 1997). In addition, SP reduces the mucosal hyperaemia which in the rat stomach is elicited by electrical stimulation of the vagus nerve (Yokotani and Fujiwara, 1985) or by capsaicin-evoked stimulation of afferent nerve fibres (Grønbech and Lacy, 1994). The inhibitory influence of SP on the capsaicin-induced hyperaemia involves liberation of mast cell proteases (Grønbech and Lacy, 1994) which may degrade CGRP, the vasodilator peptide released by capsaicin (Li et al., 1991). It must not go unnoticed here that the vascular effects of tachykinins in the stomach are subject to species differences. Unlike in the rat and rabbit, intravascular administration of SP in the dog increases blood flow through the coeliac artery (Prokopiw and McDonald,

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INNERVATION OF THE GASTROINTESTINAL TRACT

1994) and in the wall of the stomach (Yeo, Jaffe and Zinner, 1984; Ito et al., 1993). The gastric hyperaemia in response to tachykinins is attenuated by atropine but remains unaltered by vagotomy or splanchnicotomy (Ito et al., 1993). Pharmacological analysis indicates that the vasodilator response to tachykinins in the canine stomach is brought about by activation of NK1 receptors (Ito et al., 1993), which is in keeping with the presence of some NK1 and NK2 binding sites on the endothelium and smooth muscle of submucosal arterioles and venules of the canine and human gut (Gates et al., 1988; Mantyh et al., 1988). A mediator role of tachykinins in neurogenic vasodilatation of the canine and human stomach awaits to be proved. Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) PACAP has been localised to primary afferent neurons (Mulder et al., 1994b) while VIP occurs primarily in intrinsic enteric neurons of the rat stomach (Ekblad et al., 1985). Whether PACAP or VIP, which potently increases blood flow through the rat (Holzer and Guth, 1991; Holzer et al., 1993) and canine (Nakamura et al., 1996) stomach, contributes to afferent nerve-mediated hyperaemia in the stomach has not yet been examined but is worth testing particularly in the human stomach. Prostaglandins and mast cell mediators The role of prostaglandins in the capsaicin-evoked rise of GMBF is not totally clear. Although capsaicin fails to alter the ex vivo formation of prostaglandin E2, 6-oxo-prostaglandin F1 and leukotriene C4 in the rat gastric mucosa (Holzer and Sametz, 1986; Holzer et al., 1990a), indomethacin has been reported to diminish the capsaicin-induced increase in GMBF in the rat (Table 3.3). The validity of the findings with indomethacin in terms of an implication of vasodilator prostaglandins remains ambiguous, therefore, and it is worth mentioning in this respect that the capsaicin-induced rise of blood flow in the rat jejunum (Mathison and Davison, 1995) is left unaltered by indomethacin. Vasoactive mediators derived from mast cells such as histamine do not seem to contribute to the capsaicinevoked rise of GMBF under normal circumstances (Table 3.3). How the inhibitory influence of cimetidine on the capsaicin-induced hyperaemia in the rat gastric mucosa is brought about (Kang, Teng and Chen, 1996) awaits clarification.

PHYSIOLOGICAL IMPLICATIONS OF AFFERENT VASODILATOR NERVE FIBRES IN THE STOMACH Local roles in the gastric circulation The ability of afferent nerve fibres to increase GMBF raises the question as to their physiological role in gastric circulatory control. There are two reports that basal blood flow through the rat gastric mucosa, particularly in the antrum, is lowered in rats pretreated with a neurotoxic dose of capsaicin (Uchida, Yano and Watanabe, 1993; Dembinski et al., 1995) but the majority of studies holds that defunctionalisation of

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117

capsaicin-sensitive afferent neurons does not change basal blood flow in the rat gastric corpus (Table 3.5). It would seem, therefore, that peptidergic afferent nerve fibres do not play a role in regulating GMBF under basal conditions. The gastric vasodilator responses to CGRP, acetylcholine (ACh), adenosine, histamine and gastrin-17 do likewise not involve capsaicin-sensitive neurons (Table 3.5). While the gastric mucosal hyperaemia caused by pentagastrin is either reduced or unchanged in capsaicin-pretreated rats, there is consistent evidence that the rise of GMBF brought about by low doses of cholecystokinin (CCK) or CCK octapeptide depends on capsaicin-sensitive afferent neurons (Table 3.5). Further analysis indicates that the gastric vasodilator effect of CCK octapeptide is brought about by CCKB receptors (Heinemann et al., 1995) and involves CGRP and NO as vasodilator messengers (Table 3.6). The hyperaemia caused by epidermal growth factor is similarly dependent on capsaicin-sensitive afferent neurons, CGRP and NO (Konturek, Brzozowski and Sliwowski, 1996; Teng et al., 1996a). Although ablation of fine afferent neurons does not alter the gastric vasoconstrictor response to adrenaline, it does attenuate the autoregulatory escape from adrenalineinduced vasoconstriction in the rat gastric mucosa (Table 3.5), which indicates that vascular constriction is counteracted by afferent nerve-mediated vasodilatation. An analogous explanation may apply to the finding that gastric vasoconstriction caused by platelet-activating factor or blockade of NO synthesis is amplified after capsaicin-induced defunctionalisation of afferent neurons (Table 3.5). The interaction between the tonically active NO system (Piqué, Esplugues and Whittle, 1989; Walder, Thiemermann and Vane, 1990; Lippe and Holzer, 1992; Tepperman and Whittle, 1992; Holzer et al., 1993) and stimulusdriven afferent nerve fibres suggests that physiologically these two systems act in concert to provide an active dilator drive on the gastric microcirculation (Whittle, 1993). A similar relationship holds true for haemostasis which is attenuated by capsaicin-induced ablation of afferent neurons, an effect that is intensified by blockade of NO synthesis (Lippe et al., 1993). This interaction, which is due to enhancement of platelet aggregation, suggests that sensory nerve-induced facilitation of local blood flow is aided by a concomitant reduction of haemostasis (Lippe et al., 1993). Vagal efferent activation of gastric afferent vasodilator nerves Studies of the central and autonomic regulation of gastric functions indicate that efferent parasympathetic pathways in the vagus nerves interact with CGRP-containing afferent nerve fibres in the stomach. This interplay is instrumental for the ability of intracisternal injection of a stable analogue of thyrotropin-releasing hormone (TRH) to augment GMBF. The action of TRH not only requires activation of efferent pathways in the vagus nerves but also depends on an intact innervation of the stomach by capsaicin-sensitive afferents (Table 3.5) and involves ACh, CGRP and NO as mediators, whereas tachykinins acting via NK1 receptors, VIP and prostaglandins have been ruled out (Király et al., 1994a,b,c). Since the gastric hyperaemia evoked by muscarinic ACh receptor activation is also inhibited by CGRP8–37, it is evident that CGRP comes into play secondarily to ACh (Király et al., 1994a,b). This instance is reminiscent of the finding that the delayed component in the ACh-induced dilatation of the rat mesenteric vascular bed involves release of CGRP from capsaicin-sensitive nerve fibres (Takenaga et al., 1995). Analogously, the gastric vasodilator

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INNERVATION OF THE GASTROINTESTINAL TRACT

TABLE 3.5 Effect of capsaicin-induced defunctionalisation of afferent neurons on haemodynamics in the rat stomach and duodenum. Capsaicin administration

Parameter

Effect of nerve defunctionalisation

References

Systemic

Arteriolar dilatation by acetylcholine Arteriolar dilatation by adenosine Arteriolar dilatation by capsaicin Arteriolar dilatation by CGRP Basal MBFa

No change

Chen, Li and Guth, 1992

No change

Chen, Li and Guth, 1992

Suppression

Chen, Li and Guth, 1992

No change

Chen, Li and Guth, 1992

No change

Lippe, Pabst and Holzer, 1989; Piqué, Esplugues and Whittle, 1990; Raybould et al., 1990, 1992; Thiefin et al., 1990; Holzer, Livingston and Guth,1991; Holzer et al., 1991; Li et al., 1991, 1992; Matsumoto, Takeuchi and Okabe, 1991; Matsumoto et al., 1992a,b; Takeuchi et al., 1991b, 1994; Chen, Li and Guth, 1992; Leung, 1992a; Uchida, Yano and Watanabe, 1993; Király et al., 1994a Uchida, Yano and Watanabe, 1993; Dembinski et al., 1995 Leung, 1992a

Reduction Escape from adrenaline vasoconstriction Increase in MBF by acid backdiffusion

Reduction

Inhibition

No change

Holzer, Livingston and Guth, 1991; Holzer et al., 1994; Leung, 1993b; Matsumoto et al., 1992a,b; Takeuchi et al., 1993, 1994; Grønbech and Lacy, 1996 Lippe, Pabst and Holzer, 1989; Holzer et al., 1991; Matsumoto, Takeuchi and Okabe, 1991; Matsumoto et al., 1992a; Takeuchi et al., 1991b, 1994; Leung, 1992b; Wallace, McKnight and Befus, 1992; Abdel Salam et al., 1996 Konturek et al., 1995b; Heinemann et al., 1996b Mercer et al., 1996

Reduction

Brzozowski et al., 1996a

Suppression

Konturek, Brzozowski and Sliwowski, 1996; Teng et al., 1996a

No change

Konturek et al., 1995a

Increase in MBF by capsaicin

Suppression

Increase in MBF by low dose CCKb Increase in MBF by high dose CCK Increase in MBF by E. coli endotoxin Increase in MBF by epidermal growth factor Increase in MBF by gastrin-17

Reduction

CONTROL OF GASTRIC FUNCTIONS BY EXTRINSIC SENSORY NEURONS

Increase in MBF by pentagastrin Increase in MBF by histamine Increase in MBF by 1 M NaCl

Perivagal

Pericoeliacf

No change

Livingston and Holzer, 1993

Reduction

Konturek et al., 1995b

No change

Leung, 1992a

Reduction

Matsumoto et al., 1992a

119

No change

Grønbech and Lacy, 1996

Increase in MBF by 2 M NaCl Increase in MBF by intracisternal TRHc Reduction of MBF by adrenaline Reduction of MBF by PAFd Reduction of MBF by NOSe inhibitors

No change

Endoh et al., 1993; Grønbech and Lacy, 1996

Inhibition

Király et al., 1994c

No change

Leung, 1992a

Exacerbation

Piqué, Esplugues and Whittle, 1990

Exacerbation

Tepperman and Whittle, 1992; Dembinski et al., 1995

Basal MBF

No change

Increase in MBF by capsaicin Increase in MBF by acid backdiffusion Increase in MBF by vagal nerve stimulation Increase in MBF by intracisternal TRH

No change

Raybould et al., 1990, 1992; Thiefin et al., 1990; Li et al., 1991 Li et al., 1991

No change

Raybould et al., 1992

Reduction

Thiefin et al., 1990

Reduction

Raybould et al., 1990

Basal MBF

No change

Increase in MBF by capsaicin Increase in MBF by acid backdiffusion

Reduction

Li et al., 1991; Raybould et al., 1992 Li et al., 1991

Reduction

Raybould et al., 1992

a

MBF, mucosal blood flow; b CCK, cholecystokinin; c TRH, thyrotropin-releasing hormone; d PAF, plateletactivating factor; e NOS, nitric oxide synthase; f administration of capsaicin locally to the coeliac ganglion.

response to low doses of intravenous CCK octapeptide is brought about by a vagovagal reflex and depends also on ACh, CGRP and NO as vasodilator messengers (Heinemann et al., 1996b). Although histamine acting via H1 receptors does not participate in the rise of GMBF caused by intracisternal TRH, it seems as if the interface between vagal efferents and CGRP-releasing afferents is constituted by mediators released from gastric mast cells (Király et al., 1994b,c).

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INNERVATION OF THE GASTROINTESTINAL TRACT

TABLE 3.6 Evidence that physiological/pathophysiological functions of the stomach, that involve afferent neurons, are mediated by CGRP and NO. Mediator

Sensory nerve-mediated process

CGRP

Acid-induced increase in blood flow

NO

References

Li et al., 1992; Holzer et al., 1994; Gislason et al., 1995a Capsaicin-induced increase in blood flow Li et al., 1991; Chen, Li and Guth, 1992; Merchant et al., 1994: Chen and Guth, 1995 Increase in blood flow due to CCK-8 Heinemann et al., 1996b Increase in blood flow due to epidermal growth Konturek, Brzozowski and Sliwowski, 1996; factor Teng et al., 1996a Increase in blood flow due to intracisternal TRH Király et al., 1994a,b,c Inhibition of basal gastric acid output Lawson, Mantyh and Pappas, 1994; Kato et al., 1995 Capsaicin-induced inhibition of ACh and gastrin Ren et al., 1993 release Acid-induced inhibition of gastrin release Manela et al., 1995 Stimulation of basal somatostatin release Manela et al., 1995 Acid-induced release of somatostatin Manela et al., 1995 Capsaicin-induced release of somatostatin Inui et al., 1991; Ren et al., 1993 Endogenous gastroprotection Forster and Dockray, 1991a Protection by capsaicin Chiba et al., 1991; Lambrecht et al., 1993a; Peskar et al., 1993; Kato, Yang and Taché, 1994; Merchant et al., 1994 Protection by bombesin, CCK-8 and gastrin-17 Stroff et al., 1993, 1995; Stroff, Plate and Peskar, 1995a Protection by corticotropin-releasing factor Stroff, Plate and Peskar, 1995b Protection by epidermal growth factor Stroff and Peskar, 1995a Protection by NKA-related peptides Stroff et al., 1996 Protection by peptone Stroff and Peskar, 1996 Protection by intracisternal TRH Kato, Yang and Taché, 1994, 1996 Acid-induced inhibition of gastric emptying Forster and Dockray, 1991b Hyperosmolarity-induced inhibition of gastric Forster and Dockray, 1991b emptying Inhibition of gastric emptying after abdominal Plourde et al., 1993; Zittel et al., 1994a surgery Acid-induced increase in blood flow Lippe and Holzer, 1992; Holzer et al., 1994; Gislason et al., 1996 Capsaicin-induced increase in blood flow Whittle, Lopez-Belmonte and Moncada, 1992; Brzozowski et al., 1993; Podolsky et al., 1994; Chen and Guth, 1995; Merchant et al., 1995 Increase in blood flow due to CCK-8, Konturek et al., 1995b,c; Heinemann et al., 1996b gastrin-17 and pentagastrin Increase in blood flow due to E. coli endotoxin Brzozowski et al., 1996a Increase in blood flow due to epidermal growth Konturek, Brzozowski and Sliwowski, 1996 factor Increase in blood flow due to lansoprazole Murakami et al., 1996 Increase in blood flow due to intracisternal Király et al., 1994b TRH Protection by capsaicin Peskar et al., 1991; Brzozowski et al., 1993; Lambrecht et al., 1993a; Podolsky et al., 1994; Merchant et al., 1995

CONTROL OF GASTRIC FUNCTIONS BY EXTRINSIC SENSORY NEURONS

Protection by bombesin, CCK-8, gastrin-17 and pentagastrin

Protection by corticotropin-releasing factor Protection by ecabet Protection by E. coli endotoxin Protection by epidermal growth factor

Protection by hydrotalcit Protection by lansoprazole Protection by NKA-related peptides Protection by intracisternal TRH

121

Lambrecht et al., 1993b; Lambrecht, Stroff and Peskar 1993; Konturek et al., 1995b,c; Stroff et al., 1995; Stroff, Plate and Peskar, 1995a Stroff, Plate and Peskar, 1995b Kinoshita, Kume and Tamaki, 1995 Barrachina et al., 1995; Brzozowski et al., 1996a Stroff and Peskar, 1995a; Konturek, Brzozowski and Sliwowski, 1996 Lambrecht et al., 1993c Murakami et al., 1996 Stroff et al., 1996 Kato, Yang and Taché, 1994

PEPTIDERGIC AFFERENT NERVE FIBRES, VASCULAR PERMEABILITY AND LEUCOCYTE RECRUITMENT IN THE STOMACH PLASMA PROTEIN EXTRAVASATION A prominent feature of neurogenic inflammation in many tissues is an increase in venular permeability, a reaction that facilitates the extravasation of macromolecules, leucocytes and fluid (Holzer, 1992). Capsaicin-induced stimulation of afferent nerve fibres in the rat stomach does not consistently enhance leakage of Evans blue-labelled plasma albumin (Table 3.2), as studies reporting a lack of effect of capsaicin (Holzer and Lippe, 1988; Hatakeyama et al., 1995; Sann et al., 1996) are opposed by reports of moderate increases in vascular permeability caused by capsaicin (Lördal et al., 1996). At any rate, the plasma leakage which capsaicin or electrical stimulation of afferent fibres in the vagus and splanchnic nerves evokes in the stomach is small compared to the plasma protein extravasation seen in the oesophagus and distal rectum (Saria et al., 1983; Lundberg et al., 1984; Lördal et al., 1996). Afferent nerve-mediated increases in venular permeability outside the digestive system are mediated by SP and NKA acting via NK1 receptors (Holzer, 1992). Whether tachykinins mediate the erratic rise of plasma albumin leakage, that in the rat stomach is induced by capsaicin, is not known, and the effects of exogenous tachykinins on vascular permeability in the rat gut are inconsistent. Most studies hold that vascular permeability is not appreciably altered by SP (Saria et al., 1983; Lundberg et al., 1984; Lördal et al., 1996), while one study reports that high dose SP does increase plasma protein leakage in the rat stomach and duodenum (Nicolau et al., 1993). Pharmacological analysis indicates that all three tachykinin receptors control vascular permeability in the

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INNERVATION OF THE GASTROINTESTINAL TRACT

rat gut in a regionally distinct manner (Nicolau et al., 1993; Lördal et al., 1996). The action of SP in the duodenum is mediated by NK1 receptors, while both NK2 and NK3 receptors might be responsible for the extravasation response in the stomach (Nicolau et al., 1993; Lördal et al., 1996). Since the plasma leakage which SP causes in the oesophagus and mesenteric vasculature of the rat remains unaltered by blockade of histamine H1/ H2 receptors (Saria et al., 1983; Lundberg et al., 1984), it would appear that histamine does not contribute to the response, although SP-positive nerve fibres lie in close proximity to mast cells in the rat mesentery and intestinal mucosa (Skofitsch, Savitt and Jacobowitz, 1985; Stead et al., 1987; Crivellato et al., 1991) and SP can act on rat intestinal mast cells to release histamine and other factors (Shanahan et al., 1985; Marshall et al., 1994). It is important to realise that the influence of afferent nerve fibres on vascular permeability is species-dependent, since capsaicin causes distinct plasma protein extravasation in the gastrointestinal tract of the mouse (Emanueli et al., 1996). Although not yet proven, tachykinins may definitely comediate the neurogenic leakage of plasma proteins in the murine gut, because SP is able to increase vascular permeability via stimulation of NK1 receptors (Emanueli et al., 1996; Figini et al., 1996). Further evidence that endogenous tachykinins participate in the physiological control of vascular permeability in the murine gut comes from the findings that basal plasma protein extravasation is elevated in mice made genetically deficient in neutral endopeptidase, a cell-surface enzyme known to degrade SP, and that a NK1 receptor antagonist corrects the increased vascular permeability in the knockout mice (Figini et al., 1996).

LEUCOCYTE RECRUITMENT Besides increasing vascular permeability and extravasation of macromolecules and fluid, afferent nerve stimulation and afferent nerve-derived sensory neuropeptides influence the adherence, emigration, proliferation and activity of leucocytes (McGillis, Mitsuhashi and Payan, 1990; Sung et al., 1992; Zimmerman, Anderson and Granger, 1992; Smith et al., 1993; Cabaner, Boudard and Bastide, 1995). Although SP enhances the adhesion of leucocytes to the endothelium of rat mesenteric venules (Zimmerman, Gaginella and Granger, 1991) it has not yet specifically been tested whether afferent nerve-derived peptides affect leucocyte recruitment in the stomach. In nonintestinal veins it is well established that SP, CGRP and VIP stimulate the adherence of neutrophils to the endothelium (Zimmerman, Anderson and Granger, 1992) and promote the emigration of neutrophils and eosinophils (Sung et al., 1992), actions which are accompanied by rapid translocation of P-selectin and upregulation of E-selectin expression in endothelial cells (Smith et al., 1993). SP has also been shown to contribute to the granulocyte infiltration which in the rat colon is induced by trinitrobenzene sulphonic acid (McCafferty, Sharkey and Wallace, 1994) and in the rat ileum is provoked by Clostridium difficile toxin A (Pothoulakis et al., 1994). Apart from granulocytes it is also lymphocytes (Stanisz, Befus and Bienenstock, 1986; Agro and Stanisz, 1993; Cabaner, Boudard and Bastide, 1995) and monocytes/macrophages (Bost, Breeding and Pascual, 1992) that may be influenced by afferent nerve-derived

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neuropeptides, although this interaction has not yet been examined specifically in the stomach. The ability of SP immunoneutralisation to reduce lymphocyte proliferation in the inflamed small intestine of Trichinella spiralis-infected mice (Agro and Stanisz, 1993) and the upregulation of NK1 receptor expression in rat peritoneal macrophages exposed to endotoxin (Bost, Breeding and Pascual, 1992) underlines the pathophysiological potential of neuropeptide-immune system interactions.

PEPTIDERGIC AFFERENT NERVE FIBRES AND GASTRIC SECRETION IMPACT OF AFFERENT NEURONS ON GASTRIC SECRETION In view of their projections to submucosal blood vessels and the mucosa itself (Sternini, Reeve and Brecha, 1987; Green and Dockray, 1988; Sundler, Ekblad and Håkanson, 1991; Jakab et al., 1993; Bäck et al., 1994), extrinsic afferent neurons may, conceivably, influence the secretory activity of the stomach by their regulatory input on the gastric microcirculation and by a direct control of mucosal cell functions. Capsaicin-evoked stimulation of gastric afferent nerve fibres does not, however, significantly alter the basal secretion of acid in the rat and canine stomach (Table 3.2), whereas the elimination of acid from the gastric lumen is increased (Lippe, Pabst and Holzer, 1989; Matsumoto, Takeuchi and Okabe, 1991; Sullivan et al., 1992) as a result of enhanced GMBF (Holzer et al., 1990a, 1991) and bicarbonate secretion (Takeuchi et al., 1992b,c). Only under the pathological conditions of pylorus ligation in conscious rats have capsaicin and resiniferatoxin been found to reduce basal gastric acid output and to blunt the secretory responses to bethanechol, pentagastrin and histamine (Abdel Salam, Szolcsányi and Mózsik, 1994, 1995). The effects of sensory nerve stimulation on the gastric acid/ bicarbonate balance are related to capsaicin’s ability to enhance the release of somatostatin and to inhibit the release of ACh and gastrin in the rat isolated antrum, actions which are at least in part mediated by CGRP (Figure 3.5; Table 3.6). The stimulant effect of sensory nerve stimulation on gastric mucus secretion (Kang et al., 1995a) has not yet been examined systematically. Defunctionalisation of capsaicin-sensitive afferent neurons fails to change the basal secretion of gastric acid, pepsin and bicarbonate (Table 3.7). The basal secretion of mucus in the rat gastric corpus is likewise unaltered while that in the antrum is reduced (Uchida, Yano and Watanabe, 1993). The failure of capsaicin pretreatment to change basal gastric secretion is additionally reflected by its failure to change the permeability of the gastric mucosa to acid and ions (Holzer, Livingston and Guth, 1991; Matsumoto, Takeuchi and Okabe, 1991; Takeuchi et al., 1992c) and suggests that fine afferent nerve fibres do not take part in the regulation of basal gastric secretion. It cannot be ruled out, however, that chronic ablation of afferent neurons triggers compensatory mechanisms that balance the missing input from afferent neurons. This conjecture is supported by the observation that administration of the CGRP antagonist CGRP8–37 to dogs elevates basal acid secretion and amplifies the acid output induced by bombesin (Lawson, Mantyh and

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INNERVATION OF THE GASTROINTESTINAL TRACT

Figure 3.5 Schematic diagram illustrating two different roles of capsaicin-sensitive afferent neurons in the regulation of acid secretion in the rat stomach. CGRP1-R, CGRP1 receptor; CNS, central nervous system; D, gastric D (somatostatin) cells; G, gastric G (gastrin) cells; NK2-R, tachykinin NK2 receptor.

Pappas, 1994). The acid output which in the rat stomach is caused by pylorus ligation or pentagastrin infusion is likewise facilitated by CGRP8–37 or a monoclonal CGRP antibody (Kato et al., 1995). There is a multitude of data showing that capsaicin-sensitive afferent neurons participate in stimulated gastric secretion (Table 3.7), but owing to discrepancies in the reports it is at present difficult to draw an integrated hypothesis as to how physiological stimulation of gastric secretion depends on input from afferent neurons. Thus, some laboratories hold that acid secretion induced by histamine, pentagastrin or 2-deoxy-D-glucose is reduced by pretreatment of rats with a neurotoxic dose of capsaicin, whereas other groups have failed to see any change (Table 3.7). Another unresolved issue concerns the relationship between vagal efferents and capsaicin-sensitive afferents in the control of gastric acid secretion. The rise of gastric acid output evoked by electrical stimulation of the vagus nerves remains unchanged after systemic pretreatment of rats with capsaicin (Table 3.7) but is attenuated after selective ablation of capsaicin-sensitive vagal afferents (Sharkey et al., 1991). The latter finding has been taken to speculate that vagal afferents may themselves be cholinergic, given that the secretory response to vagal nerve stimulation in untreated rats is abolished by hexamethonium and atropine (Sharkey et al., 1991). A further aspect of this question is mirrored by the observation that the inhibitory effect of a low dose of intracisternal TRH on gastric acid secretion involves ACh, prostaglandins and CGRP (Király et al., 1994c). No hypothesis is at present available that would incorporate all these findings in a coherent manner. There is little controversy over the observations that acid secretion elicited by distension of the gastric wall depends significantly on an intact sensory innervation of the stomach and involves both spinal and vagal afferents (Figure 3.5; Table 3.7). Capsaicininduced ablation of sensory neurons attenuates the acid secretory response to intragastric

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125

peptone (Table 3.7), and the inhibition of peptone-stimulated gastric acid secretion, which is seen after intraduodenal administration of lipid, relies also in part on the integrity of vagal, but not spinal, afferent nerve fibres (Lloyd et al., 1993). In contrast, enterogastric inhibition of gastric acid secretion caused by intraduodenal acid is mediated by a reflex that involves both vagal and splanchnic afferents sensitive to the neurotoxin capsaicin (Saperas, Santos and Malagelada, 1995).

TABLE 3.7 Effect of capsaicin-induced defunctionalisation of sensory neurons on exocrine secretion in the rat stomach and duodenum. Capsaicin Parameter administration

Effect of nerve defunctionalisation

References

Systemic

Acid back diffusion

No change

Basal acid output

No change

Basal bicarbonate output

No change

Basal mucus output Basal mucus output Basal pepsin output Enterogastric reflexa (acid) Stimulated acid output (AChb, VNSc)

No change (corpus) Increase (antrum) No change Inhibition No change

Stimulated acid output (2-deoxy-D-glucose)

Reduction

Holzer, Livingston and Guth, 1991; Takeuchi et al., 1993, 1994 Szolcsányi and Barthó, 1981; Alföldi et al., 1986; Dugani and Glavin, 1986; Holzer and Sametz, 1986; Evangelista et al., 1989b; Lippe, Pabst and Holzer, 1989; Raybould and Taché, 1989; Esplugues et al., 1990 Takeuchi et al., 1991a, 1992c; Leung, 1993b Uchida, Yano and Watanabe, 1993 Uchida, Yano and Watanabe, 1993 Szolcsányi and Barthó, 1981 Saperas, Santos and Malagelada, 1995 Alföldi et al., 1986; Evangelista et al., 1989b; Esplugues et al., 1990; Barrachina et al., 1992 Evangelista et al., 1989b

Stimulated acid output (gastric distension) Stimulated acid output (histamine)

No change Suppression Reduction No change

Stimulated acid output (insulin) Stimulated acid output (pentagastrin)

Stimulated acid output (peptone)

No change No change

Reduction Reduction

Matsumoto et al., 1992b Esplugues et al., 1990; Barrachina et al., 1992 Alföldi et al., 1986 Esplugues et al., 1990; Ramos, Esplugues and Esplugues, 1992; Takeuchi et al., 1992a, 1994 Esplugues et al., 1990 Alföldi et al., 1986; Esplugues et al., 1990; Livingston and Holzer, 1993; Saperas et al., 1995 Dugani and Glavin, 1986 Ramos, Esplugues and Esplugues, 1992

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INNERVATION OF THE GASTROINTESTINAL TRACT

TABLE 3.7 Continued Capsaicin Parameter administration

Perivagal

Effect of nerve defunctionalisation

References

Stimulated acid output (intracisternal TRH) Stimulated bicarbonate output (acid)

Reduction

Saperas et al., 1995

Inhibition

Takeuchi et al., 1991a, 1992c; Hamlet, Jonson and Fändriks, 1992; Inada and Satoh, 1996

Basal acid output

No change

Enterogastric reflex (acid) Enterogastric reflex (lipid) Stimulated acid output (bethanechol) Stimulated acid output (VNS)

No change

Raybould and Taché, 1989; Thiefin et al., 1990; Yoneda and Raybould, 1990; Sharkey et al., 1991; Lloyd et al., 1993; Saperas, Santos and Malagelada, 1995 Saperas, Santos and Malagelada, 1995

Reduction

Lloyd et al., 1993

No change

Raybould and Taché, 1989

No change

Thiefin et al., 1990

Reduction Reduction

Sharkey et al., 1991 Raybould and Taché, 1989

Reduction

Raybould and Taché, 1989

No change

Yoneda and Raybould, 1990

No change

Raybould and Taché, 1989

Reduction

Raybould et al., 1990; Saperas, Santos and Malagelada, 1995 Raybould et al., 1990

Stimulated acid output (gastric distension) Stimulated acid output (histamine) Stimulated acid output (H2Rd agonist) Stimulated acid output (pentagastrin) Stimulated acid output (intracisternal TRHe) Stimulated 5-HTf release (intracisternal TRH) Pericoeliacg

a

No change

Basal acid output

No change

Enterogastric reflex (acid) Enterogastric reflex (lipid) Stimulated acid output (gastric distension) Stimulated acid output (intracisternal TRH)

No change

Esplugues et al., 1990; Lloyd et al., 1993; Saperas, Santos and Malagelada, 1995 Saperas, Santos and Malagelada, 1995

No change

Lloyd et al., 1993

Suppression

Esplugues et al., 1990

No change

Saperas, Santos and Malagelada, 1995

Enterogastric reflex: inhibition of stimulated gastric acid output by intraduodenal acid; b ACh, acetylcholine; VNS, vagal nerve stimulation; d H2R, histamine H2 receptor; e TRH, thyrotropin-releasing hormone; f 5-HT, 5-hydroxytryptamine; g administration of capsaicin locally to the coeliac ganglion. c

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A synopsis of the available data indicates that afferent neurons play two fundamentally different roles in the regulation of gastric acid secretion (Figure 3.5; Table 3.7). The inhibitory effect of afferent nerve stimulation on gastric acid secretion is likely to reflect the local release and action of CGRP in the stomach. In contrast, the implications of afferent neurons in the secretory responses to gastric distension, peptone and secretagogues point to a vagal reflex whose afferent reflex arc is constituted by capsaicin-sensitive sensory neurons (Figure 3.5). This also applies to the enterogastric inhibition of acid secretion, which is elicited by intraduodenal acid challenge. It is not yet clear whether the local antisecretory and the prosecretory reflex functions of afferent neurons in the stomach are activated simultaneously or separately and whether they are governed by the same or different nerve fibres. The acid-evoked output of bicarbonate from the rat stomach and duodenum is suppressed in capsaicin-pretreated rats (Table 3.7) as is the lansoprazole-induced facilitation of acid-evoked bicarbonate secretion in the rat duodenum (Inada and Satoh, 1996). One study holds that basal pepsin output is decreased by intragastric capsaicin (Brzozowski et al., 1996b) but it is not known whether afferent neurons play a role in stimulated pepsinogen secretion.

MEDIATORS BY WHICH AFFERENT NEURONS CONTROL GASTRIC SECRETION CGRP is most likely the transmitter by which afferent neurons in the rat suppress gastric acid output (Figure 3.5), an inference that is in keeping with the peptide’s high activity in inhibiting gastric acid secretion (Holzer, 1994). The antisecretory action of CGRP in the rat and canine stomach is mediated by CGRP1 receptors (Evangelista, Tramontana and Maggi, 1991; Lawson, Mantyh and Pappas, 1994; Kato et al., 1995) and related to the peptide’s ability to increase the release of somatostatin, while the release of gastrin and ACh is attenuated (Inui et al., 1991; Ren et al., 1991; Holzer, 1994; Manela et al., 1995) as is the release of histamine from enterochromaffin-like cells (Sandor et al., 1996). Tachykinins acting via NK2 receptors do not take part in the antisecretory response to CGRP (Plate et al., 1996). It is increasingly becoming clear that acid-evoked release of CGRP from the gastric mucosa (Table 3.4) represents a negative feedback mechanism (Figure 3.5) that limits excess secretion of acid when the acidity in the gastric juice rises (Kato et al., 1995; Manela et al., 1995; Ren et al., 1995). Thus, accumulation of gastric acid in the stomach seems to excite afferent nerve fibres which release CGRP, because the CGRP1 antagonist CGRP8–37 or a monoclonal CGRP antibody elevates acid output stimulated by pylorus ligation or pentagastrin infusion (Kato et al., 1995). It has moreover been demonstrated that CGRP acting via CGRP1 receptors contributes to the acid-evoked release of somatostatin and to the acid-evoked inhibition of gastrin release from the rat antral mucosa (Manela et al., 1995). An involvement of tachykinins in afferent nerve-mediated changes of gastric secretion has not yet been tested although such an implication is conceivable, given that the intraluminal

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INNERVATION OF THE GASTROINTESTINAL TRACT

release of SP is related to the acidity of the gastric juice in humans (Mueller et al., 1991). Albeit tachykinins do not influence basal acid secretion in the rat and canine stomach, SP reduces the secretory response to vagal nerve stimulation, intravascular pentagastrin or intraluminal peptone (Konturek et al., 1981; Modlin, Lamers and Walsh, 1981; Martensson et al., 1984; Yokotani and Fujiwara, 1985; Schepp et al., 1990; Coruzzi, Adami and Bertaccini, 1991; Stroff et al., 1996). In contrast, pepsinogen secretion from the canine and guinea-pig stomach is stimulated by tachykinins, an effect that is brought about by NK1 receptors located directly on the chief cells (Vigna et al., 1989; Kitsukawa et al., 1996). The effects of tachykinins on gastric endocrine secretion depend on the species under study. Thus, the secretion of histamine from rat enterochromaffin-like cells (Sandor et al., 1996) and of gastrin from the canine and rat stomach remains unaltered by SP and NKA (Konturek et al., 1981; Modlin, Lamers and Walsh, 1981; Martensson et al., 1984; McIntosh et al., 1987), while gastrin release from the isolated porcine antrum is inhibited by SP and NKA through activation of NK1 and NK2 receptors (Schmidt et al., 1996). Somatostatin release from the isolated porcine antrum is stimulated by activation of NK1 and NK2 receptors (Schmidt et al., 1996), whereas somatostatin release from the rat isolated stomach (Figure 3.5) is attenuated by tachykinins acting via NK2 receptors on gastric D cells (Chiba et al., 1980; Kwok et al., 1985, 1988). An interaction with the somatostatin system is also indicated by the ability of SP to stimulate the expression of somatostatin messenger RNA in the gastric antrum of rats pretreated with a neurotoxic dose of capsaicin, but not of intact animals (Dimaline et al., 1994), although the functional significance of this finding is not yet understood. PACAP, another peptide that occurs in capsaicin-sensitive afferent neurons (Mulder et al., 1994b), is like CGRP able to suppress basal and pentagastrin/histamine-stimulated acid secretion in the rat stomach (Mungan et al., 1995). Whether the antisecretory effect of PACAP, which may be due to a direct action on parietal cells (Mungan et al., 1995), has any bearing on afferent nerve-mediated control of gastric acid output remains to be clarified.

PEPTIDERGIC AFFERENT NERVE FIBRES AND GASTRIC MOTOR ACTIVITY MOTOR EFFECTS AND MEDIATORS OF AFFERENT NERVE STIMULATION Sensory nerve stimulation with capsaicin has been found to exert both stimulant and inhibitory effects on the motor activity of the stomach (Table 3.2). While the longitudinal muscle of the rat isolated fundus is relaxed by capsaicin (Lefebvre, De Beurme and Sas, 1991), composite effects including contraction and relaxation have been observed in the circular muscle of the rat gastric corpus in vitro (Holzer-Petsche, Seitz and Lembeck, 1989). The motor activity of the rat gastric corpus in vivo is enhanced by low intragastric doses of capsaicin while high doses of the drug inhibit gastric motility (Table 3.2). This latter action may be related to the chilli- and capsaicin-induced

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129

inhibition of gastric emptying, which has been observed in rats and humans (Table 3.2). The lower oesophageal sphincter pressure in the dog is enhanced by local administration of capsaicin which seems to activate an intrinsic neural reflex involving ganglionic transmission and muscle excitation via muscarinic ACh receptors (Sandler et al., 1993). The relaxant response of the rat stomach to capsaicin depends on extrinsic afferent neurons but does not involve SP, CGRP or VIP (Table 3.3). Tachykinins, though, participate in the capsaicin-induced increase of gastric motor activity (Table 3.3), which is consistent with the overall excitatory influence of tachykinins on gastrointestinal motility (HolzerPetsche, 1995). SP and/or NKA also mediate the capsaicin-evoked increase in lower oesophageal sphincter pressure in the dog (Table 3.3) and the contraction of the feline stomach caused by extrinsic nerve stimulation (Delbro et al., 1983). The contractions which vagal or splanchnic nerve stimulation elicits in the canine and feline stomach are believed to result from antidromic activation of extrinsic afferent neurons which through peripheral transmitter release stimulate cholinergic enteric motor neurons (Delbro et al., 1983; Lidberg et al., 1983; Nakazato et al., 1987; Ito et al., 1993). Analysis of the capsaicininduced contraction in the guinea-pig small intestine suggests that afferent nerve fibres stimulate enteric neurons through unidentified transmitters and that ACh and tachykinins are the transmitters by which enteric motor neurons cause muscle contraction (Barthó et al., 1994; Holzer and Barthó, 1996). Another mechanism by which tachykinins released from afferent nerve fibres may control gastrointestinal motility relates to their ability to depolarise sympathetic ganglion cells in the prevertebral ganglia (Otsuka and Yoshioka, 1993). Spinal afferent nerve fibres containing SP, NKA and CGRP send axon collaterals into the ganglia to make synaptic contacts with postganglionic sympathetic neurons (Hayashi et al., 1982; Matthews and Cuello, 1984; Lee et al., 1987a,b; Su et al., 1987; Green and Dockray, 1988; Kondo and Yamamoto, 1988; Lindh, Hökfelt and Elfvin, 1988), and there is evidence that slow synaptic transmission in the coeliac and mesenteric ganglia of the guinea-pig is at least in part mediated by SP and NKA (Otsuka and Yoshioka, 1993). It is hence likely that primary afferent neurons from the gut, which send tachykinin-releasing axon collaterals into prevertebral sympathetic ganglia, participate in short-loop reflexes that control gastrointestinal effector systems via stimulation of sympathetic output. Such a short-loop reflex is thought to underlie the inhibition of gastric motor activity elicited by intrajejunal acid (Cervero and McRitchie, 1982).

PHYSIOLOGICAL IMPLICATIONS OF AFFERENT NEURONS IN GASTRIC MOTOR CONTROL Capsaicin-sensitive afferents do not seem to play a role in the regulation of basal motor activity in the stomach but may contribute to physiological changes of motility (Table 3.8). Basal gastric motility, gastric emptying and gastrointestinal transit are left unaltered after systemic pretreatment of rats with capsaicin or after selective defunctionalisation of vagal or spinal afferents (Table 3.8). Furthermore, capsaicin-sensitive afferents do not

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INNERVATION OF THE GASTROINTESTINAL TRACT

TABLE 3.8 Effect of capsaicin-induced defunctionalisation of afferent neurons on motor activity in the oesophagus, stomach and duodenum. Capsaicin Parameter administration

Effect of nerve References defunctionalisation

Systemic

Swallowing reflex Basal GEa and GMAb

Attenuation No change

Basal GITc

No change

Inhibition of GE by i.g.d acid, hyperosmolarity or peptone Inhibition of GMA by intrajejunal acid Inhibition of GMA by coeliac ganglion stimulation Inhibition of GIT by peritoneal irritation Inhibition of GMA by i.p.e acid, bradykinin or capsaicin Inhibition of GE, GMA and GIT by abdominal surgery

Attenuation

Jin et al., 1994 Cervero and McRitchie, 1982; Holzer, 1986; Forster et al., 1990; Holzer-Petsche, 1991; Takeuchi et al., 1991b; Barquist et al., 1992 Holzer, 1986; Holzer, Lippe and Holzer-Petsche, 1986 Forster et al., 1990

Attenuation

Cervero and McRitchie, 1982

No change

Cervero and McRitchie, 1982

Attenuation

Holzer, Lippe and Holzer-Petsche, 1986

Attenuation

Potentiation

Holzer, Lippe and Amann, 1992; Holzer-Petsche, 1992 Holzer, Lippe and Holzer-Petsche,1986; Holzer, Lippe and Amann, 1992; Barquist et al., 1992 Holzer-Petsche, 1991

No change

Matsumoto et al., 1992b

No change

Holzer-Petsche, 1991

Basal GE and GMA

No change

Inhibition of GE by atropine Inhibition of GE and GMA by cholecystokinin Inhibition of GE and GMA by secretin Inhibition of GE by i.d.f acid Inhibition of GMA by i.d. acid Inhibition of GE by i.d. lipid or maltose Inhibition of GMA by i.d. glucose or peptone Inhibition of GMA by i.d. soybean trypsin inhibitor Inhibition of GMA by duodenal distension Inhibition of GE by abdominal surgery Stimulation of GMA by vagal nerve stimulation

No change Attenuation

Attenuation

Raybould and Taché, 1988; Raybould and Hölzer, 1992; Barquist et al., 1996 Raybould and Taché, 1988 Raybould and Taché, 1988; Raybould and Hölzer, 1993b Raybould and Hölzer, 1993b; Lu and Owyang, 1995 Raybould and Hölzer, 1993a Raybould and Hölzer, 1993a Raybould and Hölzer, 1992; Hölzer et al., 1994 Raybould, 1991

Attenuation

Raybould, 1991

Attenuation

Hölzer and Raybould, 1992

No change

Plourde et al., 1993; Barquist et al., 1996 Raybould and Taché, 1988; Lu and Owyang, 1995

Stimulation of GMA by bethanechol Stimulation of GMA by 2-deoxy-D-glucose Stimulation of GMA by substance P and neurokinin A Perivagal

Attenuation

Attenuation No change Attenuation Attenuation

No change

CONTROL OF GASTRIC FUNCTIONS BY EXTRINSIC SENSORY NEURONS

Pericoeliacg

Stimulation of GMA by intracisternal TRH

No change

Raybould et al., 1990

Basal GE and GMA

No change

Inhibition of GE by cholecystokinin Inhibition of GE by secretin Inhibition of GE by i.d. acid Inhibition of GMA by i.d. acid Inhibition of GE by i.d. lipid Inhibition of GE by i.d. maltose Inhibition of GMA by coeliac ganglion stimulation Inhibition of GMA by duodenal distension Inhibition of GE by abdominal surgery

Attenuation

Esplugues et al., 1990; Raybould and Hölzer, 1992; Hölzer et al., 1994; Barquist et al., 1996 Raybould and Hölzer, 1993b

No change No change Attenuation No change Attenuation No change

Raybould and Hölzer, 1993b Raybould and Hölzer, 1993a Raybould and Hölzer, 1993a Hölzer et al., 1994 Raybould and Hölzer, 1992 Esplugues et al., 1990

Attenuation

Hölzer and Raybould, 1992

Attenuation

Plourde et al., 1993; Zittel et al., 1994a; Barquist et al., 1996

Intraduodenal Basal GE No change Inhibition of GE by i.d., Attenuation glucose or lipid Inhibition of GMA by secretin Attenuation

Zittel et al., 1994b Zittel et al., 1994b Lu and Owyang, 1995

a GE, gastric emptying; b GMA, gastric motor activity; c GIT, gastrointestinal transit; d i.g., intragastric; e i.p., intraperitoneal; f i.d., intraduodenal; g administration of capsaicin to the coeliac ganglion.

participate in the stimulation of gastric motility caused by electrical stimulation of the vagus nerves, systemic injection of 2-deoxy-D-glucose, systemic injection of SP/NKA, or intracisternal administration of a high dose of a stable TRH analogue (Table 3.8). In contrast, capsaicin-sensitive afferent neurons contribute to a number of inhibitory gastric motor reflexes including those evoked by intragastric acid, intraduodenal/intrajejunal acid and intraperitoneal administration of acid, capsaicin, bradykinin or CCK (Table 3.8). The inhibition of gastric motor activity and emptying elicited by intraduodenal administration of peptone, hyperosmolar saline, glucose, maltose or lipid or by duodenal distension depends likewise on capsaicin-sensitive afferent neurons (Table 3.8). Further analysis has revealed that the inhibitory motor effect of intraduodenal lipid is mediated by endogenous CCK acting via CCKA receptors on afferent neurons (Hölzer et al., 1994). The inhibition of gastric motility and emptying induced by intravenous secretin or intraduodenal lipid is in part mediated by vagal, but not spinal, afferents, whereas the reverse is true for the gastroparesis caused by abdominal surgery or irritation, which involves spinal, but not vagal, afferents (Table 3.8). Pharmacological studies indicate that the inhibition of gastric emptying, which is seen after intragastric administration of acid or hyperosmolar saline or after abdominal surgery relies on CGRP as a transmitter substance (Table 3.6). CGRP has, in addition, been found to contribute to other afferent nerve-mediated relaxant responses of gastrointestinal smooth muscle (Holzer and Barthó, 1996) but the sites at which CGRP participates in these reactions have not yet been elucidated.

131

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PATHOPHYSIOLOGICAL IMPLICATIONS OF PEPTIDERGIC AFFERENT NERVE FIBRES IN THE STOMACH ABLATION OF SENSORY NEURONS COMPROMISES THE RESISTANCE OF THE GASTRIC MUCOSA TO INJURY Even before the implications of capsaicin-sensitive afferents in the neural regulation of gastric blood flow, secretion and motor activity were elucidated, it had become obvious that these neurons play a role in gastric mucosal defence against injury. The pertinent studies were fostered by the knowledge that capsaicin-sensitive afferent neurons are especially sensitive to noxious chemicals of endogenous and exogenous origin (Holzer, 1991; Wood, 1993) and can give rise to inflammatory processes which are designed to facilitate the recovery from tissue injury. The first evidence that sensory neurons strengthen the resistance of the gastric mucosa to damage was obtained by studying the effects of capsaicin-induced ablation of afferent neurons on experimentally imposed injury of the rat stomach (Table 3.9). Defunctionalisation of sensory neurons does not change the gastric mucosal potential difference (Matsumoto, Takeuchi and Okabe, 1991; Takeuchi et al., 1992c), does not cause damage by itself and fails to alter the gastric injury induced by cold and restraint stress (Table 3.9). Capsaicin-induced ablation of afferent neurons, however, leads to exacerbation of mucosal lesion formation in response to water immersion restraint stress, pylorus ligation, and a variety of injurious factors including hydrochloric acid, acetic acid, ammonia, acetylsalicylic acid (aspirin), indomethacin, platelet-activating factor, a thromboxane mimetic, endothelin-1, cysteamine, 2-deoxy-D-glucose, ethanol and taurocholate (Table 3.9). The involvement of neurons in gastric mucosal protection is further supported by the ability of the nerve conduction blocker tetrodotoxin (Esplugues, Whittle and Moncada, 1989) to enhance experimentally imposed damage to the gastric mucosa. Histological examination of the mucosa has revealed that not only the extent of macroscopically visible lesions but also the depth of damage is augmented by sensory nerve ablation (Esplugues, Whittle and Moncada, 1989, 1992; Piqué, Esplugues and Whittle, 1990; Whittle, Lopez-Belmonte and Moncada, 1990; Holzer, Livingston and Guth, 1991; Whittle and Lopez-Belmonte, 1991; Li et al., 1992; Pabst, Schöninkle and Holzer, 1993; Uchida, Yano and Watanabe, 1993). In addition to aggravating experimentally imposed damage, ablation of sensory neurons compromises the ability of certain factors to protect the gastric mucosa from injury (Table 3.10). The gastric mucosal adaptation to water immersion restraint stress is prevented by capsaicin pretreatment (Brzozowski et al., 1995) while “adaptive cytoprotection” caused by mild gastric irritants such as dilute ethanol, HCl or NaOH is either attenuated (Ko and Cho, 1995) or left unchanged (Evangelista, Maggi and Meli, 1988; Miller et al., 1989; Hatakeyama et al., 1996) by sensory nerve ablation. In contrast, the gastroprotection afforded by the antiulcer drug ecabet, the antacid hydrotalcit, the proton pump inhibitor lansoprazole, prostaglandin E2, NKA-related peptides, epidermal growth factor, corticotropin-releasing factor, intracisternal TRH, γ-aminobutyric acid, low dose endotoxin and laparotomy is reduced or abolished in capsaicin-pretreated rats (Table 3.10). Gastric mucosal protection induced by peptone, bombesin, CCK octapeptide and gastrin-17 is likewise abandoned by pretreatment of rats with capsaicin, although it need be added that

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TABLE 3.9 Effect of capsaicin-induced defunctionalisation of afferent neurons on experimental lesion formation in the rat gastroduodenal mucosa. Region

Injurious factor

Effect of nerve defunctionalisation

References

Stomach

None

Failure to cause injury

Acetic acid Acetylsalicylic acid

Aggravation of injury Delay of healing Aggravation of injury

Ammonia Cold restraint stress Cysteamine 2-Deoxy-D-glucose

Aggravation of injury Injury unchanged Aggravation of injury Aggravation of injury

Endothelin-1 Ethanola

Aggravation of injury Aggravation of injury

Holzer and Sametz, 1986; Holzer and Lippe, 1988; Esplugues, Whittle and Moncada, 1989; Piqué, Esplugues and Whittle, 1990; Whittle, Lopez-Belmonte and Moncada, 1990; Uchida, Yano and Watanabe, 1993; Brzozowski et al., 1995; Grønbech and Lacy, 1996 Tramontana et al., 1994a Tramontana et al., 1994a Evangelista et al., 1988; Uchida, Yano and Watanabe, 1991a; Abdel Salam, Mózsik and Szolcsányi, 1995; Brzozowski et al., 1996b Uchida, Yano and Watanabe, 1991a Dugani and Glavin, 1986; Holzer et al., 1995 Holzer and Sametz, 1986 Uchida, Yano and Watanabe, 1991a; Matsumoto et al., 1992b Whittle and Lopez-Belmonte, 1991 Holzer and Sametz, 1986; Evangelista et al., 1988; Esplugues and Whittle, 1990; Szolcsányi, 1990; Yonei, Holzer and Guth, 1990; Evangelista and Maggi, 1991; Esplugues, Whittle and Moncada, 1992; Peskar, Nowak and Lambrecht, 1992; Pabst, Schöninkle and Holzer, 1993; Uchida, Yano and Watanabe, 1993; Kato, Yang and Taché, 1994; Abdel Salam, Mózsik and Szolcsányi, 1995; Karmeli et al., 1995

Hydrochloric acid

Restitution unchanged Delay of healing Aggravation of injury

Indomethacin

Injury unchanged Delay of healing Aggravation of injury Delay of restitution Aggravation of injury

Indomethacin plus acid

Aggravation of injury

Hypertonic saline/acid

Pabst, Schöninkle and Holzer, 1993 Peskar et al., 1995 Szolcsányi and Barthó, 1981; Holzer, Livingston and Guth, 1991; Matsumoto et al., 1992b; Raybould et al., 1992 Takeuchi et al., 1994 Takeuchi et al., 1994 Grønbech and Lacy, 1996 Grønbech and Lacy, 1996 Evangelista, Maggi and Meli, 1986; Holzer and Sametz, 1986; Whittle, Lopez-Belmonte and Moncada, 1990 Gray et al., 1994

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TABLE 3.9 Continued Region

Injurious factor

Effect of nerve defunctionalisation

References

NG-monomethylL-arginine Platelet-activating factor

Induction of injury

Pylorus ligation Taurocholate plus acid

Aggravation of injury Aggravation of injury Restitutionb delayed Aggravation of injury

Whittle, Lopez-Belmonte and Moncada, 1990 Esplugues, Whittle and Moncada, 1989; Piqué, Esplugues and Whittle, 1990 Szolcsányi and Barthó, 1981 Takeuchi et al., 1993 Takeuchi et al., 1993 Brzozowski et al., 1995

Aggravation of injury Aggravation of injury Aggravation of injury

Maggi et al., 1987a Takeuchi et al., 1992a Leung, 1993b

Water immersion restraint stress Duodenum

Cysteamine Histamine Hydrochloric acid

Aggravation of injury

a

Aggravation of ethanol-induced damage depends on the concentration of ethanol and the region of the gastric mucosa – with 100% ethanol, damage in the rat gastric corpus may be so severe that sensory nerve defunctionalisation does not further exacerbate damage while aggravation of damage in the antrum region of the stomach is observed (Uchida, Yano and Watanabe, 1993; Peskar et al., 1995); b restitution measured by recovery of transmucosal potential difference.

only low doses of pentagastrin and CCK may in their gastroprotective effect depend on capsaicin-sensitive afferents (Table 3.10). The ability of CGRP, prostaglandin I2, atropine, cimetidine, terbutaline, sodium salicylate and SH-donating or SH-blocking compounds to prevent experimental damage in the rat stomach is left unaltered by defunctionalisation of capsaicin-sensitive afferent neurons (Table 3.10). There is evidence for additional relationships between afferent neurons and some systems that have a bearing on gastric mucosal integrity. Capsaicin-sensitive afferent neurons appear to synergise with sympathetic nerve fibres and substances released from the adrenal glands, since chronic adrenalectomy (Evangelista, Maggi and Meli, 1986; Evangelista et al., 1988) and chemical sympathectomy with guanethidine (Holzer and Sametz, 1986), which aggravate experimental damage on their own, prevent the ability of sensory nerve ablation to worsen experimental injury to the gastric mucosa. Mast cell mediators and free radicals may also play a role, because the capsaicin-evoked exacerbation of gastric damage is suppressed by ketotifen and tempol (Karmeli et al., 1995). Other factors that interact with afferent nerve-mediated gastroprotection include opioid peptides and opiates. The ability of morphine to exacerbate gastric mucosal damage is halted by sensory nerve ablation which aggravates experimental damage by itself (Esplugues, Whittle and Moncada, 1989, 1992; Esplugues and Whittle, 1990). These observations suggest that morphine weakens the resistance of the gastric mucosa to injury by inhibiting the gastroprotective role of capsaicin-sensitive afferent nerve fibres, a conclusion that is consistent with the ability of opioids to inhibit transmitter release from afferent neurons (Maggi, 1991; Holzer, 1992). A further important interaction takes place between capsaicin-

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TABLE 3.10 Effect of capsaicin-induced defunctionalisation of afferent neurons on mucosal protection in the rat stomach. Protective agent

Effect of nerve ablation on gastroprotection

References

Atropine

No change

Bombesin Calcitonin gene-related peptide Capsaicin

Inhibition No change

Szolcsányi and Mózsik, 1984; Holzer and Sametz, 1986 Stroff, Plate and Peskar, 1995a Gray et al., 1994

Cholecystokinin Cholecystokinin octapeptide Cimetidine

No change Inhibition No change

Corticotropin-releasing factor Ecabet E. coli endotoxin Epidermal growth factor

Inhibition Inhibition Inhibition Inhibition

γ-Aminobutyric acid Gastrin-17

Inhibition Inhibition No change Inhibition Inhibition Inhibition Inhibition No change Inhibition Inhibition No change

Hydrotalcit (antacid) Lansoprazole Laparotomy Neurokinin A4–10 Pentagastrin Peptone Prostaglandin E2 Prostaglandin I2 SH-donating/-blocking compounds Sodium salicylate Terbutaline Intracisternal thyrotropin-releasing hormone analogue

Abolition

No change

Holzer and Lippe, 1988; Holzer et al., 1991; Takeuchi et al., 1991b; Uchida, Yano and Watanabe, 1991b; Gray et al., 1994 Konturek et al., 1995b; Mercer et al., 1996 Evangelista and Maggi, 1991; Stroff et al., 1993 Szolcsányi and Mózsik, 1984; Holzer and Sametz, 1986 Stroff, Plate and Peskar, 1995b Kinoshita, Kume and Tamaki, 1995 Barrachina et al., 1995; Brzozowski et al., 1996a Stroff and Peskar, 1995a; Konturek, Brzozowski and Sliwowski, 1996; Teng et al., 1996a,b Ren et al., 1996 Stroff et al., 1995 Konturek et al., 1995a Lambrecht et al., 1993c Murakami et al., 1996 Yonei, Holzer and Guth, 1990 Stroff et al., 1996 Konturek et al., 1995b Stroff and Peskar, 1996 Esplugues, Whittle and Moncada, 1992 Szolcsányi and Mózsik, 1984; Abdel Salam, Mózsik and Szolcsányi, 1995 Stroff and Peskar, 1995b

No change No change Inhibition

Stroff and Peskar, 1995b Holzer and Sametz, 1986 Kato, Yang and Taché, 1994

sensitive afferent nerve fibres and the NO system. NO synthase inhibitors, which do not cause damage in normal rats, give rise to extensive acid injury in capsaicin-pretreated rats (Whittle, Lopez-Belmonte and Moncada, 1990). This finding indicates that NO acts in concert with afferent neurons in maintaining the resistance of the gastric mucosa to injurious factors.

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STIMULATION OF SENSORY NEURONS STRENGTHENS THE RESISTANCE OF THE RAT AND CANINE GASTRIC MUCOSA TO INJURY Szolcsányi and Barthó (1981) were the first to propose a protective function of capsaicinsensitive sensory neurons in the gastric mucosa, when they observed that intragastric administration of small quantities of capsaicin, to stimulate sensory nerve fibres, protected from injury induced by acid accumulation in the stomach. In subsequent studies it was found that acute intragastric administration of capsaicin prevented experimental injury in the rat stomach induced by pylorus ligation, water immersion restraint stress, haemorrhagic shock or injurious factors such as hydrochloric acid, aspirin, indomethacin, ethanol and acidified taurocholate (Table 3.11). The gastric damage caused by hydrochloric acid or taurocholate in the canine stomach is also suppressed by sensory nerve stimulation with capsaicin (Table 3.11). Numerous studies have shown that the protective effect of capsaicin results in a reduction of the depth of injury while surface damage is not prevented (Holzer, Pabst and Lippe, 1989; Holzer et al., 1990a, 1991; Lambrecht et al., 1993a; Kang et al., 1995a). In contradiction of traditional views, intragastric capsaicin does not irritate the gastric mucosa and does not in any way weaken the gastric mucosal barrier as has been assumed for some red pepper spices which contain many substances other than capsaicin. Capsaicin fails to enhance backdiffusion of gastric acid (Table 3.2), does not appreciably enhance vascular permeability in the gastric wall (Table 3.2), leaves the transmucosal potential difference unchanged (Table 3.2) and fails to cause any macroscopic or histological damage on its own (Table 3.11). It can be ruled out, therefore, that capsaicin strengthens mucosal resistance to injury by virtue of an irritant action on the mucosa. The gastroprotective effect of capsaicin is shared by chilli (Table 3.11). Acute or chronic administration of chilli powder (200 mg as single dose or 200 mg daily for 4 weeks) protects the rat gastric mucosa from ethanol injury (Kang et al., 1995a). Resiniferatoxin is likewise able to reduce the mucosal damage caused by ethanol (Szolcsányi, 1990; Tramontana et al., 1994b; Abdel Salam et al., 1995b), acidified aspirin (Abdel Salam et al., 1995b) and acidified salicylate (Abdel Salam et al., 1995a) in pylorusligated rats.

STIMULATION OF SENSORY NEURONS STRENGTHENS THE RESISTANCE OF THE HUMAN GASTRIC MUCOSA TO INJURY Importantly, the protective effects of capsaicin and chilli powder in the rat stomach are reproduced in the gastric mucosa of humans (Table 3.11). Thus, there is epidemiological evidence that dietary chilli ingestion has a protective action against peptic ulcer disease (Kang et al., 1995b). Careful examination of the multiracial population in Singapore has revealed that the Chinese who eat less chilli than the Malays and Indians have a higher frequency of peptic ulcer disease and that, within the Chinese population, the frequency of ulcer disease correlates inversely with the amount of chilli intake (Kang et al., 1995b). The gastroprotective effect of chilli was directly proven by the ability of 20 g chilli powder to reduce damage of the human gastroduodenal mucosa exposed to 600 mg aspirin

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TABLE 3.11 Effect of afferent nerve stimulation by capsaicin, resiniferatoxin or chilli on experimental lesion formation in the mucosa of the oesophagus, stomach and duodenum. Species/region

Injurious factor

Effect of nerve stimulation

References

Rabbit oesophagus

Ethanol

Protection

Rat stomach

Acidified acetylsalicylic acid

Protection

Acidified indomethacin Acidified salicylate Acidified taurocholate

Protection

Holzer and Lippe, 1988; Holzer, Pabst and Lippe, 1989; Holzer et al., 1990a, 1991; Lippe, Pabst and Holzer, 1989; Takeuchi et al., 1992b Holzer, Pabst and Lippe, 1989; Abdel Salam et al., 1995b; Hatakeyama et al., 1995 Gray et al., 1994

Protection Protection

Ethanol

Protection

Haemorrhagic shock Hydrochloric acid

Protection Protection

Abdel Salam et al., 1995a Mercer, Ritchie and Dempsey, 1994; Merchant et al., 1994, 1995; Podolsky et al., 1994 Holzer and Lippe, 1988; Holzer et al., 1990a, 1991; Szolcsányi, 1990; Peskar et al., 1991, 1993; Peskar, Nowak and Lambrecht, 1992; Takeuchi et al., 1991b; Uchida, Yano and Watanabe, 1991b; Brzozowski et al., 1993; Lambrecht et al., 1993a; Tramontana et al., 1994b; Abdel Salam et al., 1995b; Kang et al., 1995a; Stroff et al., 1996 Teng et al., 1996b

Pylorus ligation Water immersion restraint stress

Protection Protection

Takeuchi et al., 1994; Abdel Salam, Mózsik and Szolcsányi, 1995; Hatakeyama et al., 1996 Szolcsányi and Barthó, 1981 Brzozowski et al., 1996b

Canine stomach

None Acid Acidified taurocholate

Failure to cause injury Protection Protection

Sullivan et al., 1992 Sullivan et al., 1992 Sullivan et al., 1992

Human stomach

None Acetylsalicylic acid

Failure to cause injury Protection

Graham et al., 1988; Yeoh et al., 1995 Yeoh et al., 1995

Rat duodenum

Hydrochloric acid

Protection

Leung, 1993b

(Table 3.11) and examined by endoscopy and biopsy (Yeoh et al., 1995). Ingestion of chilli alone fails to cause any macroscopic and microscopic injury of the human gastric mucosa (Yeoh et al., 1995), which is also true for ingestion of jalapeno-seasoned food (Graham et al., 1988).

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PATHWAYS OF SENSORY NERVE-MEDIATED GASTRIC MUCOSAL PROTECTION The protective effect of intragastric capsaicin involves afferent nerve fibres because it is prevented by defunctionalisation of extrinsic sensory neurons (Table 3.3). Another indication that neurons are involved is the observation that capsaicin’s ability to reduce ethanol injury is suppressed by nerve conduction blockade with tetrodotoxin (Holzer et al., 1991) or lidocaine (Mercer, Ritchie and Dempsey, 1994). However, the pathways underlying sensory nerve-mediated gastric mucosal protection in the stomach have not yet been fully delineated. Atropine, acute (Abdel Salam et al., 1995a) and chronic (Miller et al., 1989) vagotomy have been reported to prevent capsaicin and resiniferatoxin from protecting the gastric mucosa from experimental injury. Another study, though, has shown that acute vagotomy, acute extirpation of the coeliac ganglion and acute ligation of the blood vessels to the adrenal glands are devoid of any influence on capsaicin’s gastroprotective effect (Holzer and Lippe, 1988). The adaptation to water immersion stress-induced gastric mucosal injury and vasoconstriction is likewise mediated by capsaicin-sensitive afferent neurons of nonvagal origin (Brzozowski et al., 1995). These discrepant data might be accommodated in the conjecture that the ability of capsaicin and resiniferatoxin to strengthen gastric mucosal resistance to injury depends on the local release of transmitters such as CGRP from sensory nerve fibres within the gastric wall, a process in which vagal efferent pathways play a permissive role. Evidence for such an interaction comes from studies with low doses of a stable TRH analogue injected into the cisterna magna (Kato, Yang and Taché, 1994, 1996) or dorsal nucleus of the vagus (Kato, Yang and Taché, 1995b). The gastroprotective action of centrally administered TRH involves activation of parasympathetic efferent neurons in the vagus nerves, ACh, CGRP, NO and prostaglandins (Kato, Yang and Taché, 1994, 1995b, 1996). It is not yet clear, though, whether ACh, CGRP, NO and prostaglandins interact with each other in parallel or in series, and in which order, but the possibility has to be considered that ACh increases the formation of NO, which in turn stimulates CGRP-releasing sensory nerve fibres via a prostaglandin link, as has been shown to occur in the rat skin (Holzer, Jocic and Peskar, 1995). The protective effect of intragastric peptone depends likewise on capsaicinsensitive afferent and cholinergic efferent fibres in the vagus nerve (Stroff and Peskar, 1996). Whether enteric neurons, which can be stimulated by transmitters released from extrinsic afferent nerve fibres (Barthó and Holzer, 1985; Holzer and Barthó, 1996), are implicated in sensory nerve-mediated gastroprotection awaits further investigation. MEDIATORS OF SENSORY NERVE-MEDIATED GASTRIC MUCOSAL PROTECTION CGRP There is compelling evidence that afferent nerve-mediated gastroprotection is mediated by nonadrenergic noncholinergic neurotransmitters (Holzer and Lippe, 1988) among which CGRP plays a central role (Figure 3.4; Tables 3.3 and 3.6). Close arterial administration of this peptide to the rat stomach prevents gastric damage induced by ethanol, acidified aspirin or endothelin-1 (Lippe, Lorbach and Holzer, 1989; Whittle and Lopez-Belmonte,

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139

1991; Evangelista, Tramontana and Maggi, 1991; Lambrecht et al., 1993a; LopezBelmonte and Whittle, 1993). Intravenous (Clementi et al., 1993, 1994; Kato, Yang and Taché, 1994), subcutaneous (Maggi et al., 1987b) and intracisternal (Taché, 1992) administration of the peptide has also been reported to protect against gastric injury. The effect of CGRP to reduce ethanol-induced damage is mediated by CGRP1 receptors as it is prevented by the CGRP1 receptor antagonist CGRP8–37 (Lambrecht et al., 1993a; Kato, Yang and Taché, 1994), whereas the ability of CGRP to inhibit aspirin-induced injury is not antagonised by CGRP8–37 (Evangelista, Tramontana and Maggi, 1991). Conclusive pharmacological evidence for an implication of CGRP in afferent nervemediated gastric mucosal protection comes from the findings that the protective effects of intragastric capsaicin are blocked by the CGRP receptor antagonist CGRP8–37 (Lambrecht et al., 1993a; Kato, Yang and Taché, 1994; Merchant et al., 1994, 1995) and by immunoneutralisation of CGRP with polyclonal (Lambrecht et al., 1993a) and monoclonal (Peskar et al., 1993) antibodies to the peptide (Table 3.3). This important role of CGRP in gastric mucosal defence is corroborated by the observation that active immunisation of rats against CGRP exacerbates ethanol-induced injury in the stomach (Forster and Dockray, 1991a). CGRP has in addition been found to mediate the gastroprotective effects of NKArelated peptides, CCK octapeptide, gastrin-17, bombesin, corticotropin-releasing factor, epidermal growth factor and peptone (Table 3.6). The gastroprotective action of capsaicin is suppressed by NO synthase inhibitors (Table 3.3). A role of NO in the gastroprotection afforded by other factors that involve afferent nerve stimulation and CGRP release, such as CCK octapeptide, pentagastrin, gastrin-17, bombesin, corticotropin-releasing factor, epidermal growth factor, the antacid hydrotalcit, the proton pump inhibitor lansoprazole, and low dose endotoxin, has also been demonstrated (Table 3.6). Since the ability of CGRP to strengthen gastric mucosal defence against injurious factors is likewise abrogated by NO synthase inhibitors (Lambrecht et al., 1993a; Clementi et al., 1994) it would appear that stimulation of sensory nerve fibres by capsaicin results in the release of CGRP which, via formation of NO, augments the resistance of the gastric mucosa against experimental injury (Figure 3.4). Such a role of NO is consistent with its ability to prevent gastric mucosal injury and with the effect of NO synthase inhibitors to enhance gastric mucosal vulnerability (Whittle, 1993). Tachykinins The role of SP and NKA in afferent nerve-mediated gastroprotection has long escaped recognition, because SP is devoid of a protective action (Evangelista, Maggi and Meli, 1987b; Evangelista et al., 1989a) and has in fact been found to exaggerate gastric mucosal damage caused by acidified taurocholate or ethanol (Soper and Tepperman, 1986; Karmeli et al., 1991). The deleterious effect of SP involves activation of NK1 and NK3 receptors, mast cell degranulation and subsequent release of histamine, platelet-activating factor, leukotriene B4 and leukotriene C4 (Karmeli et al., 1991, 1993). The discharge of peptidedegrading proteases from mast cells is thought to explain why in the rat gastric mucosa exogenous SP inhibits the CGRP-mediated hyperaemic response to capsaicin (Grønbech and Lacy, 1994). Endogenous tachykinins share this adverse action of exogenous SP, since tachykinin antagonists reduce ethanol-induced injury (Karmeli et al., 1991, 1993;

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Cho, Ko and Tang, 1994; Hayashi et al., 1996). That tachykinins contribute to ethanol damage is, in addition, suggested by the ability of ethanol to release SP into the gastric lumen and by the action of SP to constrict mucosal and submucosal venules (Karmeli et al., 1993; Katori, Ohno and Nishiyama, 1993; Hayashi et al., 1996). While NK1 receptor activation impairs mucosal homeostasis, NK2 receptor stimulation is beneficial for the gastric mucosa, at least under certain experimental conditions. Thus, NKA and related NK2 receptor agonists inhibit ethanol-induced injury to the rat gastric mucosa in spite of a decrease in gastric mucosal blood flow (Evangelista et al., 1989a, 1990; Heinemann et al., 1996a; Stroff et al., 1996). The protective effect of NK2 agonists depends on stimulation of afferent nerve fibres (Table 3.10), release of CGRP and formation of NO (Figure 3.4; Table 3.6). Since the NK2 receptor antagonist MEN-10,627 attenuates the protective action of intragastric capsaicin, it follows that endogenous tachykinins acting via NK2 receptors participate in the sensory nerve-mediated maintenance of gastric mucosal integrity (Stroff et al., 1996). The effect of NKA on gastric mucosal integrity, though, depends on the experimental model under study. While ethanol injury is reduced, damage caused by acid backdiffusion through a disrupted mucosal barrier is exacerbated by NKA, because this tachykinin suppresses the gastric hyperaemia that is evoked by acid backdiffusion and that is important to counteract the deleterious consequences of acid influx into the mucosa (Heinemann et al., 1996a). This antivasodilator action of NKA is mediated by NK2 receptors and thus is independent of the tachykinin’s vasoconstrictor action which in vivo is due to NK1 receptor activation (Heinemann et al., 1996a). Taken together, these data show that both CGRP acting via CGRP1 receptors and tachykinins acting via NK2 receptors are transmitters of afferent nerve-mediated gastroprotection, both transmitters using NO as second messenger (Figure 3.4; Tables 3.3 and 3.6). In addition, there are complex interactions between CGRP and tachykinins because, on the one hand, the gastroprotective effect of NK2 receptor agonists depends on the integrity of the afferent innervation of the stomach and involves release of CGRP (Stroff et al., 1996) and, on the other hand, mucosal protection due to CGRP is blunted by a NK2 receptor antagonist (Plate et al., 1996). Prostaglandins As the data are controversial, there is no conclusive evidence that eicosanoids play a messenger role in sensory nerve-mediated gastric mucosal protection. Intragastric capsaicin does not affect the ex vivo formation of prostaglandin E2, 6-oxo-prostaglandin F1α and leukotriene C4, and indomethacin fails to alter the gastroprotective effect of capsaicin (Holzer et al., 1990a). The ability of CGRP to enhance the resistance of the gastric mucosa to injury remains likewise unaltered by indomethacin (Lambrecht et al., 1993a). Some investigators, though, have found that the gastroprotective action of capsaicin is reduced by indomethacin (Table 3.3), but it has not been ascertained in these studies whether the effect of indomethacin is indeed due to inhibition of prostaglandin synthesis. It is worth considering in this context that prostaglandins may contribute to the activation of afferent nerve fibres under appropriate conditions (Dray and Perkins, 1993), as prostacyclin has been found to release CGRP and SP from rat sensory neurons (Hingtgen and Vasko, 1994).

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MECHANISMS OF SENSORY NERVE-MEDIATED GASTRIC MUCOSAL PROTECTION Gastric mucosal hyperaemia From the available information it is obvious that CGRP and NO play an important mediator role both in the protective and in the hyperaemic response to capsaicin-evoked stimulation of afferent nerve fibres in the stomach (Figure 3.4). The identity of the messengers involved in the two processes points to a close relationship between the increase in GMBF and the increase in mucosal resistance to injury. The rise of GMBF is indeed also seen when capsaicin is administered together with an injurious concentration of ethanol (Holzer et al., 1991). Capsaicin prevents the ethanol-induced constriction of submucosal arterioles (Ohno et al., 1994), and there is a remarkable correlation between the hyperaemic response to capsaicin and the concomitant reduction of gross damage and deep haemorrhagic erosions (Holzer et al., 1991; Brzozowski et al., 1993). However, this parallelism does not prove that hyperaemia is the primary mechanism by which afferent nerve stimulation strengthens gastric mucosal defence against injury. Evidence is in fact accumulating that protective mechanisms other than vasodilatation are operated by afferent nerve fibres. Hyperaemia, nevertheless, can support a wide range of endogenous mechanisms of gastroprotection as seen with many vasodilator drugs (Guth, Leung and Kauffman, 1989; Holzer, Livingston and Guth, 1994). This interrelationship applies particularly to the gastric output of bicarbonate which is stimulated by sensory nerve stimulation (Takeuchi et al., 1992b,c) and which depends on an adequate blood flow to deliver bicarbonate to the surface mucus layer (Allen et al., 1993; Guttu et al., 1994). Other mechanisms of gastroprotection may be facilitated by afferent nerve-mediated hyperaemia or take place independently of blood flow alterations (Figure 3.6). Hyperaemia-independent mechanisms Several lines of evidence indicate that mechanisms other than a rise of GMBF play a significant part in the gastroprotective action of sensory nerve stimulation (Figures 3.4 and 3.6).

Figure 3.6 Summary of the mediators and presumed mechanisms by which afferent nerve stimulation enhances the resistance of the stomach to injury. CGRP1-R, CGRP1 receptor; NK2-R, tachykinin NK2 receptor.

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The most conclusive hint has come from studies with the NK2 receptor antagonist MEN10,627 which attenuates the gastroprotective action of capsaicin but fails to inhibit the concomitant rise of GMBF (Stroff et al., 1996). This observation is consistent with the ability of NKA-related NK2 receptor agonists to enhance gastric mucosal resistance to experimental injury despite a marked reduction of GMBF (Stroff et al., 1996) and implies that the component of afferent nerve-mediated gastroprotection that involves endogenous tachykinins acting via NK2 receptors is independent of gastric mucosal hyperaemia. There are a number of other findings that indicate a dissociation of the gastroprotective and vasodilator activity of sensory nerve stimulation. For instance, the hyperaemic response to capsaicin in the mucosa of the canine gastric corpus is blocked by lidocaine whereas the protective effect of capsaicin is left unaltered by the local anaesthetic (Sullivan et al., 1992). In a rat gastric chamber model, capsaicin causes a prolonged increase in the resistance of the mucosa to injury that outlasts the increase in GMBF (Morris et al., 1993). Conversely, ablation of capsaicin-sensitive afferent neurons exacerbates the mucosal damage, that is caused by exposure of the rat stomach to hypertonic saline, but fails to abrogate the concurrent gastric vasodilatation (Grønbech and Lacy, 1996). Finally, CGRP counteracts the injurious influence of endothelin-1 and, at the same time, inhibits the hyperaemic response to endothelin-1 in the rat gastric mucosa (Lopez-Belmonte and Whittle, 1993). This observation suggests that CGRP enhances gastric mucosal resistance by protecting the vascular endothelium fom injury (Figure 3.6) rather than by causing vasodilatation (Lopez-Belmonte and Whittle, 1993). The nature of the hyperaemia-independent gastroprotective mechanisms which are operated by capsaicin-sensitive afferent neurons has not yet been identified with certainty although a number of possibilities have been envisaged (Figure 3.6). Inhibition of gastric emptying (Table 3.2) together with a possible increase in fluid secretion may result in dilution of the injurious factors in the gastric juice (Figure 3.6) and thus afford protection of the gastric mucosa (Hatakeyama et al., 1995). Whether the ability of sensory nerve stimulation to modulate gastric acid secretion (Tables 3.2 and 3.3) has any bearing on gastroprotection is not known. For the time being it seems more likely that the capsaicin-induced attenuation of acid output (Abdel Salam, Szolcsányi and Mózsik, 1994, 1995; Brzozowski et al., 1996b) reflects increased bicarbonate secretion (Takeuchi et al., 1992b,c) and acid elimination (Lippe, Pabst and Holzer, 1989) rather than reduced acid secretion. The resiniferatoxin-evoked decrease in acid output in pylorusligated rats (Abdel Salam et al., 1995a,b) may also reflect an increase in bicarbonate secretion rather than an antisecretory effect, given that in other studies capsaicin has failed to attenuate gastric acid secretion (Lippe, Pabst and Holzer, 1989; Matsumoto, Takeuchi and Okabe, 1991; Matsumoto et al., 1992a; Sullivan et al., 1992). Another protective mechanism is reflected by the ability of capsaicin to stimulate the secretion of gastric mucus (Kang et al., 1995a). This activity of capsaicin is mimicked by NO (Brown et al., 1993), whereas the actions of CGRP and tachykinins on gastric mucus production have not yet been studied. Finally, afferent nerve-derived peptide transmitters could modulate gastric mucosal integrity through their influence on the adherence, emigration, proliferation and activity of leucocytes (Figure 3.6) (Stead, Bienenstock and Stanisz, 1987; Sharkey, 1992), a possibility that has not yet been explored experimentally.

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INFLUENCE OF SENSORY NEURONS ON RESTITUTION AND HEALING OF GASTRIC LESIONS There is mounting evidence that not only acute defence against pending injury but also repair of the injured gastric mucosa is facilitated by the sensory innervation of the stomach (Table 3.9). If the damage involves the superficial epithelium only, the mucosa is quickly repaired by migration of mucous cells over the denuded areas of the lamina propria (Allen et al., 1993). This process of rapid restitution is independent of afferent neurons, because restitution of the ethanol-injured gastric mucosa in the absence of luminal acid remains unchanged in rats treated with a neurotoxic dose of capsaicin (Pabst, Schöninkle and Holzer, 1993). Restitution, however, is promoted by the acid-induced hyperaemia that depends on afferent neurons, as has been observed in the rat stomach exposed to hypertonic saline plus acid (Grønbech and Lacy, 1996). The proper healing of gastric lesions is consistently facilitated by capsaicin-sensitive neurons, as the rate of repair of gastric ulcers induced by hydrochloric acid, acetic acid and ethanol is delayed after sensory nerve ablation (Table 3.9). It seems as if the afferent nerve-promoted acceleration of healing also depends on the sustained hyperaemia that afferent neurons trigger in response to luminal acid (Takeuchi et al., 1994). The retarded healing of gastric lesions seen in capsaicin-pretreated rats is not due to changes in prostaglandin formation, NO generation (Peskar et al., 1995) or gastric acid secretion (Takeuchi et al., 1994) but is associated with increased invasion of polymorphonuclear leucocytes into the gastric wall (Peskar et al., 1995). Particularly worth noting is the observation that capsaicin-pretreated, but not control, rats develop antral ulcers after gastric challenge with ethanol or excess hydrochloric acid (Uchida, Yano and Watanabe, 1993; Takeuchi et al., 1994; Peskar et al., 1995). The formation of antral ulcers has been discussed to arise from a decrease in GMBF and mucus secretion, which following afferent nerve ablation occurs in the gastric antrum but not corpus (Uchida, Yano and Watanabe, 1993). In keeping with a role of afferent neurons in ulcer healing is the observation that sensory nerve stimulation with low dose capsaicin promotes the healing of acetic acid-induced ulcers in the rat stomach (Kang, Teng and Chen, 1996). Interestingly enough, the ulcerhealing effect of capsaicin is counteracted by cimetidine which, unlike capsaicin, decreases GMBF (Kang, Teng and Chen, 1996). Whether the antagonistic influence of cimetidine on capsaicin-evoked promotion of ulcer healing is due to its antivasodilator effect has not directly been shown, and it may also be argued that it is the antisecretory action by which cimetidine removes acid as a factor that stimulates sensory nerve fibres in the stomach. The precise mechanisms by which afferent neurons promote healing of the injured gastric mucosa remain to be determined. It is relevant here to keep in mind that the sensory nerve-derived peptides CGRP, SP and NKA may exert trophic effects (Table 3.7) as they are capable of stimulating the proliferation of fibroblasts, vascular smooth muscle and endothelial cells (Nilsson, von Euler and Dalsgaard, 1985; Payan, 1985; Haegerstrand et al., 1990; Ziche et al., 1990a,b). Indirect evidence suggests that afferent nerve fibres may also exert a trophic influence on gastric mucosal cells. Thus, chronic intake of chilli at doses that are expected to stimulate sensory nerve fibres in the rat stomach enhances gastric mucosal mass but does not change crypt cell proliferation or experimental

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carcinogenesis (Kang et al., 1992). Conversely, ablation of capsaicin-sensitive afferent neurons reduces the weight of the stomach, the gastric synthesis of DNA and the gastric content of DNA and RNA, effects which are exacerbated by concomitant inhibition of NO synthesis (Dembinski et al., 1995).

SENSORY NEURONS SIGNAL FOR AN INCREASE IN GASTRIC MUCOSAL BLOOD FLOW IN THE FACE OF PENDING ACID INJURY Neural pathways underlying the acid-evoked gastric hyperaemia The implications of peptidergic afferent neurons in the regulation of GMBF and gastric mucosal resistance to injury are put into pathophysiological perspective if their role in the gastric responses to acid challenge is considered. Acid backdiffusion through a disrupted gastric mucosal barrier has long been known to cause a prompt increase in GMBF (Whittle, 1977; Bruggeman, Wood and Davenport, 1979; Oates, 1990). Disruption of the rat gastric mucosal barrier with 15% ethanol in the presence of 0.15 M HCl in the gastric lumen causes a 2.5–4 fold rise of blood flow in the left gastric artery (Figure 3.7) and through the gastric mucosa (Holzer, Livingston and Guth, 1991; Holzer et al., 1994). The prompt vasodilator response requires rapid signalling between the acid-threatened surface of the mucosa and resistance vessels in the submucosa. Indeed, an involvement of neurons has been demonstrated by the observations that the acid-evoked rise of gastric blood flow is inhibited by tetrodotoxin (Holzer, Livingston and Guth, 1991) or pretreatment of rats with a neurotoxic dose of capsaicin (Table 3.5). This holds true both for the vasodilator response, which is seen after the gastric mucosal barrier has been disrupted by ethanol (Holzer, Livingston and Guth, 1991; Holzer et al., 1994) or taurocholate (Takeuchi et al., 1993) in the presence of a high luminal acid concentration, and for the hyperaemic

Figure 3.7 Recording of the acid-evoked hyperaemia in the left gastric artery of an urethane-anaesthetised rat. The response was elicited by disruption of the gastric mucosal barrier with ethanol (EtOH, 15%) in the presence of luminal acid (HCl, 0.15 M). The traces from the top to the bottom show heart rate (HR), mean arterial blood pressure (MAP) and blood flow in the left gastric artery (BF/LGA). Data from Holzer et al. (1994).

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response to excess acid alone, which is seen in the chambered mucosa preparation only (Matsumoto et al., 1992a,b; Takeuchi et al., 1994). Inhibition of the hyperaemic reaction to gastric acid backdiffusion by tetrodotoxin, capsaicin pretreatment, morphine (Holzer, Livingston and Guth, 1991; Raybould et al., 1992), CGRP8–37 (Li et al., 1992), the bradykinin receptor antagonist HOE-140 (Pethö, Jocic and Holzer, 1994) and NKA or SP (Heinemann et al., 1996a) is associated with an aggravation of gross and histological damage to the mucosa. The nerve fibres involved in the acid-induced hyperaemia pass through the coeliac ganglion and hence originate from spinal ganglia (Raybould et al., 1992). The inhibitory effects of tetrodotoxin (Holzer, Livingston and Guth, 1991) and the autonomic ganglion blocking drug hexamethonium (Holzer and Lippe, 1992; Wachter et al., 1995) suggest that the increment of gastric blood flow in the face of pending acid injury results from a reflexlike mechanism (Figure 3.8). However, the individual arcs of this reflex and the possible autonomic or enteric pathways and relays which they use are not yet understood (Holzer, 1992). It is known that the acid-evoked rise of gastric blood flow relies on intact pathways in the splanchnic nerves and through the coeliac/superior mesenteric ganglion complex (Holzer and Lippe, 1992; Wachter et al., 1995), although a contribution from sympathetic noradrenergic neurons (Holzer and Lippe, 1992) and postganglionic parasympathetic or enteric cholinergic neurons (Bruggeman, Wood and Davenport, 1979; Holzer, Livingston and Guth, 1991) has been ruled out. The involvement of ganglionic transmission and pathways running in the splanchnic nerves is difficult to reconcile with the hypothesis that the gastric hyperaemic response to acid backdiffusion results from an axon reflex between mucosal and submucosal collaterals of afferent neurons (Holzer, Livingston and Guth, 1991; Holzer, 1992; Li et al., 1992). The main support for this conjecture comes from the evidence that the vasodilator transmitter of the acid-evoked gastric hyperaemia is CGRP (Table 3.6) which in the rat stomach occurs in extrinsic afferents only (Green and Dockray, 1988; Sternini, 1992). The axon

Figure 3.8 Effect of the CGRP1 receptor antagonist CGRP8–37 and the NO synthase inhibitor NG-nitro-Larginine methylester (L-NAME) on the gastric mucosal hyperaemia caused by gastric acid backdiffusion. D-NAME, inactive enantiomer of L-NAME. Data from Holzer et al. (1994).

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reflex theory is also backed by the ability of acidification to cause a calcium-dependent release of CGRP from rat gastric tissue (Table 3.4), which is in line with other findings showing that hydrogen ions or factors generated in the tissue in response to acidification can stimulate capsaicin-sensitive afferent neurons (Cervero and McRitchie, 1982; Forster et al., 1990; Bevan and Yeats, 1991; Holzer, Lippe and Amann, 1992; Holzer-Petsche, 1992). The axon reflex concept does not explain, however, why hexamethonium, acute extirpation of the coeliac ganglion and acute splanchnic nerve transection inhibit the hyperaemic response to gastric acid backdiffusion (Holzer and Lippe, 1992; Holzer et al., 1994). Since gastric acid backdiffusion does not appear to activate afferent pathways in the spinal cord (Schuligoi et al., 1996), it is hypothesised that the acid-induced increase in GMBF is relayed by a peripheral neural circuitry (Figure 3.8) that depends on an excitatory or inhibitory input from splanchnic and/or enteric neurons (Holzer, 1992). Mediators of the acid-evoked gastric hyperaemia The acid-evoked vasodilatation in the rat stomach depends on CGRP or a related peptide acting via CGRP1 receptors (Figure 3.9), because the CGRP1 receptor antagonist CGRP8–37 depresses the acid-evoked rise of blood flow after disruption of the mucosal barrier with ethanol (Li et al., 1992; Holzer et al., 1994) or taurocholate (Merchant et al., 1995). The vasodilatation which in the rabbit oesophagus is seen after exposure to acidified bile salts is also inhibited by CGRP8–37 (McKie et al., 1994). Like the vasodilator response to exogenous CGRP, the acid-evoked rise of GMBF is blocked by NO synthase inhibitors (Figure 3.9) in an enantiomer-selective manner (Lippe and Holzer, 1992; Holzer et al., 1994). It would appear, therefore, that the gastric mucosal hyperaemia due to acid backdiffusion is mediated by release of CGRP from perivascular afferent nerve fibres (Table 3.6). CGRP in turn stimulates the formation of NO which acts as the final vasodilator messenger

Figure 3.9 Summary of the gastric vasodilator and somatic vasoconstrictor responses to acid backflux in the gastric mucosa. BRAD, bradykinin; 5-HT, 5-hydroxytryptamine.

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(Figures 3.4 and 3.8). It is obvious that, as regards CGRP and NO, the vasodilator mediators of the hyperaemic response to acid backdiffusion in the rat stomach are identical with those of the hyperaemic response to sensory nerve stimulation with capsaicin. It is important to realise, though, that the vasorelaxant mediator role of NO is confined to the gastric microcirculation (Holzer et al., 1994), since NO synthase inhibitors block the acid-evoked rise of blood flow in the gastric mucosa but not that in the left gastric artery (Holzer et al., 1994). Thus, the effector mechanisms of neurogenic vasodilatation differ in the various levels of the gastric arterial tree. Tachykinin NK1 or NK2 receptor antagonists do not inhibit the hyperaemic response to gastric acid backdiffusion (Holzer et al., 1994; Heinemann et al., 1996a). SP and NKA, however, inhibit the acid-evoked rise of GMBF via activation of NK2 receptors (Heinemann et al., 1996a). The inhibitory effect of NKA on the gastric vasodilator response to acid backdiffusion remains unaltered by ketotifen and seems to be independent of the vasoconstrictor effect of the peptide which in vivo is brought about by stimulation of NK1 receptors (Heinemann et al., 1996a). The significance of the inhibitory effect of tachykinins on the acid-induced vasodilatation in the stomach is not yet clear but may reflect a negative feedback control mechanism. Histamine acting via histamine H1 receptors (Holzer, Livingston and Guth, 1991), vasodilator prostanoids (Lippe and Holzer, 1992), ACh acting via muscarinic receptors (Holzer, Livingston and Guth, 1991) and transmitters of noradrenergic neurons (Holzer and Lippe, 1992) have been ruled out as mediators of the hyperaemic reaction to acid challenge of the rat stomach. Bradykinin, to the contrary, plays some role, given that the bradykinin B2 antagonist HOE-140 (Pethö, Jocic and Holzer, 1994) attenuates the hyperaemia and enhances the mucosal damage elicited by barrier disruption in the presence of a high concentration of intraluminal acid (0.15 M HCl). It would hence seem that amounts of bradykinin sufficient to raise GMBF are formed only during severe acid challenge of the rat gastric mucosa (Pethö, Jocic and Holzer, 1994) but it remains to be examined whether bradykinin causes vasodilatation by its own or by stimulating the neural vasodilator system. Model and species differences in the acid-evoked gastric hyperaemia Model- and species-related differences in the mediators of the gastric hyperaemic response to acid backdiffusion become apparent when the data obtained with acidified ethanol or taurocholate in the rat are compared with those obtained with acidified hypertonic saline in the rat and cat. Sequential exposure of the rat gastric mucosa to hypertonic saline and acid has revealed that the initial hyperaemic response to hypertonic saline is independent of afferent neurons (Endoh et al., 1993; Grønbech and Lacy, 1996) whereas the sustained rise of GMBF in response to acid requires an intact sensory innervation of the stomach (Grønbech and Lacy, 1996). The participation of extrinsic afferent neurons in the hyperaemia, which in the feline stomach is elicited by exposure to hypertonic saline plus acid, has not yet been rigidly tested. Although some CGRP is released into the portal blood and makes a small contribution to the gastric vasodilatation caused by hypertonic saline plus acid (Gislason et al., 1995a), it appears as if histamine acting via histamine H1 and H2 receptors, prostaglandins (Gislason et al., 1995a,b), NO and possibly adenosine

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(Gislason et al., 1996) play a more important role. It follows that, while the gastric vasodilator response to acid backdiffusion is conserved across different species, the transmitters mediating the rise of GMBF appear to be subject to considerable variation. It is not known which neuropeptides mediate gastric mucosal hyperaemia and protection in response to sensory nerve stimulation in the human stomach. There is reason to doubt that CGRP plays a significant role, given that CGRP-containing nerve fibres are scarce in the human stomach (Tsutsumi and Hara, 1989; Sundler, Ekblad and Håkanson, 1991). Since capsaicin releases VIP, but not CGRP, from the human isolated small intestine (Maggi et al., 1989) it is conceivable that VIP, rather than CGRP, is the major vasorelaxant transmitter of extrinsic afferent nerve fibres in the human gut. Somatic vasoconstriction associated with acid-evoked gastric hyperaemia Gastric hyperaemia in response to acid backdiffusion through a disrupted gastric mucosal barrier is aided by a reduction of blood flow to somatic vascular beds (Figure 3.8), e.g. those of the femoral artery (Wachter et al., 1995). Somatic vasoconstriction and gastric vasodilatation peak at different times and represent different entities with regard to pathways, mechanisms and mediators (Wachter et al., 1995, 1998). Further analysis has shown that it is the acid-induced gastric injury which gives rise to femoral vasoconstriction, because the gastric hyperaemia evoked by intragastric capsaicin, which does not injure the mucosa, is not accompanied by a reduction of femoral blood flow (Wachter et al., 1995). Although in part dependent on the extrinsic innervation of the stomach, the gastric acid-evoked constriction of the femoral artery does not involve capsaicin-sensitive afferent neurons and remains unaltered after blockade of ganglionic transmission with hexamethonium, blockade of noradrenergic sympathetic transmission with guanethidine, and ligation of the vascular supply to the adrenal glands (Wachter et al., 1995). Attempts to identify the factors by which gastric acid injury leads to somatic vasoconstriction have ruled out the implication of prostanoids, angiotensin II acting via AT1 receptors, vasopressin acting via V1 receptors, endothelin acting via ETA and ETB receptors (Wachter et al., 1995) and histamine acting via H1 and H2 receptors (Wachter et al., 1998). It has been observed, however, that acid injury of the rat gastric mucosa is associated with luminal release of 5-hydroxytryptamine (5-HT) and that the femoral vasoconstrictor response to gastric acid injury is significantly attenuated by blockade of 5-HT2 and 5-HT3 receptors (Wachter et al., 1998). Taken all available data together, it would seem as if acid challenge of the stomach releases 5-HT from the injured mucosa and that 5-HT in turn stimulates an unknown somatic vasoconstrictor mechanism via capsaicin-insensitive extrinsic neurons of the stomach (Figure 3.8). Physiological relevance of the acid-evoked gastric hyperaemia The observation that inhibition of the hyperaemic response to acid backdiffusion by a variety of drugs (Holzer, Livingston and Guth, 1991; Li et al., 1992; Raybould et al., 1992; Pethö, Jocic and Holzer, 1994; Heinemann et al., 1996a) goes hand in hand with the formation of deep mucosal erosions attests to the important defensive nature of this vascular reaction. By facilitating the disposal of acid, this mechanism prevents the build-up of

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an injurious concentration of H+ ions in the tissue and thus limits acid damage to the surface of the mucosa (Whittle, 1977; Bruggeman, Wood and Davenport, 1979; Guth, Leung and Kauffman, 1989; Oates, 1990; Holzer, Livingston and Guth, 1994). The afferent nerve-mediated response to acid challenge thus reflects the existence of a neural emergency system which is called into operation when there is pending or actual acid injury to the stomach. In addition, this acid-evoked rise of gastric blood flow creates favourable conditions for rapid restitution (Grønbech and Lacy, 1996) and healing (Takeuchi et al., 1994) of the wounded mucosa. INVOLVEMENT OF AFFERENT NEURONS IN THE ACID-INDUCED SECRETION OF BICARBONATE Another pathophysiologically relevant implication of afferent neurons concerns the regulation of mucosal bicarbonate secretion in response to luminal acidification of the upper gastrointestinal tract (Table 3.2). Stimulation of afferent nerve fibres with capsaicin enhances the mucosal output of alkali from the rat stomach (Takeuchi et al., 1992b,c) and duodenum (Takeuchi et al., 1991a, 1992b), whereas defunctionalisation of capsaicinsensitive afferent neurons prevents these effects of capsaicin (Table 3.3) and blunts the gastric and duodenal secretion of bicarbonate in response to luminal acidification (Table 3.7). As is the case in the stomach, the ability of afferent nerve stimulation to enhance bicarbonate secretion from the duodenum is associated with hyperaemia and results in enhanced resistance to acid-induced deep mucosal damage (Leung, 1993b). Capsaicinsensitive afferent neurons are likewise involved in the effect of lansoprazole to facilitate acid-evoked bicarbonate secretion in the rat duodenum (Inada and Satoh, 1996). The pathological significance of the duodenal alkali response to luminal acidification is reflected by the observation that this process is severely compromised in duodenal ulcer patients infected with Helicobacter pylori (Hogan et al., 1996).

INVOLVEMENT OF AFFERENT NEURONS IN PATHOLOGICAL INHIBITION OF GASTRIC MOTOR ACTIVITY Abdominal surgery and peritoneal irritation or inflammation are known to inhibit gastrointestinal motor activity (Furness and Costa, 1974; Livingston and Passaro, 1990). The acute inhibition of gastric and intestinal motility after abdominal surgery or peritoneal irritation is in part relayed by a neural reflex that consists of capsaicin-sensitive afferent (Table 3.8) and sympathetic efferent (Holzer, Lippe and Holzer-Petsche, 1986; Holzer, Lippe and Amann, 1992; Holzer-Petsche, 1992) neurons. The pathways of the reflex run through the coeliac ganglion but not in the vagus nerves (Table 3.8). As CGRP8–37 and a monoclonal antibody to CGRP ameliorate surgery-induced gastroparesis, it follows that CGRP plays an important transmitter role in the process of acute postoperative motor inhibition (Table 3.6). Such a function is consistent with the ability of CGRP to inhibit gastric motility after local (Maton et al., 1988; Holzer-Petsche, Seitz and Lembeck, 1989; Katsoulis and Conlon, 1989; Lefebvre, De Beurme and Sas, 1991), systemic (Lenz, 1988; Chijiiwa et al., 1992) and intracisternal (Raybould, Kolve and Taché, 1988) administration.

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The site at which CGRP mediates acute postoperative gastroparesis has been suggested to lie within the coeliac ganglion where it acts as a transmitter of an extraspinal intestinogastric inhibitory reflex (Zittel et al., 1994a). Tachykinins and VIP may also play a role, since preoperative administration of a tachykinin or VIP antagonist reduces acute postoperative motor inhibition in the rat and hastens the return of myoelectrical activity in the small intestine (Espat et al., 1995). How abdominal surgery activates afferent neurons to cause reflex inhibition of gastrointestinal motility is not yet fully understood. One study indicates that interleukin-1β is a contributory factor, since the inhibition of gastric emptying seen after abdominal surgery is attenuated by an interleukin-1β receptor antagonist and the ability of interleukin-1β to inhibit gastric emptying is prevented by CGRP8–37 (Coimbra and Plourde, 1996). A role of interleukin-1β in stimulating afferent neurons is consistent with the cytokine’s activity to facilitate the release of CGRP in the skin (Herbert and Holzer, 1994) and that of SP in the spinal cord (Malcangio et al., 1996). While acute shutdown of gastrointestinal motility by surgery or inflammation can be viewed as protective response which prevents movements that could be deleterious to the traumatised gut, continued blockade of motor activity gives rise to the clinical manifestations of ileus. The question as to whether peptidergic afferent neurons contribute to the prolonged, pathological form of postoperative and peritonitis-associated ileus has not yet been addressed. It may be speculated, though, that the conditions of inflammation, bacterial overgrowth and electrolyte imbalance, which are associated with ileus, may contribute to sensitisation of afferent pathways and to recruitment of otherwise inactive afferent neurons (Cervero, 1994; Mayer and Gebhart, 1994) and in this way perpetuate the reflex inhibition of gastrointestinal motility.

SUMMARY AND PERSPECTIVES CAPSAICIN-SENSITIVE AFFERENTS AS A NEURAL EMERGENCY SYSTEM The findings reviewed here prove the existence of a neural emergency system in the stomach, which is called into operation in the face of pending injury to the tissue but is not tonically active. As a result, blood flow to the stomach is greatly augmented, an effect that facilitates the delivery of bicarbonate to the surface epithelium and overlying mucus layer (Guttu et al., 1994), adds to the removal of injurious factors from the mucosa and promotes a wide range of processes that either reduce the vulnerability, or aid the repair, of the gastric mucosa (Guth, Leung and Kauffman, 1989; Oates, 1990; Allen et al., 1993; Holzer, Livingston and Guth, 1994). Apart from facilitation of gastric blood flow, augmentation of bicarbonate and mucus secretion and, under certain conditions, inhibition of acid output and motor activity have been shown to contribute to the overall protective role of peptide-containing afferent neurons. This neural emergency system, which acts in concert with other mechanisms of protection (Whittle, 1993), is operative not only in the stomach but also in other regions of the gastrointestinal system including the oesophagus (Bass et al., 1991; McKie et al., 1994), upper small intestine

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(Rózsa et al., 1986; Evangelista, Maggi and Meli, 1987a; Maggi et al., 1987a; Remak, Hottenstein and Jacobson, 1990; Hottenstein et al., 1991; Takeuchi et al., 1991a, 1992a,b; Leung, 1993b; Jacobson et al., 1994; Vanner and Surprenant, 1996) and colon (Evangelista and Meli, 1989; Leung, 1992b; Eysselein et al., 1992; Goso et al., 1993; Reinshagen et al., 1994, 1996). Although most of the homeostatic actions are brought about by release of peptide transmitters from their peripheral endings, sensory nerve fibres also constitute the afferent arc of certain autonomic reflexes that regulate secretory and motor functions. Amongst the messenger molecules of afferent neurons, CGRP acting via CGRP1 receptors and tachykinins acting via NK2 receptors have proved to play a central role in enhancing mucosal resistance to pending injury, both classes of transmitter using NO as second messenger (Figures 3.4 and 3.6; Tables 3.3 and 3.6). CGRP and NO are potent vasodilators which, by increasing GMBF, support those protective mechanisms which depend on mucosal hyperaemia. NKA, to the contrary, strengthens mucosal resistance to injury by promoting hyperaemia-independent protective mechanisms. In addition, CGRP and other afferent nerve-derived peptides participate in the postoperative inhibition of gastric motility (Plourde et al., 1993; Zittel et al., 1994a) and in the pathophysiological regulation of gastric motor activity, secretion and immunity. In the long term, afferent neurons seem to be involved in the coordination of trophic and healing processes. This function is envisaged from the delayed restitution and healing of gastric damage in rats pretreated with a neurotoxic dose of capsaicin (Table 3.9) and the ability of SP, NKA and CGRP to stimulate the proliferation of endothelial cells (Haegerstrand et al., 1990; Ziche et al., 1990b), arterial smooth muscle cells (Nilsson, von Euler and Dalsgaard, 1985; Payan, 1985) and fibroblasts (Nilsson, von Euler and Dalsgaard, 1985; Ziche et al., 1990a) and from the ability of SP to stimulate angiogenesis (Ziche et al., 1990b). PATHOPHYSIOLOGICAL CONDITIONS TO ALARM THE NEURAL EMERGENCY SYSTEM To put the functional characteristics of capsaicin-sensitive afferents into proper pathophysiological perspective, the conditions under which these neurons are activated and the functional consequences which may arise from a dysfunction of these neurons need to be considered. A variety of chemicals including hydrochloric acid (Clarke and Davison, 1978; Cervero and McRitchie, 1982; Forster et al., 1990; Bevan and Yeats, 1991; Geppetti et al., 1991; Holzer, Livingston and Guth, 1991; Holzer-Petsche, 1992; Takeuchi et al., 1994; Manela et al., 1995), acetic acid (Leung, 1992b), the bacterial peptide N-formyl-methionylleucyl-phenylalanine (Giuliani et al., 1991) and oxygen radicals (Stahl, Pan and Longhurst, 1993) as well as ischaemia (Pan and Longhurst, 1996) and hypertonicity (Forster et al., 1990; Matsumoto et al., 1992a) are able to stimulate capsaicin-sensitive afferent neurons. These factors will activate the neurons either directly or indirectly via injury-induced formation or liberation of autacoids such as histamine, 5-HT, prostanoids, leukotrienes, bradykinin (Maggi, 1991; Stebbins, Theodossy and Longhurst, 1991; Dray and Perkins, 1993; Geppetti, 1993; Cervero, 1994; Mayer and Gebhart, 1994), interleukin-1β (Herbert and Holzer, 1994; Coimbra and Plourde, 1996; Malcangio et al., 1996), platelet-activating

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factor (Piqué, Esplugues and Whittle, 1990), and endothelin-1 (Whittle and Lopez-Belmonte, 1991). The presence of some or, more likely, a combination of these factors will turn on and/or upregulate the neural emergency system which supports homeostasis by local release of protective transmitters. PRE- AND POSTJUNCTIONAL DEFECTS OF THE NEURAL EMERGENCY SYSTEM With the gastroprotective role of peptidergic afferent neurons in mind it is logical to assume that improper functioning of this neural emergency system is liable to weaken the resistance of the tissue to injurious stimuli and thus may be an aetiological factor in gastroduodenal ulcer disease. Evidence in favour of this conjecture is indeed accumulating (Figure 3.10), as there are reports that either a prejunctional dysfunction of the afferent nerve fibres (sensory neuropathy) or a postjunctional defect in the effector mechanisms disturbs gastric mucosal homeostasis. It need be emphasised, though, that sensory neuropathies resulting from excessive intake of capsaicin-containing peppers are not known. To the contrary, it rather seems as if dietary chilli intake is actually a factor that protects from peptic ulcer disease (Kang et al., 1995b). There are a number of reports that experimental or pathological injury of the stomach is associated with a change in the tissue concentration of CGRP, SP and other neuropeptides (Evangelista et al., 1992, 1993; Wattchow et al., 1992; Kaneko et al., 1993; Tramontana et al., 1994a), but it is not yet known whether these neurochemical alterations reflect changes in the functionality of the neural emergency system and whether they contribute to, or are secondary consequences of, the disease. Reflux of bile is a process that may have a bearing on gastritis and ulcer development, and it is pertinent to note that chronic treatment of rats with oral taurocholate causes a sensory neuropathy that manifests itself in a reduction of the capsaicin-evoked CGRP release and hyperaemia in the gastric mucosa (Narita et al., 1995). Analogously, chronic

Figure 3.10 Schematic diagram of the neural emergency system in the stomach and summary of the pathophysiological conditions that are associated with a malfunction of this system.

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intake of nicotine by rats has been found to suppress the increase in mucosal blood flow due to gastric acid backdiffusion (Battistel et al., 1993). This observation has been related to a postjunctional depression of the vasodilator response to acid challenge, as chronic nicotine treatment is associated with enhanced formation of vasoconstrictor leukotrienes in the rat stomach (Battistel et al., 1993). Various insults ranging from smoking (Ainsworth et al., 1993) to infection with Helicobacter pylori (Hogan et al., 1996) inhibit the acid-stimulated secretion of bicarbonate from the mucosa of the human duodenum. Since the acid-evoked secretion of bicarbonate in the rat duodenum involves capsaicin-sensitive afferent neurons (Takeuchi et al., 1991a) it would seem as if some of the adverse effects of nicotine, smoking and Helicobacter pylori on the gastroduodenal mucosa may be related to a malfunction of afferent nervecontrolled effector systems, given that in H. pylori infection the acid-induced bicarbonate secretion is blunted due to a defect in the duodenal epithelium (Hogan et al., 1996). Other studies indicate that the gastropathy associated with experimental cirrhosis (Ferraz et al., 1995; Nishizaki et al., 1996) and uraemia (Quintero et al., 1992) is associated with an inadequate rise of GMBF in response to capsaicin or acid backdiffusion through a leaky gastric mucosal barrier. Since the supply of gastric submucosal arterioles by CGRPcontaining nerve fibres is unchanged in experimental cirrhosis (Ferraz et al., 1995; Nishizaki et al., 1996) and uraemia (Quintero et al., 1992) it can be inferred that the gastric circulatory dysregulation arises from a postjunctional defect, possibly in the vascular NO/ cyclic guanosine 3′,5′-cyclic monophosphate signalling pathway (Ferraz et al., 1995). It is worth noting here that capsaicin-sensitive afferent neurons have been suggested to contribute to the development of experimental portal hypertension (Lee and Sharkey, 1993), although such an implication has been refuted in another study (Fernández et al., 1994). Apart from pathological changes in the neural emergency system of the stomach it need be considered that the functionality of the system may change with the development and ageing of the afferent nervous system (Figure 3.10) and the effector control systems which afferent neurons interact with. The capsaicin- and acid-evoked hyperaemia in the rat gastric mucosa (Grønbech and Lacy, 1995; Miyake et al., 1996) and rat superior mesenteric artery (Seno et al., 1996) declines with age, a change that is associated with a significant reduction in the density of CGRP-containing nerve fibres around arterioles in the gastric submucosa (Grønbech and Lacy, 1995). It is hence likely that a sensory neuropathy is in part responsible for the compromised ability of the aged gastric mucosa to defend itself against injurious factors. From a pharmacological/toxicological perspective it need be borne in mind that certain drugs and medicines, that influence the release, action and metabolism of CGRP, NKA and NO, will interfere with the homeostatic function of peptidergic afferent neurons in the stomach. This is particularly true for CGRP and NKA receptor antagonists and NO synthase inhibitors. At the prejunctional level it is morphine and other opioid agonists that inhibit the release of peptide transmitters from afferent nerve fibres (Maggi, 1991; Holzer, 1992). Indeed, morphine worsens the vulnerability of the gastric mucosa by ethanol, platelet-activating factor and acid through an action that involves µ-opioid receptors and that depends on an intact afferent innervation of the stomach (Esplugues, Whittle and Moncada, 1989, 1992; Esplugues and Whittle, 1990; Piqué, Esplugues and Whittle, 1990; Holzer, Livingston and Guth, 1991; Whittle and Lopez-Belmonte, 1991). The release of

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peptide transmitters from afferent nerve fibres may also be under the control of prejunctional adrenoceptors as it is the case in other tissues (Maggi, 1991; Holzer, 1992) but this possibility has not yet been explored in the gastric mucosa. A final question concerns the role that peptidergic afferent neurons play in chronic gastritis, nonulcer dyspepsia, ulcer disease and other functional disorders of the stomach. It is potentially important to note that the expression of SP is upregulated in the gastric mucosa of patients suffering from nonulcer dyspepsia with abdominal pain (Kaneko et al., 1993), but it remains to be elucidated whether this change has any bearing on gastric mucosal homeostasis. Further related to this issue is the question as to whether the local protective function of peptidergic afferent neurons in the stomach is associated with nociception. Although peptidergic afferent neurons supplying the stomach are connected to nociceptors (Cervero, 1994), gastric acid backdiffusion fails to activate spinal neurons as examined by the expression of messenger RNA for c-fos, a marker of neuronal excitation. It would seem, therefore, that the local emergency function of extrinsic afferents takes place without the concomitant transmission of afferent activity to the spinal cord (Schuligoi et al., 1996). It is in place to recall here that ulcer pain is likely to depend on the presence of inflammation in the gastric wall, which sensitises afferent pathways and recruits otherwise inactive nociceptors (Cervero, 1994; Mayer and Gebhart, 1994). The relationship between local emergency function and nociception needs hence to be examined under conditions of chronic injury and inflammation.

ACKNOWLEDGEMENTS Work performed in the author’s laboratory was supported by the Austrian Science Foundation (grants 7845, 9473, 9823 and 11834), the Austrian National Bank (grants 4207, 4905 and 6237) and the Franz Lanyar Foundation at the Medical Faculty of the University of Graz. The author is grateful to Drs. Ulrike Holzer-Petsche and Christof H. Wachter for drawing the computer graphs and to E. Fauland for secretarial help.

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4 Neural Control of the Large Intestine* Kalina Venkova, Beverley Greenwood-Van Meerveld and Jacob Krier Oklahoma Foundation for Digestive Research, Basic Science Labs, V.A. Medical Center, Oklahoma City, OK 73104, USA Efferent neurones in the extrinsic parasympathetic and sympathetic outflow from the central nervous system to the large intestine provide the reflex pathways which form and coordinate the motility patterns responsible for the storage, transport and evacuation of the colonic contents. Using experimental models of pelvic nerve and lumbar colonic nerve stimulation we have characterised the effects of selective activation of parasympathetic or sympathetic extrinsic pathways and have defined the neurotransmitters and receptors involved. In summary, we demonstrated that the extrinsic nervous control of the colon involves both prejunctional and direct smooth muscle effects. Efferent fibres to the colon provide cholinergic input to excitatory motoneurones in ganglia of the pelvic and the myenteric plexus, which induce smooth muscle contractions releasing acetylcholine and/or excitatory non-adrenergic, non-cholinergic (NANC) neurotransmitter(s). In addition, efferent parasympathetic fibres within the pelvic nerve may synapse with inhibitory motoneurones inducing muscle relaxation via the release of nitric oxide and/or inhibitory neurotransmitters such as vasoactive intestinal polypeptide and ATP. The relationship between nitric oxide-induced inhibition and a prostaglandin-dependent post-inhibitory excitation in colonic muscles is characteristic for NANC parasympathetic input to the colon. We believe that it plays a role in formation of parasympathetically controlled patterns of colonic activity. It has long been known that parasympathetic motor activity is under sympathetic influence by inhibitory α-adrenoceptors located on cholinergic nerve terminals. However, our observations have demonstrated direct motor responses in the circular muscle of non-sphincteric regions of the colon. Similar to vascular muscle innervation, postganglionic sympathetic fibres in the lumber colonic nerves co-release noradrenaline and ATP and express a presynaptic control mechanism operated by autoinhibitory α2-adrenoceptors. At the level of the muscle, the sympathetic motor response is formed by the activation of different adrenoceptor and purinoceptor subtypes i.e. α- and β-adrenoceptors and P2X receptors. KEY WORDS: colon; parasympathetic innervation; sympathetic innervation; adrenergic; cholinergic; NANC transmission.

INTRODUCTION The storage, transport and evacuation of colonic contents are accomplished by coordinated patterns of smooth muscle activity which in turn are regulated by the complex interaction between myogenic and neurogenic mechanisms. The neural regulation of colonic motility is provided by divisions of the autonomic nervous system organised in a three-rank

*Dedicated to the memory of our colleague and friend Jack Krier. 171

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hierarchy. At the local level, a network of neurones located entirely within the gastrointestinal wall forms the enteric nervous system (ENS) which mediates simple stereotyped reflexes, such as ascending excitation and descending inhibition (Furness and Costa, 1987; Furness et al., 1995). At a higher level, peristaltic reflexes coordinate the activity of widely separated intestinal regions by neurones in the prevertebral ganglia (Szurszewski and King, 1989). Colonic motility is also under the influence of the central nervous system via neural connections contained within the extrinsic parasympathetic and sympathetic nerves supplying the gut. Until recently, the concept that parasympathetic nerves are excitatory to the colon and that sympathetic nerves cause inhibition of colonic motility was the generally accepted dogma. The most distal regions of the colon and rectum, receive prominent extrinsic parasympathetic innervation, with the pelvic nerves conveying the main parasympathetic innervation (McRorie, Krier and Adams, 1991) and providing phasic excitation during defecation and inhibition of the internal anal sphincter (Bouvier and Gonella, 1981b). The proximal part of the colon receives additional parasympathetic input through distal branches of the vagal nerves. The sympathetic nerves provide the major extrinsic supply to the caecum and proximal colon (Gonella, Bouvier and Blanquet, 1987; Furness and Bornstein, 1991). Postganglionic sympathetic fibres, which control colonic contractile activity, originate in the inferior mesenteric ganglion and in paravertebral ganglia, and travel to the colon in lumbar colonic nerves (de Groat and Krier, 1979). The most distal portion of the colon and the internal anal sphincter also receive sympathetic innervation through the hypogastric nerves (Baron, Janig and McLachlan, 1985a). Although the anatomical organisation of these pathways is well known, the physiological significance of the extrinsic innervation to the colon is less well understood. Except for the parasympathetic cholinergic excitation, the neuronal pathways and neurotransmitter mechanisms, involved in the extrinsic control of colonic contractile activity are still incompletely characterised. The lack of pharmacological data and the conflicting results obtained upon sympathetic nerve stimulation are due in part to the fact that, in most of the studies, colonic motility responses to extrinsic nerve stimulation have been investigated in vivo. These types of experiments provide accurate information about the functional significance of the extrinsic nervous control, however the technique does have limitations when studying receptors and pharmacological effects of specific neurotransmitters. A different approach was taken by Gillespie and his colleagues (Gillespie and McKenna, 1961; Gillespie and Khoyi, 1977) when they introduced an isolated preparation in which a colonic segment with the extrinsic lumbarcolonic and pelvic nerves attached was placed in an organ bath. In studies using this preparation the effects of extrinsic of sympathetic and parasympathetic nerve stimulation on the intraluminal pressure in the colon segment were investigated in the presence of selective adrenoceptor antagonists. The results supported the hypothesis that parasympathetic nerves induce motor responses which are controlled by sympathetic nerves and established that the main inhibitory effect of sympathetic nerves is due to activation of presynaptic inhibitory α-adrenoceptors located on cholinergic neurones. Subsequent data demonstrated the existence of postjunctional excitatory α-adrenoceptors in the circular muscle of non-sphincteric regions of the colon in different mammalian species (Gagnon and Belisle, 1970; Anuras and Christensen, 1981; Zhang et al., 1992) and in the human colon (Gagnon, Devroede and Belisle, 1972). While it is known that these excitatory

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postjunctional receptors respond to adrenoceptor agonists with contractile responses, it is unknown whether they are activated through the release of neurotransmitter(s) from sympathetic nerve fibres. The nature of parasympathetic motor responses has also proved to be more complicated since agonist-induced activation of nicotinic cholinoceptors in the ENS of the ileo-colonic region has been found to induce a prominent relaxatory response in the presence of atropine (Boeckxstaens et al., 1991). Studies using electrical field stimulation (EFS) of intramural nerve terminals, showed that nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) are released and act as non-adrenergic, noncholinergic (NANC) inhibitory neurotransmitters in the ENS of the colon (Sanders and Ward, 1992). However, the physiological mechanisms of activation of NANC neurones in the colon and their functional significance still remains unresolved. In search of an answer, we concentrated our efforts on in vitro nerve-muscle preparations which are suitable for pharmacological studies of the mechanical responses of the colon circular or longitudinal muscle to stimulation of the extrinsic lumbar colonic (Venkova and Krier, 1993) or pelvic nerves (Kennedy and Krier, 1987a; Venkova and Krier, 1994). This chapter will review our findings related to the influence of lumbar and sacral autonomic neural pathways on the motor function of the colon with particular emphasis on the pharmacology of receptor mechanisms involved in the extrinsic neural control of colonic contractility.

PARASYMPATHETIC CONTROL OF THE COLON Preganglionic neurones in the sacral parasympathetic nucleus at intermediolateral regions of the sacral spinal cord are the source of efferent fibres that control the functions of the colon (Nadelhaft, de Groat and Morgan, 1980; de Groat et al., 1982; Nadelhaft et al., 1983). Parasympathetic efferent fibres emerge from the spinal cord in the second and third sacral ventral roots, than pass peripherally in a branch of the pelvic nerve to the pelvic plexus and to the colon, rectum and anal canal. Stimulation of sacral parasympathetic efferent fibres in animal models has led to conflicting results showing both excitation (de Groat and Krier, 1976, 1978) or inhibition (Fasth, Hulten and Nordgren, 1980) of contractility of the colon and rectum. Pelvic nerve stimulation in atropinised cats elicited a sustained contraction of the proximal colon and a relaxation of the rectum (Fasth et al., 1981). In our studies we investigated changes in colonic smooth muscle activity in response to stimulation of preganglionic pelvic nerve fibres using an isolated preparation of the cat colon. The findings revealed some new and exciting information about the extrinsic neural control of the colon (Kennedy and Krier, 1987a; Venkova and Krier, 1994). Our isolated preparation consists of a muscle strip, stripped of the mucosa and excised in the direction of the longitudinal muscle with one pelvic nerve attached to the muscle by connective tissue that carries branches of the nerve and a portion of the pelvicplexus ganglia. The preparation is mounted vertically in an organ bath to record isometric contractile activity of the longitudinal muscle. A bipolar platinum electrode is placed on the main branch of the pelvic nerve to stimulate preganglionic fibres and a pair of plate platinum electrodes, placed parallel to the muscle strip, supply transmural EFS. This preparation includes the efferent components of sacral parasympathetic pathways (preganglionic pelvic nerve fibres, pelvic plexus ganglia, colonic ganglia and colonic fibre bundles,

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neurones and plexuses of the ENS and both muscle layers) and proved to be a powerful model to study the pharmacology of muscular motor responses in the colon. In the isolated colonic longitudinal muscle preparation with the pelvic nerve attached, we found that stimulation of preganglionic fibres induces cholinergic contractions, that are blocked by hexamethonium or atropine, and modulated by Met-enkephalin (Kennedy and Krier, 1987a,b). In the colon, sacral parasympathetic neurones provide excitatory input, synapsing with nicotinic cholinoceptors on postganglionic neurones in the colonic ganglia and/or in ganglia of the myenteric plexus, which then synapse with muscarinic cholinoceptors on smooth muscle cells. The data suggest that the activity of this pathway can be regulated by endogenous opioids acting on presynaptic δ-opioid receptors at the level of both ganglionic and nerve-muscle synapses. When adrenergic nerve terminals are depleted by guanethidine and cholinergic neuroeffector junctions are blocked by atropine, electrical stimulation of the pelvic nerve induces biphasic NANC responses of the longitudinal muscle of the colon. The NANC responses involve relaxation and/or inhibition of spontaneous phasic contractions, which occur during electrical stimulation and are followed by an excitatory component that develops upon cessation of the stimulus and which we referred to as an off-contraction (Venkova and Krier, 1994). A characteristic of the longitudinal muscle of the colon is that it maintains a low level of spontaneous tone (resting tension of about 0.1–0.2 g/mm2 cross sectional area) and generates spontaneous phasic contractions when isolated and suspended under standardised optimal tension in an organ bath. If the cholinergic and adrenergic input to the muscle is eliminated in the presence of atropine and guanethidine, most of the muscle strips lose phasic activity and remain mechanically silent. Under these conditions, stimulation of the pelvic nerve as well as EFS induce only an off-contractile response with its latency being considered a measure of NANC inhibition. The amplitude and the duration of the offcontractions are considered representative of a NANC excitation. Both phases of the response are neurally mediated because they are absent in preparations treated with tetrodotoxin, a neurotoxin known to abolish axonal neurotransmission. The responses to pelvic nerve stimulation are abolished also by hexamethonium, a ganglionic nicotinic cholinoceptor antagonist, that suppresses both inhibitory and excitatory components. In contrast, the responses to EFS are resistant to hexamethonium and are blocked only by tetrodotoxin. Therefore, it was assumed that in the colon NANC neurotransmission can be involved in both excitatory and inhibitory control of colonic motility and such control can be obtained through an extrinsic parasympathetic pathway that originates in the sacral spinal cord. ROLE OF NITRIC OXIDE We studied the involvement of nitric oxide (NO) as an inhibitory neurotransmitter of NANC responses to pelvic nerve stimulation in the cat colon using inhibitors of NO synthase (NOS): N ω-nitro-L-arginine (L-NNA), N ω-nitro-L-arginine methyl ester (L-NAME) and N ω-monomethyl-L-arginine (L-NMMA). NO synthesised from L-arginine by a calciumdependent constitutive NOS (Moncada, Palmer and Higgs, 1991) has been demonstrated to be a mediator of NANC relaxation following neuronal stimulation in a wide range of intestinal preparations (Christinck et al., 1991; Sanders and Ward, 1992) including the human colon (Boeckxstaens et al., 1993). We found that all NOS inhibitors reduced

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the latency of the off-contractions to both pelvic nerve and intramural stimulation, however neither of the NOS inhibitors was able to “shift” the contractile phase of the response to the beginning of the stimulus train i.e. bring the latency to zero (Figure 4.1), strongly suggesting that NO may not be the sole transmitter of this inhibitory response. Pretreatment with α-chymotrypsin at a concentration which abolished VIP-induced relaxation (Li and Rand, 1990), further decreased the duration of the inhibitory component of NANC responses. However, the effect of the NOS inhibitors was considered specific since it was reversed by the excess of L-arginine but not by D-arginine. Our results indicate that in the colon NO and an inhibitory peptide, most likely VIP, are released upon stimulation of the pelvic nerves and may act as co-transmitters to maintain the inhibitory phase of the smooth muscle response. To further establish the role of NO as an inhibitory neurotransmitter in the colon the distribution and abundance of NOS containing neurones have been examined using nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry and NOS immunohistochemistry (Barbiers et al., 1993; Furness et al., 1994; Timmermans et al., 1994). In muscle preparations of cat colon, with branches of the pelvic nerve attached, NADPH diaphorase histochemistry showed the existence of nitrergic neurones in the myenteric and submucosal plexuses in the distal part of the colon and rectum. Nitrergic neurones were found also in extramural parasympathetic ganglia connecting colonic branches of the pelvic nerve and in fibre bundles connecting colonic ganglia with the serosal surface of the distal colon (Venkova and Krier, unpublished observations).

Figure 4.1 Isolated cat pelvic nerve-colonic muscle preparation. Off-contractions induced by transmural stimulation (A) or stimulation of the pelvic nerve (B). Horizontal bars indicate the time of electrical stimulation. The upper traces show off-contractions elicited by EFS (2 Hz, 0.5 ms pulse duration, 30 s train duration) and pelvic nerve stimulation (8 Hz, 0.5 ms pulse duration, 45 s train duration) in the absence of NOS inhibitor. The middle traces illustrate the inhibitory effect of L-NNA (100 µM) on the latency, amplitude and duration of the off-contractile responses. A partial reversal of the inhibitory effects by L-arginine (120 µM) is shown in the lower traces. (From Venkova and Krier, 1994, with permission).

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Although there is no direct evidence about the functional role of these nitrergic neurones, we postulate that they may be inhibitory motoneurones activated by preganglionic parasympathetic input through nicotinic cholinoceptors, because the NANC inhibitory responses to stimulation of the pelvic nerve were blocked by hexamethonium (Venkova and Krier, 1992, 1994). Other more complete studies in the guinea-pig found NOScontaining cell bodies in the submucosal plexus of the colon and rectum, and NOS containing terminals in the circular muscle, including the sphincters. Within the myenteric ganglia NOS-containing terminals were extremely dense in the proximal colon and rectum. NOS neurones have been classified as: (1) inhibitory motor neurones to the circular muscle throughout the gastrointestinal tract and (2) interneurones in the ileum and colon. The existence of two distinct subpopulations of NOS-containing enteric neurones acting as interneurones or inhibitory motor neurones was confirmed also for the ENS of the developing human digestive tract (Timmermans et al., 1994). In the cat colon we have demonstrated that motoneurones releasing NO are activated by extrinsic parasympathetic neurones running within the pelvic nerves and synapsing via nicotinic cholinoceptors. ROLE OF PROSTAGLANDINS The nature of the off-contractions, which follow inhibition of smooth muscle activity, has been a matter of speculation for more than two decades (Bennett, 1966). It is now apparent that the rebound excitation phenomenon is a complex event, which appears in response to intramural or extrinsic parasympathetic stimulation. In the longitudinal muscle of the colon, the NANC off-contraction is preceded by smooth muscle inhibition, requires the synthesis of prostaglandins and may also involve the effect of an excitatory NANC neurotransmitter. The pharmacology of the neurally mediated NANC off-contraction in the colon is still a matter of speculation, although it has been suggested that rebound contractions which follow inhibitory responses result from the effect of an excitatory NANC neurotransmitter, which is masked during stimulation (Wood and Marsh, 1973). An additional myogenic rebound phenomenon following smooth muscle membrane hyperpolarisation may contribute to the generation of off-contractions. Prostaglandins have been implicated in the development of rebound excitation in the guinea-pig taenia coli (Burnstock et al., 1975), mouse colon (Fontaine, Grivegnee and Reuse, 1984) and canine proximal colon (Ward et al., 1992) because cyclooxygenase inhibitors reduce the off-contractions or post-inhibitory smooth muscle membrane hyperpolarisation. In the cat colon longitudinal muscle we hypothesised that prostaglandins may play a role in mediating a component of the off-contraction. We tested our idea using indomethacin, an inhibitor of prostaglandin synthesis that blocks cyclooxygenase. Although preincubation with indomethacin did not change the tone or the spontaneous contractions, there was a concentration-dependent reduction in amplitude and duration of NANC off-contractions evoked by EFS or pelvic nerve stimulation. The maximum inhibition of amplitude and duration of off-contractions was an average of 45–70% of control responses measured in the absence of indomethacin (Figure 4.2). In contrast, indomethacin did not change the latency of the off-contractions evoked by either EFS or stimulation of the pelvic nerve, suggesting that the release of prostaglandins is a post-stimulus event involved only in the development of the off-contraction.

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Figure 4.2 Isolated cat pelvic nerve-colonic muscle preparation. Off-contractions induced by transmural stimulation (A) or stimulation of the pelvic nerve (B). Effects of indomethacin applied at concentrations of 10 µM and 30 µM on the latency (upper panels), amplitude (middle panels) and duration (lower panels) of the off-contractile responses. Filled symbols represent time-matched control responses and open symbols represent the effect of indomethacin. Note that indomethacin significantly (*P < 0.05; **P < 0.01) reduces the amplitude and duration of the off-contraction without changing its latency. (From Venkova and Krier, 1994, with permission).

Others have reported that off-excitation also occur in the canine colon, especially in the region where electrical slow waves are generated (Smith, Reed and Sanders, 1987; Sanders and Smith, 1989). Responses to activation of intrinsic inhibitory nerves were characterised by hyperpolarisation and reduction in slow wave amplitude and duration during the period of stimulation and a rebound enhancement of the slow waves immediately following the stimulus. In the canine colon, exogenous NO mimicked the inhibitory responses to nerve stimulation inducing: hyperpolarisation of the membrane potential, inhibitions of slow waves and a period of post-inhibitory rebound excitation following the inhibitory response to NO. Pretreatment with indomethacin reduced the rebound excitation following NO application without blocking the inhibitory components of the

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responses (Ward et al., 1992). These results are compatible with the finding that in the cat colon NANC off-contractions induced by stimulation of the pelvic nerve are positively linked to the NO released during stimulation i.e. the magnitude of off-contractions decreased with the decrease of neurally released NO brought about by pretreatment with competitive NOS inhibitors (L-NAME, L-NNA and L-NMMA) or hydroquinone which enhances the formation of superoxide free radicals, shortening the life-time of NO (Venkova and Krier, 1994). In summary, motoneurones in the extrinsic parasympathetic outflow to the colon involve both cholinergic and NANC excitatory neurotransmission to induce colonic contraction. Inhibitory pathways are also activated by extrinsic parasympathetic neurones and are probably essential in coordinating colonic contractile activity to form complicated motility patterns involved in the storage and elimination of body waste. The chemistry of the inhibitory pathways remains to be elucidated though it is clear that NO plays an important role as an inhibitory motor transmitter.

SYMPATHETIC CONTROL OF THE COLON The lumbar sympathetic outflow to the large intestine consists of preganglionic fibres arising from neurones located in the autonomic nucleus in the spinal cord (L2–L4 segments), which in most species pass through the sympathetic chain of paravertebral ganglia and synapse with neurones in the prevertebral ganglia, e.g. inferior mesenteric and pelvic plexus ganglia. Postganglionic fibres arising from neurones in the inferior mesenteric ganglia divide into two principal groups: the lumbar colonic nerves and the hypogastric nerves. The lumbar colonic nerves form a diffuse network around the inferior mesenteric artery and accompany the artery to the distal colon and rectum (Costa and Gabella, 1971). Sympathetic postganglionic fibres that travel to the colon in lumbar colonic nerves innervate vascular and visceral smooth muscle (de Groat and Krier, 1979; Baron, Janig and McLachlan, 1985b). Sympathetic fibres also innervate the epithelial cells, providing adrenergic regulation of fluid and electrolyte transport in the colon (Hubel, 1985; Smith and McCabe, 1986). The most distal part of the colon and the internal anal sphincter receive sympathetic innervation through the hypogastric nerves. In most of the colon, adrenergic nerves densely innervate the circular muscle but sparsely innervate longitudinal muscle fibres. The longitudinal muscle receives dense adrenergic innervation in the most distal region of the colon and the rectum, as well as the internal anal sphincter (Howard and Garret, 1973). In general, the sympathetic supply to the large intestine is associated with a tonic inhibition of colonic contractility and the maintenance of high tone by the internal anal sphincter. However, the physiological significance of the sympathetic supply remains unclear, especially in the non-sphincteric regions of the colon. The influence of extrinsic sympathetic outflow on the contractile activity of the colon has been among the most studied but least understood mechanisms. Different models of sympathetic stimulation yielded controversial data about the sympathetic regulation of colonic motility. The classic experiments carried out in whole animals indicate relaxation of the colon and rectum and contraction of the internal anal sphincter in response to stimulation of extrinsic sympathetic pathways (Garry, 1933; Hulten, 1969). Subsequently, acute

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transection of the lumbar colonic nerves enhanced the basal intraluminal pressure and the amplitude of spontaneous phasic contractions in the non-sphincteric regions of the colon, indicating a tonic inhibitory control (de Groat and Krier, 1979). In the anaesthetised cat efferent electrical stimulation of the lumbar colonic and hypogastric nerves caused a sustained contraction of the distal colon and rectum which was inhibited by phentolamine and unaffected by hexamethonium suggesting direct α-adrenergic effect (Hedlung, Fasth and Hulten, 1984). In contrast, electrical stimulation of lumbar colonic nerves was found to decrease the basal intraluminal pressure and the amplitude of cholinergic pelvic nerveevoked contractions (Gillespie and McKenna, 1961; de Groat and Krier, 1976; Gillespie and Khoyi, 1977). Activation of lumbar sympathetic nerves caused a constriction in the sphincter region which was attributed to activation of smooth muscle α-adrenoceptors (Garret and Howard, 1975). The inhibitory effects observed in the non-sphincteric regions were related to presynaptic inhibition of cholinergic activity via α2-adrenoceptors (Gillespie and Khoyi, 1977; De Groat and Krier, 1976) and to a lesser extent to the direct activation of smooth muscle inhibitory β-adrenoceptors (Bouvier and Gonella, 1981a,b). In addition to inhibitory β-adrenoceptors on colonic muscle cells (Ek, 1985), excitatory α-adrenoceptors have been characterised in the circular muscle of non-sphincteric regions in cat and dog colon (Anuras and Christensen, 1981; Zhang et al., 1992; Venkova, Milne and Krier, 1994; Venkova and Krier, 1995) and in both circular and longitudinal muscle of the rat and human colon (Gagnon and Belisle, 1970; Gagnon, Devroede and Belisle, 1972). While it is known that activation of α-adrenoceptor is coupled to contraction of the circular muscle it is unknown whether the α-adrenoceptors are activated through the release of neurotransmitter from the lumbar sympathetic nerve fibres. To characterise the responses of the colon to stimulation of extrinsic sympathetic nerves we developed an isolated lumbar colonic nerve-muscle preparation (Venkova and Krier, 1993). The preparation includes a mucosa-free muscle strip, cut in the direction of the circular muscle, with attached branches of the lumbar colonic nerves. The spontaneous activity of the circular muscle is characterised by low resting tension (0.2–0.35 g/mm2 cross sectional area) and rhythmic low-amplitude contractions generated at a frequency of 4–5 cycles per min. Electrical stimulation of the lumbar colonic nerves causes a frequency-dependent increase in tone and in the amplitude of spontaneous contractions.

Figure 4.3 Isolated cat lumbar colonic nerve-colonic muscle strip. Inhibitory effects of guanethidine on contractile responses of the circular muscle induced by stimulation of colonic nerve fibres (2–20 Hz, 0.5 ms pulse duration, 45 s train duration). (From Venkova and Krier, 1993, with permission).

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Lumbar colonic nerve-evoked contractions occur in the presence of hexamethonium or atropine but are abolished by either tetrodotoxin or guanethidine (Figure 4.3) and by sectioning the nerve between the electrode and the muscle. Thus, the isolated lumbar colonic nerve-colonic muscle preparation presents an in vivo model for studying sympathetic responses which avoids nonselective activation of intramural nerve terminals and provides the selective activation of neurotransmitter(s) release from sympathetic nerve fibres contained in branches of the lumbar colonic nerves. NORADRENALINE AND ATP AS CO-TRANSMITTERS OF SYMPATHETIC NERVES TO THE COLON In order to further characterise the pharmacology of the sympathetic innervation to the colon, experiments were designed using the lumbar colonic nerve-circular muscle preparation. Contractile responses to stimulation (2–20 Hz, 0.5 ms) of the lumbar colonic nerves were tested in the presence of the α-adrenoceptor antagonists phentolamine or prazosin. Contractions of colonic circular muscle induced by exogenous administration of noradrenaline or phenylephrine were completely inhibited by the α-adrenoceptor antagonists. In contrast, both agonists concentration-dependently reduced the lumbar colonic nerve-evoked contractile responses to approximately 50% of control value but did not abolish them. Since the remaining component of the responses was abolished by guanethidine, an agent used to deplete the presynaptic vesicles in sympathetic neurones, it was described as reflecting the co-release of a second neurotransmitter from the sympathetic terminals. The possibility of β-adrenoceptor inhibition being part of the overall response to lumbar colonic nerve stimulation was tested in preparations treated with propranolol, a selective β-adrenoceptor antagonist. Propranolol caused a concentration-dependent increase in the amplitude of lumbar colonic nerve-evoked contractions. Similarly, the inhibition of β-adrenoceptors caused a potentiation of the contractile responses to exogenous noradrenaline but not to phenylephrine. Taken together our findings suggest that lumbar sympathetic nerves regulate the contractile activity of the circular muscle in non-sphincteric regions of the colon releasing noradrenaline, which activates both excitatory α-adrenoceptors and inhibitory β-adrenoceptors. The co-release of one or more non-adrenergic neurotransmitters inducing a contractile response in colonic circular muscle, is also evident. Although the identity of the neurotransmitter co-released with noradrenaline in the colon is unknown, ATP is considered a putative candidate since in the guinea-pig colon vasoconstriction of submucosal arterioles following stimulation of sympathetic nerves is mediated by ATP (Evans and Surprenant, 1992). In the cat colon, administration of ATP and α,β-methylene ATP were found to contract the circular muscle and these contractile responses were abolished after desensitisation with α,β-methylene ATP. Moreover, we discovered that the contractions evoked by stimulation of lumbar colonic nerves decrease in amplitude after desensitisation of P2 purinoceptors with α,β-methylene ATP. Furthermore, ANAPP3 (arylazido-aminopropyl-ATP), known to act as specific P2X purinoceptor antagonist (Hogaboom, O’Donnell and Fedan, 1980; Fedan et al., 1982; Burnstock and Kennedy, 1985) caused an irreversible reduction in the amplitude of the lumbar colonic nerve-evoked responses. In both cases the remainder of the contractile response was

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Figure 4.4 Isolated cat lumbar colonic nerve-colonic muscle strip. (A) Inhibitory effects induced by the consecutive addition of prazosin and α,β-methylene ATP (α,β-mATP) on the contractions of the circular muscle elicited by stimulation of colonic nerve fibres. Horizontal bars indicate the time of electrical stimulation. (B) Summary graph of the inhibitory effects of prazosin (3 µM, open triangles) and α,β-mATP (5 µM, filled triangles) applied on the background of prazosin. The responses are expressed as percentage of controls (filled circles) in untreated preparations for each of the stimulus frequencies. (From Venkova and Krier, 1993, with permission).

further inhibited by prazosin. When α1-adrenoceptors were blocked and P2 purinoceptors were desensitised, the colonic contractile responses to lumber colonic nerve stimulation were virtually abolished, with the exception of a low-amplitude contraction developing in response to high frequency stimulation (Figure 4.4). The order in which prazosin and

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α,β-methylene ATP were added to the bathing solution did not change the final effect suggesting that once released from the nerve terminal, noradrenaline and a nucleotide, probably ATP, interact with the smooth muscle receptors independently. The possible involvement of a third neurotransmitter responsible for a small portion of the excitatory response has not been studied. The effect of “nonadrenergic, noncholinegic, nonpurinergic” neurotransmitter is evident only for the high frequencies (10–20 Hz) of lumbar colonic nerve stimulation. The nature of such a neurotransmitter remains unknown, however its dependence to high stimulus frequencies, clearly suggests that a tachykinin or a prostaglandinlike modulatory substance may have been released. Presynaptic auto-inhibitory receptors, predominantly of the α2-adrenoceptor type, are a common feature of sympathetic junctions in vascular and visceral muscles (Starke, Gothert and Kilbinger, 1989). Therefore we aimed to establish whether the sympathetic neurotransmission to the colon is regulated by presynaptic autoinhibitory control. We examined the responses to stimulation of the lumbar colonic nerves in the presence of clonidine, a selective α2-adrenoceptor agonist or yohimbine, a selective α2-adrenoceptor antagonist. Clonidine caused a concentration-dependent reduction of lumbar colonic nerve evoked contractions, while yohimbine increased the responses. Both compounds had no effect on basal tone and spontaneous contractile activity. These results are consistent with the existence of prejunctional α2-adrenoceptors maintaining a negative feedback control of neurotransmitter release by the sympathetic terminals. However, whether α2-adrenoceptor activation is linked to inhibition of the co-release of a purinergic neurotransmitter or regulates only the release of noradrenaline remains to be determined. PURINE RECEPTORS Since 1970, when Geoffrey Burnstock postulated that ATP is an inhibitory NANC neurotransmitter in the mammalian intestine, the list of potential purinergic neurotransmitters has been expanded to include the adenine nucleotides (ATP, ADP and AMP) and the adenine nucleoside adenosine. In the past, problems of desensitisation and rapid interconversion of purine derivatives complicated investigation, however stable analogues resistant to metabolism are now available. According to the recent nomenclature the receptors for adenosine and adenine nucleotides are divided into two categories: P1 purine receptors activated by adenosine and designated as adenosine receptors (A1 and A2) and P2 purine receptors (P2X, P2Y, P2Z and P2T) activated by ATP and its analogues (Burnstock and Kennedy, 1985; Abbracchio and Burnstock, 1994). Activation of purine receptors in different smooth muscles shows a wide variety of effects including inhibition, relaxation or even biphasic responses. Furthermore, our experiments demonstrated that purine agonist induce different effects in one and the same muscle depending on the level of muscle tone. When the level of basal tone in isolated cat colon circular muscle is low ATP and the ATP analogues α,β-methylene-ATP (α,β-MeATP), β,γ-methylene-ATP (β,γ-MeATP) and 2-methylthio-ATP (2-MeSATP) induced concentration-dependent contractions with a potency order of α,β-MeAT > β,γ-MeATP > 2-MeSATP > ATP. This order of potency is considered characteristic for P2X purinoceptors as it has been defined in the urinary bladder (Hoyle, Knight and Burnstock, 1990) and in a number of blood vessels (see Burnstock, 1991). The existence of P2X purinoceptors causing contraction of the colon circular

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Figure 4.5 Schematic illustration of the extrinsic parasympathetic and sympathetic efferent pathways to colonic musculature. Preganglionic nerve fibres running with the pelvic nerves synapse with motoneurones in ganglia of the pelvic and myenteric plexus. In addition to the cholinergic contraction, the pelvic nerves induce NANC inhibitory responses. The putative inhibitory neurotransmitters released at the level of the neuromuscular junction, NO and VIP, contribute to the development of parasympathetic motor responses. Sympathetic postganglionic fibres emerging from the inferior mesenteric ganglion innervate the circular muscle of the colon and produce excitatory responses in non-sphincteric regions. There are pharmacological evidences that ATP is co-released with noradrenaline at the neuromuscular junction. The sympathetically mediated motor responses express both excitatory and inhibitory components induced by the activation of smooth muscle P2X purinoceptors and α1-adrenoceptors causing contractions and β-adrenoceptors causing relaxation. The activity of parasympathetic and sympathetic motor pathways to the large intestine is integrated by sympathetic nerve regulation via presynaptic α-adrenoceptors and α2-autoreceptors.

muscle was confirmed by the rightward shift of the concentration-effect curves in the presence of suramin, a selective and competitive antagonist of the contractile responses to α,β-MeATP and ATP in other smooth muscles (Leff, Wood and O’Connor, 1990; von Kügelgen, Bültmann and Starke, 1990). In the cat colon circular muscle suramin acted as a competitive antagonist of α,β-MeATP contractions with a pA2 value of 4.45 (Kb = 30.6 µM) and a slope of the Schild plot of 1.06. However, the contractile responses to ATP were less sensitive to suramin (Kb = 52.3 µM) suggesting that ATP may either activate a suramin-insensitive component of the P2X purinoceptor as was suggested in the mouse vas deferens (von Kügelgen, Bültmann and Starke, 1990) or may interact with another “nucleotide” receptor (O’Connor, Dainty and Leff, 1991). The latter is consistent with the development of inhibitory responses to ATP and 2-MeSATP in colonic circular muscles precontracted with noradrenaline. However, α,β-MeATP and β,γ-MeATP induced only contractile responses which were blocked by suramin, regardless of the

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initial level of smooth muscle tone. A purine receptor which differs from the P2X purinoceptor, and is activated by ATP or by adenosine derived as the product of ATP degradation was suggested to be responsible for an inhibitory effect in the cat colon. Reactive blue 2, a selective antagonist of P2Y purinoceptors (Manzini, Hoyle and Burnstock, 1986), inhibited the relaxation induced by ATP or 2-MeSATP by 80–90%. The remaining relaxation was blocked by 8-(p-sulfophenyl)-theophylline, an antagonist of adenosine P1 purinoceptors (Kennedy, 1990). Furthermore, at low levels of muscle tone, reactive blue 2 increased the amplitude of contractile responses to ATP but did not change the contractions to α,β-MeATP. Data from other experiments indicate that inhibitory P2Y purinoceptors in the gastrointestinal tract are not restricted to the circular muscle of the colon, because relaxations to ATP which are blocked by reactive blue 2 were found in different regions including the gastric fundus (Lefebvre and Burnstock, 1991), duodenum (Manzini, Maggi and Meli, 1985) and caecum (Manzini, Hoyle and Burnstock, 1986). The pharmacological analysis of purine effects in the colon proved the existence of three distinct types of smooth muscle purinoceptors: P2X purinoceptors causing contraction, P2Y purinoceptors causing relaxation and inhibitory P1 purinoceptors activated by the hydrolytic breakdown of ATP to adenosine. The distribution of receptors amongst the extrinsic sympathetic and parasympathetic pathways to the colon that has been described in this review are schematically summarised in Figure 4.5.

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Hoyle, C.H., Knight, G.E. and Burnstock, G. (1990). Suramin antagonises responses to P2-purinoceptor agonists and purinergic nerve stimulation in the guinea-pig urinary bladder and taenia coli. British Journal of Pharmacology, 99, 617–621. Hubel, K.A. (1985). Intestinal nerves and ion transport: stimuli, reflexes and responses. American Journal of Physiology, 248, G261–G271. Hulten, L. (1969). Extrinsic nervous control of colonic motility and blood flow. An experimental study in the cat. Acta Physiologica Scandinavica Supplementum, 335, 1–116. Kennedy, C. (1990). P1- and P2-purinoceptor subtypes-an update. Archives Internationales de Pharmacodynamie et de Therapie, 303, 30–50. Kennedy, C. and Krier, J. (1987a). (Met5)enkephalin acts via δ-opioid receptors to inhibit pelvic nerve-evoked contractions of cat distal colon. British Journal of Pharmacology, 92, 291–298. Kennedy, C. and Krier, J. (1987b). δ-Opioid receptors mediate inhibition of fast excitatory postsynaptic potentials in cat parasympathetic colonic ganglia. British Journal of Pharmacology, 92, 437–443. Lefebvre, R.A. and Burnstock, G. (1990). Effects of adenosine triphosphate and related purines in the rat gastric fundus. Archives Internationales de Pharmacodynamie et de Therapie, 303, 199–215. Leff, P., Wood, B.E. and O’Connor, S.E. (1990). Suramin is a slowly equilibrating but competitive antagonist of P2X-receptors in the rabbit isolated ear artery. British Journal of Pharmacology, 101, 645–649. Li, C.G. and Rand, M.J. (1990). Nitric oxide and vasoactive intestinal polypeptide mediate non-adrenergic, noncholinergic inhibitory transmission to smooth muscle of the rat gastric fundus. European Journal of Pharmacology, 191, 303–309. Manzini, S., Maggi, C.A. and Meli, A. (1985). Further evidence for involvement of adenosine-5’-triphosphate in non-adrenergic, non-cholinergic relaxation of the isolated rat duodenum. European Journal of Pharmacology, 113, 399–408. Manzini, S., Hoyle, C.H.V. and Burnstock, G. (1986). An electrophysiological analysis of the effect of reactive blue 2, a putative P2-purinoceptor antagonist, on inhibitory junction potentials of rat caecum. European Journal of Pharmacology, 127, 197–204. McRorie, J., Krier, J. and Adams, T. (1991). Morphology and projections of myenteric neurones to colonic fibre bundles of the cat. Journal of the Autonomic Nervous System, 32, 205–215. Moncada, S., Palmer, R.M. and Higgs, E.A. (1991). Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacological Reviews, 43, 109–142. Nadelhaft, I., de Groat, W.C. and Morgan, C. (1980). Location and morphology of parasympathetic preganglionic neurones in the sacral spinal cord of the cat revealed by retrograde axonal transport of horseradish peroxidase. 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American Journal of Physiology, 250, G432–G439. Smith, T.K., Reed, J.B. and Sanders, K.M. (1987). Origin and propagation of electrical slow waves in circular muscle of canine proximal colon. American Journal of Physiology, 252, C215–C224. Starke, K., Gothert, M. and Kilbinger, H. (1989). Modulation of neurotransmitter release by presynaptic autoreceptors. Physiological Reviews, 69, 864–989. Szurszewski, J.H. and King, B.F. (1989). Physiology of prevertebral ganglia in mammals with special reference to inferior mesenteric ganglion. In Handbook of Physiology, Section 6, Vol. I, edited by J.D. Wood, pp. 465–518. Bethesda, Maryland: American Physiological Society. Timmermans, J.-P., Barbiers, M., Scheuermann, D.W., Bogers, J.J., Adriensen, D., Fekete, E., Mayer, B., Van Merck, E.A. and De Groodt-Lasseel, M.H. (1994). Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract. Cell and Tissue Research, 275, 325–345. 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5 Neurons of the Gallbladder and Sphincter of Oddi Gary M. Mawe, Erin K. Talmage, Lee J. Jennings, Kirk Hillsley and Audra L. Kennedy Department of Anatomy and Neurobiology, C423 Health Science Complex, The University of Vermont, Burlington, VT 05405 USA Following a meal, a cascade of events is initiated that results in the delivery of bile from the gallbladder to the lumen of the duodenum. An important messenger in the activation of this response, cholecystokinin, acts through neural mechanisms to increase pressure in the gallbladder and decrease resistance in the sphincter of Oddi, thus facilitating the flow of bile. Only recently have the neurons residing in the gallbladder and the sphincter of Oddi been directly studied to establish their morphological, electrical and neurochemical properties; the types of inputs that they receive; and how they respond to these signals. This chapter is organized to provide an overview of (1) the basic properties of gallbladder and sphincter of Oddi neurons, (2) the inputs to these cells that have been investigated, and (3) the reflex interactions that exist between the gut and the gallbladder, the gut and the sphincter of Oddi, and the gallbladder and the sphincter of Oddi. Although the neurons that migrate to, and ultimately innervate, the gallbladder and the sphincter of Oddi share a common ancestry with the neurons of the gut tube, the neurobiology of each of these regions is uniquely adapted to carry out their particular functions. KEY WORDS: autonomic ganglia; motility; innervation; enteric nervous system; biliary tract; immunohistochemistry; electrophysiology.

INTRODUCTION Developmentally, the sphincter of Oddi (SO) and the gallbladder arise from enteric precursors. The gallbladder forms as a ventral outgrowth of the primitive bile duct, and the SO forms as a specialization at the junction between the bile duct and the duodenum. The neural precursors that colonize these accessory regions of the gastrointestinal tract are likely to be derived from the vagal crest cells which also give rise to myenteric and submucous plexuses of the gut tube (Fontaine, Le Lievre and Le Douarin, 1977). Despite their common ancestry, the neurons that develop in the walls of the gallbladder and SO ultimately express electrical, morphological and neurochemical phenotypes that are different from one another, and different from those of the neurons in the gut tube. 189

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Over the past several years, efforts in this and other laboratories have concentrated on elucidating the properties of the neurons that directly innervate the gallbladder and the SO. This chapter includes a summary of the morphological, neurochemical, and electrophysiological findings that have been reported within the past few years, as well as a discussion of how the neurons of these regions are functionally linked with the neurons of the enteric nervous system.

GALLBLADDER GANGLIA STRUCTURAL AND ELECTRICAL PROPERTIES OF GALLBLADDER NEURONS Morphological properties of gallbladder ganglia and neurons The general layout of the neural plexuses of the gallbladder have been studied in many species, including human, rhesus monkey, pig, dog, cat, marmoset, guinea-pig, North American opossums (Didelphis virginiana and Monodelphis domestica), Australian brush tailed possum (Trichosurus vultecula), and mouse (Alexander, 1940; Burnett, Gairns and Bacsich, 1964; Sutherland, 1966; Sutherland, 1967; Kyösola, 1977; Cai and Gabella, 1983a; Mawe and Gershon, 1989; Bauer et al., 1991; Talmage and Mawe, 1993; Talmage et al., 1996). Each layer of the gallbladder is innervated by a plexus of nerve fibres; the most conspicuous of which is found in the serosal layer where a prominent ganglionated network is located just outside of the muscularis. Here, a matrix of small, irregularly shaped ganglia, is interconnected by bundles of unmyelinated axons. The nerve bundles of the ganglionated plexus are contiguous with nerve bundles that follow the numerous blood vessels that exist in this layer. The neural plexus of the muscularis is largely composed of nerve fibres that travel parallel to the direction of muscle bundles. This plexus is somewhat sparse in some species such as the guinea-pig, but rather prominent in others such as the dog and human. A dense network of nerve fibres and occasional small ganglia exists in the lamina propria of the gallbladder. Nerve fibres in the mucosal plexus may pass singly or in bundles, and they often are seen in close association with the epithelium. The ultrastructural properties of the ganglionated plexus of the gallbladder have been studied in the guinea-pig (Cornbrooks, Pouliot and Mawe, 1992). Ultrastructurally, the ganglia and interganglionic connectives of the gallbladder appear to be hybrids of the enteric and non-enteric autonomic systems. Like enteric ganglia, gallbladder ganglia are surrounded by a connective tissue sheath and a layer of basal lamina that is confined to the exterior of the ganglion. The ganglia are comprised of neurons, glial cells, and a compact neuropil that is usually displaced to the periphery of the ganglia. Collagen, basal laminae, and intercellular spaces are noticeably absent from the interior of gallbladder ganglia. The neuropil of gallbladder ganglia consists of glial processes, unmyelinated axons, and nerve terminals that contain clear spherical and dense core vesicles. Unmyelinated axons in interganglionic connectives of the gallbladder are individually ensheathed by Schwann cell processes, as in non-enteric peripheral nerve bundles. Individual gallbladder neurons have been stained by intracellular injection of horseradish peroxidase or neurobiotin in guinea-pig and opossum ganglia (Mawe, 1990; Bauer et al., 1991; Cornbrooks, Pouliot and Mawe, 1992). The shapes of these neurons are

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reminiscent of parasympathetic neurons, and are simpler than those of enteric neurons. Gallbladder neurons consist of a soma and one or two long processes, with no appreciable dendritic arborization. The long processes of these cells appear to be mainly confined to the ganglionated plexus. The processes pass from their ganglion of origin into interganglionic nerve bundles where they often project for some distance and terminate. Frequently these processes exhibit large varicosities as they traverse one or more adjacent ganglia. This indicates that the circuitry for interganglionic communication exists in the ganglionated plexus of the gallbladder. It is doubtful that the intrinsic neurons of the gallbladder play a role in controlling vascular tone within the organ since processes from gallbladder ganglion cells do not project to the para- or perivascular plexuses that are associated with the rich vascular bed that exists in the serosa of the guinea-pig gallbladder. Electrical properties of gallbladder neurons Gallbladder neurons have been studied with intracellular recording techniques in the guinea-pig, North American opossum and human. In their resting state, gallbladder neurons are rather inexcitable, as spontaneous activity is only rarely observed, and they do not generate repetitive action potentials throughout the duration of a prolonged depolarizing current pulse (Table 5.1, Figure 5.1; Mawe, 1990; Bauer et al., 1991; Hillsley, Jennings and Mawe, 1998). Guinea-pig and human gallbladder neurons typically fire only one action potential at the onset of a prolonged depolarizing current pulse, though occasionally up to four spikes are recorded. In the guinea-pig, the upstroke of the action potential is largely due to a tetrodotoxin (TTX)-sensitive Na+ conductance, but a Ca2+ spike can be revealed in the presence of TTX and tetraethylammonium (TEA). The after-hyperpolarization (AHP) of guinea-pig gallbladder neurons, which lasts about 170 ms, involves two sequential Ca2+-dependent K+ events (Mawe, 1990). The AHP has a reversal potential at the equilibrium potential for potassium, and is completely abolished in a Ca2+-free Krebs solution. The early phase of the AHP is attenuated in the presence of TEA, and is likely to result from the activation of large-conductance Ca2+-activated potassium channels (BK channels), whereas the late phase of the AHP is diminished in the presence of apamin and is likely to involve small-conductance Ca2+-activated K+ channels (SK channels) (Figure 5.1B; Mawe, 1990). The late phase of the AHP apparently contributes to the rapid adaptation of TABLE 5.1 Electrical properties of guinea-pig gallbladder and sphincter of Oddi neurons. Data from Mawe, 1990; Wells and Mawe, 1993. Gallbladder

Excitability Membrane potential Input resistance AHP duration Morphology

Sphincter of Oddi

Gallbladder cell

Tonic cell

Phasic cell

AH cell

Low ≈−50 mV ≈80 MΩ ≈180 Ms Parasympathetic

High ≈−50 mV ≈70 MΩ ≈20 Ms Dogiel I

Low ≈−50 mV ≈70 MΩ ≈20 Ms Dogiel I

Low ≈−65 mV ≈45 MΩ >2 s Dogiel II

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Figure 5.1 Active electrical properties of guinea-pig gallbladder neurons. A. Four consecutive overlapping traces showing the response of a neuron to prolonged depolarizing current pulses (250 ms; 0.2 nA). As is typical of these cells, this neuron generated only one action potential at the onset of the current pulse. B. Response of a neuron to a brief current pulse illustrating a typical AHP, which is composed of an early and a late phase that are sensitive to tetraethyl ammonium and apamin, respectively. C. Elimination of the late phase of the AHP by apamin (100 nM) converts the neuron from an adaptive to a non-adaptive state, thus allowing for the generation of repetitive action potentials. (From Mawe et al., 1997).

guinea-pig gallbladder neurons since suppression of this component of the AHP with apamin causes the cells to fire action potentials repetitively throughout the duration of a depolarizing current pulse (Figure 5.1C; Mawe, 1990). The neurons of the North American opossum gallbladder are classified into two groups (Bauer et al., 1991), adaptive neurons which respond to intracellular current pulses with a short burst of action potentials, and rapidly adaptive neurons that respond to current pulses with a single action potential. The adaptive cells are more numerous, comprising about 70% of the population. Action potentials of neurons in these ganglia are TTX-sensitive, and are followed by a brief AHP lasting about 30 ms. Synaptic inputs to guinea-pig gallbladder neurons Synaptic input to gallbladder neurons, in response to stimulation of interganglionic fibre bundles, has been studied in the guinea-pig and the opossum (Mawe, 1990; Bauer et al.,

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1991; Hillsley, Jennings and Mawe, 1998). Both fast and slow excitatory postsynaptic potentials (EPSPs) can be elicited, but inhibitory synaptic potentials have not been reported. Fast EPSPs, which can be activated by low frequency (0.5 Hz) stimulation of interganglionic fibre tracts, are sensitive to hexamethonium and are reversibly eliminated when the tissue is bathed in a Ca2+-free/high Mg2+ Krebs solution. Although interganglionic communication in the form of nicotinic EPSPs is likely to exist in these ganglia, the major source of nicotinic input to gallbladder neurons is from vagal preganglionic nerve fibres (see section on Vagal efferent fibres provide the driving force for gallbladder neurons; Mawe, Gokin and Wells, 1994). Slow EPSPs can be elicited by high frequency fibre tract stimulation (10–20 Hz) in about 30% of the neurons in guinea-pig (Mawe, 1990), and 20% of the neurons in opossum (Bauer et al., 1991). In the guinea-pig, the most thoroughly studied species, these slow synaptic events are Ca2+-dependent, and are associated with a decrease in input resistance. The slow EPSP is likely to involve an activation of a non-selective cation conductance since its amplitude decreases as cells are depolarized and increases when the membrane is hyperpolarized, with an estimated reversal potential near 0 mV. Recent evidence suggests that tachykinins and calcitonin gene-related peptide (CGRP) released from extrinsic afferent fibres mediate slow EPSPs in gallbladder ganglia (see section on Extrinsic sensory fibres; Mawe, 1995).

REGULATORY INPUTS TO GALLBLADDER GANGLIA Vagal efferent fibres provide the driving force for gallbladder neurons As described above, all gallbladder neurons exhibit nicotinic fast EPSPs in response to stimulation of interganglionic fibre tracts (Mawe, 1990; Bauer et al., 1991; Hillsley, Jennings and Mawe, 1998). Since gallbladder ganglia are developmentally associated with enteric ganglia, and since synaptic inputs to enteric ganglia are mainly intrinsic (Furness and Costa, 1987; Wood, 1989; Costa et al., 1992), it could not be presumed that nicotinic inputs to gallbladder neurons are from preganglionic neurons. However, it does appear that all gallbladder neurons do receive nicotinic input from the vagal preganglionic fibres. Several lines of evidence verify a significant vagal input to gallbladder ganglia. Stimulation of the vagus nerves causes gallbladder contraction, or, in the presence of atropine, relaxation (Ryan, 1987; Dodds, Hogan and Geenen, 1989; Dahlstrand, 1990). Neurons in the dorsal motor nucleus of the vagus have been retrogradely labelled from the gallbladder (Mawe, 1990). Also, nerve fibres that are immunoreactive for the biosynthetic enzyme of acetylcholine, choline acetyltransferase (ChAT), are present in ganglia, surrounding gallbladder neurons, and in the paravascular plexus, indicating that extrinsic cholinergic nerve fibres enter the gallbladder. Data from a study involving stimulation of extrinsic inputs to the gallbladder in normal and vagotomized guinea-pigs indicate that all gallbladder neurons receive vagal input, and that the vagal input is the major source of fast synaptic input to gallbladder neurons (Mawe, Gokin and Wells, 1994). Vagotomy results in the complete elimination of extrinsic nicotinic inputs, which are normally received by all gallbladder neurons. Therefore, unlike the ganglion cells in the bowel, all gallbladder neurons receive direct input

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from the central nervous system. Following vagotomy, fast EPSPs could be elicited in some neurons by stimulation of interganglionic fibre tracts, indicating that interganglionic neural projections may exist amongst gallbladder ganglia. Sympathetic postganglionic fibres In addition to receiving input from vagal preganglionic fibres, intramural ganglia are the target of sympathetic postganglionic inputs to the gallbladder. The ganglionated plexus of the gallbladder is rich in nerve fibres that express catecholamine histofluorescence (Cai and Gabella, 1983a, 1984; Mawe and Gershon, 1989), as well as immunoreactivities for tyrosine hydroxylase and dopamine β-hydroxylase (Mawe and Gershon, 1989). It is likely that these nerve fibres arise in the celiac ganglia because celiac neurons are labelled following injection of retrograde axonal tracers in the wall of the gallbladder (Mawe and Gershon, 1989). We have established that noradrenaline can be released from catecholaminergic nerve fibres and that it acts to attenuate the release of acetylcholine from vagal terminals in gallbladder ganglia (Mawe, 1993; Mawe, Gokin and Wells, 1994). Exogenously applied noradrenaline decreases the amplitude of fast EPSPs in a concentration-dependent manner by acting on α2-adrenoceptors. The action of noradrenaline is mimicked by the α2-adrenoceptor agonist, clonidine, and is antagonized by the α2-adrenoceptor antagonist, yohimbine. When endogenous catecholamines are released from sympathetic nerves by tyramine application or by electrical stimulation of the vascular plexus, a yohimbine-sensitive decrease in fast synaptic activity is observed. Since it has been demonstrated that the major source of fast synaptic input to gallbladder neurons is vagal preganglionic fibres, and that fast EPSPs elicited by cystic nerve stimulation are sensitive to noradrenaline (Mawe, Gokin and Wells, 1994), it is likely that vagal input to the gallbladder can be attenuated by sympathetic activity. Therefore, the decrease in gallbladder tone, that can be elicited by stimulation of the splanchnic nerves (Pallin and Skoglund, 1964; Persson, 1971, 1972, 1973; Yamasato and Nakayama, 1990), may be the result of a presynaptic inhibitory effect of sympathetic nerves on the vagal terminals in gallbladder ganglia. Yamasato and Nakayama (1990) reported that subthreshold stimulation of the celiac nerve in the dog, which had no effect on gallbladder motility, markedly decreased the responsiveness of the gallbladder to vagal stimulation. It is possible that this inhibitory influence of sympathetic input on the vagal tone facilitates gallbladder filling. Furthermore, administration of sympathomimetics for treatment of low blood pressure could have the side effect of decreasing gallbladder motility by decreasing vagal output to gallbladder ganglia, thus leading to gallbladder stasis. Extrinsic sensory fibres The concept that sensory neurons can release neuroactive compounds from their peripheral processes, and initiate local reflex activity, has now been established in several organ systems, including the gallbladder. In the gallbladder, nerve fibres that are immunoreactive for substance P (SP) and CGRP are abundant in the ganglionated and vascular plexuses (Goehler, Sternini and Brecha, 1988; Maggi et al., 1989; Mawe and Gershon,

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1989). Furthermore, application of capsaicin to gallbladder strips causes contraction and the release of SP and CGRP (Maggi et al., 1989). Since gallbladder neurons do not express CGRP-immunoreactivity (Goehler, Sternini and Brecha, 1988; Mawe and Gershon, 1989; Talmage et al., 1992, 1996), the SP/CGRP-immunoreactive nerve fibres must be extrinsic and are likely to arise from thoracic sensory ganglion cells which project to the gallbladder (Mawe and Gershon, 1989). Application of tachykinins, CGRP or capsaicin to guinea-pig gallbladder neurons causes a depolarization that is similar to the slow EPSP recorded in these preparations (Mawe, 1995; Gokin, Jennings and Mawe, 1996). Both tachykinins and CGRP depolarize gallbladder neurons by activating a non-selective cation conductance, and increase the excitability of these cells. The tachykinin-mediated depolarization of gallbladder neurons has been studied with receptor-specific agonists and antagonists (Mawe, 1995). Naturally occurring tachykinins depolarize gallbladder neurons with a rank order potency that is characteristic of neurokinin-3 (NK3) receptors (neurokinin B > neurokinin A > SP). Consistent with this finding, the NK3 receptor agonist, senktide, was more potent than any of the naturally occurring tachykinins in eliciting a depolarization. Once the involvement of NK3 receptors was established, [Trp7,β-Ala8 ]-NKA(4–10), which acts as an antagonist at NK3 receptors, was used in the preparation. This compound shifted the concentration-effect curve for SP to the right, and depressed both capsaicin-induced depolarizations and stimulus-evoked slow EPSPs. Therefore, sensory fibres are likely to contribute to slow EPSPs in gallbladder ganglia by releasing tachykinins, and probably CGRP, in an axon reflex response. It is possible that such a response may contribute to gallbladder inflammation since release of neuroactive peptides from extrinsic sensory fibres is one of the initial steps in toxin A-induced inflammation in the bowel (Mantyh et al., 1996). Hormonal cholecystokinin Cholecystokinin (CCK) has long been recognized for its ability to cause gallbladder contractions (Ivy and Oldberg, 1928). However, the issue of whether CCK elicits its physiological effect through a direct action on gallbladder smooth muscle or by facilitating the neural output to the organ has been debated. Over the past 10 years, studies from several different laboratories provide strong support for the concept that, physiologically, CCK acts through neural mechanisms to cause gallbladder emptying. Studies of in vivo preparations have provided support for the view that the principle physiological effect of CCK in the gallbladder involves a neural mechanism (Grossman, 1975; Behar and Biancani, 1980, 1987; Takahashi et al., 1982; Gullo et al., 1984; Fisher, Rock and Malmud, 1985; Marzio et al., 1985; Strah et al., 1985, 1986; Pozo, Salida and Madrid, 1989; Hanyu et al., 1990b; Takahashi, May and Owyang, 1991). These studies demonstrate that gallbladder contractions elicited by feeding, or by intravascular injections of physiological post-feeding concentrations of CCK, were disrupted by atropine, hexamethonium, TTX, or vagal blockade. Similar results have been reported in an in vitro preparation by Brotschi, Pattavino and Williams, (1990). Furthermore, when applied to gallbladder muscle strips, CCK causes the release of ACh (Yau and Youther, 1984; Yamamura et al., 1986; Rakovska, Milenov and Bocheva, 1989; Galligan and Bertrand,

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1994). Taken together, these data indicate that it is likely that feeding and CCK-induced physiological contraction of the gallbladder occurs through a neural mechanism rather than a direct action of CCK on smooth muscle. It is clear that CCK receptors do exist on gallbladder smooth muscle cells, but these receptors may not normally play a role in gallbladder emptying. It is plausible that the neural effect that was described was a result of actions of CCK on vagal afferent fibres and/or vagal terminals within gallbladder ganglia since hexamethonium and vagal blockade disrupt the meal- and CCK-induced gallbladder contraction (Hanyu et al., 1990b; Takahashi, May and Owyang, 1991). Electrophysiological studies of gallbladder whole mount preparations support this view. Direct studies of the actions of CCK in gallbladder ganglia have been conducted in the guinea-pig (Mawe, 1991; Mawe, Gokin and Wells, 1994) and in the opossum (Bauer et al., 1991) using intracellular electrophysiological recording techniques. In both of these species, CCK has a profound presynaptic facilitory effect on ganglionic transmission, but does not have a direct effect on the gallbladder neurons. Upon application of CCK, the amplitude of cholinergic fast EPSPs is increased, usually converting subthreshold EPSPs to suprathreshold EPSPs (Bauer et al., 1991; Mawe, 1991). CCK increases the quantal content three-fold without altering quantal size, but does not alter the sensitivity of these neurons to exogenously applied acetylcholine, indicating that CCK acts through a presynaptic mechanism (Mawe, 1991). Most importantly, it has been shown that CCK is quite potent in its ability to promote the release of acetylcholine. The concentration-effect relationship for CCK in gallbladder ganglia peaks at 1.0 nM, and has a half-maximal effective concentration (EC50) of 33 pM; the EC50 for the direct contractile effect of CCK on gallbladder muscle is 10 nM. In the presence of 10 pM CCK, which is within the range of postprandial serum levels of CCK (Takahashi, May and Owyang, 1991), the peptide increases synaptic currents by about 20%. The nerve terminals that are sensitive to CCK are from the vagus nerve since synaptic responses to cystic nerve stimulation are sensitive to CCK, and since these inputs are eliminated following vagotomy (Mawe, Gokin and Wells, 1994). CHEMICAL CODING OF GALLBLADDER NEURONS Despite the cumulative knowledge that has been gained from motility and ganglion electrophysiological studies, it is not entirely clear what compounds are actually released from gallbladder neurons to modulate smooth muscle function. It is recognized, from muscle strip studies involving electric field stimulation, that excitatory transmitters are released to promote gallbladder emptying, and it is possible that inhibitory transmitters are released to promote gallbladder filling. Two major theories have been proposed to explain how the gallbladder fills. One theory suggests that the gallbladder undergoes a “passive” filling between meals (Ryan, 1987; Shaffer, 1991), and the other theory suggests that the gallbladder actively expands to draw hepatic bile into its lumen, much as a bellows draws air in as it is expanded (Lanzini, Jazrawi and Northfield, 1987; Jazrawi et al., 1995). In order for the gallbladder to act as a bellows, a potent inhibitory output from the ganglia of the gallbladder would be necessary to induce an active relaxation. An important step in determining how gallbladder neurons could influence gallbladder function was to identify the neurochemical phenotypes of gallbladder neurons. We and

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others have studied the chemical coding of gallbladder neurons in several species, including guinea-pig, human, dog and opossum, and have identified sets of neuroactive compounds that are co-expressed. Characteristic trends, as well as interspecies differences, have emerged from these investigations. For example, all gallbladder neurons are likely to be cholinergic since all detectable neurons express immunoreactivity for the essential biosynthetic enzyme for acetylcholine, choline acetyltransferase (Talmage et al., 1996). Furthermore, gallbladder neurons can express immunoreactivities for tachykinins, vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS) and either neuropeptide Y (NPY) (guinea-pig and human) or galanin (dog and opossum) (Keast, Furness and Costa, 1985; Talmage et al., 1992, 1996; De Giorgio et al., 1995). Species specific patterns of neuropeptide co-expression have been identified in neurons of the human, canine and guinea-pig gallbladder (Talmage et al., 1992, 1996; De Giorgio et al., 1995). These are schematically represented in Figure 5.2. In the human, the vast majority of neurons express VIP and NPY, and most of these neurons express SP as well (De Giorgio et al., 1995; Talmage et al., 1996). In the dog, where NPY appears to be replaced by galanin, most neurons express VIP and galanin, and most of these express SP as well (Talmage et al., 1996). In the guinea-pig, most neurons express SP and NPY, and a distinct subpopulation of neurons expresses VIP (Talmage et al., 1992). It is clear that gallbladder ganglia comprise neurons that express the enzymatic machinery to synthesize nitric oxide. In all species that have been studied, including the human (Talmage and Mawe, 1993; De Giorgio et al., 1995), monkey (De Giorgio et al., 1994), dog (Talmage and Mawe, 1993), opossum (Talmage and Mawe, 1993), guinea-pig (Talmage and Mawe, 1993; Grozdanovic et al., 1994; Siou, Belai and Burnstock, 1994), gerbil (Talmage and Mawe, 1993) and mouse (Grozdanovic, Baumgarten and Bruning, 1992), neurons in the gallbladder express NOS and/or NADPH-diaphorase (NADPH-DA) activity. In guinea-pig there is a direct correlation between NOS-immunoreactivity and NADPH-DA activity in gallbladder ganglia (Mawe et al., 1997). As mentioned above, all gallbladder neurons express ChAT; coexpression of ChAT and NOS is also observed in these ganglia (Figure 5.3). The physiological role of neurally released nitric oxide in the gallbladder, if there is one, is unclear at this time. However, nitric oxide can relax the gallbladder (Mourelle et al., 1993a; McKirdy, McKirdy and Johnson, 1994) and may modulate the CGRP-induced relaxation of the gallbladder (Kline and Pang, 1994).

Figure 5.2 Chemical coding in neurons in ganglia of the human, dog and guinea-pig gallbladder. Note the coexpression of neuroactive compounds that have excitatory (+) and inhibitory (–) effects on gallbladder smooth muscle. ACh, acetylcholine; VIP, vasoactive intestinal peptide; NPY, neuropeptide Y; SP, substance P; NOS, nitric oxide synthase. (Modified from Talmage et al., 1996).

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Figure 5.3 Immunoreactivities for ChAT and NOS in ganglia of the guinea-pig gallbladder and sphincter of Oddi. Immunoreactivities in these double stained preparations illustrate the co-expression of ChAT and NOS in a subset of gallbladder neurons, and the exclusive expression of ChAT or NOS by sphincter of Oddi neurons. Note that in the micrographs of the SO ganglion, the NOS-immunoreactive neurons are located in the spaces that are devoid of ChAT immunoreactivity.

Unlike the ganglia of the bowel, ganglia of the gallbladder lack CGRP-immunoreactive neurons. Immunohistochemical studies in the human (De Giorgio et al., 1995; Talmage et al., 1996), dog (Talmage et al., 1996), pig (Sand, Tainio and Nordback, 1993), guineapig (Goehler, Sternini and Brecha, 1988; Mawe and Gershon, 1989; Talmage et al., 1992), Australian possum (Padbury, 1990), Monodelphis domesticus opossum (Talmage et al., 1996), and toad (Davies and Campbell, 1994) failed to identify neurons in the gallbladder wall that expressed immunoreactivity for CGRP. However, CGRP-immunoreactive nerve fibres have been described in the gallbladders of all of these species, where they are most abundant in the ganglionated plexus and in association with blood vessels, primarily in the paravascular plexus. In the human, dog, opossum, Australian possum, guinea-pig, and toad gallbladders these fibres have been shown to co-express SP-immunoreactivity, and probably originate in sensory ganglia (Goehler, Sternini and Brecha, 1988; Mawe and Gershon, 1989; Talmage et al., 1996). It is quite possible that CGRP can be released from these fibres as part of an axon reflex circuit (see section on Extrinsic sensory fibres of gallbladder ganglia), and exert influences on neurons, muscle and/or epithelial cells. CGRP causes a relaxation of gallbladder muscle (Kline and Pang, 1992) that is due to an activation of an ATP-dependent K+ conductance (Zhang et al., 1994). As described above, in guinea-pig gallbladder ganglia, CGRP causes a depolarization of neurons that is quite similar to the slow excitatory postsynaptic potential that occurs in these ganglia (Gokin, Jennings and Mawe, 1996).

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Together with the knowledge that all of the gallbladder neurons are cholinergic, these patterns of expression of neuroactive compounds are somewhat perplexing; as they suggest the coexpression of excitatory and inhibitory transmitters in single neurons of all of these species. In muscle strip assays, acetylcholine (Ryan, 1987), SP (Meldrum, Bojarski and Calam, 1987; Dahlstrand et al., 1988; Guo et al., 1989; Maggi et al., 1989) and NPY (Lillemoe, Webb and Pitt, 1988) have been shown to increase gallbladder tone, whereas VIP (Ryan and Cohen, 1977; Ryan and Ryave, 1978; Dahlstrand, Dahlström and Ahlman, 1989) can relax the precontracted gallbladder. The ostensibly opposing outputs from individual gallbladder neurons that express excitatory and inhibitory compounds could be explained by the following theoretical scenarios: (1) excitatory and inhibitory neuroactive compounds are separately released from a given neuron in response to distinct inputs; (2) compounds with opposing actions are released onto the same target sequentially, with one acting as a physiological antagonist of the other; or (3) both sets of compounds are co-released, but act on different targets. For example, if acetylcholine, SP and VIP were released together, acetylcholine and SP may act on the muscle to elicit a contraction, while VIP may act on epithelial cells. Neuroactive compounds may also act on adjacent nerve terminals to modulate further release. Further studies will be required to test these models.

SPHINCTER OF ODDI GANGLIA To date, many studies that have involved the direct study of SO ganglia have involved the guinea-pig model. Therefore, most of the following information is related to ganglia of the guinea-pig SO region. This is important to note because considerable interspecies variation exists with regard to the structure and function of the SO (see Mawe, 1992). In the guinea-pig, this area includes the terminal portion of the common bile duct (choledochal sphincter), an ampulla, and a terminal papilla (Dahlstrand et al., 1989; Hirose and Ito, 1991; Mawe, 1992). The studies that are reviewed here relate to neurons located throughout the anatomically defined regions of the ampulla and the choledochal sphincter. No regional differences have been observed in the electrical or morphological characteristics of neurons in these areas. STRUCTURAL AND ELECTRICAL PROPERTIES OF SPHINCTER OF ODDI NEURONS Morphological properties of sphincter of Oddi ganglia and neurons The ganglionated plexus of the guinea-pig SO includes a network of ganglia and interganglionic fibre bundles that is similar in appearance to the myenteric plexus of the small intestine. When stretched and pinned out flat, the SO region encompasses an area of approximately 20–25 mm2 containing an average of about 60 ganglia, and a total of about 2000 neurons (Wells and Mawe, 1993). The structure of individual SO neurons has been determined by injecting neurobiotin into individual neurons, from intracellular recording electrodes, and labelling the neurobiotin

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with fluorophore- or horseradish peroxidase-conjugated avidin (Wells and Mawe, 1993). Guinea-pig SO neurons conform to the morphological classification scheme described by Dogiel for myenteric neurons (see Furness and Costa, 1987). The neurons of the SO that were classified as both tonic and phasic cells (see section on Electrophysiological properties of gallbladder neurons), which together represent the majority of SO neurons, have the shape of Dogiel type I cells. These neurons typically have a single long process and several very short processes emanating from the soma. In most cells, the short processes were flattened and “club-shaped”, and the remainder of the neurons had several thin processes that were approximately the length of the somal diameter. The long process of most cells, thought to be the axon, usually projects into adjacent ganglia. These processes frequently end as expansion bulbs, indicating that they may have been severed during the dissection. Processes of some cells were followed to strands of circular muscle that had not been removed during the tissue preparation. A small proportion of the neurobiotin-filled SO neurons, which are classified electrophysiologically as AH cells, are shaped like Dogiel type II cells. These neurons have several long branched processes that terminated both within the ganglionated plexus as well as within the muscularis. Calbindin-immunoreactive neurons in SO ganglia have a shape similar to that of the AH cells. Electrical properties of sphincter of Oddi neurons Unlike the guinea-pig gallbladder, where only one electrically classified type of neuron exists, three classes of neurons have been identified in the guinea-pig SO, on the basis of their active and passive electrical properties as determined by intracellular voltage recordings (Table 5.1, Figure 5.4; Wells and Mawe, 1993). The two most prevalent cell types, which together comprise about 95% of the total neuronal population, are called tonic and phasic cells. Tonic cells are very excitable; they generate spontaneous action potentials and elicit action potentials continuously throughout a prolonged suprathreshold depolarizing current pulse. The tonic cells of the SO are comparable to the S/type 1 cells of the small intestine (Wood, 1989) and rectum (Tamura and Wood, 1989), and the gastric Type I (Schemann and Wood, 1989) and colonic type 1 cells (Wade and Wood, 1988). Phasic cells have passive membrane characteristics that are similar to tonic cells, but differ in their active properties. Phasic cells do not exhibit spontaneous activity, and when directly

Figure 5.4 Typical responses of sphincter of Oddi tonic and phasic cells to a prolonged depolarizing current pulse. Tonic cells, which often exhibit spontaneous activity, generate action potentials throughout the duration of a depolarizing current pulse. Phasic cells, which are not spontaneously active, generate action potentials only at the onset of the current pulse, regardless of its amplitude or duration.

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stimulated, they adapt rapidly, typically firing only 1–3 action potentials at the onset of a prolonged depolarizing current pulse. This type of neuron has not been reported in the duodenum or small intestine; however neurons with similar properties have been described in the stomach (gastric type II; Schemann and Wood, 1989; Tack and Wood, 1992) and the distal colon (colonic type 4; Wade and Wood, 1988). The remainder of SO neurons, comprising about 5% of the population, are called AH cells. These cells have properties identical to those of the Type 2/AH cells of the bowel (Wood, 1989). Action potentials in these cells, which are TTX-insensitive, have a broadened shoulder during the repolarization, and the AHP of these cells lasts several seconds. Synaptic inputs to sphincter of Oddi neurons When interganglionic fibre bundles are stimulated, three different types of synaptic potentials can be elicited: fast EPSPs, slow EPSPs and inhibitory postsynaptic potentials (IPSPs). The fast excitatory input, which can be activated by low frequency stimulation, is present in most tonic and phasic cells, and some AH cells. These events are reversibly eliminated when the tissue is bathed in a Ca2+-free/high Mg2+ Krebs solution, and they are sensitive to hexamethonium. Potential sources of fast synaptic input to SO neurons include other SO neurons, neurons of the duodenum, and vagal preganglionic fibres. In some SO neurons, membrane depolarizations and hyperpolarizations can be activated by trains of high-frequency (10–20 Hz, 0.5 ms pulse) fibre tract stimulation (Wells and Mawe, 1993). These responses are eliminated by exposure to Ca2+-free Krebs, and therefore are presumed to be slow EPSPs and IPSPs. The sources and ionic mechanisms of slow excitatory inputs have not been evaluated, but SO neurons could receive modulatory input from other SO neurons, duodenal neurons, and/or extrinsic sensory fibres. Inhibitory synaptic inputs to SO neurons probably involves the activation of a K+ conductance, and these inputs are provided by sympathetic postganglionic input from the coeliac ganglion (see below). REGULATORY INPUTS TO SPHINCTER OF ODDI GANGLIA As indicated above, not as much is known about the neural control of the SO relative to other regions of the gut, including the gallbladder. Of the potential physiological modulators of SO ganglion function, two that have been studied directly in SO ganglia are sympathetic postganglionic inputs and CCK. Sympathetic inputs to sphincter of Oddi ganglia The action of sympathetic input to this region appears to increase SO tone. Activation of the sympathetic input to the feline SO, in vivo, by stimulating the splanchnic nerves or postganglionic nerve bundles, increases the tone of the sphincter musculature (Persson, 1972, 1973), whereas in non-sphincter regions of the gut, sympathetic input results in decreased motility (see Furness and Costa, 1987). Although the muscularis of the SO does contain catecholamine-histofluorescent nerve fibres, the area of most dense catecholaminergic innervation lies within the intrinsic

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ganglionated neuronal plexus (Baumgarten and Lange, 1969; Mori, Azuma and Fujiwara, 1971; Cai and Gabella, 1983b). In the ganglionated plexus of the SO, immunoreactivities for tyrosine hydroxylase and dopamine β-hydroxylase are abundant, and coexistent in nerve fibres, but these synthetic enzymes are not expressed by SO neurons (Wells and Mawe, 1994). The actions of noradrenaline and endogenous catecholamine release have been studied in the guinea-pig SO (Wells and Mawe, 1994). Exogenous noradrenaline reversibly decreases the amplitude of fast EPSPs evoked by stimulation of interganglionic fibre tracts. Since noradrenaline does not alter the responsiveness of the neurons to acetylcholine, the action of noradrenaline in SO ganglia is thought to be presynaptic. The α2-adrenoceptor agonist, UK14304, mimics the noradrenaline-induced effect, and this effect is attenuated by the α2-adrenoceptor antagonist, Idazoxan. Agonists of α1- and β-adrenoceptor have no detectable effect in SO ganglia. Results of experiments involving application of exogenous noradrenaline are supported by the finding that release of endogenous catecholamines, by tyramine, causes an idazoxan-sensitive decrease in the fast EPSP. In SO neurons that exhibit IPSPs, noradrenaline causes hyperpolarization of the membrane potential. The IPSP and the noradrenaline-induced hyperpolarization have reversal potentials near the K+ equilibrium potential, and are inhibited by α2-adrenoceptor antagonists. Further evidence for the concept that sympathetic nerves mediate IPSPs is that desipramine, a catecholamine reuptake inhibitor, reversibly increases the amplitude of the IPSP. The findings from morphological and electrophysiological studies indicate that sympathetic nerves target SO ganglia, and that within these ganglia, noradrenaline can act both pre- and postsynaptically as an inhibitory neurotransmitter. Therefore, ganglia of the SO can be added to the growing list of enteric and parasympathetic ganglia that receive interganglionic input from sympathetic postganglionic nerves. Although noradrenaline has actions in SO ganglia that are similar to actions in other gastrointestinal ganglia, the net effect of sympathetic input to the sphincter is contraction rather than relaxation. The activation of divergent net effects through common mechanisms supports the view that precise circuitry exists within ganglia in various regions of the gut. Sympathetic pre- and postsynaptic activity is likely to have the net effect of decreasing the release of inhibitory substances and/or increasing the release of excitatory neuroactive compounds. Since the effects of noradrenaline are inhibitory it is likely that it decreases the release of inhibitory compounds. It is not clear whether the IPSPs occur selectively on inhibitory neurons of the SO, but this type of circuitry could contribute to the increased SO tone that results from sympathetic nerve stimulation. Actions of CCK on sphincter of Oddi neurons At the level of the SO, the stimulatory effect of CCK on bile flow appears to involve a neural mechanism. Several studies, in various species, have shown that the actions of CCK on the SO can be attenuated by muscarinic blockade with atropine and/or complete neural blockade with TTX (Behar and Biancani, 1987; Vogalis, Bywater and Taylor, 1989; Hanyu et al., 1990a). Furthermore, CCK-induced release of neuroactive compounds such as acetylcholine (Harada, Katsuragi and Furukawa, 1986), VIP (Wiley, O’Dorisio

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and Owyang, 1988; Dahlstrand et al., 1990) and nitric oxide (Pauletzki et al., 1993) have been reported. The actions of CCK on guinea-pig SO neurons have been investigated by application of CCK while recording from SO neurons with intracellular recording electrodes (Gokin, Hillsley and Mawe, 1997). CCK has a direct, excitatory effect on tonic and phasic cells in SO ganglia. The depolarization that is elicited by CCK likely involves the activation of a non-selective cation conductance since it reverses near 0 mV, and it is significantly reduced in a low Na+ solution. The CCK-induced depolarization of tonic and phasic cells involves the CCK-A receptor subtype; it is significantly reduced by the CCK-A antagonist, PD 140,548 N-methyl-D-glucamine, but not by the CCK-B receptor antagonist, PD 135,158 N-methyl-D-glucamine. Although SO neurons express CCK receptors, and CCK can act on these receptors to depolarize SO neurons, it is disputable whether hormonal CCK could act physiologically at this site to alter SO tone. This is because of the discrepancy between the sensitivity of SO neurons for CCK and the peak concentrations of CCK in the serum following a meal. The lowest concentration of CCK that results in a detectable response in SO neurons is 1 nM, and the EC50 in these neurons is 10 nM. Since peak serum concentrations of CCK following a meal are in the low picomolar range, hormonal CCK would be delivered at subthreshold concentrations. It is possible that CCK could have a paracrine effect on SO neurons due to the close proximity of the SO to the duodenum, where CCK is released. Alternatively, CCK may alter SO tone through vagal and/or duodenum-SO neural circuits (see section NEURAL INTERACTION BETWEEN THE GUT AND THE SPHINCTER OF ODDI). CHEMICAL CODING OF SPHINCTER OF ODDI NEURONS To identify potential chemical coding in sphincter of Oddi neurons, immunohistochemistry and histochemistry have been employed to test for the presence of putative neurotransmitters and related synthetic enzymes. In the guinea-pig SO, the pattern of chemical coding is less ambiguous than that found in the gallbladder; there is a distinction between the expression of excitatory and inhibitory compounds in SO neurons (Figure 5.3). In the guinea-pig SO, about 70% of the neurons are immunoreactive for ChAT, indicating that they are likely to synthesize acetylcholine and most of these neurons are also immunoreactive for SP and enkephalin (Wells, Talmage and Mawe, 1995; Talmage et al., 1997). Since acetylcholine, SP and opiates increase SO tone (Azuma and Fujiwara, 1973; Kudoh et al., 1981, Behar and Biancani, 1984; Dahlstrand et al., 1985, 1988), and since nerve fibres that are immunoreactive for SP and ChAT are abundant in the muscularis of the SO (Talmage et al., 1997), it is likely that the SP/enkephalin/ChAT-immunoreactive population includes excitatory motor neurons. A distinct population of guinea-pig SO neurons express NOS, and some of the NOS-positive neurons express VIP, whereas others express NPY. The NOS/VIP and NOS/NPY neurons are likely to be inhibitory neurons because both nitric oxide (Allescher et al., 1992; Kaufman et al., 1993; Mourelle et al., 1993b; Pauletzki et al., 1993) and VIP (Behar and Biancani, 1980; Wiley, O’Dorisio and Owyang, 1988; Dahlstrand et al., 1990) have been shown to decrease SO tone. To determine whether a correlation exists between the electrical and chemical coding properties of SO neurons, we conducted a study combining intracellular recording and dye

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injection, along with chemical coding. Results of this study clearly demonstrated that there is no relationship between the electrical properties of SO neurons (Tonic versus Phasic) and their chemical coding patterns (cholinergic versus nitrergic) (Hillsley and Mawe, 1998).

REFLEX INTERACTIONS BETWEEN ENTERIC AND BILIARY NEURONS Since the beginning of the twentieth century, when Langman, Bayliss and Starling, and Trendelenberg were evaluating the neural activity of the bowel, it has been recognized that peristaltic reflex responses could be observed in the absence of the central nervous system. Evidence also exists for reflex interactions between the duodenum and the gallbladder and sphincter of Oddi, and between the gallbladder and sphincter of Oddi. Although these gutSO-gallbladder interconnections have not been evaluated as extensively as the intrinsic circuits of the gut, it is clear that the circuitry exists for reflex interactions between duodenal and biliary tract neurons.

NEURAL INTERACTIONS BETWEEN THE GUT AND THE GALLBLADDER In 1933, DuBois and Kistler found that stimulation of the duodenal ampulla causes the gallbladder to contract. This contractile response to ampulla stimulation was eliminated by transection of the bile duct, but gallbladder responses to vagal stimulation and stimulation of the cut end of the bile duct persisted. On the basis of these findings, DuBois and Kistler proposed that a direct neural connection exists between the gut and the gallbladder, and that these axons pass along the cystic duct. Morphological evidence from retrograde labelling experiments in the guinea-pig and Australian possum, show that the gallbladder receives extrinsic neural projections from several sources, and support the concept of a direct gut-gallbladder projection (Mawe and Gershon, 1989; Padbury et al., 1993). Sources of input to the gallbladder include the dorsal motor nucleus of the vagus nerve, nodose ganglia, coeliac ganglia, and thoracic dorsal root ganglia. In addition, when retrograde tracers were injected into the wall of the gallbladder, neurons of the duodenal myenteric plexus and the ganglia of the SO were labelled. These data indicate that, in addition to being regulated in conventional reflex responses that involve central nervous system processing, the circuitry exists for the gallbladder to receive direct inputs from the bowel. The evidence described above indicates that gut neurons project to the gallbladder where they exert an excitatory influence. However, the physiological relevance, if there is any, of such a pathway, and the precise origins and targets of the gut-gallbladder projection have not been resolved. According to the DuBois and Kistler study, activation of this circuit has an excitatory influence on gallbladder motor activity; however it is not known whether the target of this input is on gallbladder ganglia, on the smooth muscle cells, or at both of these locations. When retrograde tracers were injected into the gallbladder they could have been taken up by axon terminals in any layer of the organ.

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It is doubtful that the gut–gallbladder projection contributes to the fast EPSPs that have been studied in gallbladder ganglia. Following vagotomy, nicotinic input to gallbladder neurons, in response to stimulation of the nerve bundles that pass along the cystic duct, are completely eliminated (Mawe, Gokin and Wells, 1994). However, if this pathway is similar to the projection from enteric neurons to the pancreas (Kirchgessner and Pintar, 1991), there may be a serotonergic projection from the gut to the ganglia of the gallbladder. Nerve fibres that are immunoreactive for serotonin do exist in the ganglia of the gallbladder (Mawe and Gershon, 1989), and when serotonin is applied to gallbladder neurons, it causes a prolonged depolarization that is associated with an increase in the excitability of gallbladder neurons (Mawe, 1990). One potential physiological role of an excitatory neural pathway from the gut to the gallbladder would be to activate the gallbladder contractions that occur in coordination with the migrating myoelectric complex. During the interdigestive period, the gallbladder undergoes periods of increased intraluminal pressure, in phase with the migrating myoelectric complex, that are accompanied by a delivery of bile from the gallbladder to the lumen of the duodenum (Itoh et al., 1983; Ryan, 1987). It is thought that the migrating myoelectric complex serves a “housekeeping function”. According to this model, increased motor activity would advance undigested food from the proximal bowel toward the large intestines, and the associated delivery of bile into the intestinal lumen would facilitate the overall digestive process. NEURAL INTERACTIONS BETWEEN THE GUT AND THE SPHINCTER OF ODDI Duodenal distension and electrical field stimulation causes changes in SO tone in the dog, cat, and Australian possum (Wyatt, 1967; Thune, Jivegård and Svanvik, 1989; Saccone et al., 1994). In the possum, these responses are abolished both by crushing the duodenum between the site of stimulation and the SO and by TTX, but not by vagotomy. This indicates that neural projections from duodenal neurons to the SO are responsible for this response. Also in the Australian possum, Padbury and colleagues (1993) have demonstrated that injections of retrograde tracers into the SO resulted in retrograde labelling of duodenal myenteric neurons. In the guinea-pig, SO ganglia are richly innervated by calbindin-immunoreactive nerve fibres, but these ganglia contain few, if any, calbindin-immunoreactive neurons (Wells and Mawe, 1993). Since a high proportion of neurons in the nearby myenteric ganglia of the duodenum are calbindin-positive, the duodenum-myenteric projection neurons represent a likely source of calbindin-positive fibres in SO ganglia. This suggestion is supported by findings from studies demonstrating that the calbindin-immunoreactive neurons project from the myenteric plexus of the duodenum to SO ganglia (Kennedy and Mawe, 1998). Evidence from retrograde labelling studies validates the existence of a neural projection between the duodenum and the SO in the guinea-pig and Australian possum. Injection of DiI into the wall of the SO, in the Australian possum, in vivo, results in retrograde labelling of neurons in the duodenal myenteric plexus (Padbury et al., 1993). In vitro retrograde label studies in the guinea-pig have demonstrated that specific subpopulations of guineapig duodenal myenteric neurons project to the ganglionated plexus of the SO in guinea-pig (Kennedy and Mawe, 1998). An average of 110 neurons project from the duodenum to the

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SO in the guinea-pig, and these neurons express ChAT-, but not NOS- or calretininimmunoreactivity. Importantly, many (~20%) of these projection neurons are calbindinpositive. This is a critical detail because calbindin is a marker for intrinsic primary afferent neurons in the guinea-pig enteric nervous system, and calbindin-positive neurons in the myenteric plexus send projections to the mucosa. The duodenum-SO projection neurons are depolarized by CCK, indicating that they are capable of responding to CCK released from the mucosa. Because calbindin-IR myenteric neurons send a projection to the mucosa, and postprandial release of CCK also occurs in the mucosa, it is possible that the duodenum-SO neural circuit is activated by postprandial release of mucosal CCK. To examine the nature of the synaptic signals received by SO neurons from neurons in the duodenum, membrane potential recordings were obtained from SO neurons with intracellular microelectrodes while duodenal nerve bundles and villi were stimulated (Kennedy and Mawe, 1999). Focal stimulation of interganglionic fibre bundles in the myenteric plexus of the duodenum, or duodenal mucosal villi, elicits nicotinic fast synaptic potentials in SO neurons. In these studies, antidromic action potentials were also detected in SO neurons indicating that SO neurons project to the duodenum. This reciprocal projection was verified morphologically by retrograde labelling studies demonstrating that SO neurons are labelled when DiI is applied to the duodenal myenteric plexus, in vitro. Thus, it is clear that duodenal neurons provide input to SO neurons and, at least in the guinea-pig, this input is in the form of nicotinic fast EPSPs through a reflex that can be activated at the duodenal mucosa. This circuit could play a role in any or all of the SOs functions. For example, it is possible that the neurons that project to the SO are activated, directly or through other neurons, by the release of CCK. According to this model, CCKinduced changes in SO tone would involve a local neural circuit rather than a direct action of hormonal CCK on SO neurons. This is consistent with the finding, described above, that SO neurons are not sensitive to the levels of CCK that exist in the serum. Another possible role of the duodenum-SO circuit could be to cause contraction of the SO in coordination with contractions of the duodenum. This would help prevent the reflux of lumenal contents into the biliary and pancreatic ducts. Finally, the gut-SO circuit could be involved in coordinating the relaxation of the SO in concert with the gallbladder contractions and delivery of bile that occur in phase with the migrating myoelectric complex. NEURAL INTERACTIONS BETWEEN THE GALLBLADDER AND THE SPHINCTER OF ODDI In addition to reflex interactions between the gut and the biliary tract, there exists a neural reflex circuit that links the gallbladder to the SO. In dogs, cats and humans, distension or electrical stimulation of the gallbladder results in a decreased motility, or flow resistance, in the SO (Wyatt, 1967; Thune, Thorness and Svanvik, 1986; Thune, Jivegård and Svanvik, 1989; Thune et al., 1991). In the cat, this response was eliminated by TTX or application of local anaesthetics to the bile ducts (Thune, Thorness and Svanvik, 1986). The mechanisms for the reflex between the gallbladder and the SO are unknown. Authors of these reports have suggested that the reflex may involve a direct neural link between the gallbladder and the SO; however, these studies have not ruled out the possibility of a vagal reflex mechanism.

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CONCLUDING REMARKS The neurons of the gallbladder have characteristics that are more similar to parasympathetic neurons than those of enteric neurons. To release their neuroactive compounds onto the effector tissues of the organ, gallbladder neurons must be stimulated, and the major source of excitatory input to these cells is vagal preganglionic fibres. Modulatory inputs that can up- or down-regulate the efficacy of this nicotinic ganglionic transmission include CCK and noradrenaline, which have presynaptic excitatory and inhibitory effects on vagal terminals, respectively. Furthermore, sensory fibres can release SP and CGRP in gallbladder ganglia to depolarize and increase the excitability of gallbladder neurons. The concept of how gallbladder neurons, which express acetylcholine plus an array of excitatory and inhibitory neuromodulators, conduct unambiguous signals to the smooth muscle and epithelial cells of the gallbladder needs to be resolved in future studies. The neurons of the SO represent a more heterogeneous population than those of the gallbladder. Electrically, SO ganglia comprise excitable neurons with spontaneous activity, less excitable neurons that do not exhibit spontaneous activity, and a small contingent of AH cells. Neurochemically, SO ganglia include neurons that appear to be either excitatory or inhibitory; the excitatory neurons express ChAT, SP and enkephalin, and the inhibitory neurons express NOS and VIP or NPY. Although the extrinsic inputs to SO ganglia have not been extensively studied as of yet, mounting evidence suggests that significant regulatory inputs to SO ganglia originate in the myenteric plexus of the duodenum. Future studies will focus on the neurochemical mediators of duodenal-SO interactions and the physiological activators of this pathway.

ACKNOWLEDGEMENTS The studies performed in the Mawe laboratory have been supported by NIH grants DK 45410 and NS 26995. We thank the alumni and active members of the Green Mountain Gallbag Company, including Wendy Pouliot, Ellen Cornbrooks, David Wells, Lei Zhang and Alex Gokin for their contributions to many of the studies that have been described here.

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6 Pharmacology of the Enteric Nervous System Marcello Tonini, Fabrizio De Ponti, Gianmario Frigo and Francesca Crema Department of Internal Medicine and Therapeutics, Section of Clinical and Experimental Pharmacology, University of Pavia, Piazza Botta 10, I-27100 Pavia, Italy The intestine is capable of complex behaviours which allow mixing, propulsion, digestion and absorption of food. The mechanism whereby the intrinsic neurons of the enteric nervous system and the circular smooth muscle coordinate their activities to produce propagated contractions have been the subject of intensive investigations for almost a century. Since the intestine contains a large number of neuroactive substances and an equally large number of receptors for a variety of chemicals, preparations of longitudinal muscle with attached myenteric plexus have been widely used by pharmacologists and therefore have provided detailed information about the effect of drugs on motor neurons of the gastrointestinal tract. The complexity of the chemical coding of enteric neurons mirrors the complexity of the receptors involved in neurogenic intestinal functions. This chapter is intended to review the findings that associate any pharmacologically induced change in enteric nerve activity with the appropriate receptor type or subtype recognized by means of selective agonists and/or antagonists. KEY WORDS: enteric nervous system; chemical coding; cholinergic transmission; adrenergic transmission; NANC transmission; serotonergic transmission; tachykinins.

INTRODUCTION An explosive growth has recently characterized the pharmacology of the enteric nervous system, thanks to the improved anatomical, electrophysiological and functional knowledge of the various classes of neurons intrinsic to the intestinal wall and of their chemical coding (Costa and Brookes, 1994; Furness et al., 1994; Costa et al., 1996). This gave impetus to the search for highly selective agonists and antagonists for the pharmacological characterization of the receptor subtypes involved in fast and slow transmission between neurons, in the excitatory and inhibitory transmission to smooth muscle cells, and in the modulation of transmitter release by presynaptic or prejunctional mechanisms (Starke, Göthert and Kilbinger, 1989; Fuder and Muscholl, 1995). 213

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In spite of the fact that enteric neural pathways are involved in the regulation of several important functions, such as absorption/secretion, maintenance of vascular tone and motility, those involved in the regulation of gastrointestinal motility (i.e. the myenteric plexus) have been investigated much more extensively. This led to an outstanding development of the pharmacology of receptors mediating the effects of neurotransmitters on intestinal motility, which will be one of the main topics of the present chapter. Myenteric neurons can be divided into several functional classes that include sensory neurons, ascending and descending interneurons, ascending and descending (both short and long) motor neurons to the circular muscle, and excitatory motor neurons to the longitudinal muscle coat (Costa et al., 1996). All these neurons contain a wide array of neuroactive substances, which can be co-stored in and co-released from the same neuron upon appropriate stimulation. They include acetylcholine (ACh), tachykinins and opioid peptides (usually enkephalins) in the ascending pathways, and ACh, serotonin (5-hydroxytryptamine: 5-HT), somatostatin, vasoactive intestinal peptide (VIP), nitric oxide (NO) and dynorphin in descending interneurons, while descending motor neurons may contain VIP, NO, pituitary adenylate cyclase-activating peptide, gastrin-releasing peptide, dynorphin/ enkephalin and, as a result of pharmacological evidence, ATP. Some other substances are present in extrinsic pathways innervating the intestine (e.g. noradrenaline) and in mucosal endocrine cells (e.g. 5-HT) and mast cells. The complexity of the chemical coding of enteric neurons mirrors the complexity of the receptors (in terms of their subtypes, distribution, density, ionic and transduction mechanisms) involved in neurogenic intestinal functions. The presence of some of these receptors in the gut has been documented only from a pharmacological point of view and their functional significance is debated. In fact, it is a general rule that, for any given endogenous substance, a number of receptor subtypes exists even in the same tissue, leading either to similar or contrasting effects. Furthermore, it is now well documented that disease states can lead to alterations in the level of expression of a given receptor subtype. This concept of inducible receptors (Donaldson, Hanley and Villablanca, 1997) can of course profoundly affect drug action in vivo. In some cases, compounds behaving as antagonists on receptors activated by endogenous ligands offer a widely used pharmacological tool to assess the contribution of a given neurotransmitter in mediating a biological response. Indeed, if a given receptor is under tonic control by an endogenous transmitter, administration of an antagonist will elicit effects that are opposite to those of the endogenous ligand. This is based on the assumption that antagonists, according to the classical definition, lack efficacy. However, evidence has recently begun to accrue that, in the case of G protein-coupled receptors, some antagonists not only bind to the receptor, but also induce a conformational change that favours uncoupling of the receptor from its G protein (Schütz and Freissmuth, 1992; Kenakin, 1994). This implies that, in some in vitro systems with constitutive receptor activity, antagonists may show a property termed negative efficacy (or inverse agonism). Antagonists may thus be classified into compounds with no intrinsic activity and those with negative intrinsic activity (i.e. those reducing constitutive receptor activity, such as timolol or ICI 118551; Chidiac et al., 1994; Samama et al., 1994). These considerations cast some doubts on data obtained with receptor antagonists simply with the assumption that they prevent agonists binding. However, it should be noted that, while constitutive

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activity of the unliganded receptor is a well-established phenomenon in reconstituted systems with purified components, its physiological relevance is still unknown (Schütz and Freissmuth, 1992; Milligan, Bond and Lee, 1995) and, at the present state of knowledge, the effect observed with antagonists in vivo can be tentatively ascribed to antagonism of the endogenous ligand, unless evidence for inverse agonism is obtained. With these basic concepts in mind, this chapter is mainly intended to review those findings that associate any pharmacologically induced change in enteric nerve activity with the appropriate receptor type or subtype recognized by means of selective agonists and/or antagonists. Because of the complexity of the subject, this chapter should have had the size of a book. We hope the reader will forgive us if substantial information had to be neglected due to space limitations. Whenever possible, reference to extensive reviews on the subject has been provided.

PHARMACOLOGY OF CHOLINERGIC TRANSMISSION MUSCARINIC RECEPTORS Acetylcholine is the major transmitter in the enteric nervous system (Furness and Costa, 1987). Inhibition of muscarinic receptors by hyoscine was found to inhibit peristalsis (Tonini et al., 1981), although a weaker hyoscine- or atropine-resistant peristalsis may develop over time (Tonini et al., 1981; Barthó et al., 1982b; Schwörer and Kilbinger, 1988). One of the most popular intestinal preparations used to demonstrate ACh release from cholinergic nerve endings is the longitudinal muscle with attached myenteric plexus (LM-MP) from the guinea-pig small intestine (Paton and Zar, 1968). ACh is released from unstimulated LM-MP preparations and in greater amounts in response to electrical field stimulation. The postjunctional target of neuronally released ACh is a muscarinic receptor associated with membrane phosphoinositide breakdown, which causes the effector cell to contract (Eglen et al., 1994). Recently, this receptor has been recognized as the M3 receptor subtype using the M3-selective antagonist hexahydrosiladifenidol (HHSiD) in both the longitudinal (Giraldo et al., 1988) and circular muscle (Dietrich and Kilbinger, 1995). The prejunctional target of neuronally released ACh, as well as of applied muscarinic agonists, are inhibitory muscarinic autoreceptors, which depress, through a negative feed-back mechanism, the release of ACh itself. Indeed, muscarinic receptor antagonists were found to increase the electrically stimulated ACh release (Starke, Göthert and Kilbinger, 1989; Vizi et al., 1989). The evidence that muscarinic autoreceptors were antagonized with low affinity by the M1-selective antagonist pirenzepine (Kilbinger et al., 1984) and the M2-selective antagonist AF-DX 116 (Dammann et al., 1989) and with high affinity by the M3-selective antagonists HHSiD (Fuder, Kilbinger and Müller, 1985) and 4-DAMP (Kilbinger et al., 1984) suggests that these prejunctional sites belong to the M3 subtype. However, since HHSiD and 4-DAMP possess high affinity also for the M4 receptor, the participation of this site in negative feed-back mechanisms cannot be ruled out (Eglen and Watson, 1996). Furthermore, there is also evidence that cholinergic ganglionic transmission is regulated by presynaptic inhibitory muscarinic autoreceptors characterized so far as M2 subtypes (North, Slack and Surprenant, 1985).

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In contrast with the longitudinal muscle, prejunctional autoreceptors that inhibit ACh release in the circular muscle of the guinea-pig ileum belong to the M1 subtype (Dietrich and Kilbinger, 1995). This is of particular relevance, since M1 receptors located on the cell bodies of myenteric S neurons (i.e. interneurons and motor neurons) cause a slow membrane depolarization (North and Tokimasa, 1982; Morita, North and Tokimasa, 1982) and stimulate resting ACh output (Kilbinger and Nafziger, 1985; Vizi et al., 1989) through a mechanism inhibited by pirenzepine. Functionally, M1 receptors on myenteric (inter) neurons are activated during the ascending excitatory reflex in response to localized gut wall distension. In fact, in a partitioned organ bath, administration of pirenzepine at the site of distension inhibited the reflex contraction recorded in the oral compartment. Conversely, administration of pirenzepine at the site of reflex recording was ineffective (Tonini and Costa, 1990). Previous experiments by Schwörer and Kilbinger (1988) had indicated that M1 receptors also play a role in peristalsis since nanomolar concentrations of pirenzepine were found to enhance peristalsis, an effect apparently in contrast with that observed in the ascending excitatory reflex (Tonini and Costa, 1990). Pirenzepine-induced facilitation of peristalsis was explained in terms of antagonism of M1 receptors activating an inhibitory pathway. Based on recent findings, however, this effect can be easily explained by the removal of a tonic M1 receptor mediated inhibition of ACh release from the circular muscle (Dietrich and Kilbinger, 1995). The existence of muscarinic heteroceptors, i.e. modulating release of neurotransmitters other than ACh (e.g. noradrenaline), is also documented (Alberts and Stjärne, 1982; Manber and Gershon, 1979; Yokotani and Osumi, 1993; Marino et al., 1997). NICOTINIC RECEPTORS Nicotinic receptors are ligand-gated ion channels which are present on certain subclasses of myenteric neurons, where they cause rapid membrane depolarization (fast excitatory post-synaptic potentials, fast EPSPs) leading to fast communication between neurons. Stimulation of these receptors evokes release of numerous transmitters including ACh (Yau, Dorsett and Parr, 1989), NO (Jin and Grider, 1992), somatostatin (Grider, 1989) and L-glutamate (Wiley, Lu and Owyang, 1991). A polysynaptic chain of cholinergic ascending interneurons are involved in the distension-evoked ascending excitatory reflex of the ileal circular muscle. The ACh released from these neurons acts on nicotinic receptors on interneurons and motor neurons. Inhibition of these receptors by hexamethonium added to the oral, intermediate or anal compartment of a partitioned organ bath, markedly reduced the amplitude of the reflex contraction (Tonini and Costa, 1990), indicating that fast ganglionic nicotinic transmission exerts a crucial role in such responses. In contrast, probably because ACh is not handled by all classes of descending interneurons, the descending inhibitory reflex is reportedly less sensitive to nicotinic receptor blockade (Smith and Furness, 1988; Smith, Bornstein and Furness, 1990; Smith and Robertson, 1998). Nicotinic ganglionic transmission is also crucial for normal peristalsis, since hexamethonium abolishes propulsion both in the ileum and colon (Fontaine, Van Nueten and Janssen, 1973; Barthó, Holzer and Lembeck, 1987; Barthó et al., 1989; Kadowaki, Wade and Gershon, 1996; Tonini et al., 1996). In the ileum, weaker peristalsis can be obtained

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using greater distending volumes, providing naloxone is present to block the inhibitory effects of endogenous opioids (Barthó, Holzer and Lembeck, 1987; Barthó et al., 1989). Thus, non-nicotinic (i.e. hexamethonium-resistant) ganglionic transmission also occurs during peristalsis. This form of transmission is inhibited by endogenous opioids. Typically, nicotinic receptors are localized to the somatodendritic region of excitatory interneurons and motor neurons. Recently, however, prejunctional and presynaptic nicotinic receptors have been described in myenteric plexus neurons of the guinea-pig small intestine, where they mediate the release of excitatory non-cholinergic (tachykinergic) transmitters (Galligan, 1999; Schneider and Galligan, 2000; Schneider, Perrone and Galligan, 2000).

PHARMACOLOGY OF NON-CHOLINERGIC EXCITATORY (TACHYKINERGIC) TRANSMISSION Tachykinins represent a family of peptides which have been isolated from mammals (substance P, neurokinin A, neurokinin B) and amphibians (physalaemin, phyllomedusin, uperolein, eledoisin, kassinin) and whose role in intestinal motility has been reviewed recently (Holzer and Holzer-Petsche, 1997a,b). In the gut, although tachykinins may be present in the enterochromaffin and immune cells of the mucosa, most of them are found in intrinsic enteric neurons and extrinsic primary afferent nerve fibres (Holzer and HolzerPetsche, 1997b). In the myenteric plexus of the guinea-pig small intestine, substance P (SP) immunoreactivity has been found in excitatory motor neurons projecting to the circular (CM) and longitudinal muscle (LM) layers, ascending interneurons and intrinsic primary afferent neurons (Llewellyn-Smith et al., 1988; Brookes and Costa, 1990; Brookes, Steele and Costa, 1991a,b, 1992; Brookes et al., 1992; Costa et al., 1996). SP and neurokinin A (NKA) have been found in synaptic vesicles from enteric neurons where they are co-released with ACh, whereas neurokinin B (NKB) is absent (Deacon et al., 1987; McDonald et al., 1988; Regoli, Bondon and Fouchère, 1994; Lippi et al., 1998).

TACHYKININ RECEPTORS Three distinct receptors mediate the biological effects of endogenous tachykinins in the gastrointestinal tract. They are known as tachykinin NK1, NK2 and NK3 receptors, which have some preferential (though sometimes negligible) affinity for SP, NKA and NKB, respectively (Maggi, 1995). Since NKB is not expressed in the gastrointestinal tract, NK3 receptors are agonized by SP and NKA, which may act as full agonists at these sites (Maggi, 1995). Nevertheless, the pharmacological characterization of tachykinin receptors was made possible by the development of potent and selective peptide-derived agonists, e.g. NK1 receptor: SP methyl ester, [Sar9]-SP sulphone; NK2 receptor: [β-Ala8]-NKA(4–10), [Nleu10]-NKA(4–10); NK3 receptor: senktide, [MePhe7]-NKB, and peptide (FK-888; MEN11420; PD-161182) or non-peptide antagonists (SR-140333; SR-48968; SB-142801) (Holzer and Holzer-Petsche, 1997a).

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Effects of tachykinin receptor stimulation on neuromuscular preparations The NK1 and NK2 subtypes are located on smooth muscle cells, where they mediate contraction (Holzer and Lembeck, 1980; Costa et al., 1985; Maggi et al., 1990a, 1994a) and on neurons. In the guinea-pig ileum and colon LM, the direct contractile response to SP is mediated by the NK1 subtype (Lee et al., 1982; Briejer et al., 1993a), while in the CM both NK1 and NK2 receptors are involved (Maggi et al., 1990a, 1994a). In the colon, NK1 and NK2 receptors produce a non-adrenergic non-cholinergic (atropine-resistant) excitation of CM to nerve stimulation, which show a remarkable specialization. NK1 receptors mediate a “fast” excitatory transmission, which depend on the activation of voltage-sensitive calcium channels, whereas NK2 receptors mediate a “slow” excitatory transmission that is largely independent from calcium channels (Maggi, Zagorodnyuk and Giuliani, 1994b). Species-related pharmacological differences detected by variable affinities of competitive antagonists have emerged for both NK1 and NK2 receptors (Maggi, 1995). NK1 receptors are expressed on both ascending and descending neuronal pathways and on the interstitial cells of Cajal (Sternini et al., 1995; Legat et al., 1996; Portbury et al., 1996b; Johnson, Bornstein and Burcher, 1998; Lomax, Bertrand and Furness, 1998; Lecci et al., 1999; Bian et al., 2000). In CM strips from the guinea-pig small intestine, contractions mediated by NK1 receptors are atropine-resistant, but partially sensitive to tetrodotoxin (TTX) (Maggi et al., 1990a; Bian et al., 2000), suggesting that NK1 receptors are present, at least in part, on excitatory neurons, where they stimulate release of a non-cholinergic transmitter. In the same preparation, use of GR-73632, a selective NK1 receptor agonist, produced a TTX-sensitive decrease of spontaneous contractility, due to release of NO from inhibitory motor neurons (Lecci et al., 1999). NK1 receptor-mediated, TTX-sensitive relaxations due to release of NO have been also demonstrated in the colonic circular muscle (Bian et al., 2000). Lastly, prejunctional inhibitory NK1 receptors operating a feedback inhibition of ACh release have been described in LM-MP preparations (Kilbinger et al., 1986; Loeffler et al., 1994). Neuronal NK2 receptors have been described in descending pathways only (Portbury et al., 1996a; Zagorodnyuk and Maggi, 1995). In the guinea-pig colon CM strips, NKA and the NK2 receptor-selective synthetic agonist [β-Ala8]-NKA(4–10), in addition to causing a direct excitation, were found to activate inhibitory non-adrenergic non-cholinergic motor neurons with consequent release of NO and an apamin-sensitive inhibitory transmitter (Zagorodnyuk and Maggi, 1995). NK3 receptors are predominantly neuronal in origin. They are located on intrinsic primary afferent neurons (Mann et al., 1997a), and on ascending and descending pathways (Maggi et al., 1990a, 1993, 1994c; Legat et al., 1996; Johnson et al., 1996; Johnson, Bornstein and Burcher, 1998; Jenkinson et al., 1999). Intrinsic sensory neurons are connected to each other via synapses communicating with NK3 receptors, which therefore contribute to the reinforcement of perceptive signal (Furness et al., 1998). In ascending pathways, activation of NK3 receptors by senktide or NKB promotes release of tachykinins (SP and NKA) and ACh from the myenteric plexus of the guinea-pig ileum (Guard and Watson, 1987; Yau et al., 1992). NK3 receptors located on descending pathway induce release of NO and inhibition of circular muscle activity in both the ileum and colon (Maggi et al., 1993; Maggi, Zagorodnyuk and Giuliani, 1994c).

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Electrophysiological effects of tachykinins on neurons Substance P depolarizes 85% of AH neurons and 76% of S neurons in the myenteric plexus when applied electrophoretically. The depolarization is associated with an increase in input resistance due to inactivation of a resting potassium conductance (Katayama, North and Williams, 1979). Slow EPSPs in S and AH neurons evoked by repeated pulse stimuli are due to the release of a non-cholinergic neurotransmitter and can be abolished by desensitization to SP (Katayama and North, 1978; Johnson et al., 1981). Thus, tachykinins are likely to mediate some slow EPSPs in myenteric neurons. On pharmacological grounds, it might be predicted that NK3 (and NK1), but not NK2, receptors are involved (Hanani et al., 1988; Willard, 1990; Bertrand and Galligan, 1994, 1995). Role of endogenous tachykinins Electrically induced, nerve-mediated, non-cholinergic contractions of the CM are reduced in amplitude by antagonists at the NK1 and NK2 receptor subtypes (Costa et al., 1985; Barthó et al., 1992). This indicates the participation in the contractile response of both SP and NKA, which are co-released upon electrical stimulation (Lippi et al., 1998). The ascending reflex contraction of the CM produced by radial distension of the gut wall is abolished by hyoscine at low distensions, but partly reduced at higher distension volumes. Under the latter conditions, the reflex contraction is reduced by tachykinin NK1 and NK2 receptor antagonists (Costa et al., 1985; Holzer, 1989), and in particular, by those acting at the NK2 receptor subtype (Barthó et al., 1992; Maggi et al., 1994d). Exposure of guinea-pig ileum CM to SP produces depolarization and action potentials (Niel, Bywater and Taylor, 1983a). The depolarization is associated with decreased membrane resistance. Noncholinergic fast excitatory junction potentials in the intestinal CM are abolished by desensitization to SP and by SP receptor antagonists acting at NK1 and NK2 receptors (Niel, Bywater and Taylor, 1983a; Taylor and Bywater, 1986; Serio et al., 1998). Taken together, these results indicate that endogenous tachykinins mediate non-cholinergic excitatory transmission to the CM. This transmission is mainly mediated by the NK2 receptor subtype, although NK1 receptors participate in this event. There is now convincing evidence that tachykinins play a role in peristalsis. Figure 6.1 illustrates the distribution of tachykinin receptors on effector cells and intrinsic neuronal pathways (i.e. myenteric plexus), which mediate propulsive activity in the gastrointestinal tract. SP is released into venous effluent in response to distension of the small intestine during peristalsis. This release is blocked by TTX and enhanced by the nicotinic receptor agonist dimethylphenylpiperizine (DMPP) (Donnerer et al., 1984; Donnerer, Holzer and Lembeck, 1984). SP stimulates peristalsis when the intestine is distended by a subthreshold volume (Holzer and Lembeck, 1979) and increases the frequency of emptying (Barthó et al., 1982a). Furthermore, atropine-resistant peristalsis is greatly inhibited by tachykinin receptor antagonists (Barthó et al., 1982b), whereas hexamethonium-resistant peristalsis is abolished by spantide (Barthó et al., 1989). Analysis of the receptors by which tachykinins influence peristalsis has shown that activation of NK1 receptors inhibits, while activation of NK2 and NK3 facilitates propulsion (Holzer, Schluet and Maggi, 1995). The NK1 receptor-mediated depression of peristalsis involves descending motor pathways utilizing

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Figure 6.1 Scheme illustrating the neuronal pathways regulating standing reflexes and peristalsis in the gastrointestinal tract. Radial distension of the gut wall by an intraluminal bolus excites ascending and descending pathways leading to excitatory and inhibitory reflexes. All three tachykinin receptor subtypes (NK1,2,3) are expressed on neurons where they mediate excitatory (+) or inhibitory effects (–) on propulsion. Tachykinin receptors located on smooth muscle cells mainly belong to the NK1 and NK2 subtypes and are excitatory in nature.

NO as a transmitter (Portbury et al., 1996b; Lecci et al., 1999), since the inhibitory effect was prevented by a nitric oxide synthase (NOS) inibitor (Holzer, 1997). On the other hand, blockade by SR-140333 of NK1 receptors markedly reduced the amplitude of inhibitory junction potentials evoked in the circular muscle by activation of descending pathways during motility reflexes. This clearly indicates that endogenous tachykinins acting via NK1 receptors partly mediate transmission to inhibitory motor neurons (Johnson, Bornstein and Burcher, 1998). Use of antagonists at NK1 (SR-140333), NK2 (SR-48968) and NK3 (SR-142801) receptors made it possible to establish that endogenous tachykinins acting via NK1 and NK2 receptors contribute to maintain intestinal peristalsis when cholinergic ganglionic and neuromuscular transmission via muscarinic receptors is suppressed. SR-142801 lacked a major influence on peristalsis, indicating that NK3 receptors play little role in peristalsis generation (Holzer et al., 1998). Conversely, NK3 receptors have been found to play a substantial role in transmission from intrinsic sensory neurons and from ascending (and descending) interneurons to excitatory motor neurons during motility reflexes (Johnson et al., 1996; Johnson, Bornstein and Burcher, 1998). It is interesting to point out that distension-evoked standing reflexes of the circular muscle subserve but cannot be identified with motor propulsive events (Tonini et al., 1996). In the guinea-pig isolated colon, NK1 and NK2 receptors contribute to peristalsis (FoxxOrenstein and Grider, 1996). In another study, blockade of NK1, NK2 and NK3 receptors inhibited propulsion. NK1 and NK2 receptors showed a synergistic interaction with muscarinic receptors, an effect observed also in the ileum (Holzer and Maggi, 1994), whereas inhibition produced by NK3 receptor blockade was additive with the decelerating effect

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caused by nicotinic receptor blockade. Simultaneous blockade of all the three receptors produced a 50% inhibition of the velocity of propulsion indicating that endogenous tachykinins acting at NK1, NK2 and NK3 receptors play an important role in maintaining propulsion (Tonini et al., 2001). In the rabbit isolated distal colon, NK2 and NK3 receptors located on descending pathways decelerate propulsion by activating a NO-dependent pathway. Both receptors, however, may also accelerate propulsion via distinct mechanisms. NK2 receptors mediate a facilitatory effect by a postjunctional synergistic interaction with muscarinic receptors, while NK3 receptors located on ascending pathways facilitate propulsion through a synergistic interaction with nicotinic receptors (Onori et al., 2000, 2001).

PHARMACOLOGY OF ADRENERGIC TRANSMISSION Noradrenaline is well established as the transmitter of postganglionic sympathetic neurons supplying the gastrointestinal tract, since it meets all the criteria for identification of a substance as a neurotransmitter (Furness and Costa, 1987). However, sympathetic neurotransmission may not be exclusively noradrenergic, as indicated, for instance, by the fact that adrenoceptor antagonists and reserpine pretreatment do not inhibit neurogenic vasoconstriction in submucosal arterioles, at least in the guinea-pig (Surprenant, 1994). In this species, noradrenergic axons supplying intestinal arterioles and submucous ganglia also contain neuropeptide Y and somatostatin, respectively, while sympathetic neurons reaching the myenteric plexus and the circular muscle coat contain neither peptide (Furness and Costa, 1987). The chemical coding of motility-inhibiting adrenergic neurons is different in the rat, a species where these neurons contain neuropeptide Y (McConalogue and Furness, 1994). Noradrenergic axons in the gut are most numerous in the myenteric and submucous ganglia and around arterioles, but there is also evidence of a sparse noradrenergic supply to the circular muscle and to the mucosa (Gabella, 1979). Concerning the sympathetic fibers reaching directly the muscle layer, in general the adrenergic innervation of the longitudinal muscle is less abundant than that of the circular layer (Gabella, 1979). Histochemical studies show that the noradrenergic innervation is particularly rich in the muscle coat of the so-called sphincteric regions such as the gastroesophageal junction, the pyloric region and the anal canal (Gabella, 1979; Furness and Costa, 1987). With few exceptions, stimulation of the sympathetic supply to sphincteric muscle is excitatory (Furness and Costa, 1974) and is thought to be due to a direct effect of noradrenaline on smooth muscle α-adrenoceptors. Electrophysiological studies have shown that noradrenergic fibers supplying the myenteric ganglia cause presynaptic inhibition of cholinergic transmission (Hirst and McKirdy, 1974a), while stimulation of noradrenergic nerves at frequencies of up to 50 Hz causes no changes in the membrane properties of myenteric neurons (Hirst and McKirdy, 1974b), although exogenous α2- and β1-adrenoceptor agonists can respectively hyperpolarize (Morita and North, 1981; Surprenant and North, 1985; Galligan and North, 1991; Wells and Mawe, 1994) or depolarize (Schemann, 1991; Tack and Wood, 1992) some neurons. Exogenous noradrenaline was shown to act presynaptically at different gut levels

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inhibiting fast EPSPs (Nishi and North, 1973; Schemann, 1991; Tack and Wood, 1992; Mawe, 1993). The most direct evidence supporting the concept of a tonic sympathetic inhibition on gut motility would be provided by electrophysiological studies showing a tonic discharge of sympathetic efferent neurons supplying the myenteric plexus and/or smooth muscle cells. Unfortunately, while release of noradrenaline from sympathetic terminals accounts for the inhibitory post-synaptic potential (IPSP) through activation of postsynaptic α2-adrenoceptors in submucosal neurons, little information is available on IPSPs in myenteric neurons, where IPSPs are seldom recorded, and there are only a few data on the possible transmitter(s) involved (Surprenant, 1994). Although noradrenaline can hyperpolarize myenteric neurons, hyperpolarization of myenteric neurons in response to sympathetic activity has never been shown (McConalogue and Furness, 1994), while, in the case of secretomotor neurons, neuronal hyperpolarization seems to be the main mode of action of the sympathetic input (North and Surprenant, 1985). In a recent study, designed to test whether sphincter of Oddi ganglia are a target of sympathetic input (Wells and Mawe, 1994), noradrenaline, when applied to neurons exhibiting IPSPs (which were, however, a minority), induced membrane hyperpolarization. Interestingly, α2-adrenoceptor blockade inhibited, whereas application of desipramine increased the amplitude of the IPSPs, an observation consistent with the hypothesis put forward by the authors that noradrenaline may mediate IPSPs at least in this preparation. ADRENOCEPTOR SUBTYPES At present, adrenoceptors are classified into three major classes: α1, α2 and β, each further divided into several subtypes (Bylund et al., 1994; Alexander and Peters, 2000), but only fragmentary information is available on the distribution of adrenoceptor subtypes in the enteric nervous system (Table 6.1). On a pharmacological basis, α1-adrenoceptors are defined as receptors selectively stimulated by methoxamine, cirazoline or phenylephrine and competitively blocked by low concentrations of prazosin, WB-4101 or corynanthine, while α2-adrenoceptors are characterized by a high sensitivity to agonists such as clonidine, xylazine, UK14 304 and antagonists such as rauwolscine, yohimbine and idazoxan (Ruffolo and Hieble, 1994). Concerning β-adrenoceptors, although many experimental data can be explained in terms of the existence of β1- and β2-adrenoceptors, a β-adrenoceptor with “atypical” pharmacological characteristics was described in some tissues such as the adipose tissue, gastrointestinal tract and skeletal muscle (Arch et al., 1984; Bond and Clarke, 1987; Croci et al., 1988; Bianchetti and Manara, 1990; Molenaar et al., 1991) and is now classified as a β3-adrenoceptor (Alexander and Peters, 2000). This β-adrenoceptor displays atypically low pA2 values for conventional antagonists such as propranolol, ICI 118551 and CGP 20712A and high sensitivity to agonists such as BRL 37344 and SR 58611A (Arch and Kaumann, 1993). Among conventional β-adrenoceptor antagonists, alprenolol is one of the compounds displaying the highest affinity (Blue et al., 1990), although its pA2 value for β3-adrenoceptors is rather low (Arch and Kaumann, 1993). Molecular biological approaches have confirmed the existence of at least three human genes encoding different β-adrenoceptors corresponding to the β1-, β2- and β3-subtypes (Frielle et al., 1987;

Phenylephrine, methoxamine, cirazoline

Prazosin, corynanthine, WB4101

Gq/11

Selective agonists

Selective antagonists

Predominant effectors

CGP20712A, atenolol Gs

Gi/o

Xamoterol, prenalterol

Smooth muscle relaxation

Smooth muscle, enteric neurons (?)

β1-adrenoceptors

Yohimbine, idazoxan

Clonidine, UK14304

Presynaptic inhibition, smooth muscle contraction

Adrenergic neurons (autoreceptors), myenteric neurons (heteroceptors), smooth muscle

α2-adrenoceptors1

Gs

ICI118551

Terbutaline, salbutamol, ritodrine

Smooth muscle relaxation, facilitation of transmitter release2

Smooth muscle, peripheral noradrenergic neurons (?)

β2-adrenoceptors

Gs

SR59230A

BRL37344, SR58611A, CL316243

Smooth muscle relaxation

Smooth muscle

β3-adrenoceptors

α1- and α2-adrenoceptors are further divided into the following subtypes, respectively: α1A, α1B, α1D and α2A, α2B, α2C (Alexander and Peter, 2000), but only fragmentary information is available at present on their distribution in the gut; 2 This effect has been reported in peripheral organs other than the gastrointestinal tract (Langer, 1981; Brunn et al., 1994).

Smooth muscle contraction or relaxation, neuronal depolarization (gastric neurons)

Functional response

1

Smooth muscle, gastric neurons

Distribution in the gut

α1-adrenoceptors1

TABLE 6.1 Synopsis of adrenoceptors in the gut

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Kobilka et al., 1987; Emorine et al., 1989). More recently, the development of selective β3-antagonists has provided a new tool for β3-adrenoceptor identification in functional tests (Manara et al., 1995; De Ponti et al., 1996; De Ponti, 1997). ROLE OF PREJUNCTIONAL AND POSTJUNCTIONAL ADRENOCEPTORS Adrenoceptors of both α- and β-subtype have been recognized at different levels of the gastrointestinal tract where they are involved in the regulation of motility and secretion. In particular, α1-adrenoceptors are located postsynaptically on smooth muscle cells and, to a lesser extent, on intrinsic neurons, while α2-adrenoceptors may be present both pre- and postsynaptically (Table 6.1). At a presynaptic level, α2-adrenoceptors may act either as autoreceptors inhibiting noradrenaline release from adrenergic nerves (Langer, 1977; Starke, Göthert and Kilbinger, 1989), or as heteroceptors modulating release of other neurotransmitters (especially ACh) from nerve terminals (Paton and Vizi, 1969; Vizi, 1979). An important postsynaptic location of α2-adrenoceptors is that on enterocytes, where they control water and electrolyte absorption. β1- and β2-adrenoceptors are found mainly on smooth muscle cells, but the former may be present on enteric neurons (Bülbring and Tomita, 1987; Ek, Bjellin and Lundgren, 1986) (Table 6.1). Prejunctional adrenoceptors An electrophysiological study on guinea-pig antral myenteric neurons provided evidence for a concentration-dependent inhibition of cholinergic fast EPSPs and non-cholinergic slow EPSPs by application of noradrenaline or clonidine (Tack and Wood, 1992). Yohimbine and phentolamine, but not prazosin, reversed this effect, suggesting an involvement of α2-adrenoceptors. Interestingly, when noradrenaline was applied on cell bodies, no response was observed, in line with the notion that axo-axonal synaptic mechanisms are responsible for the adrenergic inhibitory effect (Tack and Wood, 1992). Another study carried out on myenteric neurons of the guinea-pig gastric corpus (Schemann, 1991) also found inhibition by noradrenaline of fast EPSPs, probably by activation of α2-adrenoceptors on extrinsic vagal fibers. Besides α2-adrenoceptors, it has been suggested that also α1- and β-adrenoceptors may be present on gastric neurons (Allescher et al., 1989; Schemann, 1991; Tack and Wood, 1992). Application of noradrenaline depolarized a small fraction of antral myenteric neurons and enhanced their excitability probably by activation of α1-adrenoceptors on somal membranes (Tack and Wood, 1992). The authors suggested that this α1-adrenoceptor may be located on non-cholinergic inhibitory neurons. In myenteric neurons of the guinea-pig gastric corpus, application of noradrenaline resulted in prolonged excitation in 40% of the neurons tested (Schemann, 1991). This excitatory effect, which is probably mediated by α1-adrenoceptors, seems peculiar to the stomach, since neuronal depolarization is not usually reported in other parts of the gut. Concerning the small bowel, many reports are available on presynaptic α2-adrenoceptors inhibiting neurotransmitter release at this level (Paton and Vizi, 1969; Wikberg, 1978; Drew, 1978; Vizi, 1979; Bauer and Kuriyama, 1982; Reese and Cooper, 1984; Wessler et al., 1987), while the presence of presynaptic β-adrenoceptors having a facilitatory effect

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on neurotransmitter release is less well documented (Alberts, Ögren and Sellstöm, 1985; Kotska et al., 1989). Evidence of a neuronal location of α2-adrenoceptors in the guinea-pig small intestine was provided by binding and functional studies (Drew, 1978; Wikberg and Lefkowitz, 1982): in the denervated longitudinal muscle, [3H]-clonidine binding was virtually abolished, indicating that denervation eliminated α2-adrenoceptors (Wikberg and Lefkowitz, 1982). Electrophysiological studies with intracellular recordings of guinea-pig myenteric neurons showed α2-adrenoceptor-mediated hyperpolarizations (resulting from an increase in potassium conductance) (Morita and North, 1981; Surprenant and North, 1985; Galligan and North, 1991) and inhibition of fast EPSPs (Galligan and North, 1991). Recent studies suggest that α2-autoreceptors and α2-heteroceptors modulating ACh release in the small bowel belong to the α2A/D subtype (Funk et al., 1995; Liu and Coupar, 1997). In the colon, sympathetic nerve stimulation reduces ACh release (Beani, Bianchi and Crema, 1969; Del Tacca et al., 1970) as well as the response to pelvic nerve stimulation (Gillespie and Khoyi, 1977) by activation of presynaptic α2-adrenoceptors (Marcoli et al., 1985), similarly to what has been described above for the small bowel. Indeed, clonidine is one of the most potent inhibitors of the colonic peristaltic reflex (Marcoli et al., 1985), an event requiring the integrity of neural circuits. Presynaptic α2-adrenoceptors can also modulate the release of the non-adrenergic, non-cholinergic transmitter, as indicated by inhibition of inhibitory junction potential amplitude in circular smooth muscle of guineapig caecum (Reilly, Hoyle and Burnstock, 1987) and by the contractile effect observed in colonic longitudinal muscle strips in mice (Fontaine, Grivegnée and Reuse, 1984). Postjunctional adrenoceptors Catecholamines can affect gastric motility through direct activation of smooth muscle adrenoceptors, which may belong to the α1-, α2-, β1-, β2- and β3-class (El-Sharkawy and Szurszewski, 1978; Verplanken, Lefebvre and Bogaert, 1984; Bülbring and Tomita, 1987; Coleman, Denyer and Sheldrick, 1987; McLaughlin and MacDonald, 1991; Cohen et al., 1995). β-mediated responses are usually inhibitory, α2-responses are excitatory, while the responses to α1-adrenoceptor stimulation are variable: both relaxations and contractions have been reported (Sahyoun, Costall and Naylor, 1982; Costall, Naylor and Tan, 1983; Chihara and Tomita, 1987; Bülbring and Tomita, 1987; MacDonald, Kelly and Dettmar, 1990; Mandrek and Kreis, 1992). In circular muscle strips of the porcine pyloric ring, the excitatory responses to catecholamines were unaffected by propranolol or yohimbine but were completely antagonized by prazosin and phentolamine, indicating the involvement of α1-adrenoceptors (Mandrek and Kreis, 1992). Binding studies with [3H]-prazosin and [3H]-yohimbine support the existence of both α1- and α2-adrenoceptors in the guinea-pig stomach, although they do not make it possible to distinguish between neural and smooth muscle receptors (Taniguchi et al., 1988). In some species, such as the rainbow trout, noradrenaline and adrenaline have been reported to induce smooth muscle contraction mainly by a direct action on muscular α2-adrenoceptors (Kitazawa, Kondo and Temma, 1986). Although functional responses can be obtained in the isolated stomach by stimulation of postjunctional α1- and α2-adrenoceptors, their physiological relevance in vivo is still unknown.

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Both α- and β-adrenoceptors have been detected at the postjunctional level in the small bowel. Excitatory α1-adrenoceptors are found especially in the terminal ileum (Bauer, 1981, 1982; Bauer and Kuriyama, 1982), although inhibitory responses probably mediated by these receptors have also been described (Bülbring and Tomita, 1987). Postjunctional α2-adrenoceptors have been detected in a binding study (Ahmad et al., 1991), but their function is still disputed, although Bauer and Kuriyama (1982) suggested an inhibitory effect by observing reduction or suppression of the generation of excitatory junction potentials by clonidine. Postjunctional β-adrenoceptors mediating smooth muscle relaxation were initially characterized as belonging to the β1-subtype (Grassby and Broadley, 1984; Mian, Malta and Raper, 1984; Sim and Lim, 1983). In the rabbit ileum, motor inhibition by perivascular nerve stimulation was reduced by 75% by the β1-adrenoceptor antagonist atenolol, while the remainder of the response was blocked by the β2-adrenoceptor antagonist butoxamine (Greenwood, Davison and Dodds, 1990). Several reports showing low pA2 values of conventional β-blockers have now provided evidence in favour of the presence of β3-adrenoceptors (Salimi, 1975; Bond and Clarke, 1987; Bond and Clarke, 1988; Grassby and Broadley, 1987; Norman and Leathard, 1990; Taneja and Clarke, 1992; Growcott et al., 1993a; Growcott et al., 1993b; MacDonald et al., 1994), which, at least in some preparations, seem to contribute to a significant extent to isoprenaline-induced relaxation (Tesfamariam and Allen, 1994). Postjunctional α-adrenoceptors may belong to the α1- and α2-subtype: the former may be both inhibitory (Fontaine, Grivegnée and Reuse, 1984; Dettmar, Kelly and MacDonald, 1986; Reilly, Hoyle and Burnstock, 1987) and excitatory (Venkova, Milne and Krier, 1994), while activation of α2A-adrenoceptors, at least in the circular smooth muscle of the canine colon, results in a significant increase in contractile force, probably mediated by a decrease in cyclic AMP levels (Zhang et al., 1992). In line with the general rule of a postjunctional location of β-adrenoceptors, most colonic β-sites mediate relaxation by a direct action on smooth muscle cells (Ek, Jodal and Lundgren, 1987; Taniyama, Kuno and Tanaka, 1987; Kwon et al., 1993; Smith et al., 1993), except for a minor population of neuronal β1-adrenoceptors which have been proposed to inhibit the activity of cholinergic neurons (Ek and Lundgren, 1982; Ek, 1985). This hypothesis of a neuronal location was based on the observation that the effect of the partial β1-agonist prenalterol was markedly reduced by the neurotoxin tetrodotoxin and 6-hydroxydopamine pretreatment (Ek, Bjellin and Lundgren, 1986). Binding studies performed in rat colon with [125I]-(−)-pindolol and [3H]-dihydroalprenolol revealed a small population of β1- (20–25%) and a large β2-adrenoceptor population (Ek and Nahorski, 1986; Landi et al., 1992). Although binding studies have failed to detect β3-sites because of the lack of selective ligands (Landi et al., 1992), several functional studies have provided evidence in favour of the presence of β3-sites. Grivegnée, Fontaine and Reuse (1984), studying the relaxatory effect of isoprenaline on the canine colon, concluded that the inhibitory effect of conventional β-blockers on the agonist-induced relaxation was “weak even at high concentrations”. Since this is now an established hallmark for the presence of β3-adrenoceptors, several investigators have assessed their role in the modulation of colonic motility in vitro (Bianchetti and Manara, 1990; McLaughlin and MacDonald, 1990; Landi, Croci and

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Manara, 1993; MacDonald and Lamont, 1993; Koike et al., 1994; De Ponti et al., 1995, 1996) and found concentration-dependent relaxations by using selective β3-agonists such as SR 58611A or CGP 12177 (a partial agonist).

PHARMACOLOGY OF NON-ADRENERGIC, NON-CHOLINERGIC INHIBITORY TRANSMISSION Intrinsic inhibitory transmission in the gut has much attracted the attention of investigators, leading to the recognition that non-adrenergic, non-cholinergic (NANC) inhibitory neural responses can be demonstrated in virtually all sections of the gut. The nature of the transmitter(s) responsible for NANC inhibitory transmission has been a matter of debate: over the years, ATP, VIP-like peptides and NO have been proposed as the main mediators of NANC relaxatory responses. Recently, evidence has begun to accrue that the hypotheses about the identity of the transmitter are not mutually exclusive and that multiple inhibitory mechanisms may coexist (Maggi and Giuliani, 1993; Lefebvre, Smits and Timmermans, 1995; Smits and Lefebvre, 1996; Selemidis, Satchell and Cocks, 1997) and may operate at different gut levels in response to mechanical, chemical or electrical stimuli. NANC inhibitory neurons are involved in the relaxation of sphincter regions, in the accommodation processes in the gastric fundus, small and large intestine, as well as in the descending inhibition in response to localized gut wall distension and peristalsis (Ciccocioppo et al., 1994; Tonini et al., 2000). VIP-LIKE PEPTIDES The VIP family of peptides includes VIP, peptide histidine isoleucine (PHI), pituitary adenylate cyclase-activating peptide (PACAP), glucagon, secretin and gastrin-releasing factor. There is considerable evidence in favour of a transmitter role for VIP in the intestine (Furness and Costa, 1987; McConalogue and Furness, 1994). In the guinea-pig small intestine, VIP is found in short and long descending inhibitory CM motor neurons and cholinergic and non-cholinergic descending interneurons (Furness and Costa, 1979a; Costa et al., 1980a, 1996; Jessen et al., 1980; Schultzberg et al., 1980; Costa and Furness, 1983; Llewellyn-Smith et al., 1988; Brookes, Steele and Costa, 1991b). A small population of VIP-immunoreactive LM motor neurons may exist (Schultzberg et al., 1980; Costa and Furness, 1983). VIP-immunoreactive fibres form synapses on both VIP- and NOSimmunoreactive myenteric neurons (Llewellyn-Smith, Furness and Costa, 1985; Costa et al., 1996). PHI is derived from the same precursor protein as VIP, is colocalized with VIP in vesicles (Agoston et al., 1989), and is thus found in the same populations of neurons as VIP. PACAP was originally isolated from ovine hypothalamus. Two forms occur: a 39-amino acid peptide and a 27-amino acid peptide which show 68% homology with VIP (Miyata et al., 1989; Miyata et al., 1990). PACAP nerve fibres are found in the smooth muscle layers of all regions of the rat gastrointestinal tract (Köves and Arimura, 1990; Nagahama et al., 1998). PACAP-immunoreactive fibres are found in the myenteric plexus and muscularis

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externa of the guinea-pig small and large intestine (Sundler et al., 1992; Portbury et al., 1995). While high affinity PACAP binding sites in the central nervous system do not bind VIP, in the periphery PACAP and VIP appear to share high affinity binding sites (Gottschall et al., 1990; Mao et al., 1998; Teng et al., 1998). Effects of VIP-like peptides on neuromuscular preparations VIP, at concentrations greater than 0.1 nM, enhances the cholinergic twitch and produces contraction of guinea-pig small intestine LM-MP preparations (Kusunoki et al., 1986). The contraction is partly blocked by hyoscine (Kusunoki et al., 1986), suggesting that VIP stimulates ACh and possibly tachykinin release (Katsoulis et al., 1992) from LM motor neurons. More direct evidence has shown that VIP causes increased release of [3H]-ACh from LM-MP preparations, isolated myenteric ganglia and from LM-MP synaptosomes (Yau, Youther and Verdun, 1985; Kusunoki et al., 1986; Yau, Dorsett and Youther, 1986b; Yau, Dorsett and Parr, 1989). Intra-arterial injection of VIP or PACAP1–27 or PACAP1–38 promotes ACh release from canine ileal circular muscle (Fox-Threlkeld et al., 1999). In the guinea-pig ileum CM, VIP produces concentration-dependent relaxations through a direct myogenic effect, which is resistant to apamin (Costa, Furness and Humphreys, 1986). PACAP causes relaxation of rat stomach, duodenum, jejunum, ileum and colon, through a direct action on the LM and CM (Mungan et al., 1992). In the human sigmoid colon, PACAP and VIP concentration-dependently inhibit phasic myogenic contractions of the LM. The effect of PACAP but not VIP is reduced by 80% by apamin. The inhibitory effect of VIP, but not PACAP, was attenuated by 70% by TEA (Schwörer et al., 1992). Similar findings were obtained in the rat colon (Ekblad, 1999), where the potassium channels involved in the VIP action are also sensitive to charybdotoxin (Kishi et al., 2000). These results suggest that PACAP and VIP act at different receptor subtypes which are coupled to different potassium channels, as also observed in the guinea-pig taenia coli (Jin et al., 1994). The receptors involved belong to the PAC1 subtype in the dog ileum (Fox-Threlkeld et al., 1999) and in the rat colon (Ekblad, 1999), whereas only the VIP2 /PACAP3 receptor (now VPAC2 receptor) is apparently expressed in gastric smooth muscle cells of rabbit and guinea-pig (Teng et al., 1998). Electrophysiological effects of VIP-like peptides on neurons and enteric smooth muscle cells VIP depolarizes myenteric neurons and mimics the slow EPSP in AH neurons (Williams and North, 1979; Zafirov et al., 1985). By combining electrophysiological and immunohistochemical methods, Katayama, Lees and Pearson (1986) demonstrated that all VIPimmunoreactive neurons belong to the S type. Non-cholinergic slow EPSPs could be evoked in 77% of VIP-immunoreactive neurons by stimulating orally or anally to the recording site. Thus, VIP neurons receive both ascending and descending inputs. PACAP1–27 or PACAP1–38 were found to depolarize 96% of AH neurons and 36% of S type neurons, respectively (Christofi and Wood, 1993).

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In the rat stomach, the apamin-resistant inhibitory junction potential (IJP) is mediated by VIP (Ito et al., 1990), whereas both VIP and PACAP are involved in the IJP generation in the longitudinal muscle of distal colon (Kishi et al., 1996). VIP produces a slow hyperpolarization of the CM of the guinea-pig ileum, antagonized by VIP10–28 (Crist, He and Goyal, 1992). In the murine gastric fundus circular muscle, the slow component of the IJP is also blocked by VIP10–28. This effect is apparently mediated by blockade of neuronal VIP receptors, which in turn cause release of NO as final transmitter (Mashimo et al., 1996). Role of endogenous VIP-like peptides VIP is a recognized transmitter of inhibitory motor neurons of the gut in a number of mammalian species, which is released upon a variety of physiological stimuli (Grider and Makhlouf, 1988b). In the rat and guinea-pig colon and human jeunum, distension-induced reflex relaxation is associated with increased VIP release and is blocked by VIP10–28 or VIP antibodies (Grider and Makhlouf, 1986, 1988b; Grider and Rivier, 1990; Foxx-Orenstein and Grider, 1996; Grider, Foxx-Orenstein and Jin, 1998). Release of PACAP from rat colon during reflex relaxation has also been documented (Grider et al., 1994). PACAP participates in the inhibitory transmission of guinea-pig teaenia coli, internal anal sphincter of opossum and in the canine ileal circular muscle (Jin et al., 1994; Chadker and Rattan, 1998; Fox-Threlkeld et al., 1999). Antibodies to somatostatin reduce both VIP release and the relaxation; exogenous somatostatin stimulates VIP release (Grider, Arimura and Makhlouf, 1987). Grider and Makhlouf (1986) have therefore proposed that somatostatin interneurons stimulate release of VIP from inhibitory motor neurons in response to distension. NITRIC OXIDE Nitric oxide is now known to act as a transmitter in the central and autonomic nervous system. The synthetic enzyme for NO, nitric oxide synthase (NOS) has been localized immunohistochemically in both central and peripheral neurons (Bredt, Hwang and Snyder, 1990). The previously characterized enzyme, NADPH diaphorase (Hope and Vincent, 1989), appears to be the same as NOS in some tissues and has therefore been used as a marker for NOS-containing neurons (Dawson et al., 1991; Hope et al., 1991). NOS catalyzes the synthesis of NO and L-citrulline from L-arginine via a Ca2+/calmodulindependent mechanism, which also requires the presence of tetrahydrobiopterin and NADPH as cofactors (Bredt and Snyder, 1989; Knowles et al., 1989; Mayer, John and Böhme, 1990). In some studies, concentrations of L-citrulline are used as an index of NOS activity (Currò, Volpe and Preziosi, 1996). NOS immunoreactivity has been demonstrated to be colocalized with VIP in discrete populations of myenteric neurons: descending inhibitory motor neurons and descending interneurons to other myenteric ganglia and submucous ganglia (Costa et al., 1991, 1996). Generally, the colocalization NOS/VIP has been recognized throughout the gastrointestinal tract of various species, from the stomach (Tonini et al., 2000) to the internal anal sphincter. Double immunostaining in the canine bowel revealed NOS and VIP in the same nerve varicosities, but never in the same organelles (Berezin et al., 1994).

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The “receptor” for NO is the soluble form of guanylate cyclase. NO binds to the haeme group of this enzyme, producing a conformational change to the active state. Thus, NO stimulates synthesis of cGMP, which in turn is believed to regulate protein kinases, phosphodiesterases and ion channels (Goy, 1991; Vincent and Hope, 1992). A recent study (Franck et al., 1997) using a novel inhibitor of guanylate cyclase (ODQ), suggested that electrical and mechanical effects of endogenous and exogenous NO in the canine colon are largely due to cGMP synthesis, while no evidence was found in support of a cGMPindependent mechanism of NO action. NO is now believed to act as a NANC inhibitory transmitter in several regions of the gastrointestinal tract in different species. The following sections will concentrate largely on the effects of exogenous and endogenous NO in the guinea-pig small intestine. Effects of NO on neuromuscular preparations Electrically induced NANC relaxations of the CM in the guinea-pig ileum are reduced by approximately 50% by apamin, while the remaining relaxation is blocked by L-N Gnitroarginine methyl ester (L-NAME). Alone, L-NAME reduces the relaxation by 71% and this inhibition can be reversed by L-arginine (Humphreys, Costa and Brookes, 1991). These results, suggest that NO-induced relaxation depends in part on an apamin-resistant mechanism and in part on an apamin-sensitive one. Sodium nitroprusside (SNP), a nitric oxide donor, causes relaxation of the guinea-pig ileum (Osthaus and Galligan, 1992) which is partly apamin-sensitive (which means involvement of small conductance calcium-activated potassium channels). Furthermore, relaxations can be mimicked by 8-bromo-cGMP (Osthaus and Galligan, 1992). The exact mechanism whereby cGMP causes relaxation is not clear, although it is known to vary according to the tissue and species. In smooth muscle cells isolated from canine colon, cGMP increases the probability of opening of potassium channels (Thornbury et al., 1991). The resultant hyperpolarization reduces voltage-dependent calcium influx into the muscle cells, thereby reducing tone. However, not all cGMP-mediated relaxation is associated with hyperpolarization. In vascular smooth muscle, cGMP has been shown to stimulate Ca2+-ATPase activity, thus reducing cytoplasmic calcium concentrations (Lincoln, 1989). Grider and Makhlouf (1992) proposed a complex mechanism for relaxation of intestinal tissue: they reported that NO could be synthesized in isolated myenteric ganglia from the guinea-pig small intestine in response to DMPP and gastrin-releasing peptide (GRP) (Jin and Grider, 1992). Circular smooth muscle cells from rat colon and guinea-pig gastric fundus could also synthesize NO in response to VIP (Grider and Makhlouf, 1992; Grider et al., 1992). L-N G-nitroarginine (L-NNA) inhibited VIP-induced relaxation and suppressed NO production. In a further study, Jin et al. (1992) reported that relaxation induced by SNP could be abolished by a cGMP-dependent protein kinase (protein kinase G) inhibitor, whilst relaxation due to VIP was reduced by 42%. Furthermore, VIP-induced relaxation was reduced by 33% by an inhibitor of cAMP-dependent protein kinase (protein kinase A), whereas the response to SNP was unaffected. Grider et al. (1992) have therefore proposed that VIP is released from inhibitory motor neurons and acts on smooth muscle receptors leading to stimulation of adenylate cyclase and NO production. NO in turn stimulates guanylate cyclase and also feeds back to further stimulate VIP release from the

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inhibitory motor neuron. In the canine colon, both VIP and NO appear to be responsible for the relaxation. Huizinga, Tomlinson and Pintin-Quezada (1992) found that exogenous VIP produced relaxation of the CM which was partly TTX-sensitive. L-NNA also partly blocked the relaxation. The effect of L-NNA was not significantly different from that produced by TTX or TTX and L-NNA, indicating that NO is responsible for the nervemediated component of the relaxation to VIP. Similarly, relaxation produced by 5-HT in the canine terminal ileum and ileocolonic junction is inhibited by L-NNA (Bogers et al., 1991) and VIP- and electrically evoked relaxations of the opossum internal anal sphincter are blocked by L-NNA (Rattan and Chakder, 1992). However, the relaxation produced by VIP in the canine lower oesophageal sphincter (De Man et al., 1991), guinea-pig taenia coli (Grider et al., 1992), and pig, canine and human gastric fundus strips (Lefebvre, Smits and Timmermans, 1995; Bayguinov et al., 1999; Tonini et al., 2000) are not affected by L-NNA. Thus, the degree to which NO mediates relaxation produced by VIP and other transmitters varies according to species and region of intestine.

Electrophysiological effects of NO on neurons and enteric smooth muscle cells In the canine jejunum CM, a single brief pulse of NO produces transient membrane hyperpolarization and relaxation (Stark, Bauer and Szurszewski, 1991). When NO is applied repetitively, the initial hyperpolarization runs down, although spontaneous phasic contractions are still inhibited (Irons, Stark and Szurszewski, 1992). This suggests that NO relaxes the intestine by two mechanisms: one involving hyperpolarization and the other independent of hyperpolarization. Transmural electrical stimulation of guinea-pig ileum in the presence of atropine and SP desensitization produces a fast and slow IJP in the CM (Niel, Bywater and Taylor, 1983b; Bywater and Taylor, 1986). The fast IJP is apamin-sensitive, whereas the slow IJP is apamin-resistant (Niel, Bywater and Taylor, 1983b). L-NNA, a NOS inhibitor, has no effect on the fast IJP (Watson, Bywater and Taylor, 1992), but blocks the slow IJP and this effect is partly reversed by L-arginine (He and Goyal, 1992; Lyster, Bywater and Taylor, 1992), suggesting that NO is involved. The slow IJP is also blocked by VIP10–28 (Crist, He and Goyal, 1992). Furthermore, He and Goyal (1992) demonstrated that the slow hyperpolarizing response to VIP in CM is inhibited by L-NNA, suggesting that NO mediates the response to VIP. Thus, VIP released from inter- or motor neurons may stimulate NO synthesis in inhibitory motor neurons which in turn relaxes and hyperpolarizes the CM. This mechanism has also been proposed for the generation of slow IJPs in circular muscle strips of murine gastric fundus (Mashimo et al., 1996).

Role of endogenous NO The hypothesis that NO may serve as an inhibitory transmitter in the gastrointestinal tract is based on a great deal of evidence accumulated in the last decade. These findings generally indicate that neurogenic relaxations evoked by electrical field stimulation from gastrointestinal specimens of several animal species, including humans, depend on

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a combination of NO and VIP release. Compared to VIP, NO is preferentially released at low frequency of stimulation (Tonini et al., 2000). NO seems to play an important role in maintaining tonic neural inhibition and compliance of the circular muscle. In fact, NO could be viewed as the primary inhibitory transmitter initiating accommodation in the guinea-pig ileum, rabbit colon and human gastric fundus (Waterman, Costa and Tonini, 1994; Ciccocioppo et al., 1994; Tonini et al., 2000). Apart from its action as an inhibitory transmitter at a postjunctional site, NO may also act as a modulator of excitatory transmission. Several reports now indicate that NO may inhibit cholinergic as well as noncholinergic transmitter release in several models (Knudsen and Tøttrup, 1992; Wiklund et al., 1993; Hryorenko, Woskowska and Fox-Threlkeld, 1994; Yunker and Galligan, 1996; Holzer-Petsche et al., 1996; Tonini et al., 2000). ADENOSINE TRIPHOSPHATE The presence of ATP in all cells makes it difficult to identify which neurons use ATP as a transmitter, although a recent study (McConalogue et al., 1996) provided evidence that ATP and its major metabolites are released from a neuronal source in the guinea-pig taenia coli. In other studies (Matsuo et al., 1997) ATP seems to derive mainly from smooth muscle cells. The best evidence that ATP may have a transmitter role in the enteric nervous system comes from studies using synaptosomes from LM-MP preparations. In these preparations, ATP is released in response to stimulation by high potassium, veratridine, ACh, or 5-HT (White, 1982; White and Leslie, 1982; White and Al-Humayyd, 1983; Al-Humayyd and White, 1985). Since the release is calcium-dependent, it is likely to represent a transmitter rather than a metabolic pool (White and Leslie, 1982). ATP is also released from guinea-pig taenia coli in response to electrical field stimulation. Some of this release is TTX-sensitive (Rutherford and Burnstock, 1978; McConalogue et al., 1996) and is therefore neuronal in origin. Relaxations in this preparation, like in many other gastrointestinal tissues, are mediated by P2Y purinoceptors modulating a subset of calcium-activated potassium channels (Kong, Koh and Sanders, 2000). Nevertheless, most of the measurable ATP is not neuronal in origin, since ATP can be released by smooth muscle relaxants such as papaverine and nitroglycerine (Kuchii, Miyahara and Shibata, 1973). The use of synaptosomes overcomes this problem. A further complication, however, is that ATP release from myenteric synaptosomes is reduced by 50–90% when the guinea-pigs are pretreated in vivo with 6-hydroxydopamine, suggesting that the majority of released ATP comes from noradrenergic nerve terminals of extrinsic nerves (White and Al-Humayyd, 1983; Al-Humayyd and White, 1985). ATP depolarizes S neurons, mimicking slow EPSPs, and hyperpolarizes AH neurons, mimicking slow IPSPs (Katayama and Morita, 1989). These effects are likely to be mediated by calcium-dependent potassium channel opening and closing, respectively (Katayama and Morita, 1989). Recent data also indicate that, in addition to ACh, ATP contributes to fast synaptic transmission through P2X receptors in myenteric neurons (Galligan and Bertand, 1994; LePard, Messori and Galligan, 1997). This transmission, which is antagonized by the P2X receptor antagonist PPADS (Lambrecht et al., 1992), predominantly occurs between descending interneurons and inhibitory motor neurons in descending inhibitory reflex pathways of guinea-pig ileum (Bian et al., 2000). Other

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electrophysiological evidence supports convincingly a role for endogenous ATP as an inhibitory transmitter. Single pulse transmural stimulation of the guinea-pig ileum produces a fast IJP which is TTX- and apamin-sensitive (Niel, Bywater and Taylor, 1983b; Crist, He and Goyal, 1992). This IJP, like the fast hyperpolarization induced by exogenous ATP, is antagonized by Reactive blue 2 and desensitization to α,β-methyleneATP, but is unaffected by VIP10–28 (Crist, He and Goyal, 1992). It is likely therefore, that ATP or a related nucleotide mediates the apamin-sensitive component of non-adrenergic, non-cholinergic inhibitory transmission. More recently, ATP was found to mediate fast relaxations or IJPs in response to electrical stimulation in the circular muscle of guinea-pig proximal colon and internal anal sphincter, the fast component of IJPs in the circular muscle of murine gastric fundus, and the IJPs in the circular muscle of porcine ileum via mechanisms sensitive to apamin, desensitization to α,β-methyleneATP, Reactive blue 2, and suramin, a non-selective P2 receptor antagonist (Maggi and Giuliani, 1996; Rae and Muir, 1996; Mashimo et al., 1996; Fernandez et al., 1998). Desensitization to ATP does not significantly alter peristalsis in the guinea-pig ileum (Weston, 1973) and rabbit colon (Tonini et al., 1982). It is therefore not clear whether endogenous ATP plays a role in peristalsis.

PHARMACOLOGY OF SEROTONERGIC TRANSMISSION Serotonin (5-HT) is present in enterochromaffin cells and neurons in the guinea-pig small intestine (Furness and Costa, 1982). Non-neuronally located 5-HT can be released in a calcium-dependent manner in response to increased intraluminal pressure (Bülbring and Lin, 1958; Bülbring and Crema, 1959; Schwörer, Racké and Kilbinger, 1987) or to vagal stimulation (Furness and Costa, 1987). 5-HT is localized in descending interneurons which project to other myenteric ganglia and/or submucous ganglia (Costa et al., 1982; Furness and Costa, 1982; Brookes, Steele and Costa, 1992). These neurons are also immunoreactive for cholineacetyl transferase. Ultrastructural studies indicate that 5-HT neurons form close contacts with other 5-HT neurons and with non 5-HT-immunoreactive Dogiel type I and type II neurons (Young and Furness, 1991). No obvious classification of enteric neurons has been found based on the number of 5-HT inputs they receive (Young and Furness, 1991). Release of 5-HT from these neurons can be demonstrated in response to depolarizing stimuli (Holzer and Skofitsch, 1984). 5-HT has a bewildering range of effects in the intestine, largely due to the presence of multiple receptor subtypes which appear to be present on several classes of myenteric neurons and smooth muscle cells (Table 6.2). The following section summarizes recent studies in which more selective agonists and antagonists have been used. Earlier work has been reviewed on numerous occasions (e.g. Costa and Furness, 1979; Furness and Costa, 1982, 1987). EFFECTS OF 5-HT ON NEUROMUSCULAR PREPARATIONS 5-HT contracts the ileal LM, producing a biphasic concentration-response curve (Buchheit et al., 1985; Eglen et al., 1990; Kilbinger and Wolf, 1992). The first phase has an EC50 for

8-OH-DPAT, buspirone

(±)WAY100635

Gi/o

Selective agonists

Selective antagonists

Effectors

Ketanserin, SB200646 MDL100907 SB204741

Gq/11

GR127935 (5-HT1B/D), SB216641 (5-HT1Bselective), BRL15572 (5-HT1D-selective)

Gi/o

Gq/11

α-Me-5-HT, BW723C86

α-Me-5-HT

sumatriptan

Longitudinal smooth muscle

5-HT2B

Contraction

Smooth muscle

5-HT2A

Contraction

Facilitation of Peristalsis, contraction

Myenteric neurons, Circular smooth muscle2

5-HT1B/D

Ligand-gated ion channel

Ondansetron granisetron

2-Me-5-HT, m-CPBG

Enhanced transmitter release

Myenteric neurons

5-HT3

Gs

Slow EPSP

Myenteric neurons

5-HT1P1

Gs

Go

5-HTP-DP

5-Carboxamido- Hydroxylated tryptamine indalpines

Relaxation

Smooth muscle

5-HT7

SB204070, Methiotepin, GR113808, GR125487 metergoline, SB258719

Renzapride, tegaserod (HTF919), ML10302, BIMU8

Enhanced transmitter release, relaxation

Myenteric neurons, smooth muscle

5-HT4

5-HT1P receptors are not included in the IUPHAR nomenclature of 5-HT receptors; 2 Sumatriptan is the only 5-HT1B/D receptor agonist so far tested and only fragmentary data exist on the possible involvement of these receptor subtypes in the gut.

Reduced transmitter release

Functional response

1

Myenteric neurons

Distribution in the gut

5-HT1A

TABLE 6.2 Synopsis of major 5-HT receptor subtypes in the gut.

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5-HT of 15 nM, indicating the involvement of a high affinity receptor (Eglen et al., 1990; Buchheit et al., 1985). This phase is selectively blocked by high concentrations of ICS 205–930 and mimicked by 5-MOT, BIMU 1, BIMU 8, cisapride and DAU 6236 (all 5-HT4 receptor agonists; Schuurkes et al., 1985; Eglen et al., 1990; Kilbinger and Wolf, 1992; Rizzi et al., 1992). These characteristics suggest that 5-HT is acting via the 5-HT4 receptor subtype. The first phase is also blocked by atropine, TTX, morphine and [D-Pro4, 7,9 D-Trp ]SP4–11 and augmented by physostigmine (Buchheit et al., 1985; Schuurkes et al., 1985). In the presence of antagonists to block all but the 5-HT4 receptor subtype, 5-HT and 5-MT enhance electrically evoked ACh release and associated twitch, and increase basal ACh release (Kilbinger and Pfeuffer-Friederich, 1985; Craig and Clarke, 1990; Fox and Morton, 1990; Kilbinger and Wolf, 1992). Cisapride also increases electrically evoked ACh release and the associated contraction (Taniyama et al., 1991). Cisapride has no effect on the contraction produced by exogenous ACh (Schuurkes et al., 1985). Consequently, the 5-HT4 receptor is likely to be located on LM motor neurons, where its activation stimulates the release of ACh and SP in a TTX-sensitive manner. The second phase of the concentration-response curve has an EC50 for 5-HT of 1.3 mM, indicating the involvement of a low affinity receptor (Buchheit et al., 1985; Eglen et al., 1990). This phase is blocked by low concentrations of ICS 205–930 (Buchheit et al., 1985; Eglen et al., 1990), suggesting that the 5-HT3 receptor subtype is involved. The contraction is inhibited by TTX, morphine, [D-Pro4, D-Trp7,9]SP4–11 and atropine and enhanced by physostigmine (Buchheit et al., 1985; Fox and Morton, 1989; Eglen et al., 1990). Activation of 5-HT3 receptors enhances basal ACh outflow (Kilbinger and Wolf, 1992). Thus, activation of 5-HT3 receptors may stimulate ACh and SP release from LM motor neurons. 5-HT also stimulates calcium-dependent, TTX-sensitive γ-aminobutyric acid (GABA) release through an action at 5-HT3 receptors (Shirakawa et al., 1989). At high concentrations (>10 µM), 5-HT acts at 5-HT2 receptors (formerly called D receptors) which are present on the smooth muscle and stimulate contraction (Engel et al., 1984; Buchheit et al., 1985; Richardson et al., 1985; Fox and Morton, 1989; Table 6.2). 5-HT produces a TTX-sensitive relaxation of precontracted LM in the presence of antagonists to 5-HT2, 5-HT3 and 5-HT4 receptors (Bill, Dover and Rhodes, 1990; Elswood and Bunce, 1992). This action is mimicked by carboxamidotryptamine and sumatriptan, suggesting that 5-HT1A and 5-HT1D receptors are involved (Bill, Dover and Rhodes, 1990; Elswood and Bunce, 1992). This relaxation may be due to inhibition of excitatory LM motor neurons, since activation of 5-HT1A receptors reduces the amplitude of the tachykinin-mediated twitch by a prejunctional mechanism (Galligan, 1992). It has also been reported that 5-HT1A agonists inhibit electrically evoked ACh outflow (Fozard and Kilbinger, 1985; Kilbinger and Wolf, 1992; Dietrich and Kilbinger, 1996) and reduce cholinergic twitch responses (Fozard and Kilbinger, 1985; Craig and Clarke, 1990). However, these effects may be due in part to an action of the agonists at histamine and muscarinic receptors (Galligan, 1992). 5-HT1A receptors also mediate inhibition of GABA release (Shirakawa et al., 1989). Some authors suggest caution in considering the involvement of 5-HT1A receptors in mediating the effects of 5-HT in the gut on the basis of data obtained with 8-OH-DPAT, a compound previously considered a selective 5-HT1A receptor agonist. After the discovery that 5-HT7 receptors mediate relaxation probably by a direct action on the smooth muscle

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(Carter et al., 1995; Prins et al., 1999) and that 8-OH-DPAT is also a partial agonist at the 5-HT7 receptor, some functional data should probably be reconsidered (Vanhoenacker, Haegeman and Leysen, 2000). Agonists at 5-HT4 receptors (BIMU 8, cisapride and 5-HT) cause a concentrationdependent increase in the amplitude of submaximal cholinergic twitch responses. This effect is inhibited by micromolar concentration of ICS 205–930 and DAU 6285 (Tonini et al., 1992a), confirming that 5-HT4 receptors are involved. In addition to enhancing electrically evoked twitch responses, cisapride enhances the ascending reflex contraction produced by balloon distension of the ileum (Tonini, 1992). Since the drug was added to the anal compartment of a partitioned organ bath, which contains the sensory neurons and some interneurons involved in the reflex, the effect of cisapride may be due to enhancement of transmission between these neuronal classes. Electrophysiological studies (see below) indicate that cisapride enhances nicotinic transmission. The ability of 5-HT to enhance peristalsis has been known for many years (Bülbring and Crema, 1958; Bülbring and Lin, 1958). The receptor involved in this process has been the subject of several investigations. Renzapride (a 5-HT4 receptor agonist) and 5-HT initiate peristalsis in the guinea-pig ileum and this effect is blocked by high concentrations of ICS 205–930 (Craig and Clarke, 1991). Using a slightly different preparation, Tonini, Galligan and North (1989) and Rizzi et al. (1992) showed that the frequency of emptying was increased by cisapride, BIMU 1, BIMU 8 and DAU 6236; on close inspection of the recordings, the compliance of the intestinal wall also appears to have been decreased. These effects were inhibited by high concentrations of ICS 205–930, but not by ondansetron, indicating the involvement of 5-HT4 receptors. There was no effect of 5-HT4 receptor agonists on LM muscle contraction during the preparatory phase or on the maximal ejection pressure (Tonini, Galligan and North, 1989a; Rizzi et al., 1992). 5-HT4 receptor agonists could conceivably reduce the threshold volume required to trigger emptying of the intestine and the compliance of the intestinal wall, by stimulating excitatory motor neuron pathways or inhibiting inhibitory motor neuron pathways (Waterman, Costa and Tonini, 1992). The former hypothesis seems more likely, since 5-HT4 receptor agonists can enhance neuronal release of ACh and SP (Kojima and Shimo, 1996), while the inhibitory effects of 5-HT4 receptor agonists on an inhibitory pathway is less well documented. However, other mechanisms may be involved: first, enhancement of ACh and SP release from LM-MP preparations does not necessarily mean that these agonists enhance transmitter release from CM motor neurons. Second, enhancement of the cholinergic twitch response in CM by 5-HT4 agonists could equally well be due to removal of an inhibitory input to the muscle as to stimulation of excitatory motor neurons. Therefore, even though the net effect is prokinetic, it is unclear whether the underlying mechanism is one of excitation or inhibition. Interestingly, there are animal (Grider, Kuemmerle and Jin, 1996) and human (FoxxOrenstein, Kuemmerle and Grider, 1996) data suggesting that 5-HT released by mucosal stimulation initiates a peristaltic reflex by activating 5-HT4 receptors on sensory neurons containing calcitonin gene-related peptide (CGRP). These effects are mimicked by mucosal application of selective 5-HT4 receptor agonists (prucalopride and tegaserod: Grider, Foxx-Orenstein and Jin, 1998). Experimental evidence for this mechanism in humans is so far limited to the small bowel.

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Important differences may exist among different species/different gut levels. 5-HT4 receptor-mediated prokinesia may result from increased release of ACh (and tachykinins) from excitatory neurons and may operate in human small bowel and stomach (SakuraiYamashita et al., 1999; Schuurkes et al., 1991), whereas this pathway does not seem to operate in the human colonic circular muscle (Burke and Sanger, 1988; Burleigh and Trout, 1985). In addition, it should be noted that, in contrast with what observed in the one of the most widely used models [the guinea-pig colon, where neuronal 5-HT4 receptors mediate contractile responses that are mainly cholinergic in nature (Briejer et al., 1993b; Wardle and Sanger, 1993)], human colonic circular muscle strips are endowed with 5-HT4 receptors located on smooth muscle cells, where they mediate relaxation (McLean and Coupar, 1996; Prins et al., 2000b; Sakurai-Yamashita et al., 2000; Tam et al., 1995). A recent report (Prins et al., 2000a) suggests the presence of 5-HT4 receptors on cholinergic neurons supplying the longitudinal muscle in the human colon. Figure 6.2 illustrates the distribution of 5-HT4 receptors on effector cells and intrinsic neuronal pathways (i.e. myenteric plexus), which mediate propulsive activity in the gastrointestinal tract. 5-HT1B/D receptors are now emerging as possible targets of 5-HT action in the gut (Borman and Burleigh, 1997; Coulie et al., 1997; Coulie et al., 1999; Tack et al., 2000). However, sumatriptan is the only 5-HT1B/D receptor agonist so far tested and, to the best of our knowledge, no formal assessment of the involvement of this receptor subtype by the use of selective antagonists has been performed (Cipolla et al., 2001). A study carried out

Figure 6.2 Modulation of intestinal motility by serotonergic 5-HT4 receptors. These receptors, which are distributed at multiple neuronal sites, have an excitatory effect (+) on enteric neurons leading to transmitter release and facilitation of propulsion. 5-HT4 receptors are also located on smooth muscle cells where they produce relaxation (–).

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in the human ileum suggested that, in the circular muscle, 5-HT-induced contraction is mediated via a receptor of the 5-HT1D subtype, whereas a receptor of the 5-HT2B subtype mediates the contractile response to 5-hydroxytryptamine of longitudinal muscle layer (Borman and Burleigh, 1997). Selective 5-HT1D receptor antagonists were not available at the time of this study to confirm the hypothesis. Finally, preliminary data suggest that 5-HT1B/D receptors are involved in mediating the facilitatory effect of sumatriptan on gastric accommodation in an in vivo canine model (De Ponti, 2000). ELECTROPHYSIOLOGICAL EFFECTS OF 5-HT ON NEURONS The electrophysiological effects of 5-HT on enteric neurons has been reviewed by Galligan (1995). 5-HT1A receptors are located on the cell bodies of AH neurons and on nerve terminals of cholinergic neurons (Galligan et al., 1988). Agonists at these receptors produce membrane hyperpolarization in AH neurons, but not S neurons, due to an increase in potassium conductance (Galligan et al., 1988; Galligan and North, 1991). These receptors also mediate presynaptic inhibition of ACh release which is recorded electrophysiologically as an inhibition of fast EPSPs (North et al., 1980; Galligan et al., 1988; Galligan and North, 1991). The amplitude of slow EPSPs is also reduced (Galligan et al., 1988; Galligan and North, 1991). 5-HT1P receptors are present on AH neurons where they mediate a slow depolarization associated with an increase in input resistance (Mawe, Branchek and Gershon, 1986). These receptors have been reported to be specifically antagonized by dipeptides of 5-hydroxytryptophan (5-HTP-DP) (Takaki et al., 1985a; Mawe, Branchek and Gershon, 1986). Activation of 5-HT3 receptors by 2-methyl-5-HT produces a fast depolarization in AH neurons which is associated with a decrease in input resistance, due to the opening of a cation channel (Mawe, Branchek and Gershon, 1986; Surprenant and Crist, 1988; Derkach, Surprenant and North, 1989; Mawe, Branchek and Gershon, 1989). This effect is blocked by low concentrations of ICS 205–930 (Mawe, Branchek and Gershon, 1986). Cisapride increases the amplitude of fast EPSPs through a presynaptic action, since it has no effect on the fast depolarization produced by exogenous ACh. This is likely to be due to stimulation of ACh release (Tonini, Galligan and North, 1989a). Cisapride had no effect on the resting membrane potential in several studies (Mawe, Branchek and Gershon, 1989; Tonini, Galligan and North, 1989a), but was reported to produce depolarization or hyperpolarization in a minority of cells (Nemeth et al., 1985; Nemeth and Gullikson, 1989). Cisapride does not alter non-cholinergic slow EPSPs in AH neurons (Tonini, Galligan and North, 1989a). ROLE OF ENDOGENOUS 5-HT Although 5-HT is present in and can be released from enterochromaffin cells and neurons in the guinea-pig ileum, it is difficult to prove its physiological role. 5-HT3 receptors are ligand-gated cation channels and mediate rapid depolarization of myenteric neurons (Surprenant and Crist, 1988; Derkach, Surprenant and North, 1989; Mawe, Branchek and Gershon, 1989). It is conceivable therefore, that at least some of the

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fast EPSPs recorded in the myenteric plexus are due to the action of an endogenous 5-HT-like substance on 5-HT3 receptors rather than the action of ACh at nicotinic receptors. This possibility has received little attention. However, Tonini, Galligan and North (1989a) found that the 5-HT3 receptor antagonists, ICS 205–930 and ondansetron, had no effect on the amplitude of fast EPSPs. It has been proposed that 5-HT mediates some or all slow EPSPs in the myenteric plexus. The latter hypothesis is unlikely, since lesioning 5-HT inputs to myenteric ganglia does not significantly alter the probability of recording slow EPSPs (Bornstein et al., 1984). However, 5-HT may be responsible for a proportion of slow EPSPs. Takaki et al. (1985b) and Mawe, Branchek and Gershon, (1986, 1989) reported that electrically evoked slow EPSPs could be blocked by 5-HTP-DP and renzapride but not by low concentrations of ICS 205–930. Some slow EPSPs may therefore be mediated by the action of 5-HT-like substances at 5-HT1P receptors. Since exogenous 5-HT can elicit fast and slow EPSPs, it is reasonable to propose that endogenous 5-HT might be responsible for non-cholinergic transmission. Such transmission has been shown to occur in the ascending excitatory reflex response to distension (Holzer, 1989; Tonini and Costa, 1990). However, endogenous 5-HT-like substances acting at 5-HT1, 5-HT1P, 5-HT3 and 5-HT4 receptors do not mediate the non-cholinergic component of ganglionic transmission in this reflex (Tonini et al., 1992b). Nevertheless, it is not known whether 5-HT may play a role in modulating cholinergic transmission in this reflex pathway through an action at presynaptic receptors. Endogenous 5-HT may play a role in the ascending excitatory reflex response to mucosal distortion, rather than distension. Bülbring and Crema (1958) and Bülbring and Lin (1958) originally proposed that 5-HT is involved in sensitizing mucosal processes of sensory neurons in the intestine. This was based on these pieces of evidence: first, 5-HT is present in enterochromaffin cells which are near the mucosal processes of presumed sensory neurons. Second, 5-HT could be released into the lumen of the intestine and this release is increased by increasing intraluminal pressure. Third, mucosal application of 5-HT stimulates peristalsis. More recently, Kirchgessner, Tamir and Gershon (1992) reported that stimulation of the mucosa with puffs of nitrogen gas results in c-fos expression in some myenteric and submucous neurons. Expression of c-fos is inhibited by dipeptides which are believed to inhibit 5-HT1P receptors. These investigators also demonstrated that antibodies to 5-HT1P receptors label submucosal calbindin-immunoreactive neurons, the proposed sensory neurons. The investigators therefore suggested that the response to nitrogen puffs may be due to release of 5-HT from enterochromaffin cells, which stimulates sensory neurons through an action at 5-HT1P receptors. However, these results await to be confirmed by other research groups. Furthermore, even if the conclusion drawn by Kirchgessner, Tamir and Gershon (1992) is correct, it does not necessarily mean that this mechanism operates during peristalsis. In fact, studies by other investigators, provide evidence that 5-HT is not necessarily involved. Schwörer, Racké and Kilbinger (1989) measured 5-HT release from enterochromaffin cells in the guineapig ileal mucosa in response to increased pressure in the lumen. If the intestine was infused with fluid at 22° C instead of 37° C, release of 5-HT was inhibited. However, there were no concomitant changes in peristalsis, thereby suggesting that 5-HT released from mucosal enterochromaffin does not play a significant role in peristalsis. Furthermore,

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antagonists at 5-HT3 and 5-HT4 receptors do not appear to have any effect on peristalsis (Craig and Clarke, 1991), suggesting that endogenous 5-HT has little, if any, role in peristalsis through an action at these receptors. However, the facilitatory effect of mucosally applied 5-HT to reduce the peristaltic threshold in the guinea-pig ileum is probably mediated by 5-HT3 receptors located on the mucosal side (Tuladhar, Kaisar and Naylor, 1997), and in particular on the terminals of intrinsic primary afferent neurons (Bertrand et al., 2000).

PHARMACOLOGY OF AMINO ACIDS AS ENTERIC NEUROTRANSMITTERS γ-AMINOBUTYRIC ACID (GABA) The presence of GABA in the guinea-pig small intestine has been demonstrated by immunohistochemistry (Jessen, Hills and Saffrey, 1986; Saito and Tanaka, 1986; Pompolo and Furness, 1990) and autoradiography (Krantis and Kerr, 1981). GABA is contained in short inhibitory CM motor neurons and LM motor neurons (Krantis, Kerr and Dennis, 1986; Saito and Tanaka, 1986; Hills, Jessen and Mirsky, 1987; Furness et al., 1989b). Myenteric neurons also express the synthetic enzyme for GABA, glutamic acid decarboxylase (GAD) (Miki et al., 1983; Williamson et al., 1995) and a high affinity uptake mechanism for the neurotransmitter (Krantis and Kerr, 1981; Saffrey et al., 1983; Krantis, Kerr and Dennis, 1986). GABA is released from myenteric synaptosomes of the guinea-pig small intestine in response to depolarization by high potassium and veratridine in a calcium-dependent manner (Yau and Verdun, 1983). The release of GABA from LM-MP preparations of the guinea-pig small intestine has also been demonstrated during either high potassium or electrically-evoked stimulation (Taniyama et al., 1983a; Wiley, Lu and Owyang, 1991). Somatostatin, SP, 5-HT, neurotensin and cholecystokinin (CCK) can stimulate GABA release from LM-MP preparations (Kerr and Krantis, 1983; Taniyama et al., 1983a; Tanaka and Taniyama, 1985; Nakamoto, Tanaka and Taniyama, 1987; Sano, Taniyama and Tanaka, 1989; Shirakawa et al., 1989; Takeda et al., 1989). This release is blocked by TTX and low Ca2+ concentrations. GABA release can be inhibited presynaptically by agonists at M1 receptors and α2-adrenoceptors (Hashimoto, Tanaka and Taniyama, 1986). Immunohistochemical evidences have been provided for the presence of both GABAA and GABAB receptors in the enteric nervous system (Krantis et al., 1995; Nakajima et al., 1996). In particular, GABAA receptor immunoreactivity has been detected on the soma of myenteric ganglion cells in the rat distal colon (Krantis et al., 1995). In addition, molecular biological investigations have demonstrated an abundant and widespread distribution of different GABAA receptor subunit mRNAs in the rat’s small and large intestines (Zeiter, Li and Broussard, 1996; Poulter et al., 1999). GABAB immunoreactivity was found on the soma of neurons in both submucosal and myenteric ganglia at different levels in the rat gut (Nakajima et al., 1996).

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Effects of GABA on neuromuscular preparations GABA is not effective on nerve-free preparations. In the LM of the guinea-pig small intestine, GABA produces a transient contraction followed by a relaxation (Inouye et al., 1960; Krantis et al., 1980; Giotti et al., 1983; Taniyama et al., 1983b). Both effects are blocked by TTX (Krantis et al., 1980), indicating that GABA is not directly acting on the LM. GABA-induced contractions are mimicked by muscimol and associated with an increase in ACh release (Kleinrok and Kilbinger, 1983; Tonini et al., 1987). The enhancements both in tension and in ACh release are blocked by bicuculline, picrotoxin, hyoscine and atropine, suggesting the involvement of GABAA receptors. Hexamethonium reduced the phasic GABAA-mediated contraction by 20% in one study (Tonini et al., 1987), suggesting that GABA is capable of stimulating cholinergic interneurons. GABA has also been shown to enhance ACh release from synaptosomes isolated from LM-MP preparations of the guinea-pig small intestine, suggesting that the stimulatory effect of GABA is due, at least in part, to an action on nerve terminals (Yau and Verdun, 1983). Tonini et al. (1987) found that GABA-induced contractions were also reduced by [D-Pro4, D-Trp7,9]SP4–11, indicating that GABAA receptors are also involved in the stimulation of tachykinin release from motor neurons and interneurons in the LM pathways of the guinea-pig ileum. The presence of GABAA receptors is also reported on inhibitory neurons and their stimulation induces relaxant responses (Minocha and Galligan, 1993; Boeckxstaens et al., 1991). Several reports are available to suggest that GABA may also inhibit both electricallyand chemically-evoked contractions by acting prejunctionally to reduce ACh release, in a bicuculline- and picrotoxin-insensitive manner (Hobbiger, 1958a; Hobbiger, 1958b; Florey and McLennan, 1959; Inouye et al., 1960; Kleinrok and Kilbinger, 1983; Ong and Kerr, 1983). This effect is mimicked by homotaurine (3-APS) and baclofen, but not by muscimol (Kleinrok and Kilbinger, 1983; Hills et al., 1989). δ-Aminovaleric acid (DAVA), but not bicuculline, reversed the action of both GABA and baclofen (Ong and Kerr, 1983). On the whole, these observations suggest that inhibitory effects of GABA on contractile responses are due to inhibition of ACh release from motor neurons mediated via GABAB receptors. GABA does not have any significant effect on electrically stimulated contraction of the CM, however it reduces the frequency of spontaneous CM contractions (Ohkawa, 1987).

Electrophysiological effects of GABA on neurons Ionophoretic application of GABA to myenteric neurons of the guinea-pig ileum determines a depolarizing response of AH neurons in a bicuculline-sensitive manner. This effect is associated with an increase in chloride conductance and is prone to desensitization suggesting the involvement of GABAA receptors (Grafe, Galvan and Mayer, 1979; Cherubini and North, 1984a). Data from studies in acutely isolated myenteric plexus preparations and in myenteric neurons maintained in primary culture suggest that GABAA-mediated responses are potentiated by a number of drugs acting at benzodiazepine and barbiturate binding sites (Cherubini and North, 1985a; Bertrand and Galligan, 1992; Zhou and Galligan, 2000). Electrophysiological and immunohistochemical investigations

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have recently demonstrated that myenteric neurons expressing calbindin immunoreactivity, a marker for AH neurons, have a high density of GABAA receptors (Zhou and Galligan, 2000). In addition to rapid depolarizing responses induced by ionophoretic application of GABA, superfusion of the agonist produces depolarizations in AH myenteric neurons which are mimicked by baclofen and are insensitive to bicuculline. This effect is associated with a smaller increase in conductance than that evoked by ionophoresis of GABA and does not desensitize. On the whole these observations suggest that this latter effect is mediated via activation of GABAB receptors probably located on nerve processes rather than on neuronal somata (Cherubini and North, 1984a). In isolated myenteric ganglia of the guinea pig ileum, GABA does not alter the resting membrane potential or conductance of S neurons (Cherubini and North, 1984b). However, GABA reduces the amplitude of evoked fast EPSPs without altering the response to ionophoretic application of ACh (Grafe, Galvan and Mayer, 1979; Cherubini and North, 1984b). This effect is mimicked by baclofen and resistant to bicuculline, suggesting the involvement of a GABAB receptor in the response (Cherubini and North, 1984b). In the same experimental model, GABAB receptors have also been shown to mediate the inhibition of slow EPSPs (Cherubini and North, 1984b). Thus, inhibition of both fast and slow EPSPs in S myenteric neurons is accomplished through GABAB receptors, which have been suggested to be located presynaptically to inhibit the release of both ACh and of a non-cholinergic excitatory transmitter.

Role of endogenous GABA Pharmacological studies have been described on the possible involvement of GABA receptors in the modulation of peristalsis with contrasting results. Indeed, early studies reported a GABA-mediated inhibition of peristalsis in a picrotoxin-sensitive manner (Hobbiger, 1958b; Inouye et al., 1960), whereas in a later study Schwörer and Kilbinger, (1988) could not demonstrate any significant effect of exogenous GABA on this functional parameter. In guinea-pig ileum, the amplitude of cholinergic twitches in the LM and the efficiency of peristalsis were enhanced by bicuculline (Schwörer and Kilbinger, 1988; Tonini et al., 1989b). However, these effects were not mimicked by picrotoxin or SR 95531, which also act on the GABAA receptor complex. These evidences might be explained on the basis of a non-specific effect of bicuculline (Tonini et al., 1989b), which might explain the discrepancy between an apparent effect of endogenous GABA on peristalsis (as deduced from the effect of bicuculline) and the lack of effect of exogenous GABA. However, in the guinea-pig colon, bicuculline has been shown to enhance the efficiency of peristalsis in normal but not in GABA-desensitized preparations, suggesting an action of bicuculline entirely dependent on the availability of functional GABA receptors (Frigo et al., 1987). In the same study bicuculline enhanced both cholinergic excitatory and non-adrenergic, non-cholinergic inhibitory responses to transmural stimulation and the release of ACh. These effects were completely abolished after desensitization with GABA (Frigo et al., 1987).

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SP- and neurotensin-induced stimulation of ACh release is partly reduced by bicuculline (Tanaka and Taniyama, 1985; Nakamoto, Tanaka and Taniyama, 1987). The authors concluded that endogenous GABA, which is released in response to SP and neurotensin, acts on GABAA receptors to stimulate ACh release. However, it seems likely that these effects are also due to non-specific actions of bicuculline, since the concentrations used in these studies (1–30 µM) were the same as those employed by Tonini et al. (1989b). Other evidence for non-specific effects of bicuculline has previously been reported in isolated rat atria, guinea-pig ileum and mouse vas deferens (Bartolini et al., 1985). Somatostatin-induced relaxation of the LM and the associated inhibition of ACh release, are blocked by phaclofen (Takeda et al., 1989). Somatostatin can stimulate GABA release (Takeda et al., 1989); therefore these results are likely to be due to a somatostatininduced GABA release which acts at GABAB receptors to inhibit ACh release and LM contraction. Since GABA is present only in motor neurons, and not in sensory neurons or interneurons, these results suggest that GABA may be released at the neuroeffector junction and act on local nerve terminals to inhibit (via GABAB receptors) ACh release. Exogenous GABA has a similar action on cholinergic nerve terminals in the myenteric plexus. Spontaneous neurogenic contractions of the guinea-pig ileum LM can be inhibited by atropine, bicuculline, picrotoxin, RU 5135, desensitization to GABA, 3-mercaptopropionic acid and cortisol (Ong and Kerr, 1984, 1989). Together with other results, these findings suggest that endogenous GABA is released from LM motor neurons and acts on local nerve terminals via GABAA receptors to stimulate ACh release. Immunohistochemical evidences have been provided for the presence also of inhibitory neurons co-expressing GABA and NOS immunoreactivity in the guinea-pig small intestine (Williamson, Pompolo and Furness, 1996). EXCITATORY AMINO ACIDS The concept that glutamate, the major excitatory neurotransmitter in the central nervous system (Ozawa, Kamiya and Tsuzuki, 1998), plays a role as a neurotransmitter also in the enteric nervous system is gaining increased acceptance (Liu et al., 1997; Sinsky and Donnerer, 1998). Immunoreactivity for glutamate has been detected in subsets of neurons in both the myenteric and submucosal plexus of the rat and guinea-pig ileum (Liu et al., 1997). In particular, glutamate immunoreactivity was found in enteric cholinergic neurons colocalized with substance P and/or calbindin (Liu et al., 1997). Immunohistochemical evidence has also been provided for glutamate storage into terminal axonal varicosities of myenteric neurons in the guinea-pig ileum (Liu et al., 1997). At this level, L-glutamate can be synthesized from the precursor, L-glutamine (Wiley, Lu and Owyang, 1991) and released during high potassium (Wiley, Lu and Owyang, 1991) or electrical-evoked (Sinsky and Donnerer, 1998) depolarization in a Ca2+-dependent manner. A recent study has demonstrated that both N-type and P-type calcium channels control most of stimulated glutamate release from LM-MP preparations of the guinea-pig ileum (Reis et al., 1999). Both ionotropic and metabotropic glutamate receptors have been found to be abundantly expressed in the enteric nervous system. Immunoreactivity for ionotropic receptors of the

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N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5methyl-4-isoxazole propionic acid (AMPA) and of the kainate type, and for group I metabotropic receptors of the mGluR5 type has been detected in both the myenteric and submucosal plexus of the guinea-pig ileum (Kirchgessner, Liu and Alcantara, 1997; Liu et al., 1997; Liu and Kirchgessner, 2000). In addition, expression of mRNAs encoding for NMDA and mGluR2/3 metabotropic receptors has been demonstrated within enteric ganglia at different levels in the guinea-pig and rat gut (Broussard et al., 1994, Burns, Stephens and Benson, 1994; Larzabal et al., 1999). Enteric neurons are also endowed with a glutamate-inactivating mechanism, represented by the high affinity neuronal transporter, EAAC1 whose expression along the gut is particularly extensive (Liu et al., 1997). Effects of excitatory amino acids on neuromuscular preparations Evidence from functional studies suggests that L-glutamate mediates contraction of the longitudinal muscle in the guinea-pig ileum through activation of NMDA receptors, probably located on excitatory cholinergic motor neurons (Luzzi et al., 1988; Shannon and Sawyer, 1989; Wiley, Lu and Owyang, 1991; Sinsky and Donnerer, 1998). Indeed, in LM-MP preparations of the guinea-pig ileum, L-glutamate, L-aspartate and NMDA determine contractile responses which are competitively antagonized by NMDA receptor antagonists and noncompetitively blocked by Mg2+ (Moroni et al., 1986; Luzzi et al., 1988; Shannon and Sawyer, 1989; Wiley, Lu and Owyang, 1991). An NMDA receptor has been suggested to be involved in the contraction of the guinea-pig ileum also since the action of glutamate is potentiated by glycine in a strychnine-insensitive manner (Luzzi et al., 1988). In addition, glutamate-induced contractions are inhibited by TTX and hyoscine, but not by hexamethonium (Moroni et al., 1986; Luzzi et al., 1988; Wiley, Lu and Owyang, 1991). On the whole, this evidence suggests that L-glutamate stimulates ACh release from LM motor neurons which in turn causes the contraction via activation of postjunctional muscarinic receptors. Indeed, pharmacological evidence has been provided for an NMDA-mediated enhancement of ACh release from myenteric neurons in the guinea-pig ileum and colon (Wiley, Lu and Owyang, 1991; Cosentino et al., 1995). At this latter level, application of NMDA enhances also the release of an inhibitory neurotransmitter such as noradrenaline. This effect has been correlated to the NMDA-mediated inhibition of peristalsis in the colon (Cosentino et al., 1995). The possible involvement of AMPA and kainate receptors in the modulation of motor function in the gastrointestinal tract has not yet been completely clarified. AMPA and kainate receptors do not seem to mediate a contractile response in the guinea-pig ileum, since neither quisqualate nor kainate are effective in producing contractions of LM-MP preparations at concentrations up to 1 mM (Moroni et al., 1986; Luzzi et al., 1988; Shannon and Sawyer, 1989). Recent pharmacological investigations have demonstrated that activation of AMPA receptors inhibits both electrically-induced contractions of the circular muscle and ACh release in the guinea-pig distal colon (Giaroni et al., 2000). In the same study, AMPA has been described to enhance the efficiency of peristalsis. On the basis of these apparently contrasting results, the authors suggested that AMPA receptors might have a more prominent role in the modulation of inhibitory inputs to the colon (Giaroni et al., 2000). Furthermore, in accordance with the inability of kainate to induce LM-MP

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contractions in the guinea-pig ileum (Luzzi et al., 1988), this compound was ineffective in modulating both ACh release and peristalsis in the colon of the same species (Giaroni et al., 2000). Electrophysiological effects of excitatory amino acids on neurons Electrophysiological studies of glutamate effects on enteric neurons have been described with contrasting results for the occurence of depolarizing responses and identification of glutamate receptors involved in the response. Grafe, Galvan and Mayer (1979) found no significant electrophysiological effect of L-glutamate in seven myenteric neurons studied, whereas Liu et al. (1997) found that application of glutamate evokes depolarizing responses in myenteric neurons that had fast and slow components. The fast component was mimicked by AMPA, whereas the slow component was mimicked by NMDA. In the same study, glutamate and ACh have been suggested to represent excitatory co-transmitters, both contributing to mediate fast ganglionic neurotransmission in AH/type 2 neurons. However, contrasting data have been recently reported suggesting that glutamate evokes only slowly activating depolarizing responses in submucous and myenteric neurons of the guinea-pig small intestine by activation of group I metabotropic receptors (Ren et al., 2000).

PHARMACOLOGY OF HISTAMINERGIC TRANSMISSION Histidine decarboxylase (HDC, a marker for histamine containing neurons) and its mRNA have been demonstrated in neurons in the rat central and peripheral nervous systems (Watanabe et al., 1984; Karhula et al., 1990). HDC immunoreactivity has been demonstrated in myenteric neurons in the rat small intestine (Ekblad et al., 1985). However, since the antibody used in these studies cross-reacts with guinea-pig L-dopa decarboxylase, the distribution of HDC in the myenteric neurons in the guinea-pig small intestine could not be determined (Ando-Yamamoto et al., 1986). In the intestine, histamine interacts with three types of receptor, namely H1, H2 and H3 receptors, for which there are currently available selective agonists and antagonists (Hill et al., 1997). H1 receptors are located postjunctionally where they mediate contraction (Zavecz and Yellin, 1982; Trzeciakowski, 1987; Barocelli et al., 1993). H2 receptors are present on LM motor neurons of guinea-pig small intestine where they facilitate the release of ACh and tachykinins (Zavecz and Yellin, 1982; Barker and Ebersole, 1982; Poli, Loruzzi and Bertaccini, 1990). These receptors excite myenteric AH neurons partly by enhancing chloride conductance (Starodub and Wood, 2000). H3 receptors are mainly prejunctional and inhibit the release of ACh and tachykinins from LM motor neurons (Trzeciakowski, 1987; Hew, Hodgkinson and Hill, 1990; Menkveld and Timmerman, 1990; Taylor and Kilpatrick, 1992). In addition, H3 receptors have been demonstrated to mediate inhibition of noradrenaline release from intestinal sympathetic nerves (Blandizzi et al., 2000; Liu et al., 2000). The effects mediated by histamine receptors have been recently assessed in distensionevoked ascending excitatory transmission in the guinea-pig ileum. Histamine acting at H1

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receptors depressed synaptic transmission at low concentrations, whereas increased it at high concentrations. Activation of H2 receptors by the agonist dimaprit was associated with facilitation of transmission, whereas activation of H3 receptors by α-methylhistamine inhibited synaptic transmission (Izzo et al., 1998). Histamine is known to exist in mast cells and basophils in the intestinal mucosa, but the role of endogenous histamine in intestinal motility is unknown.

PHARMACOLOGY OF PEPTIDES AS ENTERIC NEUROTRANSMITTERS OPIOID PEPTIDES The guinea-pig small intestine contains peptides derived from proenkephalin and prodynorphin but not pro-opiomelanocortin (Steele et al., 1992). The presence of enkephalinlike and dynorphin-like immunoreactivity in the myenteric plexus has been reported by a number of researchers (Furness and Costa, 1987). Opioids are found in orally and anally directed interneurons, excitatory and inhibitory CM motor neurons and rarely in LM motor neurons (Brookes, Steele and Costa, 1991b, 1992; Steele et al., 1992; Costa et al., 1996). Recently, the endogenous opioid endomorphin-1 and endomorphin-2 have been detected in brain (Zadina et al., 1997) and spinal cord of various species, including man. However, endomorphins have not been found yet in the gut. Ligand binding and functional studies indicate that µ and κ receptors, and to a lesser extent δ receptors, are present in the myenteric plexus of mammals. The presence of δ receptors has been detected in the ileal submucosal plexus, at least in the guinea-pig. cDNA encoding an “orphan” receptor has been identified which has a high degree of homology to the classical opioid receptors. This receptor has been named ORL1 (opioid receptor like) (Mollereau et al., 1994; Nicholson et al., 1998). The endogenous peptide agonist for ORL1, nociceptin referred also as orphanin FQ, has been detected in porcine, rat and guinea-pig gastrointestinal tract (Osinski et al., 1999; Yazdani et al., 1999; O’Donnell et al., 2001). In the guinea-pig myenteric plexus, nociceptin immunoreactivity is expressed preferentially in excitatory motor neurons projecting to the longitudinal and circular muscle layers, as well as a small subgroup of descending interneurons (O’Donnell et al., 2001). Effects of opioids on neuromuscular preparations In the guinea-pig ileum, agonists at µ (morphine, normorphine, enkephalins, DAMGO, and endomorphin-1,2), κ receptors (dynorphin and U-50488H), and ORL1 receptors (nociceptin) inhibit electrically-induced longitudinal muscle cholinergic contractions by presynaptic inhibition of ACh release (Greenberg, Kosterlitz and Waterfield, 1970; Chavkin and Goldstein, 1981; Vizi et al., 1984; Kojima et al., 1994; Tonini et al., 1998; Sternini et al., 2000; O’Donnell et al., 2001). Opioids acting at µ and κ receptors have been shown directly to decrease ACh release evoked by substance P, neurotensin and caerulein (Yau, Verdun and Youther, 1983; Yau, Verdun and Youther, 1983; Yau, Dorsett and Youthe, 1986b; Huidobro-Toro et al., 1984). They can inhibit hyoscine-resistant,

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substance P-mediated contractions (Gintzler and Scalisi, 1982; Barthó et al., 1982b) and have been shown to reduce substance P release (Holzer, 1984). The action of agonists at ORL1 receptors is region specific as observed in the rat gastrointestinal tract. In this species, nociceptin inhibits cholinergic transmission in the stomach and small intestine, whereas it stimulates colonic contractions by inhibiting a neurogenic inhibitory pathway within the myenteric plexus (Yazdani et al., 1999). No specific effect of δ receptor agonists on excitatory LM motor neurons has been demonstrated. The agonists DPLPE and DPDPE were found to reduce LM contraction by 50% (Mosberg et al., 1983; Galligan et al., 1984; Ersparmer et al., 1989) or to be ineffective (Kojima et al., 1994). Whilst these effects were reversed by naloxone (Mosberg et al., 1983; Galligan et al., 1984) and the µ receptor antagonist CTP, (Shook et al., 1987) they were not antagonized by the specific δ receptor antagonist, ICI 174864 (Shook et al., 1987). Agonists at µ and κ receptors inhibit CM contraction in the whole wall, CM-MP and CM-axons preparations evoked by 5-HT (Harry, 1963; Johnson et al., 1987; Johnson, Costa and Humphreys, 1988). The δ agonist, DPLPE, partly inhibited circular muscle contraction at very high concentrations (3 mM); however this effect was not antagonized by the δ antagonist, ICI 174864. Morphine inhibits ascending excitatory reflex contractions when added to the oral, intermediate or anal compartments of a partitioned organ bath (Tonini et al., 1992c). This suggests that µ receptors are present on CM motor neurons and interneurons, where they inhibit transmitter release. Selective agonists at µ and κ receptors (PLO17 and U-10488, respectively) concentration-dependently inhibit the ascending excitatory reflex response in the rat ileum, whereas the δ agonist DPDPE is ineffective (Allescher et al., 2000). However, δ receptors seem to be involved together with µ receptors in the timing of the reflex response. The effects of opioid receptor stimulation on NANC inhibitory transmission have been investigated in rat, guinea-pig, canine and human gastrointestinal tract. Morphine was found to inhibit electrically evoked NANC inhibitory responses in rat gastric fundus strips (Dehpour et al., 1994), whereas loperamide (a non-selective µ receptor agonist) inhibited the 5-HT-induced NANC relaxation in isolated intact stomach from guinea-pig (Meulemans, Helsen and Schuurkes, 1993). Agonists acting at µ and κ receptors decreased the amplitude of NANC IJPs evoked by transmural nerve stimulation in the circular muscle of canine duodenum (Bauer and Szurszewski, 1991). In the circular muscle of guinea-pig and human colon, NANC inhibitory responses are reduced by activation of δ receptors (Hoyle et al., 1990; Zagorodnyuk and Maggi, 1994). Electrophysiological effects of opioids on neurons Early studies involving intracellular recordings of myenteric neurons showed that opioid agonists acting at µ receptors, such as met- and leu-enkephalin, morphine and normorphine hyperpolarize cell membranes (North and Tonini, 1977; Sakai, Hymson and Shapiro, 1978; North, Katayama and Williams, 1979; Cherubini and North, 1985b) via stereospecific and naloxone-reversible mechanisms, whereas opioids that are selective for κ receptors (dynorphin, U-50488H) do not (Cherubini and North, 1985b). Opioids acting at both receptor subtypes caused presynaptic inhibition of ACh release in the myenteric

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plexus, depressing the amplitude of fast EPSPs (Cherubini and North, 1985b). However, µ and κ opioids inhibit transmitter release by different presynaptic inhibitory mechanisms; µ receptor activation results in hyperpolarization due to increased potassium conductance, whereas κ receptor activation may depress the release of ACh by directly reducing calcium entry into the nerve terminals (Cherubini and North, 1985b). A comprehensive study of the electrophysiological effects of DAMGO was undertaken by Pillai and Johnson (1991). This study demonstrated that 42% of S neurons are hyperpolarized by DAMGO. The magnitude of the hyperpolarization increased with increasing concentrations of DAMGO and was prevented by pre-application of naloxone. Fast EPSPs were significantly diminished in amplitude in 48% of S cells; this effect occurred in some cells which had not been hyperpolarized by DAMGO. This effect is probably due to a presynaptic action of opioids, since fast EPSPs evoked by exogenous nicotine are unaffected (Cherubini and North, 1985b; Cherubini, Morita and North, 1985). In AH cells, there was no effect of DAMGO on the resting membrane potential, input resistance, action potential duration or amplitude, nor on the amplitude or duration of the fast and slow after-hyperpolarizations. Propagation of action potentials to the soma of AH neurons was also unaffected by DAMGO. Ito and Tajima (1980) provided electrophysiological evidence that µ agonists do not act directly on the muscle. Morphine reduced the amplitude of the excitatory junction potential, but did not change the membrane potential, resistance or electrical threshold for activation of spikes in circular or longitudinal muscle. Thus the site of action of morphine was prejunctional. There is no evidence for inhibition of inhibitory junction potentials by opioids (Ito and Tajima, 1980). Role of endogenous opioids Trendelenburg (1917) was the first to demonstrate inhibition of peristalsis by morphine in the guinea-pig small intestine in vitro. Morphine, fentanyl and met-enkephalin inhibit both longitudinal and circular muscle contraction during the preparatory and emptying phases respectively (Schaumann, 1955; Kosterlitz and Robinson, 1957; Fontaine, Reuse and Van Nueten, 1973; Van Nueten, Van Ree and Vanhoutte, 1977). Fontaine, Reuse and Van Nueten (1973) reported a preferential effect of morphine and fentanyl on longitudinal muscle contraction. At submaximal doses, morphine, fentanyl and met-enkephalin increased the pressure at threshold, reduced longitudinal and circular muscle activity, increased filling of the intestine, reduced the volume expelled per wave and reduced the frequency of peristaltic waves (Fontaine, Reuse and Van Nueten, 1973; Van Nueten, Van Ree and Vanhoutte, 1977). Similarly, Kromer and co-workers (Kromer, Pretzlaff and Woinoff, 1981; Kromer, Steigemann and Shearman, 1982) have shown a dose-dependent inhibitory effect of normorphine in terms of the frequency of peristaltic waves. Inhibition was also produced by D-Ala2-Leu-enkephalinamide, β-casomorphin, DAMGO, ketocyclazocine and dynorphin (Kromer, Pretzlaff and Woinoff, 1980; Kromer, 1990). All of these results were obtained using the Trendelenburg preparation. However, peristalsis was also abolished by dermorphin, morphine, D-Ala2-D-Met5-enkephalin, FK 33–824 and dynorphin in the vascularly-perfused preparation (Holzer and Lembeck, 1979; Barthó et al., 1982a; Donnerer and Lembeck, 1985). At low concentrations, these opioids reduced

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both the frequency of peristaltic waves and the maximal ejection pressure. Barthó et al. (1982a) also demonstrated that FK 33–824 could abolish atropine-resistant peristalsis. A more recent study (Waterman, Costa and Tonini, 1992) has demonstrated that µ opioid receptor agonists reduce the compliance of the intestinal wall, the LM contraction during the preparatory phase and increase the threshold volume required to trigger emptying. Kappa receptor agonists reduce the maximal ejection pressure during the emptying phase and LM contraction and increase the threshold volume. Delta receptors also modulate peristalsis in the guinea-pig ileum. In agreement with studies by Kromer (1990), Waterman, Costa and Tonini (1992) demonstrated that δ agonists reduce the maximal ejection pressure, LM contraction during the preparatory phase and increase the threshold volume. In 1977, Schulz et al. reported that material with enkephalin-like activity was released from LM-MP strips. Furthermore, the enkephalin content of LM-MP strips was reduced by high frequency stimulation (1 and 10 Hz) – but not low frequency (0.1 Hz) – stimulation (Hughes, Kosterlitz and Sosa, 1978). Oka and Sawa (1979) showed that this release was calcium-dependent. The first direct measurement of met-enkephalin release from LM-MP strips was not achieved until 1986 (Glass, Chan and Gintzler, 1986). Enkephalin release was detectable following electrical stimulation at frequencies between 5 and 80 Hz. The release was tetrodotoxin-sensitive and calcium-dependent. Enkephalin release was enhanced in the presence of naloxone, but reduced by morphine pretreatment. This suggests that endogenous opioids can modulate their own release (Sublette and Gintzler, 1992). Preparations of synaptosomes from guinea-pig LM-MP strips have been shown to release met- and leu-enkephalin. Release increased following depolarization by potassium. Met-enkephalin release was detectable only in the presence of naloxone or atropine. Leu-enkephalin release was detectable only in the presence of naloxone, atropine or a purine receptor antagonist (1,3-dipropyl-8-p-sulphophenylxanthine) (Christofi, McDonald and Cook, 1990). Waterfield and Kosterlitz (1975) reported that naloxone stereospecifically increased the release of ACh in response to low frequency (0.017 Hz) electrical stimulation of the LM-MP preparation. The effect was less marked at a stimulation frequency of 10 Hz. Puig et al. (1977) combined different stimulation frequencies and showed that longitudinal muscle contraction evoked by stimulation at 0.1 Hz was reduced following stimulation at 10 Hz for 5 min. This inhibitory effect was antagonized in a dose-dependent manner by naloxone and naltrexone. Similar results have since been reported by Horàcek and Kadlec (1984). Gintzler and Scalisi (1982) reported that naloxone increased atropine-resistant contractions of the longitudinal muscle evoked by electrical stimulation at 20 Hz. This effect was unaltered by the addition of hexamethonium, but blocked by prior desensitization of the preparation to SP. Barthó et al. (1982b) similarly showed that naloxone enhanced atropineresistant contractions of the longitudinal muscle. These studies indicate that endogenous opioid peptides released by high frequency electrical stimulation can inhibit the release of SP from excitatory longitudinal muscle motor neurons. More direct evidence for this was provided by Holzer (1982). He directly measured SP release from LM-MP strips and showed that this was increased in the presence of naloxone. Research by Garzon et al. (1985, 1987) also indicated that endogenous opioid peptides inhibit the release of ACh and SP. They showed that contraction of LM-MP strips in response to a variety of peptides

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(corticotropin-releasing factor, CCK, neurotensin, bombesin, angiotensin II, bradykinin and SP) was enhanced by naloxone in the presence and absence of atropine. In the guinea-pig colon, naloxone was found to increase resting and electrically evoked ACh release and electrically evoked noradrenaline release, and to enhance NANC relaxations in the circular muscle. The functional relevance of endogenous opioids appears to be enhanced after chronic sympathetic denervation (Marino et al., 1993). In the guinea- pig small intestine, naloxone enhances the amplitude of ascending excitatory reflex contractions when added to the oral compartment of a partitioned organ bath (Tonini et al., 1992c). Naloxone increases the rate of peristaltic contractions in the rat small intestine (Coupar, 1995). In the same preparation, CTOP and nor-binaltorphimine (nor-BNI), selective antagonists at µ and κ receptors respectively, increase the ascending excitatory reflex, whereas the selective δ antagonist ICI 174864 is ineffective (Allescher et al., 2000). In the rat colon, naloxone enhances distension evoked descending relaxation and VIP release (Grider and Makhlouf, 1987a). Together these findings suggest that endogenous opioids acting preferentially at µ and κ receptors modulate the release of excitatory and inhibitory transmitters in the gastrointestinal tract. Peristaltic activity rapidly “fatigues” when the Trendelenburg preparation is used. In this preparation, the oral end of the intestine is occluded and fluid enters the anal end of the intestine. The intestine empties fluid against the inflow head of pressure. Van Nueten, Janssen and Fontaine (1976) showed that the opioid receptor antagonist, naloxone, restored peristaltic activity in fatigued preparations. Furthermore, the organ bath solution surrounding a fatigued preparation inhibited peristalsis in non-fatigued segments. The latter effect was antagonized by naloxone. Van Nueten and colleagues therefore concluded that fatigue was due to the release of endogenous opioid peptides. In a similar study, Kadlec and Horàcek (1980) demonstrated that inhibition of peristalsis by a “stress stimulus” (12 cm H2O intraluminal pressure and 3 g longitudinal tension) was reversed by naloxone. Davison and Najafi-Farashah (1982) demonstrated that naloxone restored peristaltic activity in fatigued segments of intestine. At higher distending pressures, when intermittent myogenic activity was recorded, naloxone also restored continuous peristaltic activity. In a series of studies, Kromer and colleagues have shown that naloxone stereospecifically increases the frequency of peristaltic waves, reduces the number and duration of peristalsis-free intervals and transiently reduces the volume expelled (Kromer and Pretzlaff, 1979; Kromer and Woinoff, 1980; Kromer, Pretzlaff and Scheiblhuber, 1980; Kromer, Pretzlaff and Woinoff, 1980). Increasing the extracellular calcium concentration attenuated the effects of naloxone (Kromer, Scheiblhuber and Illes, 1980). These effects were observed in segments of ileum and to a lesser extent in the duodenum and jejunum from adult, fetal and pregnant guinea-pigs (Kromer and Pretzlaff, 1979; Kromer, Pretzlaff and Woinoff, 1980). A similar study (Kromer, Steigemann and Shearman, 1982) showed that peristaltic activity was also enhanced by the µ antagonist and proposed σ receptor agonist, SKF 10047. However more recently, Kromer (1990) reported that the µ antagonist, CTOP, did not significantly alter peristaltic activity. Instead, peristalsis was enhanced by the κ antagonist, nor-BNI. In more physiological preparations, in which both ends of the intestine are open and fluid is infused through the oral end, intra-arterial administration of naloxone has no effect

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on peristalsis (Holzer and Lembeck, 1979; Donnerer and Lembeck, 1985). Nevertheless, dynorphin is released into the venous effluent of the vascularly-perfused guinea-pig small intestine during peristalsis (Donnerer, Holzer and Lembeck, 1984). Dynorphin release increases during peristalsis. Donnerer, Holzer and Lembeck (1984) also showed that naloxone enhances the release of substance P and somatostatin during peristalsis, but not under resting conditions. Thus endogenous opioids may inhibit the release of SP and somatostatin during peristalsis. Another study, using more physiological conditions than the Trendelenburg technique, found that the power generated by the intestine when emptying was increased in the presence of nor-BNI, under certain conditions (Waterman, Costa and Tonini, 1992). Thus, endogenous opioids acting at κ receptors, inhibited peristalsis when the intestine was forced to empty against a high resistance. SOMATOSTATIN Somatostatin is a tetradecapeptide which is present in neuronal cell bodies in the myenteric and submucous plexuses of the guinea-pig small intestine (Costa et al., 1977, 1980b; Furness et al., 1980; Schultzberg et al., 1980; Endo, Uchida and Kobayashi, 1986; Portbury et al., 1995; Song et al., 1997). In the myenteric plexus, somatostatin is contained in 4% of neurons, which project up to 70 mm in an anal direction and form very long interneuronal chains (descending interneurons) that connect with both myenteric and submucous neurons (Song et al., 1997; Mann et al., 1997b). At the electron microscope level, the neurons can be seen to form axosomatic synapses with somatostatin-positive and -negative neurons (Endo, Uchida and Kobayashi, 1986). Somatostatin-containing neurons do not project to either the circular or longitudinal muscle layers (Costa et al., 1977, 1980b; Endo, Uchida and Kobayashi, 1986). Somatostatin is colocalized with ChAT, SP and CCK in descending interneurons (Brookes, Steele and Costa, 1992; Portbury et al., 1995; Costa et al., 1996; Mann et al., 1997b; Hens et al., 2000). Effects of somatostatin on neuromuscular preparations Somatostatin reduces the amplitude of the cholinergic twitch of the LM evoked by electrical field stimulation in LM-MP and in whole wall preparations of guinea-pig ileum (Guillemin, 1976; Cohen et al., 1978, 1979; Furness and Costa, 1979b; Jhamandas and Elliot, 1980). Contractions induced by neurotensin and caerulein, but not by SP, are also inhibited (Monier and Kitagbi, 1981; Teitelbaum et al., 1984; Yau, Lingle and Youther, 1983; Yau, Dorsett and Youther, 1986b). Somatostatin has no effect on LM contractions elicited by exogenous ACh (Guillemin, 1976; Cohen et al., 1978), carbachol (Furness and Costa, 1979b) or histamine (Cohen et al., 1979), indicating that somatostatin acts by inhibiting the release of ACh rather than by interfering with its action on the smooth muscle. Inhibition of resting and evoked ACh release by somatostatin has been measured directly (Yau, Lingle and Youther, 1983; Yau, Dorsett and Youther, 1986b; Teitelbaum et al., 1984; Takeda et al., 1989; Milenov and Atanassova, 1993). More recently, somatostatin was found to possess both excitatory and inhibitory effects on myenteric cholinergic transmission, which partly involve the release of endogenous GABA. This transmitter, in turn, may enhance or inhibit ACh release via activation of GABAA or GABAB receptors,

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respectively (Roberts, Hasler and Owyang, 1993). The effect of somatostatin on the CM of the guinea-pig ileum has not been studied in detail. Kromer and Woinoff (1981) reported that somatostatin did not have any effects on the circular muscle in the presence of TTX, atropine, hexamethonium or 5-HT desensitization. Thus, somatostatin does not act directly on the CM. In the guinea-pig colon, somatostatin stimulates inhibitory motor neurons, since it produces a TTX-sensitive relaxation of the LM (Furness and Costa, 1979b). This relaxation is unaffected by guanethidine, and is therefore mediated by non-adrenergic neurons (Furness and Costa, 1979b). It is not clear whether somatostatin stimulates both the apamin-sensitive and -insensitive components of inhibitory transmission. In isolated smooth muscle cells from the stomach and colon of guinea-pig, somatostatin was found to possess a direct contractile activity (Corleto et al., 1997). In these cells, five somatostatin (SST) receptors have recognized (Corleto, Weber and Jensen, 1999), but the contractile effect was predominantly mediated by SST3 receptors in gastric cells and by SST5 receptors in colonic cells (Corleto et al., 1997). Electrophysiological effects of somatostatin on neurons Extracellular recordings of unit activity in the myenteric plexus indicate that somatostatin reduces the firing frequency of neurons (Williams and North, 1978). This effect is independent of extracellular calcium levels. Thus the action of somatostatin is likely to be a direct one on the neurons whose activity was being recorded. Intracellular recording demonstrated that somatostatin can depolarize and hyperpolarize both AH and S type neurons. The proportion of S neurons depolarized varied from 17–34% and those hyperpolarized varied from 20–31%, depending on whether the somatostatin was applied by perfusion or iontophoretically. Similarly, the proportion of AH neurons depolarized and hyperpolarized respectively was 9–29% and 8–20%. Depolarization was associated with an increase in input resistance and had a reversal potential near the equilibrium potential of potassium, suggesting that somatostatin acts by closing a potassium channel. The somatostatin-induced hyperpolarization was associated with an decrease in input resistance (Katayama and North, 1980; Liu et al., 2000). Role of endogenous somatostatin Endogenous somatostatin is released into the venous effluent during peristalsis in various species (Donnerer, Holzer and Lembeck, 1984; Schmidt, Rasmussen and Holst, 1993). This release was partly TTX-sensitive and therefore nerve mediated. However, a significant proportion is likely to represent release from mucosal endocrine cells. Somatostatin release could be increased by DMPP in a TTX-sensitive manner and by CCK-8 in TTXinsensitive manner. Somatostatin is released from isolated myenteric ganglia in response to nicotinic receptor stimulation by DMPP (Grider, 1989). The amount of somatostatin release is decreased by VIP and can be augmented by a VIP receptor antagonist (VIP10–28; Grider, 1989). This suggests that the release of somatostatin may be inhibited by endogenous VIP. Somatostatin has been reported to have a variety of effects on peristalsis. Holzer and Lembeck (1979) found that somatostatin had no immediate effect when infused intra-

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arterially. However, 12 minutes after the end of infusion, peristalsis became increasingly irregular and the volume of fluid expelled was decreased. This effect appeared to be irreversible (Holzer and Lembeck, 1979). Kromer and Woinoff (1981) studied peristalsis using the Trendelenburg method, in which the intestine “fatigues” and ceases to undergo normal peristalsis. They found that somatostatin stimulated peristalsis when it had been highly intermittent and inhibited it when it had been less intermittent. In the rat colon, somatostatin release increases along with VIP in response to the descending inhibitory reflex (Grider, Arimura and Makhlouf, 1987). Anti-somatostatin antibodies reduce VIP release and the reflex relaxation, whereas exogenous and endogenous somatostatin enhances both VIP/NO release and the reflex relaxation (Grider, 1994a). The latter effects are partly achieved through a decrease of met-enkephalin release, which exerts a tonic inhibitory influence on descending pathways. GASTRIN, CHOLECYSTOKININ AND CAERULEIN Cholecystokinins consist of a group of peptides which are C-terminal fragments of CCK-33 (Williams, 1982). Cholecystokinins, gastrin, caerulein (also known as ceruletide) and phyllocaerulein share a common pentapeptide sequence at their C-terminus and act with varying affinities at the same receptors, i.e. CCKA and CCKB receptors (Alexander and Peters, 2000). Immunohistochemical studies have demonstrated that CCK is localized in specific subpopulations of myenteric neurons in the guinea-pig small intestine: anally directed interneurons in the myenteric plexus, neurons projecting to the mucosa, intestinofugal neurons and neurons projecting from the myenteric to the submucous plexus (Furness and Costa, 1987; Brookes, Steele and Costa, 1992). In descending interneurons, CCK is colocalized with somatostatin, ChAT and SP. CCK is not present in motor neurons in either the CM or LM. Electrophysiological and immunohistochemical studies have demonstrated the presence of both CCKA and CCKB receptors in rat and guinea-pig myenteric neurons (Schutte, Akkermans and Kroese, 1997; Sternini et al., 1999a; Sayegh and Ritter, 2000). CCKA receptors are predominantly expressed in neurons containing VIP, but they are also expressed in neurons containing SP (Sternini et al., 1999a). Effects of CCK-like peptides on neuromuscular preparations Gastrin produces a contraction of the guinea-pig LM which is unaffected by hexamethonium but abolished or reduced by atropine, suggesting that the agonist acts on cholinergic LM motor neurons (Bennett, 1965; Yau et al., 1974; Yau, 1978). Caerulein and CCK-8 induce a pronounced and sustained TTX-sensitive contraction of the guinea-pig ileum LM. This contraction is partly inhibited by atropine and potentiated by eserine, suggesting that these peptides also stimulate release of ACh from LM motor neurons (Del Tacca et al., 1970; Hedner, 1970; Yau et al., 1974; Zetler, 1979; Hutchison and Dockray, 1981; Garzón et al., 1985). Increased ACh release in response to CCK-8, pentagastrin, gastrin and caerulein has been confirmed by direct measurement (Del Tacca, Soldani and Crema, 1970; Vizi et al., 1972, 1973; Yau, Lingle and Youther, 1983; Teitelbaum et al., 1984; Sano, Taniyama and Tanaka, 1989; Milenov and Atanassova, 1993). The

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atropine-resistant contraction is abolished by SP desensitization (Hutchison and Dockray, 1981), suggesting that SP or a related tachykinin is involved. Circumstantial evidence in favour of this hypothesis was provided by Holzer (1984), who showed that CCK-8 stimulates the release of SP from LM-MP preparations in a TTX-sensitive manner. In some experiments, the hyoscine-resistant contraction is reduced by about 20–35% by hexamethonium (Del Tacca, Soldani and Crema, 1970; Hedner, 1970), indicating that caerulein and CCK-8 also stimulate cholinergic interneurons. Sano, Taniyama and Tanaka (1989) found that CCK-induced release of ACh from guinea-pig LM-MP preparations was inhibited by 42% by bicuculline, suggesting that part of the effect of CCK is indirect, via release of GABA which acts on GABAA receptors. Nevertheless, neurally-mediated effects may be contractile (via release of ACh/tachykinins) or relaxant (via release of VIP/NO) as demonstrated in the CCK-mediated relaxation of gastric fundus and emptying (Grider, 1994b). CCK also appears to act directly on the LM, since higher concentrations (0.1 mM) can produce contractions in the presence of TTX. A direct effect of CCK has been demonstrated in gastric fundus and in caecal smooth muscle cells where are present both receptor subtypes, which mediate a contractile response (Grider and Makhlouf, 1987b; de Weerth et al., 1997; Motomura et al., 1997). Caerulein induces TTX-sensitive, rhythmic contractions superimposed on increased tone of the guinea-pig ileum CM (Holzer, Lembeck and Donnerer, 1980; Barthó et al., 1987a). These contractions are blocked by atropine but not hexamethonium (Holzer, Lembeck and Donnerer, 1980), suggesting that caerulein acts by stimulating ACh release from CM motor neurons. The contractions are partly or completely inhibited by desensitization to SP, indicating that tachykinin release mediates part of the contractile response. Caerulein and pentagastrin lower the threshold distension required to trigger emptying of the gut and consequently increase the frequency of emptying when the intestine is infused with fluid at a constant rate (Frigo et al., 1971; Chijikwa and Davison, 1978; Tonini et al., 1989b). Similarly, CCK-8 produces a concentration-dependent increase in the frequency of peristaltic waves and in the volume of fluid expelled (Barthó et al., 1982a). Caerulein can initiate peristalsis in the undistended intestine (Holzer and Lembeck, 1979) and restore peristalsis which has been impaired by atropine or hexamethonium (Frigo et al., 1971; Barthó et al., 1989). This effect is likely to be due to the ability of CCK to stimulate release of ACh from CM motor neurons. Electrophysiological effects of CCK-like peptides on neurons The electrophysiological effects of CCK-like peptides in the guinea pig ileum have received little attention. CCK-8 mimics slow EPSPs in 68% of S neurons and 52% of AH neurons (Nemeth, Zafirov and Wood, 1985). CCK-8 also hyperpolarizes 17% of AH neurons and 5% of S neurons. These responses were unaffected by proglumide. Caerulein had similar effects to CCK-8, although hyperpolarizing responses were not recorded (Nemeth, Zafirov and Wood, 1985). Pentagastrin had no significant effect on AH neurons, and was not tested on S neurons (Nemeth, Zafirov and Wood, 1985). In another study, CCK-8 was found to depolarize 83% of S neurons. Depolarization was also obtained with the CCKB receptor agonist CCK-8NS. Use of CCKA and CCKB selective antagonists indicated that some neurons possess exclusively the CCKA and CCKB receptor subtype,

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but others possess both subtypes (Schutte, Akkermans and Kroese, 1997). Since selective antagonists for CCKA and CCKB (L-364,78 and L-365,260, respectively) were found to inhibit slow EPSPs in myenteric neurons, this provides evidence that neurally released CCK is involved in the mediation of slow EPSPs via CCKA and CCKB receptors (Schutte et al., 1997). Role of endogenous CCK-like peptides CCK-8 is released into the venous effluent in response to distension of the intestine during peristalsis (Donnerer et al., 1985). The nicotinic receptor agonist, DMPP, did not produce a significant increase in release above basal levels (Donnerer et al., 1985). It is therefore unlikely that CCK is released from neurons during peristalsis in response to excitation by cholinergic nicotinic transmission. The role of endogenous CCK in peristalsis is not known. Peristalsis was not significantly altered by the CCK receptor antagonist, CR 1409 (Barthó et al., 1987a). Similarly, lorglumide did not produce any consistent, significant effects on hexamethonium-resistant peristalsis other than an increase in the pressure at threshold (Barthó et al., 1989). The latter may reflect a decrease in the compliance of the intestinal wall, due to either a decrease in inhibitory neuronal tone or an increase in excitatory neuronal activity (Waterman, Costa and Tonini, 1992). Thus, endogenous CCK may stimulate inhibitory CM motor neurons or inhibit excitatory CM motor neurons. Both effects are possible, given the ability of CCK to depolarize and hyperpolarize myenteric neurons. In the duodenum of rat, use of selective CCKA and CCKB receptor antagonists (L-364,718 and L-365,260, respectively) had no effect on the ascending excitation to electrical field stimulation of the mucosa, but abolished the inhibitory effect of CCK-8 on electrical stimulation (Giralt and Vergara, 2000). The role of endogenous CCK in the descending inhibitory reflex has not been investigated. Since CCK is present in descending interneurons and not in motor neurons (Furness and Costa, 1987; Brookes, Steele and Costa, 1992), the most likely role for CCK would be in the descending inhibitory reflex, rather than the reflex pathways studied previously. NEUROPEPTIDE Y (NPY) AND PEPTIDE YY (PYY) NPY is a 36 amino acid peptide which has some sequence homology to PYY and pancreatic polypeptide (PP) (Tatemoto, Carlquist and Mutt, 1982). NPY and PYY are found in 5% of myenteric neurons, including anally projecting interneurons and short descending inhibitory motor neurons to the CM (Furness and Costa, 1987; Brookes, Steele and Costa, 1991b, 1992). NPY-immunoreactive nerve terminals synapse on non NPY-immunoreactive somata and fibres (Fehér and Burnstock, 1986). PYY-like immunoreactivity was not detectable by radioimmunoassay of LM-MP synaptosome samples (McDonald et al., 1988). In the guinea-pig gastric fundus and corpus, NPY has been localized on both ascending and descending pathways projecting to the mucosa (Pfannkuche et al., 1998; Reiche and Schemann, 1999). NPY is also present in secremotor neurons in the guineapig small intestine (Lomax, Bertrand and Furness, 1998). Five functional NPY receptors (Y receptors) have been recognized so far.

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Effects of NPY on neuromuscular preparations NPY inhibits contractions of the LM induced by CCK-8 or electrical field stimulation (Garzón, Höllt and Sánchez-Blázquez, 1986; Holzer et al., 1987). This is not due to a direct inhibitory effect on the LM, since NPY does not alter concentration-response curves to ACh or carbachol in the presence of TTX (Garzón, Höllt and Sánchez-Blázquez, 1986; Holzer et al., 1987). Instead, NPY inhibits the release of ACh and a noncholinergic transmitter, probably a tachykinin, from LM motor neurons (Holzer et al., 1987; Takahashi, Yamamura and Utsunomiya, 1992). Similar results have been found in rabbit ileum, in which inhibition of ACh release is mediated by the Y5 receptor (Pheng et al., 1997). In the guinea-pig colon, PYY and NPY inhibit the twitch contractions mediated by the stimulation of cholinergic neurons and the resulting release of ACh. The inhibitory effect of PYY is mediated by a receptor located directly on cholinergic neurons, whereas the effect of NPY is mediated by noradrenaline release due to stimulation of a receptor located on adrenergic neurons (Sawa et al., 1995). NPY abolishes phasic TTX-sensitive contractions of the CM induced by DMPP or caerulein (Holzer et al., 1987). However, TTX-insensitive contractions evoked by carbachol are unaffected. Thus, NPY inhibits CM contractions through an action on excitatory CM motor neurons rather than by a direct effect on the CM. Such an action may also underlie the reduction in basal CM tone produced by NPY in some preparations (Holzer et al., 1987). NPY inhibits the ascending excitatory reflex contraction of the CM in response to balloon distension (Holzer et al., 1987). Both the atropine-sensitive and atropine-resistant components of the response are inhibited. Although these effects are likely to be due to inhibition of excitatory CM motor neurons, NPY needs to be tested in a partitioned organ bath to rule out the possibility that it also inhibits interneurons or sensory neurons. The inhibitory effect of NPY on the ascending excitatory reflex is significantly reduced by apamin (Holzer et al., 1987). NPY may therefore inhibit neurons by opening a calcium-dependent potassium channel in the neuronal membrane. Alternatively, NPY may stimulate inhibitory CM motor neurons which relax the muscle through an apamin-sensitive mechanism. At variance with the effects observed in the guinea-pig intestine, NPY enhances ACh release in the rat colon by acting on Y2 and Y4 receptors (Pheng et al., 1999). Y2 receptors have been also characterized in colonic smooth muscle cells (Feletou et al., 1998). Electrophysiological effects of NPY and related peptides on neurons NPY and PPY and bovine PP suppress nicotinic fast EPSPs (and ACh release) in guineapig gastric myenteric neurons via a mechanism insensitive to α-adrenoceptor blockade (Schemann and Tamura, 1992). These peptides have been found to inhibit fast EPSPs in myenteric neurons of descending colon through a mechanism involving activation of presynaptic Y2 receptors (Browning and Lees, 2000).

CALCITONIN GENE-RELATED PEPTIDE CGRP is a 37-aminoacid peptide localized in blood vessels and nerves of the gastrointestinal tract. Based upon the differential biological activity of various CGRP analogs, CGRP

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receptors have been classified into two major classes, namely the CGRP1 and CGRP2 subtypes (van Rossum, Hanisch and Quirion, 1997). In the guinea-pig ileum, CGRP is present in intrinsic and extrinsic fibres. The intrinsic fibres supply only the mucosa (Costa, Furness and Gibbons, 1986). Fibres innervating the myenteric plexus which are immunoreactive for CGRP are of extrinsic origin (Costa, Furness and Gibbons, 1986a). During peristalsis, CGRP may potentially be released from both sources. In fact, at least in the rat colon, extrinsic sensory pathways, which mediate the peristaltic response to muscle stretch, and intrinsic sensory pathways, which are involved in the peristaltic response to muscular stimulation, utilize CGRP as a sensory transmitter (Grider, 1994c). In the guinea-pig small intestine, however, extrinsic sensory neurons do not participate in the neural pathway mediating peristalsis, but stimulate peristaltic activity following capsaicin stimulation (Barthó and Holzer, 1995). CGRP produces a transient contraction followed by a longer relaxation of the guineapig ileum LM-MP preparation (Barthó et al., 1987b; Sun and Benishin, 1991). The contraction is blocked by TTX, atropine, 2-chloroadenosine, mepyramine and clonidine (Tippins et al., 1984; Sun and Benishin, 1991), suggesting that the contraction is mediated by ACh released from LM motor neurons and acting at muscarinic receptors. Histamine may also be involved in this response. The CGRP-induced relaxation is partly nerve-mediated, since it is partly blocked by TTX. However, CGRP also has a direct relaxing effect (Barthó et al., 1987b; Sun and Benishin, 1991) which is not antagonized by apamin (Holzer et al., 1989). CGRP has also been shown to inhibit electrically induced cholinergic contractions of the guinea-pig ileum LM-MP preparation (Barthó et al., 1987b) via CGRP1-like receptor activation (Tomlinson and Poyner, 1996). In smooth muscle cells isolated from the circular and longitudinal layers of guinea-pig ileum, CGRP triggers different intracellular pathways to induce relaxation: cAMP is involved in cells from both layers while NO is involved only in relaxation of circular smooth muscle cells (Rekik et al., 1997). CGRP induces phasic contractions of the guinea-pig ileum CM by stimulating ACh release from cholinergic motor neurons (Holzer et al., 1989). Part of the action of CGRP is likely to be on cholinergic interneurons, since the phasic contractions are reduced by hexamethonium (Holzer et al., 1989). CGRP modulates peristalsis by increasing the pressure which occurs at threshold (Holzer et al., 1989). This probably reflects a decrease in the compliance of the intestinal wall and increased CM tone due to either an increase in excitatory neuronal activity or a decrease in inhibitory neuronal activity. The former is more likely given the excitatory effect of CGRP on cholinergic motor neurons mentioned previously and that CGRP has not been reported to inhibit neurons. In agreement with this, CGRP was shown to enhance the amplitude of the ascending excitatory reflex (Holzer et al., 1989). In guinea-pig distal colon CGRP inhibits electrically-evoked NANC contractions through a postjunctional mechanism (Kojima, 1997). In electrophysiological studies, CGRP mimicked the slow EPSP in all myenteric AH neurons when applied by superfusion and pressure ejection. Depolarization was associated with an increase in input resistance, suppression of post-spike hyperpolarization and enhanced excitability (Palmer et al., 1986a). This effect may be mediated by a decrease in

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the calcium-activated potassium conductance which is active in resting AH neurons (Palmer et al., 1986a). NEUROTENSIN Neurotensin immunoreactivity in the guinea-pig small intestine occurs mainly in the mucosa (Helmstaedter et al., 1977; Sundler et al., 1977; Schultzberg et al., 1980; Leander et al., 1984). In these studies, neurotensin-like immunoreactivity was not found in myenteric neurons of the guinea-pig ileum. However, other researchers have reported neurotensin-like immunoreactivity in these neurons (Reinecke et al., 1983; Tange, 1983). Neurotensin has been measured by radioimmunoassay in the external muscle layers of the guinea-pig ileum by one research group (Holzer et al., 1982) but was not found by another (Furness et al., 1982). It is possible that the discrepancies relate to the specificity of the antibodies used. Consequently, a peptide similar to neurotensin rather than authentic neurotensin may be present in the guinea-pig small intestine. Neurotensin binding sites have been demonstrated in the CM (Goedert, Hunter and Ninkovic, 1984). Recently, two neurotensin receptors have been identified in various tissues and selective antagonists for neurotensin-1 (NTS1) receptors have been developed (Vincent, Mazella and Kitabgi, 1999). Pharmacological experiments have demonstrated that neurotensin induces relaxation and then contraction of the guinea-pig LM-MP preparation (Kitagbi and Freychet, 1978, 1979a; Monier and Kitagbi, 1980; Goedert, Hunter and Ninkovic, 1984; Wagner and Wahl, 1986). The contraction is nerve-mediated, since it is TTX-sensitive (Kitagbi and Freychet, 1978, 1979a; Zetler, 1980; Garzón et al., 1985) and may be mediated by the release of ACh (Kitagbi and Freychet, 1978, 1979b; Zetler, 1980; Monier and Kitagbi, 1981; Huidobro-Toro and Way, 1982; Yau, Verdun and Youther, 1983; Teitelbaum et al., 1984; Garzón et al., 1985; Yau, Dorsett and Youther, 1986b; Nakamoto, Tanaka and Taniyama, 1987; Rakovska, 1993) and SP (Monier and Kitagbi, 1980). Neurotensin stimulates the release of ACh and SP from LM-MP preparations in a TTX-sensitive and calciumdependent manner (Yau, Verdun and Youther, 1983; Holzer, 1984; Yau, Dorsett and Youther, 1986b; Nakamoto, Tanaka and Taniyama, 1987). The neurotensin enhancement of ACh release is inhibited by stimulation of α2-adrenoceptor agonists (Rakowska, 1993). Stimulation of ACh release is unaffected by hexamethonium, hyoscine or SP receptor antagonists (Kitagbi and Freychet, 1979b; Nakamoto, Tanaka and Taniyama, 1987), suggesting that neurotensin may directly stimulate excitatory LM motor neurons. The action of neurotensin is likely to be at least partly at the level of the nerve terminals, because neurotensin is capable of stimulating ACh release from myenteric synaptosomes (Yau, Lingle and Youther, 1983). Neurotensin-induced contraction is partly inhibited by bicuculline. Since neurotensin also stimulates GABA release, it has been proposed that neurotensin may stimulate ACh release directly and also via release of GABA (Nakamoto, Tanaka and Taniyama, 1987). Neurotensin produces a direct relaxation of the guinea-pig ileum LM (Kitagbi and Freychet, 1978, 1979a; Goedert, Hunter and Ninkovic, 1984). This effect does not appear to involve cAMP or cGMP, since levels of these nucleotides are unaffected by neurotensin (Kitagbi and Freychet, 1979a). The relaxation is however, inhibited by apamin (Holzer

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et al., 1989), suggesting that neurotensin may open small conductance calcium-activated potassium channels. Neurotensin induces a direct relaxation of the guinea-pig ileum CM, which is apaminsensitive and blocked by antibodies to neurotensin. Furthermore, relaxation induced by nerve stimulation is partly blocked by neurotensin antibodies (Goedert, Hunter and Ninkovic, 1984). This led to the proposal that neurotensin is a NANC inhibitory transmitter in the guinea-pig ileum. In the rat stomach strips, neurotensin causes a contractile response, which results from direct activation of smooth muscle receptors (Nguyen et al., 1997). In canine ileal circular muscle devoid of myenteric plexus, neurotensin causes an initial hyperpolarization associated with inhibition of contractile activity, followed by an excitatory phase. The neurotensin-induced inhibitory effect is mediated by activation of apamin-sensitive, calcium-dependent potassium channels (Christinck, Daniel and Fox-Threlkeld, 1992). In rat proximal colon, neurotensin causes a summation of excitatory and inhibitory effects, which are dependent on the influx of Ca2+ via L-type channels (Mule and Serio, 1997). Neurotensin contracts human colonic circular smooth muscle strips. The insensitivity of this contractile effect to atropine, levocablastine (a NTS2 receptor agonist) or TTX and the blockade with SR 48692 and SR 142948 (two selective NTS1 receptor antagonists) is consistent with the involvement of non-neuronal NTS1 receptors (Croci et al., 1999). Neurotensin infused intravascularly increases the frequency of peristaltic waves and the volume of fluid emptied per unit time in the guinea-pig ileum (Barthó et al., 1982a). However, atropine-resistant peristalsis is initially stimulated and then inhibited by neurotensin (Barthó et al., 1982a). These results were interpreted by the authors to represent the stimulatory effect of neurotensin on ACh and SP release and its subsequent direct inhibitory effect on smooth muscle. The non peptide NTS1 receptor antagonist SR 48692 abolished both effects. The inhibitory action of neurotensin apparently involves an apamin-sensitive mechanism (Ohashi et al., 1996). Electrophysiological studies demonstrated that neurotensin mimics the slow EPSP in 48% of S neurons and 26% of AH neurons in the guinea-pig myenteric plexus. Calciumdependent hyperpolarization was recorded in 18% of AH neurons but not in S neurons (Williams, Katayama and North, 1979). MOTILIN Motilin is a 22-aminoacid residue polypeptide, which is released at about 100-min intervals during the interdigestive state, while the presence of nutrients in the duodenum strongly suppresses its endogenous release. Although, strictly speaking, motilin is a gastrointestinal hormone rather than a neurotransmitter, it will be briefly dealt with in this chapter in light of the interesting developments on motilin agonists as gastrointestinal prokinetics (Peeters, 1993; Clark et al., 1999; Siani et al., 2000). For the history of the discovery of motilin and erythromycin derivatives as prokinetics, the reader is referred to the reviews by Itoh (1997) and Peeters (1993). In this context, it will suffice to remember that erythromycin and its derivatives, which are devoid of antibacterial activity such as EM574 (motilin-like macrolides or motilides), are strong motilin

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agonists and profoundly stimulate gut motility. This effect is brought about both through a direct action on smooth muscle motilin receptors (Depoortere, Peeters and Vantrappen, 1991; Hasler, Heldsinger and Owyang, 1992) and through neural receptors (Peeters, 1993; Van Assche, Depoortere and Peeters, 1995). Smooth muscle contraction is induced through a nifedipine-sensitive mechanism (which therefore involves entry of extracellular calcium), but there is evidence that the intracellular calcium pool may also have a role (Matthijs, Peeters and Vantrappen, 1989; Peeters, Matthijs and Vantrappen, 1991; Depoortere and Peeters, 1995; Farrugia et al., 1995). A neurally mediated effect of motilides in the gastrointestinal tract is indicated, at least in some models, by the observation that their effects are antagonized by atropine, hexamethonium or ondansetron (Peeters, 1993; Mizumoto et al., 1993; Fiorucci, Santucci and Morelli, 1993; Shiba et al., 1995; Van Assche, Depoortere and Peeters, 1995; Parkman, Pagano and Ryan, 1996; Boivin et al., 1997). Indeed, vagal and non-vagal cholinergic pathways as well as serotonergic pathways seem to play an important role in mediating the motor effect of motilides (Itoh, 1997; Inatomi et al., 1996). There are also data documenting the presence of motilin receptors in the central nervous system (Itoh, 1997). Inhibitory effects sometimes observed with motilides have been ascribed to stimulation of inhibitory neural pathways or Ca2+-channel blockade (Parkman, Pagano and Ryan, 1996; Minocha and Galligan, 1991; Furness et al., 1999). These effects, however, usually occur at high concentrations and are resistant to motilin receptor antagonists (Furness et al., 1999). GASTRIN-RELEASING PEPTIDE AND NEUROMEDIN B GRP is a 27-amino acid peptide which has been found in a variety of mammals along with the related peptide, neuromedin B (Spindel, 1986; Furness and Costa, 1987; Ohki-Hamazaki, 2000). Bombesin and alytesin are tetradecapeptides, isolated from amphibia, which share a similar sequence to the C-terminus of GRP (Ersparmer et al., 1972; Ersparmer and Melchiorri, 1980). Ranatensin is an endecapeptide which is also related to GRP (Ersparmer et al., 1972; Nakajima, 1981). GRP-immunoreactive neurons are found in the myenteric and submucous plexuses in the guinea-pig small and large intestine (Schultzberg et al., 1980; Hutchison, Dimaline and Dockray, 1981; Messenger, 1993). Myenteric neurons containing GRP include descending interneurons and long descending inhibitory motor neurons to the CM (Costa et al., 1984; Messenger, 1993). These neurons are also immunoreactive for VIP, NOS and dynorphin (Brookes, Steele and Costa, 1992). GRP has also been identified in LM-MP synaptosomes by radioimmunoassay (McDonald et al., 1988). In the guinea-pig gastric fundus, GRP is present in ascending excitatory muscle motor neurons and in neurons involved in the regulation of mucosal functions (Pfannkuche et al., 2000). Neuromedin B and GRP receptor mRNAs have been detected in human and rabbit colonic smooth muscle cells (Bitar and Zhu, 1993). In the cat, GRP and neuromedin B evoke concentration-dependent contractions in circular strips of esophagus and fundus (Milusheva et al., 1998). Similar responses have been detected in longitudinal strips of the duodenum and ileum longitudinal muscles (Kortezova et al., 1994; Milusheva et al., 1998).

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Bombesin, GRP and neuromedin B produce TTX-sensitive, tonic contractions of the guinea-pig ileum LM-MP preparation (Zetler, 1980; Pfannkuche, 1992). The contractions are inhibited by atropine, suggesting that these peptides stimulate the release of ACh from LM motor neurons (Ersparmer et al., 1972; Zetler, 1980; Garzón et al., 1985). Bombesin also stimulates SP release from LM-MP preparations in a TTX-sensitive manner (Holzer, 1984). Bombesin-, GRP- and neuromedin B-induced contractions are not altered by mepyramine, nicotine or methysergide (Ersparmer et al., 1972; Pfannkuche, 1992), suggesting that the peptides do not stimulate ACh release from cholinergic interneurons and that the contraction is independent of histamine and 5-HT. Bombesin increases the tone of guinea-pig ileum CM and induces rhythmic contractions (Holzer, Lembeck and Donnerer, 1980) in a TTX-sensitive way. However, this effect is not inhibited by tropicamide or hexamethonium (Holzer, Lembeck and Donnerer, 1980), suggesting that bombesin does not stimulate cholinergic CM motor neurons or interneurons. GRP modulates the mechanical activity of the human ileocaecal region in vitro. Longitudinal strips show a concentration-dependent increase in the rhythmic activity, whereas the circular strips react with a small decrease in tone. These effects are not affected by TTX, suggesting a direct action of GRP on smooth muscle probably mediated by distinct receptors (Vadokas et al., 1997). A study by Barthó et al. (1982a) demonstrated that intra-arterial infusion of bombesin in vitro stimulates peristalsis. This effect has not been investigated further. However, it is possible that the stimulation of peristalsis is a result of slow depolarization of excitatory CM motor neurons, causing increased excitability and increased release of a non-cholinergic transmitter. Bombesin and GRP mimic slow EPSPs in AH neurons. Depolarization is associated with an increase in input resistance which may be due to the closing of a calcium-dependent potassium channel (Zafirov et al., 1985). GALANIN Galanin is a 29-amino acid peptide originally isolated from porcine intestine (Tatemoto et al., 1983). Galanin immunoreactivity in guinea-pig ileal myenteric neurons is localized in specific subpopulations: neurons projecting to the mucosa, descending inhibitory motor neurons to the CM, descending interneurons and LM motor neurons (Melander et al., 1985; Furness et al., 1987). Galanin has also been identified by radioimmunoassay and high pressure liquid chromatography in LM-MP synaptosomes (McDonald et al., 1988). Galanin is present in anally projecting pathways in the guinea-pig colon (Messenger, 1993) and colocalizes with NOS and VIP in canine antrum, small bowel and colon (Wang et al., 1998). Galanin does not alter basal activity of the LM of guinea-pig ileum (Kuwahara, Ozaki and Tanaihara, 1989) and has no effect on neurally-evoked twitch responses (Kuwahara, Ozaki and Tanaihara, 1989, 1990). More recently, however, galanin was found to possess a marked inhibitory effect on twitch responses, probably via activation of GAL1 receptors (Sternini et al., 1999b), and on tritiated ACh release (Mulholland, Schoeneich and Flowe, 1992). Galanin does not alter the basal activity of the CM of guinea-pig ileum, but inhibits TTX-sensitive, electrically evoked contractions (Kuwahara, Ozaki and Tanaihara, 1989,

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1990). Galanin inhibits both the hyoscine-sensitive and -resistant components of the CM twitch (Kuwahara, Ozaki and Tanaihara, 1989), suggesting that it may act by inhibiting release of ACh and a tachykinin from excitatory CM motor neurons. Yau, Dorsett and Youther (1986a) demonstrated that galanin inhibits the electrically stimulated release of [3H]ACh from the myenteric plexus. It has no effect on ACh-induced contractions in the presence of TTX (Kuwahara, Ozaki and Tanaihara, 1989). In agreement with this, Grider and Makhlouf (1988a) found that galanin alone had no contractile or relaxant effect on isolated circular smooth muscle cells. However, galanin potentiated the relaxant response to VIP, isoproteronol and dibutyryl cAMP. This effect was blocked by apamin and by tetraethylammonium at high concentrations although apamin and tetraethylammonium did not alter the response to VIP, isoprenaline or dibutyryl cyclic AMP alone (Grider and Makhlouf, 1988a). Thus galanin appears to act by opening potassium channels in the smooth muscle which in turn enhances the relaxation produced by agents increasing cyclicAMP levels. Galanin may contract ileal circular muscle of various species (pig, guineapig, rat and rabbit) and relaxes dog ileum by a direct myogenic effect (Botella et al., 1992). Recently, out of the three galanin receptors so far characterized, a single receptor subtype (i.e. the GAL2 receptor) was recognized in the rat intestine (Waters and Krause, 2000). Galanin hyperpolarizes myenteric neurons, decreases input resistance and suppresses excitability. These effects mimic slow IPSPs in myenteric neurons (Palmer et al., 1986b). Galanin also inhibits fast EPSPs in S neurons. However, this does not appear to be due to an action at a presynaptic inhibitory receptor, since galanin also inhibits the depolarization evoked by exogenous ACh (Tamura, Palmer and Wood, 1987). NEUROMEDIN U Neuromedin U was originally isolated from porcine spinal cord (Minamino, Kangawa and Matsuo, 1985), but it has also been extracted from the guinea-pig ileum (Murphy et al., 1990), and identified by radioimmunoassay and high pressure liquid chromatography (Augood, Keast and Emsom, 1988). Neuromedin U immunoreactivity is localized in Dogiel type II, calbindin-positive and -negative myenteric neurons, anally projecting myenteric neurons and myenteric neurons projecting to the submucous plexus (Furness et al., 1989a). It is not found in motor neurons. In the rat gastric fundus CM and in turtle small intestine LM, neuromedin U evokes a concentration-dependent contraction (Bockman et al., 1989; Maggi et al., 1990b; Benito-Orfila et al., 1991). Since these responses are unaffected by atropine or TTX (Bockman et al., 1989), they are likely to be due to a direct effect of neuromedin U on the muscle. However CM from frog stomach and LM from rat and frog small intestine are insensitive to neuromedin U. Similarly, neuromedin U does not have any significant effect on LM from the guinea-pig ileum (Minamino, Kangawa and Matsuo, 1985; Bockman et al., 1989). Human neuromedin U cDNA has been cloned and characterized (Austin et al., 1995). Neuromedin U mRNA is expressed in human small intestine and stomach (Hedrick et al., 2000). Recently, in the human and rat intestine, a receptor for neuromedin U has been identified, which probably corresponds to the orphan G protein-coupled receptor FM3 (Raddatz et al., 2000; Szerekes et al., 2000; Fujii et al., 2000).

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BRADYKININ Bradykinin receptors of the B2 but not B1 subtype are located on the LM and CM but not the myenteric plexus of guinea-pig ileum (Manning et al., 1982; Haasemann et al., 1991; Tousignant et al., 1991; Ransom et al., 1992), and on colonic CM (Hasler et al., 1995). The B2 receptor has been cloned and expressed in Xenopus oocytes; it is a G proteincoupled receptor that appears to stimulate a chloride current (McEachern et al., 1991). Bradykinin induces contraction of LM-MP preparations from the guinea-pig ileum (Garzón et al., 1985) which is largely unaffected by atropine (Khairallah and Page, 1961, 1963; Garzón et al., 1987). However, bradykinin reverses the inhibitory effects of opioids on the cholinergic twitch in a hyoscine-sensitive manner (Goldstein et al., 1983). Thus bradykinin appears to stimulate ACh release from cholinergic motor neurons to a variable degree. The non-cholinergic component of the contraction may be mediated by the release of a non-cholinergic transmitter or by a direct action of bradykinin on the smooth muscle. Bradykinin produces a biphasic response in the guinea-pig ileum CM, i.e. contraction followed by relaxation (Calixto and Medeiros, 1991). Neither response is mimicked by a B1 receptor agonist. Both the contraction and relaxation are unaffected by atropine, prazosin, yohimbine, propranolol, pyrilamine, [Leu8]BK1–8, TTX, glibenclamide or phorbol ester (Calixto and Medeiros, 1991). Thus neither response involves the action of endogenous transmitters at muscarinic, α1-adrenoceptor, α2-adrenoceptor, β-adrenoceptor, H1 or B1 receptors. Since the effect of bradykinin is TTX-insensitive, it is likely to be a direct effect on the smooth muscle. Furthermore, bradykinin does not open ATP-sensitive potassium channels or act via stimulation of protein kinase C activity. Bradykinin induces colonic smooth muscle contraction via B2 receptors, with phosphoinositide turnover activation and adenylate cyclase inhibition (Hasler et al., 1995). In this preparation, the bradykinin-induced contraction is mediated by influx of extracellular Ca2+ via non-selective cation channels and, in part, by the release of Ca2+ from loosely bound internal store (Zagorodnyuk, Santicioli and Maggi, 1998). In the taenia caeci, bradykinin produces a relaxation followed by a contraction. This response is inhibited by the B2 receptor antagonist Hoe 140. Since both phases of the response are TTXinsensitive, it is likely that they represent a direct effect on smooth muscle (Field et al., 1994). In the opossum lower oesophageal sphincter, bradykinin induces relaxation which is apamin-sensitive, followed by a contraction (Saha, Sengupta and Goyal, 1991). The contraction is inhibited by nifedipine, but not TTX, suggesting a direct effect of bradykinin on the muscle. In the rat isolated duodenum, bradykinin induces a biphasic response (relaxation followed by contraction), which is mediated by activation of B2 receptors (Rhaleb and Carretero, 1994). Previous evidence demonstrated that the B2 receptor-mediated relaxation was apamin sensitive (Hall and Morton, 1991; Greisbacher, 1992). Longitudinal strips of postmortem human ileum display a strong contractile response to bradykinin. This effect is mediated by constitutive B2 receptors and inducible B1 receptors (Zuzack et al., 1996).

ANGIOTENSIN Immunohistochemical studies suggest that the octapeptide angiotensin is present in enteric neurons of the guinea-pig ileum, although angiotensin has not been extracted from the external musculature (Furness and Costa, 1982).

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Angiotensin contracts the LM of the guinea-pig ileum. This effect was originally believed to be due to, in part to, stimulation of ACh release from LM motor neurons for several reasons. The contraction was reduced by 60–85% by atropine and enhanced by anticholinesterase inhibitors (Khairallah and Page, 1961; Robertson and Rubin, 1962; Khairallah and Page, 1963; Blair-West and McKenzie, 1966). Hexamethonium does not inhibit the angiotensin-evoked contraction (Khairallah and Page, 1961; Robertson and Rubin, 1962; Khairallah and Page, 1963). However Paiva et al. (1976) demonstrated that the concentrations of atropine used in previous studies resulted in non-specific inhibition of contraction produced by a variety of agonists including histamine and bradykinin, both of which are believed to act directly on the muscle. The mechanism whereby angiotensin contracts the LM has therefore yet to be elucidated, but a direct effect on smooth muscle cells (as also observed in CM: Shimuta et al., 1999) is likely (Smith, Taylor and Whiting, 1994). In addition, angiotensin was found to produce a concentration-dependent inhibitory effect on twitch responses in LM-MP preparations (Smith, Taylor and Whiting, 1994). By contrast, in rat colonic preparations, angiotensin was found to facilitate ACh release at high concentrations (Voderholzer, Allescher and Muller-Lissner, 1995). The pharmacological and electrophysiological effects of angiotensin on myenteric neurons are unknown. ENDOTHELIN-LIKE PEPTIDES The endothelin (ET) family of peptides was originally isolated from cultured porcine aortic endothelial cells (Yanagisawa et al., 1988; Saida, Mitsui and Ishida, 1989). However, endothelins have also been found in human intestine and in human and porcine spinal cord neurons (Giaid et al., 1989; Shinmi et al., 1989; Egidy et al., 2000). ET-1, ET-2, ET-3 and vasoactive intestinal contractor, a related peptide, produce contraction of the isolated guinea-pig longitudinal smooth muscle cells through an action on the ETB receptor subtype. This contraction is dependent on intracellular and extracellular calcium (Yoshinaga et al., 1992). Apart from a direct effect on the smooth muscle, endothelins inhibit nerve-evoked LM contractions and ACh release (Maggi et al., 1989; Wiklund et al., 1989). This effect may be mediated by the ETA receptor subtype (Maggi et al., 1989). Endothelin receptors are present in enteric neurons and seem to play an important role in the development of the enteric nervous system (Gershon, 1997). ETA receptor activation stimulates, whereas ETB receptor activation inhibits peristalsis in guinea-pig isolated small intestine. The ability of BQ-788, a selective ETB receptor antagonist, to facilitate peristalsis per se, points to a physiological role of ETB receptors in peristaltic motor regulation (Shahbazian and Holzer, 2000).

CONCLUSIONS The intestine is capable of complex behaviours which allow mixing, propulsion, digestion and absorption of food. The mechanism whereby the intrinsic neurons of the enteric nervous system and the circular smooth muscle coordinate their activities to produce propagated contractions has been the subject of intensive investigations for almost a century. Peristalsis was originally described in 1899 and studied by a few investigators

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at the turn of the century. A burst of research activity occurred in the 1950s and 1960s. Since the 1980s interest has been focussed on the characterization of neuronal circuitries and mechanisms underlying peristalsis. An integrated approach to study a variety of parameters of intestinal function has shed some light on the factors which determine the threshold distension for triggering emptying, the strength of the contraction, the mechanism whereby the contraction propagates anally and the speed of propagation. The intestine contains a large number of neuroactive substances and an equally large number of receptors for a variety of chemicals. As a consequence, the LM-MP preparation of the guinea-pig ileum has been widely used by pharmacologists to test new drugs. We therefore have quite detailed information about the effect of drugs on LM motor neurons and the LM. Conversely, drugs are tested less frequently for their effects on CM motor neurons or CM activity. However, the irony is that the role of the LM in intestinal motor function is less understood than that of the CM. Furthermore, although we have detailed information on the effects of exogenous agents on a layer of muscle which appears to have a scarce role in peristalsis, we know little of the role of most endogenous chemicals. It is clear therefore, that future studies need to address this imbalance. The development of selective antagonists to receptors for neuropeptides, in particular, will greatly assist the ultimate goal of understanding the neuronal basis of peristalsis.

ACKNOWLEDGMENTS The authors wish to thank Dr. Amalia Di Nucci and Dr. Cristina Giaroni for their helpful suggestions during drawing up of the chapter, and Ms. Elena Giovanetti for assistance in preparing the reference list.

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