The Eicosanoids

The Eicosanoids

The Eicosanoids Edited by

Peter Curtis-Prior Cambridge Research Institute, Cambridge and Anglia Polytechnic University, UK

Copyright u 2004

John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, England Telephone (+44) 1243 779777

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Contents List of Contributors vii Preface xi Preface from Prostaglandins Acknowledgements xvii Foreword xix

12 Time-resolved Fluoroimmunoassay in Eicosanoid Analysis 123 Werner Schlegel and Harald John

xiii

SECTION THREE BIOCHEMICAL AND MOLECULAR PHARMACOLOGY 129

SECTION ONE BIOSYNTHESIS AND METABOLISM 1 1

2 3 4 5 6 7

Perspectives on the Biosynthesis and Metabolism of Eicosanoids 3 Robert C. Murphy, Rebecca C. Bowers, Jennifer Dickinson and Karin Zemski Berry Control of Eicosanoid Production by Cellular and Secreted Phospholipase A2 17 Marise Andreani, Jean-Luc Olivier and Gilbert Be´re´ziat Mechanisms of PGH Synthase-1 (COX-1) Activity and Role of Radical States 29 Carol Deby and Ginette Deby-Dupont Regulation and Function of Prostaglandin Synthase-2/ Cyclooxygenase II 43 Harvey R. Herschman Mammalian Lipoxygenases 53 Shozo Yamamoto, Hiroshi Suzuki, Natsuo Ueda, Yoshitaka Takahashi and Tanihiro Yoshimoto Biosynthesis and Biological Effects of 5-oxo-ETE and Other Oxoeicosatetraenoic Acids 61 William S. Powell Synthetic Eicosanoids 69 Norrie. H. Wilson

SECTION TWO

ANALYTICAL METHODS

95

8 Perspectives of Analytical Methods for Eicosanoids 97 Jay Y. Westcott and Angelo Sala 9 Enzyme Immunoassays of Metabolites and Enzymes Using Acetylcholinesterase as Label 103 Christophe Cre´minon and The Late Jacques Maclouf 10 Bioassay of Eicosanoids 111 Robert L. Jones 11 Gas Chromatography and Mass Spectrometry in Eicosanoid Analysis 117 Michinao Mizugaki, Takanori Hishinuma, Naoto Suzuki and Junichi Goto

13 Perspectives and Clinical Significance of the Biochemical and Molecular Pharmacology of Eicosanoids 131 Subhash P. Khanapure and L. Gordon Letts 14 Eicosanoid Antagonists 163 Kiyoshi Yasui and Akinori Arimura 15 Biosynthesis and Degradation of Anandamide, an Endogenous Ligand of Cannabinoid Receptors 179 Natsuo Ueda and Dale G. Deutsch 16 Inhibitors of Eicosanoids 189 K. D. Rainsford 17 Biology and Chemistry of Products of the Isoprostane Pathway 211 L. Jackson Roberts II and Jason D. Morrow 18 Insight into Prostanoid Functions: Lessons from Receptor-knockout Mice 219 Yukihiko Sugimoto, Shuh Narumiya and Atsushi Ichikawa

SECTION FOUR IMMUNOLOGY, ENDOCRINOLOGY AND METABOLIC REGULATION 227 19 Perspectives and Clinical Significance of Eicosanoids in Immunology, Endocrinology and Metabolic Regulation 229 Milan R. Henzl 20 Prostaglandins and the Immune Response 237 Diana C. Fleming and Rodney W. Kelly 21 Leukotrienes in Aspirin-intolerant Asthma 247 Anthony P. Sampson and Stephen T. Holgate 22 Essential Fatty Acids 257 Yoeju Min and Michael A. Crawford 23 Endothelial Secretory Function and Atherothrombosis 267 Stefan Chlopicki and Richard J. Gryglewski

vi

CONTENTS

24 Molecular Regulation of Pancreatic Islet Prostaglandin Synthesis and its Relevance to Diabetes Mellitus 277 R. Paul Robertson 25 Prostaglandins, Leukotrienes and Bone 289 Carol C. Pilbeam and Lawrence G. Raisz 26 Ageing and Prostaglandins 299 A. Hornych SECTION FIVE

INFLAMMATION

319

27 Perspectives and Clinical Significance of Eicosanoids in Pain and Inflammation 321 Burkhard Hinz and Kay Brune 28 Antiinflammatory Steroids 327 Franc¸oise Russo-Marie 29 Eicosanoids and Algesia in Inflammation 333 Lynne Murray, Henry Sarau and Kristen E. Belmonte 30 Cyclooxygenase-2 in Cancer 341 Ovidiu C. Trifan and Jaime L. Masferrer 31 Cytokines and Eicosanoids in Arthritis 347 K. D. Rainsford SECTION SIX

CIRCULATORY SYSTEM

359

32 Perspectives and Clinical Significance of Eicosanoids in the Circulatory System 361 The Late James B. Lee 33 Aspirin and Activated Platelets 373 Artur-Aron Weber 34 Generation of Vasoactive Prostanoids by the Cyclooxygenase-2 Pathway in the Cardiovascular System of the Rat 387 P. J. Kadowitz, S. R. Baber, M. M. Mazim, M. Keebler, H. C. Champion, T. J. Bivalacqua, D. B. McNamara and A. L. Hyman 35 Eicosanoid Generation and Effects in Cardiac Muscle and Coronary Vessels 393 Karsten Schro¨r SECTION SEVEN

DIGESTIVE SYSTEM

405

36 Perspectives and Clinical Significance of Eicosanoids in the Digestive System 407 Chi Hin Cho, Joshua Ka Shun Ko and Marcel Wing Leung Koo 37 Eicosanoids and Liver Regeneration 415 David A. Rudnick and Louis J. Muglia 38 Eicosanoids and the Intestine 423 Klaus Bukhave and Jørgen Rask-Madsen 39 Eicosanoids and Stomach Physiology 431 Brigitta M. Peskar SECTION EIGHT

NERVOUS SYSTEM

445

40 Perspectives and Clinical Significance of Arachidonic Acid Release, Action and Metabolism in the Nervous System 447 Christopher D. Breder

41 Eicosanoid Pathways in the Ageing of the Central Nervous System 457 Hari Manev and Tolga Uz 42 Arachidonate Metabolites in the Neurophysiological System: the Fever Pathway 463 Ji Zhang and Serge Rivest 43 Prostanoids in Pain 473 Tony L. Yaksh, Patrick W. Mantyh and Camilla I. Svensson 44 Eicosanoids: Roles in the Pathophysiology of Cerebral Ischaemia 481 Robert W. Hickey and Steven H. Graham 45 NSAIDs in the Treatment of Alzheimer’s Disease 487 Paul S. Aisen 46 Prostaglandins and Eicosanoids in Mental Illness 493 A.I.M. Glen and B.M. Ross 47 Essential Fatty Acids: Eicosanoid Precursors in the Treatment of Huntington’s Disease 499 Krishna Vaddadi

SECTION NINE

REPRODUCTIVE SYSTEM

507

48 Perspectives and Clinical Significance of Eicosanoids in Obstetric and Gynaecological Practice 509 I. Z. MacKenzie 49 Prostaglandins and Male Reproductive Physiology 517 Rodney W. Kelly 50 Prostaglandin F2a: the Luteolytic Hormone 525 John A. McCracken 51 Prostaglandins in Implantation 547 Norman L. Poyser 52 Parturition and the Clinical Interruption of Pregnancy 559 S. Cowan and A.A. Calder 53 Foetal and Neonatal Ductus Arteriosus 569 Kazuo Momma

SECTION TEN CONCLUSIONS AND CORRELATIONS 583 54 Biochemical Interactions of Platelet-activating Factor with Eicosanoids 585 Joseph T. O’Flaherty and Robert L. Wykle 55 Eicosanoid Precursors as Pharmaceuticals 593 The Late David F. Horrobin 56 Pharmaceutical Exploitation: Cyclooxygenase and Lipoxygenase Inhibitors 599 Paola Patrignani and Maria G. Sciulli 57 Pharmaceutical Exploitation: Eicosanoids and their Analogues 613 David F. Woodward and June Chen Epilogue Index

617

619

List of Contributors Paul S. Aisen Department of Neurology, Georgetown University Medical Center, 1 Bles Building, 3800 Reservoir Road NW, Washington, DC 20007, USA Marise Andreani Laboratoire de Physiologie et de Physiopathologie, University Pierre and Marie Curie, 9 Quai Saint Bernard, 75252 Paris Cedex 05, France Akinori Arimura Discovery Research Laboratories, Shionogi & Co. Ltd, 3-1-1 Futaba-cho, Toyonaka, Osaka 561-0825, Japan S. R. Baber Department of Pharmacology, Tulane University Health Sciences Center, New Orleans, LA 70112, USA Kristen E. Belmonte Respiratory and Inflammation, Centre for Excellence in Drug Discovery, GlaxoSmithKline, UW2532, 709 Swedeland Road, King of Prussia, PA 19406, USA Gilbert Be´re´ziat University Pierre and Marie Curie, Case 256 Batiment a` 5 Etage, 7 Quai Saint Bernard, 75252 Paris Cedex 05, France T. J. Bivalacqua Department of Pharmacology, Tulane University Health Sciences Center, New Orleans, LA 70112, USA Rebecca C. Bowers Division of Cell Biology, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206, USA Christopher D. Breder Division of Neuroscience, Clinical Design and Evaluation, Bristol-Myers Squibb Co., Wallingford, CT, USA Kay Brune Department of Experimental and Clinical Pharmacology and Toxicology, Friedrich Alexander University Erlangen-Nu¨rnberg, Fahrstrasse 17, 91054 Erlangen, Germany Klaus Bukhave The Royal Veterinary and Agricultural University, Copenhagen, Denmark

June Chen Department of Biological Sciences, Allergan Inc., 2525 Dupont Drive, Irvine, CA 92612, USA Stefan Chlopicki Department of Pharmacology, Jagiellonian University Medical College, Grzego´rzecka 16, 31531 Krako´w, Poland Chi Hin Cho Department of Pharmacology, Faculty of Medicine, The University of Hong Kong, Hong Kong, China S. Cowan Department of Obstetrics and Gynaecology, University of Edinburgh, UK Michael A. Crawford Institute of Brain Chemistry and Human Nutrition, London Metropolitan University, 166–220 Holloway Road, London N7 8DB, UK Christophe Cre´minon CEA, Service de Pharmacologie et d’Immunologie, De´partement de Recherche Me´dicale, CEA-Saclay, 91191 Gif-sur-Yvette Cedex, France Carol Deby Centre for Oxygen Research and Development, Institut de Chimie, B6a, Universite´ de Lie`ge, 4000 Lie`ge, Belgium Ginette Deby-Dupont Centre for Oxygen Research and Development, Institut de Chimie, B6a, Universite´ de Lie`ge, 4000 Lie`ge, Belgium Dale G. Deutsch Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, NY 11794-5215, USA Jennifer Dickinson Division of Cell Biology, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206, USA Diana C. Fleming Medical Research Council Human Reproductive Science Unit, University of Edinburgh Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9ET, UK

A. A. Calder Department of Obstetrics and Gynaecology, University of Edinburgh, UK

Junichi Goto Department of Pharmaceutical Sciences, Tohoku University Hospital, 1-1 Seiryo-machi Aoba-ku, Sendai 980-8574, Japan

H. C. Champion Department of Pharmacology, Tulane University Health Sciences Center, New Orleans, LA 70112, USA

A. I. M. Glen Ness Foundation, UHI Millennium Institute, Ness House, Dochfour Business Centre, Inverness IV3 8GY, UK

viii

CONTRIBUTORS

Steven H. Graham Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Richard J. Gryglewski Department of Pharmacology, Jagiellonian University Medical College, Grzego´rzecka 16, 31-531 Krako´w, Poland Milan R. Henzl Stanford University Medical School, 4210 Ynigo Way, Palo Alto, CA 94306, USA Harvey R. Herschman Department of Biological Chemistry, David Geffen School of Medicine at UCLA, 341 Boyer Hall, 611 Charles E. Young Drive East, Los Angeles, CA 90095-1570, USA Robert W. Hickey Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Burkhard Hinz Department of Experimental and Clinical Pharmacology and Toxicology, Friedrich Alexander University Erlangen-Nu¨rnberg, Fahrstrasse 17, 91054 Erlangen, Germany Takanori Hishinuma Department of Pharmaceutical Sciences, Tohoku University Hospital, 1-1 Seiryomachi, Aoba-ku, Sendai 980-8574, Japan Stephen T. Holgate Respiratory Cell and Molecular Biology, Division of Infection, Inflammation and Repair, Southampton University School of Medicine, Southampton, UK

Philip J. Kadowitz Department of Pharmacology, Tulane University Health Sciences Center, New Orleans, LA 70112, USA M. Keebler Department of Pharmacology, Tulane University Health Sciences Center, New Orleans, LA 70112, USA Rodney W. Kelly Medical Research Council Human Reproductive Science Unit, University of Edinburgh Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9ET, UK Joshua Ka Shun Ko Department of Pharmacology, Faculty of Medicine, The University of Hong Kong, Hong Kong, China Subhash P. Khanapure Nitro Med Inc, 12 Oak Park Drive, Bedford, MA 01730, USA The Late James B. Lee* School of Medicine and Biomedical Sciences, University at Buffalo, USA Gordon Letts NitroMed Inc., 12 Oak Park Drive, Bedford, Massachusetts 01730, USA Marcel Wing Leung Koo Department of Pharmacology, Faculty of Medicine, Hong Kong Baptist University, Hong Kong, China I. Z. MacKenzie Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK

A. Hornych Department of Nephrology, European Hospital George Pompidou, 20 Rue Leblanc, 75908 Paris Cedex 15, France

The Late Jacques Maclouf U348 INSERM I.F.R. Vaisseaux-Lariboisie`re, Hoˆpital Lariboisie`re, 8 Rue Guy Patin, 75475 Paris Cedex 10, France

The Late David F. Horrobin{ Chairman, Laxdale Ltd, Kings Park House, Laurelhill Business Park, Stirling FK7 9JQ, UK

Hari Manev The Psychiatric Institute, Department of Psychiatry, University of Illinois at Chicago, 1601 West Taylor Street, Chicago, IL 60612, USA

A. L. Hyman Department of Pharmacology, Tulane University Health Sciences Center, New Orleans, LA 70112, USA

Patrick W. Mantyh Department of Preventive Sciences, University of Minnesota, Minneapolis, MN 55455, USA

Atsushi Ichikawa Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences and Department of Pharmacology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 6068501, Japan

Jaime L. Masferrer Oncology Department, AA5C, Pharmacia Corporation, 700 Chesterfield Parkway North, Chesterfield, MO 63198, USA M. M. Mazim Department of Pharmacology, Tulane University Health Sciences Center, New Orleans, LA 70112, USA

Harald John IPF Pharmaceuticals GmbH, 30625 Hannover, Germany

John A. McCracken Department of Animal Science, University of Connecticut, Storrs, CT 06269-4040, USA

Robert L. Jones Department of Pharmacology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, NT, Hong Kong

D. B. McNamara Department of Pharmacology, Tulane University Health Sciences Center, New Orleans, LA 70112, USA

{ Correspondence to Dr Crispin Bennett, Research Information Manager ([email protected])

*Correspondence to Professor Peter Curtis-Prior, Editor ([email protected]

CONTRIBUTORS

ix

Yoeju Min Institute of Brain Chemistry and Human Nutrition, London Metropolitan University, 166–220 Holloway Road, London N7 8DB, UK

K. D. Rainsford Biomedical Research Centre, School of Science and Mathematics, Sheffield Hallam University, Howard Street, Sheffield S1 1WB, UK

Michinao Mizugaki Department of Pharmaceutical Sciences, Tohoku University Hospital, 4-4-1 Komatsushima, Aoba-ku, Sendai, 981-8558 wcl, Japan

Lawrence G. Raisz University of Connecticut Health Center, 63 Farmington Avenue, Farmington, CT 06030-2806, USA

Kazuo Momma Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan

Jørgen Rask-Madsen Department of Medical Gastroenterology, Herlev Hospital, University of Copenhagen, Denmark

Jason D. Morrow Departments of Pharmacology and Medicine, Vanderbilt University, Nashville, TN 37232, USA Louis J. Muglia Department of Pediatrics, Washington University School of Medicine, St Louis, MO 63110, USA Robert C. Murphy Division of Cell Biology, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206, USA

Serge Rivest Laboratory of Molecular Endocrinology, CHUL Research Center and Department of Anatomy and Physiology, Laval University, 2705, Boul. Laurier, Que´bec, Canada G1V 4G2 L. Jackson Roberts II Departments of Pharmacology and Medicine, Vanderbilt University, Nashville, TN 37232, USA R. Paul Robertson Pacific Northwest Research Institute, Seattle, WA 98122, USA

Lynne Murray Imperial College School of Medicine, SAF Building, Exhibition Road, Leukocyte Biology, BMS Division, South Kensington, London SW7 2AZ, UK

B. M. Ross Ness Foundation, UHI Millennium Institute, Ness House, Dochfour Business Centre, Inverness IV3 8GY, UK

Shuh Narumiya Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences and Department of Pharmacology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 6068501, Japan

David A. Rudnick Department of Pediatrics, Washington University School of Medicine, St Louis, MO 63110, USA

Joseph T. O’Flaherty Department of Internal Medicine, Section on Infectious Disease, Wake Forest University School of Medicine, Winston Salem, NC, USA

Angelo Sala Department of Pharmacological Sciences, Via Balzaretti, 20133 Milan, Italy

Jean-Luc Olivier Laboratory of Medical Biology, Faculty of Medicine of Nancy, France Paola Patrignani Department of Medicine and Aging, Division of Pharmacology, ‘‘G. D’Annunzio’’ University of Chieti School of Medicine, 66013 Chieti, Italy Brigitta M. Peskar Department of Experimental Clinical Medicine, Ruhr-University of Bochum, Bochum, Germany Carol C. Pilbeam University of Connecticut Health Center, 63 Farmington Avenue, Farmington, CT 06030-2806, USA William S. Powell Meakins-Christie Laboratories, Department of Medicine, McGill University, 3626 St Urbain Street, Montreal, Quebec, Canada H2X 2P2 Norman L. Poyser Division of Biomedical and Clinical Laboratory Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK

Franc¸oise Russo-Marie BIONEXIS, CEA Saclay Baˆtiment 520, 91191 Gif-sur-Yvette Cedex, France

Anthony P. Sampson Respiratory Cell and Molecular Biology, Division of Infection, Inflammation and Repair, Southampton University School of Medicine, Southampton, UK Henry Sarau Respiratory and Inflammation, Centre for Excellence in Drug Discovery, GlaxoSmithKline, UW2532, 709 Swedeland Road, King of Prussia, PA 19406, USA Werner Schlegel Universita¨tsklinikum Mu¨nster, Klinik und Poliklinik fu¨r Frauenheilkunde und Geburtshilfe, Albert-Schweitzer-Strasse 33, D-48149 Mu¨nster, Germany Karsten Schro¨r Institut fu¨r Pharmakologie und Klinische Pharmakologie, Universita¨tsklinikum Du¨sseldorf, Moorenstrasse 5, D-40225 Du¨sseldorf, Germany Maria G. Sciulli Department of Medicine and Aging, Division of Pharmacology, ‘‘G. D’Annunzio’’ University of Chieti School of Medicine, 66013 Chieti, Italy Yukihiko Sugimoto Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences and

x

CONTRIBUTORS

Department of Pharmacology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 6068501, Japan Hiroshi Suzuki Department of Biochemistry, Tokushima University, School of Medicine, Kuramoto-cho, Tokushima 770-8503, Japan Naoto Suzuki Department of Pharmaceutical Sciences, Tohoku University Hospital, 1-1 Seiryo-machi, Aobaku, Sendai 980-8574, Japan Camilla I. Svensson Department of Anesthesiology, University of California, San Diego, La Jolla, CA 92093, USA Yoshitaka Takahashi Department of Molecular Pharmacology, Kanazawa University Graduate School of Medicine, 13-1 Takara-machi, Kanazawa 920-8640, Japan Ovidiu C. Trifan Oncology Department, AA5C, Pharmacia Corporation, 700 Chesterfield Parkway North, Chesterfield, MO 63198, USA Natsuo Ueda Department of Biochemistry, Kagawa University School of Medicine, Miki-cho, Kita-gun, Kagawa 761-0793, Japan Tolga Uz The Psychiatric Institute, Department of Psychiatry, University of Illinois at Chicago, 1601 West Taylor Street, Chicago, IL 60612, USA Krishna Vaddadi Department of Psychological Medicine Monash Medical Centre, (Monash University), 246 Clayton Road, Clayton 3186, Victoria, Australia Artur-Aron Weber Institut fu¨r Pharmakologie und Klinische Pharmakologie, Universita¨tsklinikum Du¨sseldorf, Moorenstrasse 5, D-40225 Du¨sseldorf, Germany

Jay Y. Westcott National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206, USA Norrie H. Wilson Division of Biomedical Sciences, University of Edinburgh, 1–3 George Square, Edinburgh EH8 9XD, UK David F. Woodward Department of Biological Sciences, Allergan Inc., 2525 Dupont Drive, Irvine, CA 92612, USA Robert L. Wykle Department of Biochemistry, Wake Forest University School of Medicine, Winston Salem, NC, USA Tony Yaksh Department of Anesthesiology, University of California, San Diego, La Jolla, CA 92093, USA Shozo Yamamoto Department of Food and Nutrition, Faculty of Home Economics, Kyoto Women’s University, Imakumano, Higashiyama-ku, Kyoto 605-8501, Japan Kiyoshi Yasui Discovery Research Laboratories, Shionogi & Co. Ltd, 3-1-1 Futaba-cho, Toyonaka, Osaka 561-0825, Japan Tanihiro Yoshimoto Department of Molecular Pharmacology, Kanazawa University Graduate School of Medicine, 13-1 Takara-machi, Kanazawa 920-8640, Japan Karin Zemski Berry Division of Cell Biology, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206, USA Ji Zhang Laboratory of Molecular Endocrinology, CHUL Research Center and Department of Anatomy and Physiology, Laval University, 2705 Boul. Laurier, Que´bec, Canada G1V 4G2

Preface As the year 2000 approached, there developed in people around the world a sense of anticipation, positive for some but negative for others. However, in both groups of people there was a deep desire to mark such a rare moment in history. It was at this time that it was first suggested to me as the ideal moment for a new, comprehensive volume on the biomedical significance of polyunsaturated acids and their derivatives, bringing together the important discoveries and developments which had taken place in this area since the publication of a previous book with which I had been involved (Curtis-Prior 1988). It was that prompting which has led to this latest book, The Eicosanoids (see also Acknowledgements). So, although what might be considered as ‘‘. . . important discoveries and developments . . .’’ is to some extent a subjective exercise and certainly every chapter in this book reveals developments of fascinating new knowledge in its field of specialism, there has been one landmark advance during these fifteen years which, among much else, has reconciled the seeming paradox that prostaglandins may be both harmful and beneficial. This was, of course, the identification and characterization of an alternative cyclooxygenase enzyme (Fu et al 1990; Masferrer et al 1990; Herschman et al 1993), i.e. the proposal of the coexistence of the known enzyme responsible for basal, constitutive prostaglandin synthesis (COX-1) together with a new enzyme (designated COX-2) implicated in various inflammatory and ‘‘induced’’ settings (Funk 2001; see also Chapter 4, this volume, Regulation and Function of Prostaglandin Synthase-2/Cyclooxygenase II). This revelation provoked a whole new wave of discovery research activity in acadaemia and in the pharmaceutical industry. There followed a race to develop a new class of COX-2 selective inhibitors which inhibited prostaglandin production in inflammatory cells without the, hitherto, inevitable undesirable interference on the production of COX-1-generated prostaglandins fulfilling their physiological regulatory roles in the gastrointestinal tract (Warner et al 1999) in renal function and in the reproductive and haemostatic systems as is presented in superb detail elsewhere in this book (see for example Chapters 13, this volume, Perspectives and Clinical Significance of the Biochemical and Molecular Pharmacology of Eicosanoids and Chapter 16 Inhibitors of Eicosanoids). Some of the early fruits of this burst of scientific creativity were presented at specialized conferences in Bangkok and Boston and recorded in a state of the art ‘‘book-of-the-meetings’’ edited by Vane and Botting (1998), and this was later succeeded by their comprehensive monograph on the subject, entitled Therapeutic Roles of Selective COX-2 Inhibitors (Vane and Botting 2001). The Pharmacia drug Celebrex (celecoxib) was the first of this new generation of COX-2 selective inhibitors to receive marketing authorization (by the US FDA) for the treatment of inflammatory conditions (Penning et al 1997) and the second was Merck Sharp & Dohme’s Vioxx (rofecoxib) (Chan et al 1999; Prasit et al 1999). In a pivotal trial, when celecoxib was compared with ibuprofen and diclofenac in a 6 months-long arthritis safety study (CLASS), it was reported (Silverstein et al 1999) that the COX-2 inhibitor was associated with a lower incidence of symptomatic ulcers and

ulcer complications than the traditional COX-1 non-steroidal antiinflammatory drugs, as had been the result anticipated. However, it appears that there was some incongruity (Okie 2001; Berg et al 2001; Wright et al 2001) between this account from Silverstein and his co-workers and ‘‘. . . the complete information available to the United States Food and Drug Administration . . .’’ (Juni et al 2002) in what has been described as a ‘‘. . . failure in the therapeutic chain as a cause of drug ineffectiveness . . .’’ (Figueras and Laporte 2003). However, it should be noted that a subsequent major study (designated VIGOR) to compare the incidence of upper gastrointestinal events provoked by MSD’s selective COX-2 inhibitor rofecoxib and the non-selective NSAID naproxen, in patients with rheumatoid arthritis, demonstrated significant clinical advantages for rofecoxib (Bombardier et al 2000). Whatever might be the eventual outcome of this particular and complex situation, where we well know that the worldwide clinic is the ultimate challenge of any therapeutic product, the drug target remains fascinatingly attractive, especially for industry, in the light of a global market for analgesics estimated at US $10 billion.* Interestingly, there have also been reports of a herbal preparation called NexrutineTM (Lavelle 2003). This is a new patent-pending dietary supplement containing an extract of the plant Phellodendron amurense and has been reported to be useful in the treatment of inflammatory diseases. It possesses COX-2inhibitory qualities, but also protects the gastrointestinal tract against ulceration. In clinical studies, it was shown that Nexrutine inhibits COX-2 without interfering with the activity of COX-1. In animal studies, Nexrutine has proved to be as effective as naproxen in reducing pain and inflammation. However, its mechanism of action has been reported to be different from that of what might be termed true COX-2 inhibitors, since it does not act directly on the cyclooxygenase, but inhibits the gene responsible for the production of COX-2, as well as other inflammatory mediators. Other novel developments treated in this book include: endogenous ligands of cannabinoid receptors, referred to as endocannabinoids (Mechoulam et al 1998; see also Chapter 15, this volume, Biosynthesis and Degradation of Anandamide, an Endogenous Ligand of Cannabinoid Receptors); identification of the noxious roles of cysteinyl leukotrienes in aspirin-intolerant asthma (Samson and Holgate 1999; see also Chapter 21, this volume, Leukotrienes in Aspirin-intolerant Asthma); prostaglandin receptor knock-out mice (Negishi and Katoh 2003; see also Chapter 18, this volume, Insight into Prostanoid Functions: Lessons from Receptor-knockout Mice); the biosynthesis and roles of isoprostanes (Morrow et al, 1990; see also Chapter 17, this volume, Biology and Chemistry of Products of the Isoprostane Pathway) and their utility in the assessment of oxidative stress status in vivo, in the clinic. Finally there is in this volume (see also below) a wealth of new knowledge shared over a broad range of the actions and *Current nomenclature has been adopted, taking 1 billion (bn) as 109 rather than the traditional value of 1012.

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PREFACE

interactions of eicosanoids in biomedicine and physiology, from immunology to central nervous system disorders. The book is divided into thematic sections (see Contents) each of which begins with a chapter designated ‘‘Perspectives and clinical significance . . .’’ The intention is for these ‘‘Perspectives’’ chapters to provide an integrated synopsis of the subject area covered in the respective Section, indicating how the different specialist chapters (strands of the theme) relate to one another in the overall context of the theme. In this way, it is hoped that THE EICOSANOIDS may interest, encourage and inspire all who are fascinated by the biosciences in seeking further to understand more about the myriad roles of essential fatty acids to the benefit of human wellbeing. Notwithstanding the unpredictability of the peaks and troughs in the evolution and development of any discipline, it appears valid, still, to look forward to even more fascinating discoveries ahead through the immortal words from Robert Browning, cited on a previous occasion (Curtis-Prior 1983): Grow old along with me The best is yet to be . . .

Cambridge 2004 Peter Curtis-Prior [email protected] REFERENCES Berg Hrach J and Mora M (2001) Reporting of 6-month vs. 12-month data in a clinical trial of celecoxib. J Am Med Assoc, 286, 2398. Bombardier C, Laine L, Reicin A et al (2000) Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis. N Eng J Med, 343, 1520–1528. Chan CC, Bouce S, Brideau C et al (1999) Rofecoxib [Vioxx, MK-0966; 4(4’-methylsulfonylphenyl)-3phenyl-2-(5H)-furanone]: a potent and orally active cyclooxygenase-2 inhibitor. Pharmacological and biochemical profiles. J Pharmacol Exp Therapeut, 290, 551–560. Curtis-Prior PB (ed.) (1988) Prostaglandins: Biology and Chemistry of Prostaglandins and Related Eicosanoids. Edinburgh: Churchill Livingstone. Curtis-Prior PB (1983) Trends in prostaglandin studies [Orientations des e´tudes actuelles sur les prostaglandines]. Curr Clin Concepts, 1, 9–19. Di Marzo V (1998) Endocannabinoids and other fatty acid derivatives with cannabimimetic properties: biochemistry and possible physiopathological relevance. Biochim Biophys Acta, 1392, 153–175. Figueras A and Laporte J-R (2003) Failures of the therapeutic chain as a cause of drug ineffectiveness. Br Med J, 326, 895–896. Fu JY, Masferrer JL, Seibert K et al (1990) The induction and suppression of prostaglandin H2 synthase (cyclooxygenase) in human monocytes. J Biol Chem, 265, 16737–16740. Funk CD (2001) Prostaglandins and leukotrienes: advances in eicosanoid biology. Science, 294, 1871–1875.

Herschman HR, Fletcher BS and Kujubu DA (1993) TIS10, a mitogeninducible glucocorticoid-inhibited gene that encodes a second prostaglandin synthase/cyclooxygenase enzyme. J Lipid Mediat, 6, 89–99. Juni P, Rutjes AWS and Dieppe PA (2002) Are selective COX-2 inhibitors superior to traditional non-steroidal antiinflammatory drugs? Adequate analysis of the CLASS trial indicates that this may not be the case. Br Med J, 324, 1287–1288. Lavelle JB (ed.) (2003) The COX-2 Connection: Natural Breakthrough Treatments for Arthritis, Alzheimer’s and Cancer. Rochester VT: Healing Arts Press. Masferrer JL, Zweifel BS, Seibert K et al (1990) Selective regulation of cellular cyclooxygenase by dexamethasone and endotoxin in mice. J Clin Invest, 86, 1375–1379. Mechoulam R, Fride E and Di Marzo V (1998) Endocannabinoids. Eur J Pharmacol, 359, 1–18. Morrow JD, Hill KE, Burk RF et al (1990) A series of prostaglandin F2like compounds are produced in vivo in humans by a noncyclooxygenase, free radical-catalyzed mechanism. Proc Natl Acad Sci USA, 87, 9383–9387. Negishi M and Katoh H (2002) Cyclopentanone prostaglandin receptors. Prostagland Lipid Mediat, Special Issue: Molecular Biology of the Arachidonate Cascade, Guest Eds S Yamamoto and WL Smith, 68–69, 611–617. Okie S (2001) Missing data on Celebrex. Full study altered picture of drug. Washington Post, 5 (Aug), A11. Penning TD, Talley JJ, Bertenshaw SR et al (1997) Synthesis and biological evaluation of the 1,5-diarylpyrazole class of cyclooxygenase2 inhibitors: identification of 4-[5-(4-methyl phenyl)-3-(trifluoromethyl)1H-pyrazol-1-yl]benzenesulfonamide (SC-58635, Celecoxib). J Med Chem, 40, 1347–1365. Prasit P, Wang Z, Brideau C et al (1999) The discovery of rofecoxib [MK966, Vioxx, 4-(methylsulfonyl phenyl)3-phenyl-2(5H)-furanone], an orally active cyclooxygenase-2 inhibitor. Bioorg Med Chem Lett, 9, 563–567. Sampson AP and Holgate ST (1999) Leukotriene Modifiers in Asthma Treatment. London: Martin Dunitz. Silverstein FE, Faich G, Goldstein JL et al (2000) Gastrointestinal toxicity with celecoxib vs. non-steroidal antiinflammatory drugs for osteoarthritis and rheumatoid arthritis: the CLASS study: a randomized controlled trial. Celecoxib Long-term Arthritis Safety Study. J Am Med Assoc, 284, 1247–1255. Vane JR and Botting RM (1998) Clinical Significance and Potential of COX-2 Inhibitors. London: William Harvey Press. Vane JR and Botting RM (2000) Therapeutic Roles of Selective COX-2 Inhibitors. London: William Harvey Press. Warner TD, Giuliano F, Vojnovic I et al (1999) Non-steroid drug selectivities for cyclooxygenase-1 rather than cyclooxygenase-2 are associated with human gastrointestinal toxicity: a full in vitro analysis. Proc Natl Acad Sci USA, 96, 7563–7568. Wright JM, Perry TL, Bassett KL and Chambers KG (2001) Reporting of 6-month vs. 12-month data in a clinical trial of celecoxib. J Am Med Assoc, 286, 2398.

Preface from Prostaglandins: Biology and Chemistry of Prostaglandins and Related Eicosanoids, edited by P. B. Curtis-Prior (1988) My purpose in preparing this volume was to provide, in a single source, a comprehensive collection of information on prostaglandins and related eicosanoids.* The 57 chapters presented here are divided into ten sections, according to subject, and are the work of a total of almost 80 authors from over a dozen countries, distributed among four of the world’s continents. Although the chapters have been edited I have tried, as far as possible, to retain the original style of each author, or group of authors, in order to present a richer, international, intercultural final product. The book has been over 5 years in the making and, in spite of the more recent possibility for authors to carry out updating procedures on their contributions, must remain less than comprehensive owing, in part, to the constant influences of man’s innate curiosity and creativity providing new knowledge more quickly even than the old can be recorded. However, it is perhaps germane at this time to recall the maxim below, suggested by Dr John A. McCracken (The Worcester Foundation, Shrewsbury, Massachusetts), with which many of the ‘‘initiated’’ in this field will, no doubt, readily identify: The one who places the last stone and steps across to the terra firma of accomplished discovery gets all the credit. Only the initiated know and honor those whose patient integrity and devotion to exact observation have made the last step possible. Hans Zinsser

The demonstration of the essentiality of dietary linoleic acid, and the coining of the term ‘‘essential fatty acid (EFA)’’ by Burr and Burr (1930), the subsequent dawning of the real significance of this observation, plus the preliminary clinical studies of Kurzrok and Lieb (1930) showing the uterine muscle-stimulating properties of human semen, followed shortly afterwards by the observations of hypotensive actions of semen and acidic lipid extracts thereof by Goldblatt (1935) and von Euler (1935), really set the scene for the story recounted in this volume, a story which is certainly not yet completed. Since the 1930s there has been an enormous growth and development of interest and investigation which, however, has not been a continuum. Indeed, following the initial spate of papers of the ‘past historic’ phase of attention to prostaglandins (CurtisPrior 1985), there was a clear arrest of devotion during the years of the Second World War, prior to a resurgence—including the first isolation of prostaglandins (Bergstrom and Sorjvall 1957) in the postwar years, since when there has developed a rate of issue of 4.5–5.06103 publications each year, i.e. approaching 100 per

*‘‘Eicosanoids’’ is a useful term coined by Corey EJ, Nuira H, Falek JR, Mioskowski C, Arai Y, Marfat A (1980) Recent studies on the chemical synthesis of eicosanoids. Advances in Prostaglandin and Thromboxane Research, 6: 19–25 . . . ‘‘to describe the broad group of compounds derived from C20 fatty acids . . .’’

week.{ The origin of this present and continuing interest in prostanoids is not entirely clear, but probably stems to a large degree from the independent and virtually simultaneous findings relating to the actions of non-steroidal anti-inflammatory drugs on prostaglandin biosynthesis in France and in Britain, especially of the effects of the most widely-used drug substance the world has yet known: aspirin, as was surmised previously by Collier (1971). At L’Institut Pasteur in Paris, Vargaftig and co-workers had spent several years examining phospholipases A and C, and showed subsequently that aspirin could block the actions of vasoactive substances normally formed by phospholipases from guinea-pig lung (Vargaftig and Dao 1971). Meanwhile, Willis and Smith at the Royal College of Surgeons of England in London had observed that the production of prostaglandins by blood platelets was presented in the presence of aspirin. The significance of these observations was quickly recognized, with legendary vision, by Vane (also at the Royal College), who corroborated them using guinea-pig lung homogenate; this led to the famous trilogy of papers published in Nature that year (Ferreira et al 1971; Smith and Willis 1971; Vane 1971). Subsequently, the work of Morley and Williams (1973) demonstrated an enhancement of paw oedema by prostaglandins which entirely corroborated the association of prostaglandins and inflammation. Although the greatest interest and the major proportion of published data relate to arachidonic acid and its metabolites, there is increasing attention being paid also to eicosanoids of the 1-series and of the 3-series. Considerable public interest has been provoked latterly in health food preparations of seed oil of the evening primrose, an oleaginous (willow herb) plant of the genus Oenothera misnamed because of the resemblance of its bright yellow flowers to those of real primroses. This plant’s seed is rich in linoleic acid (72%) and contains also g-linolenic acid (c.9%). It is the latter component which is proposed as the active principle through conversion to prostaglandins of the 1-series (mainly E1) in remarkably wideranging therapeutic claims (Graham 1984). This is compatible with the wide-held belief that our main source of arachidonic acid is probably the meat of our diet (i.e. other animals’ bodies) and, since on the one hand the 5-desaturase enzyme catalysing the further transformation of dihomo-g-linolenic acid to arachidonic acid is of minor importance in man, and on the other hand that activity of the 6-desaturase responsible for the formation of glinolenic acid from linoleic acid is much reduced by our modern lifestyle, as well as the pathologies of severe hepatic insufficiency, undernutrition, insulin-dependent diabetes and senescence (Paccalin et al 1984), it is tacitly assumed that additional dietary glinolenic acid is converted mainly to prostaglandins of the 1-series. { These data were kindly supplied by the Information Services (Corporate Technical Library), Upjohn Company, Kalamazoo, USA and calculated under the revised system, 1983.

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However, the identification of other angiosperm plant seeds containing increased amounts of g-linolenic acid, like the blackcurrant (Ribes nigrum) and borage (Borago officianalis), and the recent discovery, in several parts of the world, of the very important nutritional potential of the blue-green alga of the genus Spirulina (Chan et al 1981; Ciferri 1983; Devi and Venkataraman 1983) containing much higher concentrations of g-linolenic acid synthesized by direct desaturation of linoleic acid, suggest they may shortly eclipse the present fashionable interest in ‘‘primrose oil’’. Indeed, recent advances in biotechnology could replace agricultural seed oil production altogether as a source of glinolenic and other polyunsaturated fatty acids of therapeutic potential (Ratledge 1987). The origins of man’s cognisance of the potential therapeutic importance of prostaglandins of the 3-series and their precursor polyunsaturated fatty acids has been indicated recently by Sinclair (1985).* About 3500 years ago, the Israelites were bored by making bricks without straw, and Moses (or perhaps Aaron) hit the sea with a rod—a poor tool for that purpose but the Lord was on his side. All the fish died. Shortly thereafter Pharoah had an infarct followed by an epidemic of hardening of the heart amongst the Egyptians. This is the earliest indication of the importance of fish in preventing myocardial infarction.

More recently, the extremely low incidence of myocardial infarction and a tendency to bleed attributed to an Eskimo diet has emphasized the apparent protective aspects of fish oils (Dyerberg et al 1975), although in rats fed cod liver oil PGI3 was not detectable (Hornstra and Nugteren 1981). While this observation has thrown doubt on a thromboregulatory role for 3series prostaglandins, it cannot dispute the observed non-genetic low cardiovascular morbidity and mortality of Eskimo populations and may indicate the identity of the protective principle as the polyunsaturated fatty acid (potential prostaglandin precursor) eicosapentanoic acid (Dyerberg et al 1978). However, it may be significant that furanoid or urofuran acid compounds which have been shown to be minor components of fish oils (Glass et al 1975) may also be credited with a possible cardiovascular protective role in view of their potent hypolipidaemic properties in animal models in reducing both blood cholesterol and triglycerides (Hall 1985). The recently-discovered arachidonic acid metabolites lipoxins, formed by human leukocytes stimulated with calcium ionophore (Serhan et al 1984), appear to be involved in inflammation and their possible interactions and/or associations with other eicosanoids are discussed in several contexts in this volume. Apart from the arachidonic acid metabolites, phospholipase A2 may give rise also to another lipid mediator, platelet activating factor (PAF, PAF-acether or acetyl-glyceryl-ether-phosphorylcholine) (Benveniste et al 1972), which was first recognized by its degranulating activity of rabbit platelets (Siragnanian and Osler 1971), implicated as a mediator of anaphylaxis (Henson and Pinckard 1977) and subsequently implicated in most aspects of cellular inflammation (Benveniste and Arnoux 1983). Because of the unceasing evidence of the intimate associations of eicosanoids with PAF in pathophysiological processes, a sign-post chapter (Ch. 55) has been incorporated in the final part of this volume. The recognition of the cytokine peptide interleukin-1 (IL-1) as an inflammatory mediator inducing fever, the release of acute phase proteins and cartilage degradation led naturally to the

*This is quoted with permission from a pre-publication script kindly supplied by Professor Hugh Sinclair of his pre-dinner presentation entitled: ‘‘History of EFA and their prostanoids: some personal reminiscences’’, on the occasion of the Second International Congress on Essential Fatty Acids and their Eicosanoids.

supposition of prostaglandin involvement in these pathophysiological processes. However, it is only recently that IL-1 has been clearly shown to activate cellular phospholipase A2 and thus suggests the whole gamut of arachidonic acid metabolites and PAF as its potential secondary mediators (Chang et al 1986). Superimposed on this are the complicating preliminary observations that PAF itself may IL-1 production from human macrophages (Barrett et al 1987). The early implication of eicosanoids in inflammation, referred to above, is evidently the visible tip of a complex metaphorical iceberg wherein the indicated interrelationship of prostanoids/ eicosanoids with PAF, lipoxins and cytokines and kinins are only now beginning to be unravelled. These exciting developments in inflammation and the body homeostatic defence mechanisms, those related to cardiovascular disease and diet, recent implications of PAF in nidation and a potential further means of regulating fertility (O’Neil 1986), and the most recent suggestion of a role for eicosanoids as second messengers in the nervous system (Piorrelli et al 1987) are but a few of the potentially exciting avenues awaiting further elucidation in the future of ‘‘prostaglandins’’, and may explain why often ‘‘the true innovatory researcher cannot define the eventual length or cost of a research programme’’ (Celsus 1985). P.B.C-P. Cambridge, 1988 REFERENCES Barrett ML, Lewis GP, Ward S and Westwick J (1987) Platelet activating factor induces interleukin-1 production from human adherent macrophages. Br J Pharmacol, 90, 113P. Benveniste J and Arnoux B (1983) Platelet-activating Factor and Structurally-related Ether Lipids. Amsterdam: Elsevier. Benveniste J, Henson PM, Cochrane CG 1972 Leukocyte-dependent histamine release from rabbit platelets. The role of IgE, basophils and a platelet-activating factor. J Exp Med, 136, 1356–1376. Bergstrom S and Sjovall J (1957) The isolation of prostaglandin. Acta Chem Scand, 11, 1086. Burr GO, Burr MM (1930) On the nature and the role of fatty acids essential in nutrition. J Biol Chem, 86, 587–621. Chang J, Gilman SC and Lewis AJ (1986) Interleukin 1 activates phospholipase A2 in rabbit chondrocytes: a possible signal for IL 1 action. J Immunol, 136, 1283–1287. Celsus (1985) Primroses and prostaglandin. Pharm J, 235, 607–608. Chen L-C, Chen J-S and Tung T-C (1981) Effects of Spirulina on serum lipoproteins and its hypocholesterolemic effect. J Formosan Med Assoc, 80, 934–942. Ciferri O (1983) Spirulina, the edible microorganism. Microbiol Rev, 47, 551–578. Collier HOJ (1971) Prostaglandins and aspirin. Nature New Biol, 231, 17– 19. Curtis-Prior PB (1985) Trends in prostaglandin studies. Curr Clin Concepts, 1, 9–19. Dyerberg J, Bang HO and Hjorne N (1975) Fatty acid composition of the plasma lipids in Greenland eskimos. Am J Clin Nutrit, 28, 958–966. Dyerberg J, Bang HO, Stofferson E et al (1978) Eicosapentanoic acid and prevention of thrombosis and atherosclerosis. Lancet, ii, 117–119. Devi MA and Venkaturaman LV (1983) Hypocholesterolemic effect of blue green algae Spirulina platensis in albino rats. Nutrit Rep Int, 28, 519–530. von Euler US (1935) A depressor substance in the vesicular gland. J Physiol, 84, 21P. Ferreira SM, Moncada S and Vane JR (1971) Indomethacin and aspirin abolish prostaglandin release from the spleen. Nature, 231, 237–239. Glass, RL, Krick TP, Sand DM et al (1975) Furanoid fatty acids from fish lipids. Lipids, 10, 695–702. Goldblatt MW (1935) A depressor substance in seminal plasma. J Physiol, 84, 201–218. Graham J (1984) Evening Primrose Oil. Wellingborough, UK: Thorsons.

PREFACE FROM PROSTAGLANDINS Hall H (1985) The hypolipidaemic activity of furanoic acid and furyl acrylic acid derivatives in rodents. Pharm Res, 5, 233–238. Henson PM and Pinckard RN (1977) Basophil derived platelet activating factor (PAF) as an in vivo mediator of acute allergic reactions. Demonstration of specific desensitization of platelets to PAF during IgE-induced anaphylaxis in the rabbit. J Immunol, 119, 1279–1286. Hornstra G and Nugteren D (1981) Fish oil feeding does not result in the endogenous formation of PGI3 in rats. Progr Lipid Res, 20, 911. Kurzrok R and Lieb CC (1930) Biochemical studies of human semen. The action of semen on the human uterus. Proc Soc Exp Biol Med, 28, 268– 272. O’Neill C (1986) PAF in early pregnancy. Conference papers: an International Seminar on the Therapeutic and Commercial Potential of Drugs Affecting Platelet Activating Factor (London). Paccalin J, Dabadie H, Bernard M et al (1984) Interet d’une nouvelle plante oleagineuse: l’onagre (Oenothera biennis ou larmarkiania) apport en acide g-linolenique et troubles de la desaturation en pathologie. Med Nutr, xxi, 132–136. Piorrelli D, Volterra A, Dale N et al (1987) Lipoxygenase metabolites of arachidonic acid as second messengers for presynaptic inhibition of Aplysia sensory cells. Nature, 328, 38–43.

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Ratledge C (1987) Oleaginous yeasts and moulds. Conference papers: International Conference on Biotransformations (Cambridge). Serhan CN, Hamberg M and Samuelsson B (1984) Trihydroxytetraenes: novel series of oxygenated derivatives formed from arachidonic acid in human leukocytes. Biochem Biophys Res Commun, 118, 943–949. Siraganian RP and Osler AG (1971) Destruction of rabbit platelets in the allergic response of sensitized leukocytes. 1. Demonstration of a fluid phase intermediate. J Immunol, 106, 1244–1251. Smith JB and Willis AL (1971) Aspirin selectively inhibits prostaglandin production in human platelets. Nature, 231, 235–237. Vane JR (1971) Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature, 231, 232–235. Vargaftig BB and Dao NN (1971) Release of vaso-active substances from guinea-pig lungs by ‘‘slow-reacting substance C’’ and arachidonic acid. Its blockade by non-steroidal antiinflammatory agents. Pharmacology, 6, 99–108. Williams TJ and Morley J (1973) Prostaglandins as potentiators of increased vascular permeability in inflammation. Nature, 246, 215–217.

Acknowledgements Editing this substantial volume has required, above all, a great commitment of that most valuable commodity . . . time. Time taken out of a life is not easily replaced, so I thank my wife for her enabling me to devote so much time to this exciting editorial adventure. The stimulus for this book, The Eicosanoids, grew out of suggestions from several colleagues and especially Professor Kim Rainsford (Biomedical Research Centre, Sheffield Hallam University, UK). It was suggested that a new book in this area would be a fitting and appropriate marking of the new millennium and enable recent scientific discoveries and developments to be drawn together into a work of reference, which a number of other colleagues believed would be timely. It was, thus, through these encouragements that the idea finally took on form and became a reality. The Editor wishes to express his appreciation to the publishers, John Wiley & Sons, for their patience and to the indefatigable and complementary Charlotte Brabants and Layla Paggetti from the London office, who have been such absolute stalwarts during these several years and to Monica Twine and her team at the Chichester Office for wisdom and support during the production phase. It is also my great pleasure to acknowledge the consistently reliable support from staff of the Medical Library in Cambridge University, Addenbrooke’s Hospital in Cambridge. I am most grateful to Nobel Laureate Professor Bengt Samuelsson (Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden) for agreeing to contribute a Foreword to this new volume. His pioneering work, together with that of his co-workers, in their pivotal role in elucidating eicosanoid structures, truly laid the foundations for the subsequent development in our understanding of the biochemical pathways involving polyunsaturated fatty acids. A number of the eminent scientists who were involved in a previous multi-author book on prostaglandins (see above) kindly agreed to contribute also to this new volume. To them and to the newer, distinguished contributors (particularly those who so helpfully stepped in at the proverbial ‘‘eleventh hour’’ as replacements to fill a totally unanticipated vacancy) I wish to express my thanks and warm appreciation for the vital part they have played in the production of this exciting book. Also, I should like to make a special point of thanking Professor Elisabeth Granstrom for her help, wisdom and in sharing her extensive knowledge in the eicosanoid field. I am indebted to many colleagues who kindly gave assistance, advice or made recommendations of potential contributors and in this regard wish to thank, particularly: Prof Shozo Yamamoto, Dr Christopher D. Breder, Dr Ignatius Tavares, Prof Alan R. Brash, Dr David Horrobin, Prof Brigitta Peskar, Dr Keith Crowshaw, Dr Bob

Coleman, Dr Howard R. Knapp, Dr Stephen Cunnane, Prof Michael Crawford, Dr A. Kirchenbaum, Dr Andrew J. Dannenberg, Prof Alastair Watson, Dr Joel K. Elmquist, Dr Denis McCarthy, Dr Martin L. Ogletree, Dr Frank Fitzpatrick, Prof Simonetta Nicosia, Prof Colin C. Funk, Dr R. Michael Garavito, Dr Krister Green, Prof Constantino Iadecola, Prof Richard J. Kulmacz, Prof William E. M. Lands, Prof W. L. Smith, Dr John A. McCracken, Prof Stephen Smith, Prof John C. McGiff, Prof Daniele Piomelli, Prof Rodolpho Paoletti, Prof Carlo Patrono, Dr Malcolm Peet, Prof Brendan J. Whittle, Dr Norman L. Poyser, Prof Ingvar Bjarnason, Prof Clifford B. Saper, Prof Flavio Cocceani, Prof K. Schroer, Prof Jorge Capdevila, Prof A. Barry Kay, Prof Tak H. Lee, Dr Jonathan P. Arm, Dr Crispin Bennett, Dr Patrick Loll, Dr Marie Therese Droy-Lefaix, Dr John Wallace and Professor K. C. Nicolaou. Sadly, I have to report that during the gestation period of this book from inception to receipt of the manuscripts several wouldbe contributors, who were research scientists of international repute . . . teachers . . . industrialists . . . colleagues and friends to many who will read this book, have died. Some of them had completed their contribution to this book, but all their names appear below, as a mark of respect for their different, but very significant contributions to the current pool of knowledge in the field of eicosanoids:

IN MEMORIAM Docteur Jacques Maclouf INSERM, Hoˆpital Lariboisie`re, Paris, France Doctor Robin Hoult Division of Pharmacology and Therapeutics, GKT School of Biomedical Sciences, London, UK Professor Emeritus Alan Bennett, DSc Academic Department of Surgery, GKT School of Medicine, London, UK Professor Simonetta Nicosia Department of Pharmacological Sciences, University of Milan, Milan, Italy Professor of Medicine Emeritus James B. Lee, MD University at Buffalo, New York, USA Doctor David Horrobin Chairman, Laxdale Ltd, Kings Park House, Stirling, UK

Foreword Professor Bengt Samuelsson, Nobel Laureate Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden

This book edited by Dr Curtis-Prior is entitled The Eicosanoids, which term, introduced by one of the major contributors to the field, Dr E. J. Corey, includes all derivatives of 20-carbon-atom fatty acids. The emphasis in this book is of course on arachidonic acid and products formed by oxidation and further transformation. Structure and function have always played important roles in the eicosanoid field, as well as in many other areas. It was the knowledge of the structures of prostaglandins E1, E2 and E3 (Bergstro¨m et al 1963a; Samuelsson et al 1963a) that paved the way for the discovery of the precursor product relationship between essential fatty acids like arachidonic acid and prostaglandins (Bergstro¨m et al 1964b; van Dorp et al 1964). And this finding made it possible to discover the inhibition of the transformation of arachidonic acid into prostaglandins by aspirin and other NSAIDs in 1971 (Vane 1971). At that time cyclooxygenase had not been discovered. This term was introduced in 1974 (Hamberg et al 1974a) after the endoperoxide structures PGH2 and PGG2 had been discovered. Since cyclooxygenase and COX-1/2 have become household words, I am citing from the 1974 paper (Hamberg et al 1974a): ‘‘. . . It seems likely that PGG2 is the first stable compound formed from arachidonic acid by the ‘prostaglandin synthetase’. By the isolation of PGG2 it was demonstrated for the first time that introduction of the oxygen function at C-15 of the prostaglandin occurs by a dioxygenase reaction. We propose the name fatty acid cyclooxygenase for the enzyme that catalyzes the conversion of arachidonic acid into PGG2 by oxygenation at C-11 and C-15 . . .’’

The availability of the endoperoxides PGG2 and PGH2 was a prerequisite in the discovery of the transformation product thromboxane A2 with pro-aggregatory and vasoconstrictor effects (Hamberg et al 1975b) and subsequently prostacyclin with opposite effects (Moncada et al 1976). These findings extended the cyclooxygenase pathway products from three (PGE2, PGF2a and PGD2) to five. The molecular basis of the multiple biological effects of single prostaglandins has been elucidated by the discovery of subclasses of prostaglandin receptors (Narumiya 2001). The prostaglandins and their analogues are being used as therapeutic agents for induction of labour and termination of pregnancy and also more recently in the treatment of glaucoma. The identification of the receptors will probably stimulate the development of specific prostaglandin agonists and antagonists as therapeutic agents. The isolation and cloning of cyclooxygenase constitutes another important milestone in the eicosanoid field (DeWitt et al 1988; Merlie et al 1988). Although it had been found that the cyclooxygenase can be induced (Masferrer et al 1990), it was through the advent of molecular biology that it was discovered that the induced cycloxygenase activity is due to a different chemical entity, the isozyme COX-2 (Kujubu et al 1991; Xie et al

1991; O’Banion et al 1991). This finding momentarily gave the signal to the pharmaceutical industry to develop new and specific inhibitors of COX-2. Celebra from Searle and Pharmacia and Vioxx from Merck were the first drugs of this category and achieved multibillion dollar sales in their first year. Structure and function also played an important role in the identification of the elusive ‘‘slow-reacting substance of anaphylaxis’’ (SRS-A). In 1976 Borgeat, Hamberg and Samuelsson discovered the 5-lipoxygenase pathway for transformation of arachidonic acid (Borgeat et al 1976). Work on the mechanism of formation of a dihydroxy acid (LTB4) led to the identification of an unstable intermediate (LTA4). This finding played a crucial role in determining the structures of the cysteinyl leukotrienes, LTC4, LTD4 and LTE4 (Murphy et al 1979; Samuelsson et al 1987b). The potent actions of the leukotrienes in inflammation and allergy stimulated the pharmaceutical industry to develop new drugs. Singulair and Accolate from Merck and Zeneca (now Astra-Zeneca), respectively, are now in use in the treatment of bronchial asthma. I have touched on some of the key findings in the eicosanoid field and especially those related to structure and function. The area is still developing rapidly. This development occurs in many disciplines, such as molecular biology, physiology, pathophysiology, pharmacology and clinical applications. New compounds formed by the transformation of arachidonic acid by other lipoxygenases or a combination of lipoxygenases known as the lipoxins (Samuelsson et al 1987b; Serhan et al 1999) are still being discovered. This book edited by Curtis-Prior provides an important update in many different fields of eicosanoid research. It is indeed a very useful summary of the state of the art in a rapidly developing area of research.

REFERENCES Bergstro¨m S, Ryhage R, Samuelsson B and Sjo¨vall J (1963a) The structures of prostaglandin E1, F1a and F1b. J Biol Chem, 238, 3555– 3564. Bergstro¨m S, Danielsson H and Samuelsson B (1964b) The enzymatic formation of prostaglandin E2 from arachidonic acid. Biochim Biophys Acta, 90, 207–210. Borgeat P, Hamberg M and Samuelsson B (1976) Transformation of arachidonic acid and homo-g-linolenic acid by rabbit polymorphonuclear leukocytes. J Biol Chem, 251, 7816–7820. DeWitt DL and Smith WL (1988) Primary structure of prostglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence. Proc Natl Acad Sci USA, 85, 1412– 1416. Hamberg M, Svensson J, Wakabayashi T and Samuelsson B (1974a) Isolation and structure of two prostaglandin endoperoxides that cause platelet aggregation. Proc Natl Acad Sci USA 71, 345–349.

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Hamberg M, Svensson J and Samuelsson B (1975b) Thromboxanes. A new group of biologically active compounds derived from prostaglandin endoperoxides. Proc Natl Acad Sci USA, 72, 2994– 2998. Kujubu DA, Fletcher BS, Varnum BC et al (1991) TIS10, a phorbol ester tumor promoter-inducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/cyclooxygenase homologue. J Biol Chem, 266, 12866–12872. Masferrer JL, Zweifel BS, Seibert K and Needleman P (1990) Selective regulation of cellular cyclooxygenase by dexamethasone and endotoxin in mice. J Clin Invest, 86, 1375–1379. Merlie JP, Fagan D, Mudd J and Needleman P (1988) Isolation and characterization of the complementary DNA for sheep seminal vesicle prostaglandin endoperoxide synthase (cyclooxygenase). J Biol Chem, 263, 3550–3553. Moncada S, Gryglewski R, Bunting S and Vane R (1976) An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature, 263, 663– 665. Murphy RC, Hammarstro¨m S and Samuelsson B (1979) Leukotriene C: a slow reacting substance (SRS) from murine mastocytoma cells. Proc Natl Acad Sci USA, 76, 4275–4279.

Narumiya S (2001) Physiological and pathophysiological roles of prostanoids; lessons from receptor-knockout mice for clinical applications. In Advances in Prostaglandin and Leukotriene Research: Basic Science and New Clinical Applications. Dordrecht: Kluwer Academic, 115–120. O’Banion MK, Sadowski HB, Winn V and Young DA (1991) A serumand glucocorticoid-regulated 4-kilobase mRNA encodes a cyclooxygenase-related protein. J Biol Chem, 266, 23261–23267. Samuelsson B (1963a) Prostaglandins and related factors: the structure of prostaglandin E3. J Am Chem Soc, 85, 1878–1879. Samuelsson B, Dahle´n S-E, Lindgren JA˚ et al (1987b) Leukotrienes and lipoxins: structures, biosynthesis and biological effects. Science, 237, 1171–1176. Serhan CN, Takano T, Gronert K et al (1999) Lipoxin and aspirintriggered 15-epi-lipoxin cellular interactions anti-inflammatory lipid mediators. Clin Chem Lab Med, 37, 299–309. Van Dorp DA, Beerthuis RK, Nugteren DH and Vonkeman H (1964) The biosynthesis of prostaglandins. Biochim Biophys Acta, 90, 204–206. Vane JR (1971) Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature, 231, 232–235. Xie W, Chipman JG, Robertson DL et al (1991) Expression of a mitogenresponsive gene encoding prostaglandin synthase is regulated by mRNA splicing. Proc Natl Acad Sci USA, 88, 2692–2696.

Section One Biosynthesis and Metabolism

1 Perspectives on the Biosynthesis and Metabolism of Eicosanoids Robert C. Murphy, Rebecca C. Bowers, Jennifer Dickinson and Karin Zemski Berry National Jewish Medical and Research Center, Denver, CO, USA

INTRODUCTION The regulated oxygenation of arachidonic acid leads to a large family of metabolites which have been termed eicosanoids. During investigation of the biochemical events leading to the synthesis of this family of oxygenated lipid substances, a number of quite surprising biochemical features were revealed, some unique to the eicosanoid pathway. Other features illustrated the fundamental biochemical events taking place with polyunsaturated fatty acids within the living cell. The eicosanoids represent a class of molecules that have been termed ‘‘lipid mediators’’ because they are chemical messengers that carry information of cell activation from one cell to another. These cellular messenger molecules have a diverse set of important physiological and pathophysiological roles and coordinate events between cells so that proper tissue function can result. Furthermore, these molecules play a central role in mounting important host defence reactions to protect tissues from adverse conditions, including bacterial infections, that may affect cell viability. While we stand at the threshold of understanding a great deal about these molecules, there is still much that is yet to be learned about the biosynthesis and metabolism of these molecules that further challenges investigators in the field. The first step in the synthesis of eicosanoids, common to all the pathways including prostaglandin and leukotriene biosynthesis, is the release of free arachidonic acid from its storage site in membrane phospholipids. This seemingly simple event is itself a complex biochemical process. For several decades, we have known that arachidonic acid is conserved by the cell in a manner that sequesters this polyunsaturated fatty acid in glycerophospholipids as well as triglycerides (Irvine 1982). Since the cell very tightly regulates the quantity of arachidonic acid through activation of CoA synthase and CoA transferase, as well as phospholipid remodelling (Chilton and Murphy 1986; Walsh et al 1981), there is typically little substrate available for the initial enzyme that oxygenates arachidonic acid. This being the case, it is clear that a great deal of attention has been placed on those enzymes, in particular phospholipase A2, which release arachidonic acid from phospholipid stores (Dennis 2000). Arachidonic acid is highly concentrated at the sn-2 position of glycerophospholipids and the pioneering work of Leslie and co-workers (Alonso et al 1986; Gijon and Leslie 1999) demonstrated that a high molecular weight cytosolic phospholipase A2 (cPLA2) had a unique substrate specificity in releasing arachidonic acid from glycerophosphoethanolamine and glycerophosphocholine lipids. As described by Andreani et al (Chapter 2, this volume), the The Eicosanoids. Edited by Peter Curtis-Prior &2004 John Wiley & Sons, Ltd: ISBN 0 471 48984 0

details of cytosolic phospholipase A2, its phosphorylation, translocation and role in eicosanoid biosynthesis continue to emerge. However, other phospholipases are also present in the cell and, interestingly, they also participate in the regulation of arachidonic acid release, particularly release for eicosanoid biosynthesis (Tougui and Alaoui-El-Axher 2001). The interaction of these phospholipases, including the secreted phospholipases (Type V) and cPLA2 (Type IV), continues to be an area of active investigation and a clear picture of the role of these enzymes and auxiliary enzymes, such as transacylases (Surette and Chilton 1998), is only beginning to emerge. Nonetheless, these enzymes remain interesting therapeutic targets to control eicosanoid biosynthesis within target cells.

PGH SYNTHASE The oxygenation of arachidonic acid in the prostaglandin pathway (Figure 1.1) is mediated by the enzyme PGH synthase for which two genes exist, coding for similar yet significantly different proteins, PGH synthase-1 and PGH synthase-2, commonly referred to as the cyclooxygenases, COX-1 and COX-2 (Smith et al 2000). PGH synthase was the first enzyme studied in the metabolism of arachidonic acid and yet new information continues to emerge from studies of these proteins. Chapter 3 by Deby and Deby-Dupont (this volume) describes many of the mechanistic details of PGH synthase-1, its regulation and mechanism by which the cyclic endoperoxide PGH2 emerges. The regulation and function of PGH synthase-2 is a subject of Chapter 4 (this volume) by H. R. Herschman. Fundamental questions remain concerning these two enzymes from a more chemical point of view. The three-dimensional X-ray crystallographic structures of both PGH synthase-1 and PGH synthase-2 have now been determined and have revealed important insights into the chemical mechanism involved in the cyclooxygenase reaction, including the feature that this enzyme works as a dimer (Kiefer et al 2000; Malkowski et al 2000). These enzymes are membrane-bound and located on the endoplasmic reticulum. Arachidonic acid is drawn from the phospholipid membrane into the active site through a channel in the membrane-associated region of the PGH synthase dimer. The detailed mechanisms by which cyclooxygenase becomes activated through hydroperoxide tone are also emerging. One of the interesting suggestions is that the dimeric enzyme is a critical feature, coupling the peroxidase active site with the endoperoxide active site (Malkowski et al 2000). A detailed understanding of the chemical transformation of

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Figure 1.1 Biochemical pathway involved in the conversion of arachidonic acid into the chemically reactive endoperoxide intermediate prostaglandin H2, mediated by both PGH synthase-1 and PGH synthase-2. The subsequent conversion of PGH2 to the active prostaglandins (PGE2, TXA2 and prostacyclin) is mediated by auxiliary synthases that convert PGH2 into the biologically active prostanoid

arachidonic acid into the cyclic endoperoxide and the critical role played by the tyrosine radical intermediate in the active site have been important advances in this field in understanding the free radical events taking place during the cyclization reaction (Goodwin et al 2000; Shi et al 2000). Within the past several years, it has been discovered that arachidonic acid is not the only preferred substrate for PGH synthase-2. In fact, an ester and amide-linked arachidonic acid can be efficiently converted into prostaglandin-like molecules with the carboxylic acid group linked to ethanolamine (anandamide) or to 2-arachidonoyl glycerol (Yu et al 1997). The structures of the 2-arachidonoyl glycerol products are illustrated in Figure 1.2 and represent an unexpected family of molecules which could themselves play important roles in vivo. In part they have unique physical chemical properties and thus would be distributed quite differently from the normal prostaglandin molecules. For example, these carboxyl-linked prostanoids could be stored within membranes for release of the biochemically active prostaglandin following activation of a lipase or amidase, or they may merely represent an epiphenomenon in that they are accidental substrates for PGH synthase-2. Nevertheless, the ability of PGH synthase-2, rather than PGH synthase-1, to form these molecules is remarkable and further investigation into the role that these carboxylic acid-linked prostaglandins play within cellular biochemistry and pharmacology remains to be elucidated.

Another aspect of the endoperoxide biosynthetic pathway is illustrated by two themes common to eicosanoid biosynthesis. The first theme is that chemically reactive intermediates formed by radical reactions are the primary products during the oxygenation mechanism of arachidonic acid. For PGH synthase, this intermediate is the chemically reactive PGH2. This cyclic endoperoxide has a relatively short half-life of approximately 3 min at physiological pH (Hamberg and Samuelsson 1973; Maclouf et al 1980), yet it is transformed to a host of diverse structures, including prostaglandins D2, E2, F2a, thromboxane A2 and prostacyclin (PGI2). Each of these secondary products of PGH2 are biologically active prostanoids that have substantially less chemical reactivity (except for TXA2 and PGI2) and result from the action of secondary enzymes which process PGH2 into the corresponding prostanoid. This chemical reactivity is a feature that has escaped the attention of many investigators. Prostanoids are highly lipid-soluble and in some cases have very poor water solubility, particularly PGH2. Yet PGH2, which is made within close proximity of the lipophilic membrane of the endoplasmic reticulum, must find auxiliary enzymes that in many cases are in the cytosol. Perhaps most surprising is the fact that there is clear evidence that PGH2 can escape the cell in which it is synthesized, be transferred into another cell, and be metabolized into a unique prostanoid by a secondary cell. This process has been known for approximately two decades and was first recognized when

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Figure 1.2 Biochemical conversion of 2-arachidonoyl glycerol by PGH synthase-2 into glyceryl-prostaglandins

mixtures of platelets and endothelial cells were found to synthesize prostacyclin (a unique endothelial cell product), in spite of the fact that the stimulus leading to the release of arachidonic acid and formation of PGH2 took place within the platelet, a cell which only expresses thromboxane synthase and not prostacyclin synthase (Marcus et al 1980). Some of the current active areas of investigation include probing the mechanism by which these chemically reactive intermediates can traverse membranes within and between cells, yet remain intact. Related to this feature is a growing body of evidence that a family of unique proteins that act as transporters are present within cells and move lipophilic prostanoids from the extracellular milieu into a cell where they can either exert unique biological actions or can be the substrate for metabolic processing (Schuster 1998; Pucci et al 1999). We are only at the elementary level in understanding the facile uptake of these lipophilic molecules, their transport through membranes into various compartments within the cell, and transport into peroxisomes and mitochondria where metabolic processing can take place. A second unique theme central to prostaglandin biosynthesis is the suicide inactivation of PGH synthase (Song et al 2001) as well as some of the PGH2-processing enzymes, including prostacyclin synthase (Wade et al 1995) and thromboxane synthase (Jones and Fitzpatrick 1991). Suicide inactivation of PGH synthase was

observed and reported almost 30 years ago (Smith and Lands 1972). While the suicide inactivation of an enzyme by its substrate is certainly known in biochemistry, there are perhaps no other examples of a single biochemical pathway where so many enzymes involved in the production of the biologically active products are susceptible to suicide inactivation. The exact events and detailed chemical mechanisms of suicide inactivation are also not understood. Major questions remain as to whether or not this process is a way by which cells can regulate the full extent of prostanoid production, or whether it is merely a curious artifact of PGH synthase. Yet, it is clear that the suicide inactivation of PGH synthase can limit the extent of prostaglandin biosynthesis within cells. LEUKOTRIENE BIOSYNTHESIS The second major pathway of arachidonate metabolism in terms of production of a family of biologically active products was first described in 1979 as the leukotriene pathway (Figure 1.3). The enzyme 5-lipoxygenase (5-LO) is the first critical protein involved in the oxidation of arachidonic acid and formation of leukotrienes (Samuelsson et al 1979). Following the discovery of the leukotriene pathway and elucidation of the biological activities

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Figure 1.3 Leukotriene biosynthetic pathway involving the conversion of arachidonic acid into 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and LTA4 by 5-lipoxygenase. Subsequent enzymatic processing of the chemically reactive LTA4 into LTB4 and LTC4 occurs through LTA4 hydrolase and LTC4 synthase. The series of cysteinyl leukotrienes are derived from sequential amino acid removal from the tripeptide of LTC4

PERSPECTIVES of leukotriene B4 (Ford-Hutchinson et al 1980) and leukotriene C4 (Barnes et al 1984), detailed studies of the biochemical events taking place during the 5-LO reaction began to emerge. The most significant advances were obtained once 5-LO was sequenced, cloned and expressed so that reasonable quantities were available for detailed biochemical investigation (Rouzer et al 1986). One of the first important discoveries was that 5-LO carried out two different enzymatic steps, both involving the removal of a bisallylic hydrogen atom, initially leading to the formation of 5hydroperoxyeicosatetraenoic acid (5-HPETE) and secondly converting 5-HPETE into leukotriene A4 (LTA4), the conjugated triene epoxide (Shimizu et al 1984). The first step involved removal of a hydrogen atom from carbon atom 7 of arachidonic acid, with concomitant reduction of the non-haeme iron in 5-LO from FeIII to FeII in a radical reaction mechanism. An unexpected observation which resulted from the development of a 5-LO inhibitor, MK886 (Dixon et al 1990), was the discovery of a second membrane-bound protein that was required for biosynthesis of LTA4 in the intact cell, but not necessarily required for biosynthesis in broken cell preparations or with isolated 5-LO (Shimizu et al 1984). This protein, termed five lipoxygenase activating protein (FLAP), was similar in primary sequence to only one other protein, that being LTC4-synthase (Penrose et al 1997). Furthermore, FLAP was found to be localized in or near the nuclear envelope. This observation was consistent with studies that demonstrated translocation of 5-LO to the nucleus during activation, where it likely encountered FLAP (Rouzer et al 1986). In some cells, 5-LO is localized not in the cytosol but in the nucleus (Coffey et al 1992) and activation of such cells resulted in the translocation of the nuclear-localized 5-LO to the perinuclear membrane. An understanding of the importance of the nuclear localization of 5-LO as well as the factors which control or target 5-LO to the nuclear region are still areas of active investigation. Recent evidence has suggested that unique structural features in 5-LO may target 5-LO to this region of the cell (Kulkarni et al 2002; Jones et al 2002). Fundamental questions also remain as to the nature of FLAP and its precise biochemical role. The first stable product of 5-LO is 5-HPETE. Another family of biologically active eicosanoids (Figure 1.4) is directly derived from 5-HPETE and in some cells, e.g. the neutrophil, from the peroxidase reduction product 5-hydroxyeicosatetraenoic acid (5HETE; Skoog et al 1988). Powell and co-workers (1994) described the oxidation of 5-HETE to 5-oxo-eicosatetraenoic acid (5-oxoETE) with a substantial alteration in biological activity, most likely through the engagement of specific receptors that recognize 5-oxo-ETE (Hosoi et al in press). 5-oxo-ETE and some of the related molecules resulting from oxidation at C-15 (Powell et al 1994) or conjugation with glutathione at C-7 (Bowers et al 2000) are highly chemotactic for both eosinophils and neutrophils. Dr William Powell presents an overview of the biosynthesis and pharmacological properties of these oxo-eicosanoids, which represent a more recently discovered family of 5-LO products that have substantial biochemical relevance (Powell, Chapter 6, this volume). LTA4 hydrolase carries out the stereospecific hydrolysis of the conjugated triene epoxide, LTA4, to 5(S),12(R)-dihydroxy-6,14cis-8,10-trans-eicosatetraenoic acid, LTB4. A crystal structure of LTA4 hydrolase has now been obtained (Thunnissen et al 2001) and a more detailed understanding of the mechanism involved in directing water to C-12 of LTA4 is emerging (Rudberg et al 2002). Interestingly, LTA4 hydrolase has a fair degree of homology to zinc-containing metalloproteases and in fact can carry out this enzymatic activity (Medina et al 1991; Muskardin et al 1994). Furthermore, inhibitors of certain metalloproteases, such as bestatin, have been found to inhibit LTA4 hydrolase (Muskardin et al 1994). LTA4 hydrolase is also a suicide inactivated enzyme

7

(Orning et al 1992) and it is clear that LTA4 and LTA3 can become covalently bound to LTA4 hydrolase in the active site (Mueller et al 1995, 1996). In fact, using mass spectrometric techniques it was possible to isolate a peptide derived from intact LTA4 hydrolase to which LTA3 attached to tyrosine-383 in the sequence D371-K385 (Mancini et al 1998). The conjugation of LTA4 with glutathione results in the formation of the myotropic leukotriene, LTC4, which is the initial member of the cysteinyl leukotriene series (Figure 1.3). This enzymatic step is catalysed by a unique glutathione-S-transferase (GST), termed LTC4 synthase. This 18 kDa membrane-bound protein has sequence homology only to FLAP, as mentioned above, rather than to the ubiquitous GSTs found in the cytosol of most cells. Molecular recognition of the cysteinyl leukotrienes is mediated by at least two G-protein-linked membraneassociated receptors (Christie and Henderson 2002), termed cysLT1 and cysLT2, that have the highest affinity for the cysteinyl leukotriene formed by the action of g-glutamyl transpeptidase on LTC4 (Figure 1.3). This product, leukotriene D4, and the subsequent peptide hydrolysis product, leukotriene E4, complete the family of cysteinyl leukotrienes that are potent activators of the cysLT receptors. The transport of these unique peptidolipids has been studied by Keppler et al (1997), who have described involvement of the multi-drug resistance receptor in this important process of export of LTC4 from the synthetic cell. The biosynthesis of leukotrienes within tissues is surprisingly more complex than that revealed by the initial biochemical studies of events taking place within the cell or near the nucleus of the cell. Specifically, approximately 60–70% of the LTA4 made by the neutrophil (Sala et al 1996) is not processed within this cell, but is transferred to auxiliary cells in the process described as transcellular biosynthesis (Murphy et al 1989). Thus, when mixtures of platelets and neutrophils are prepared and neutrophils stimulated, the leukotriene produced in largest amount is LTC4 rather than LTB4 (Maclouf and Murphy 1988). Since the platelet has LTC4 synthase, but no 5-LO or LTA4 hydrolase, this clearly implicates a fundamental role of transcellular biosynthesis during cell–cell interaction. However, the exact mechanisms involved are less than clear. One of the most important steps in transcellular biosynthesis is the transfer of LTA4 from the nuclear site of biosynthesis through the cellular membranes and into a recipient cell, e.g. the platelet. LTA4 must traverse multiple membranes as well as the aqueous cytosol. Since LTA4 has a chemical half-life of less than 3 s at pH 7.4 (Fitzpatrick et al 1982), there is clearly a factor present within cells which protects LTA4 during its movement within the cell and during export from the cell. Even less is known about how LTA4 is taken up by a cell and transported intact to the site where enzymatic processing occurs. Clearly, cells are very efficient in this process, since it has been shown that eosinophils treated with exogenous LTA4 at 08C have intact LTA4 present within the eosinophil for over 1 h, so long as the temperature remains low (Lam et al 1989). Virtually nothing is known about the transport process. It is likely that binding proteins are involved but what role such proteins play in total eicosanoid, and specifically leukotriene, biosynthesis is unclear. Another interesting aspect of LTA4 biochemistry has emerged following the discovery that LTA4 can covalently bind to nucleosides, oligonucleotides (Reiber and Murphy 2000) and, specifically, DNA and RNA (Hankin and Murphy unpublished). Since LTA4 biosynthesis takes place close to the nucleus where high concentrations of DNA and RNA exist, covalent binding of LTA4 to nucleic acids raises important questions as to a potential role that such covalent modification of nucleic acids could play within cells. The concept of covalent binding of LTA4 is beginning to emerge and much remains to be done.

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Figure 1.4 Biosynthesis of 5-oxo-eicosatetraenoic acid (5-oxo-ETE), either directly, as is observed in macrophages, or through the intermediate formation of 5-hydroxyeicosatetraenoic acid (5-HETE) and subsequent oxidation catalysed by the NADP+-dependent dehydrogenase present in neutrophils. The conjugation of 5-oxo-ETE by LTC4 synthase and glutathione leads to the formation of 5-oxo-7-glutathionyl-eicosatrienoic acid (FOG7)

12-/15-LIPOXYGENASE The metabolic oxidation of arachidonic acid by 12-lipoxygenase and 15-lipoxygenase (Figure 1.5) is known to take place within several cell types (Yamamoto et al, Chapter 5, this volume). Within the platelet the major eicosanoid formed by platelet 12lipoxygenase is 12-HPETE following activation of phospholipase A2 (Yamamoto et al 1997). The exact biochemical role of 12lipoxygenase products, however, remains to be established. A similar story exists for 15-lipoxygenase (Kuhn and Borngraber 1999). Important insight into the mechanism of lipoxygenases and a better understanding of the catalytic domain that contains the histidine-coordinated iron molecules was obtained through the crystal structure of 15-lipoxygenase (Gillmor et al 1997). 15Lipoxygenase is the only lipoxygenase that can actually metabolize intact phospholipids to hydroperoxides, and a role has been

suggested for 15-lipoxygenase in cardiovascular disease, particularly atherosclerosis (Funk and Cyrus 2001). CYTOCHROME P-450 AND EPOXYGENASE PATHWAY A third major arm of arachidonate oxygenation involves a series of specific cytochrome P-450 isozymes, which include CYP1A, CYP2B, CYP2C, CYP2D, CYP2G, CYP2J, CYP2N and CYP4A (Zeldin 2001). These enzymes catalyse the formation of epoxyeicosatrienoic acid (EET) regioisomers (Figure 1.6). Other pathways have also been described, including o-oxidation of arachidonic acid, which result in formation of 20-hydroxyand 20-carboxyarachidonic acid, as well as lipoxygenase-like products, with the opposite stereochemistry formed by the specific

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Figure 1.5 Metabolism of arachidonic acid catalysed by the 12-lipoxygenase and 15-lipoxygenase into the stereo- and regiospecific monohydroperoxyeicosatetraenoic acids

lipoxygenase (Bylund et al 1998). The pharmacological properties of these cytochrome P-450 metabolites of arachidonic acid have been widely studied, but perhaps the clearest evidence for a role of these compounds in vivo involves the effects of EETs and 20HETE in the kidney (Roman et al 2000). It is likely that a unique role for this pathway of arachidonate metabolism may include modulation of ion channels, mediating sodium potassium ATPase and inhibiting the effects of arginine vasopressin in the kidney (Zeldin 2001). EICOSANOID METABOLISM The role that eicosanoids, specifically prostaglandins and leukotrienes, play within tissues is critically linked to the effective extracellular concentration of these molecules as they present themselves to specific G-protein-coupled receptors. The extracellular concentration is a combination of not only the biosynthesis and biosynthetic mechanisms leading to the eicosanoid product, but also the mechanisms involved in metabolic transformation of these lipid mediators into inactive products. Therefore, an understanding of the catabolic events that occur within specific cells and tissues is an important facet of the entire picture of eicosanoid lipid mediators. Several major pathways exist for processing these endogenous polyunsaturated and

oxygenated fatty acid compounds, with ultimate conversion of the constitutive elements to acetate and water. The metabolism of eicosanoids involves events taking place within the cytosol, the mitochondrion, peroxisome and endoplasmic reticulum (cytochrome P-450). Finally, for most eicosanoids unique pathways of metabolism exist which are specific to each covalent structure. o-OXIDATION A major pathway of prostaglandin metabolism involves o-oxidation, in which a cytochrome P-450 isozyme specifically converts the methyl-terminus of the prostanoid into a primary carbinol (Figure 1.7), a much more polar moiety (Okita and Okita 1996). A specific example is the metabolism of PGE2 in the hepatocyte, where the major metabolites are 20-hydroxy- and 20-carboxy-PGE2. In addition, oxidation of the penultimate carbon atom also occurs, with formation of 19-hydroxy-PGE2, one of the important metabolic products of PGE2 in the isolated hepatocyte (Hankin et al 1997). The metabolism of LTB4 (Figure 1.8), as well as LTC4, is mediated by specific cytochrome P-450s of the CYP4F series. There have now been 10 different enzymes identified in various tissues and animal species that can carry out the o-oxidation of leukotrienes (Kikuta et al 1999). For example, LTB4 is converted by human CYP4F2 (neutrophil) into 20-hydroxy-LTB4 (Kikuta et al 1998;

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Figure 1.6 Summary of the biosynthetic products obtained following the action of various cytochrome P-450 isozymes, using arachidonic acid as substrate with formation of four different regioisomers of epoxyeicosatrienoic acid (EET), the lipoxygenase-like reaction exemplified by formation of 12(R)-HETE and o-oxidation exemplified by 20-HETE

Shak and Goldstein 1985). In the neutrophil itself, it appears that this cytochrome P-450 further oxidizes 20-hydroxy-LTB4 into 20oxo-LTB4 and then an aldehyde dehydrogenase oxidizes this further into 20-carboxy-LTB4 (Sutyak et al 1989). However, in the hepatocyte, 20-hydroxy-LTB4 is a substrate for alcohol dehydrogenase to form 20-oxo-LTB4, which is a substrate for aldehyde dehydrogenase to form 20-carboxy-LTB4 (Baumert et al 1989). b-OXIDATION Recovery of the carbon atoms of endogenous lipids as well as the generation of ATP result during the process of b-oxidation (Diczfalusy 1994). b-Oxidation of lipids takes place not only in mitochondria, but also in the peroxisome. The peroxisomal boxidation pathway is particularly important in eicosanoid metabolism. The process of b-oxidation for a prostaglandin involves initial formation of the CoA ester of the carboxylic acid followed by removal of two and four carbon atoms, leading to the dinor and tetranor prostaglandin metabolites, as shown in Figure 1.7 for PGE2. Typically, chain-shortened metabolites are some of the more abundant metabolites of prostaglandins in urine. However, it should be noted that much of the actual quantity of prostaglandin synthesized within the intact organism is never

excreted as a urinary metabolite such as the tetranor-PGE1 species. Detailed studies of the excretion rate of TXA2 in human subjects have revealed that only 1–2% of the entire biosynthetic pool of TXA2 can be found in any specific excreted product (Patrono et al 1986). Leukotrienes are metabolized by the peroxisomal b-oxidation pathway and LTB4 (Figure 1.8) also by the mitochondrial b-oxidation pathway (Jedlitschky et al 1991). However, the b-oxidation pathway most often occurs after the initial oxidation of the o-terminus into 20-carboxy-LTB4 (Figure 1.8) or 20-carboxy-LTE4. b-Oxidation then proceeds from the o-terminus, leading to the dinor and tetranor metabolites. Under some circumstances, b-oxidation from the carboxyl-terminus has been observed (Jedlitschky et al 1991), but the evidence to date suggests that most b-oxidation is subsequent to o-oxidation of the leukotriene. CONJUGATION Only recently has evidence emerged that conjugation of prostaglandins and leukotrienes by either glucuronidation or sulphation can result in major metabolic products within certain cells. In large part, this was a consequence of the inability to analyse such molecules as intact species; however, the emergence of powerful

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Figure 1.7 Metabolism of prostaglandins by o-oxidation and b-oxidation, as exemplified by formation of specific metabolites of PGE2

techniques, specifically electrospray combined liquid chromatography–mass spectrometry (LC–MS and LC–MS–MS) have enabled direct analysis of these molecules. Conjugation is clearly one of the major metabolic pathways which leads to a large number of excreted products. For example, in Figure 1.9, some of the potential metabolites of LTB4 by the glucuronidation pathway are indicated. Thus, it is not surprising that a complex mixture of glucuronides has been observed when techniques that can specifically detect these compounds are employed (Wheelan et al 1999). The extent of conjugation by the sulphate pathway or the glucuronidation pathway remains an important unexplored area of eicosanoid biochemistry. Another important conjugation reaction of some eicosanoids is covalent binding of the tripeptide glutathione (GSH, g-glu–cys– gly) with various electrophilic intermediates formed by oxidation

of arachidonic acid. As discussed above, LTA4 is conjugated with GSH to yield LTC4, a reaction catalysed by LTC4 synthase. This same enzyme has also been found to catalyse formation of another biologically active GSH adduct, FOG7 from 5-oxo-ETE (Hevko and Murphy 2002). The epoxyeicosatrienoic acids are substrates for the cytosolic glutathione-S-transferases and form glutathione adducts as common metabolites (Spearman et al 1985). For all glutathione conjugates, sequential removal of the gglutamic acid and glycine amino acids by g-glutamyl-transpeptidase and dipeptidases is a common metabolic pathway for glutathione adducts to form cysteinyl adducts (Figure 1.3). The cysteinyl adduct is often acetylated to an N-acetyl cysteinyl adduct in what is termed the mercapturic acid pathway and exemplified by formation of N-acetyl-LTE4 (Stene and Murphy 1988; Huber et al 1990).

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Figure 1.8 Metabolism of leukotrienes as exemplified by o-oxidation and subsequent b-oxidation from the o-terminus of LTB4. The conversion of 20hydroxy-LTB4 to 20-carboxy-LTB4 involves a two-step enzymatic oxidation catalysed by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldDH). These metabolites were observed in isolated hepatocytes (Shirley and Murphy 1990. Reproduced with permission of The American Society for Biochemistry & Molecular Biology)

SPECIFIC PATHWAYS Prostaglandins, leukotrienes, and the cytochrome P-450-derived eicosanoids have specific enzymes that can chemically alter the structure of the primary eicosanoid. One of the interesting metabolic enzymes for the prostaglandins is 15-hydroxyprostaglandin dehydrogenase, which is highly expressed in the lung, particularly in the pregnant woman (Bergholte et al 1987). This enzyme converts the 15-hydroxy group into the 15-oxo group, as well as terminating the activity of the prostaglandin, since these oxidized metabolites are typically not recognized by the prostanoid receptor (Figure 1.10). Once the 15-oxo-moiety is present, the a,b-unsaturated keto moiety is recognized by prostaglandin 13,14-reductase, which reduces the 13,14 double bond. This 13,14trans double bond with the allylic hydroxyl substituent is the same structural unit recognized by two enzymes that metabolize leukotrienes (insert, Figure 1.10). The enzyme

12-hydroxyeicosanoid dehydrogenase metabolizes LTB4 into the 12-oxo form of LTB4, which can then be a substrate for the 10,11reductase, leading to a family of 10,11-dihydro-leukotrienes which have substantially diminished biological activity. The 12-hydroxyeicosanoid dehydrogenase has recently been cloned and expressed (Yokomizo et al 1996). The 6-trans-LTB4 non-enzymatic hydrolysis products of LTA4 are also metabolized by reductase in a similar manner, first forming the initial 5-oxo product followed by the dehydrogenase reaction leading to the 6,7-dihydro-5-oxo product (Powell and Gravelle 1988; Wheelan and Murphy 1995). The epoxyeicosatrienoic acids are substrates for epoxide hydrolases. These cytosolic enzymes add water to the epoxide group, leading to vicinal dihydroxyeicosatrienoic acids. Various epoxide hydrolases, both in cytosolic and microsomal locations, can catalyse this hydration reaction, but considerable regioselectivity is observed, e.g. with (14R,15S)-EET (Zeldin et al 1993). Some EETs, specifically 8,9-EET and 5,6-EET, are substrates for

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Figure 1.9 Possible glucuronide conjugates of LTB4 at various positions, including the acyl glucuronide which undergoes ester migration to other hydroxyl groups on the glucuronic acid moiety to form several isomers

PGH synthase (Carrol et al 1993) and can be converted to unique prostanoids, including 5-hydroxy-PGI1 and 5,6-epoxy-PGE1 (Oliw 1985). CONCLUSION The biosynthesis and metabolism of eicosanoids is complex, but nonetheless of considerable interest. Numerous challenges remain in developing a full understanding of the role of each of the biosynthetic steps involved in prostaglandin, leukotriene and other eicosanoid production. Much remains to be understood in terms of the different biological roles of PGH synthase-1 and PGH synthase-2. The view that the PGH synthase-1 is the normally expressed ‘‘housekeeping’’ enzyme and that PGH synthase-2 is an enzyme expressed during cellular activation, such as during inflammation, is now known to be overly simplistic and probably does not reveal the true roles for these two enzymes. The mechanism of 5-LO is still only partially understood,

including the events occurring within the active site and the overall regulation of enzymatic activity. The role of FLAP, as well as the nuclear site of LTA4 biosynthesis, remain as enigmas. Finally, the trafficking of these highly lipophilic mediators within a cell or even between cells is only now becoming a serious target of investigation. However, these events are clearly critical to the fundamental process by which eicosanoids act as lipid mediators and a full understanding of the detailed events taking place in the biosynthesis and release of these compounds will very likely reveal interesting new insights into the role of these molecules in health and disease.

ACKNOWLEDGEMENTS This work was supported, in part, by grants from the National Institutes of Health (HL25785 and HL34303).

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Figure 1.10 Metabolism of PGE2 by 15-hydroxyprostaglandin dehydrogenase, leading to formation of 15-oxo-PGE2 and subsequent reduction of the 13,14 double bond. The inset shows the structural unit common to other eicosanoids which undergoes oxidation of an allylic hydroxyl followed by reduction of the adjacent double bond

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Medina JF, Wetterholm A, Radmark O et al (1991) Leukotriene A4 hydrolase: determination of the three zinc-binding ligands by sitedirected mutagenesis and zinc analysis. Proc Natl Acad Sci USA, 88, 7620–7624. Mueller MJ, Blomster M, Oppermann UC et al (1996) Leukotriene A4 hydrolase: protection from mechanism-based inactivation by mutation of tyrosine-378. Proc Natl Acad Sci USA, 93, 5931–5935. Mueller MJ, Wetterholm A, Blomster M et al (1995) Leukotriene A4 hydrolase: mapping of a henicosapeptide involved in mechanism-based inactivation. Proc Natl Acad Sci USA, 92, 8383–8387. Murphy RC, Fitzpatrick FA and Maclouf J (1989) Transcellular biosynthesis of eicosanoids and inflammation. In The Handbook of Inflammation: Mediators of the Inflammatory Process, Henson PM, Murphy RC (eds). Elsevier Science, Amsterdam, 147–154. Muskardin DT, Voelkel NF and Fitzpatrick FA (1994) Modulation of pulmonary leukotriene formation and perfusion pressure by bestatin, an inhibitor of leukotriene A4 hydrolase. Biochem Pharmacol, 48, 131– 137. Okita RT and Okita JR (1996) Prostaglandin-metabolizing enzymes during pregnancy: characterization of NAD(+)-dependent prostaglandin dehydrogenase, carbonyl reductase and cytochrome P450-dependent prostaglandin omega-hydroxylase. Crit Rev Biochem Mol Biol, 31, 101–126. Oliw EH (1985) On the metabolism of epoxyeicosatrienoic acids by ram seminal vesicles: isolation of 5(6)epoxy-prostaglandin F1 a. Biochem Biophys Res Commun, 126, 1090–1096. Orning L, Gierse J, Duffin K et al (1992) Mechanism-based inactivation of leukotriene A4 hydrolase/aminopeptidase by leukotriene A4. Mass spectrometric and kinetic characterization. J Biol Chem, 267, 22733– 22739. Patrono C, Ciabattoni G, Pugliese, F et al (1986) Estimated rate of thromboxane secretion into the circulation of normal humans. J Clin Invest, 77, 590–594. Penrose JF, Baldasaro MH, Webster M et al (1997) Molecular cloning of the gene for mouse leukotriene C4 synthase. Eur J Biochem, 248, 807– 813. Powell WS and Gravelle F (1988) Metabolism of 6-trans isomers of leukotriene B4 to dihydro products by human polymorphonuclear leukocytes. J Biol Chem, 263, 2170–2177. Powell WS, Gravelle F and Gravel S (1994) Phorbol myristate acetate stimulates the formation of 5-oxo-6,8,11,14-eicosatetraenoic acid by human neutrophils by activating NADPH oxidase. J Biol Chem, 269, 25373–25380. Pucci ML, Bao Y, Chan B et al (1999) Cloning of mouse prostaglandin transporter PGT cDNA: species-specific substrate affinities. Am J Physiol, 277, R734–R741. Reiber DC and Murphy RC (2000) Covalent binding of LTA4 to nucleosides and nucleotides. Arch Biochem Biophys, 379, 119– 126. Roman RJ, Maier KG, Sun CW et al (2000) Renal and cardiovascular actions of 20-hydroxyeicosatetraenoic acid and epoxyeicosatrienoic acids. Clin Exp Pharmacol Physiol, 27, 855–865. Rouzer CA, Matsumoto T and Samuelsson B (1986) Single protein from human leukocytes possesses 5-lipoxygenase and leukotriene A4 synthase activities. Proc Natl Acad Sci USA, 83, 857–861. Rudberg PC, Tholander F, Thunnissen MM et al (2002) Leukotriene A4 hydrolase: selective abrogation of leukotriene B4 formation by mutation of aspartic acid 375. Proc Natl Acad Sci USA, 99, 4215–4220. Sala A, Bolla M, Zarini S et al (1996) Release of leukotriene A4 versus leukotriene B4 from human polymorphonuclear leukocytes. J Biol Chem, 271, 17944–17948. Samuelsson B, Borgeat P, Hammarstrom S and Murphy RC (1979) Introduction of a nomenclature: leukotrienes. Prostaglandins, 17, 785– 787. Schuster VL (1998) Molecular mechanisms of prostaglandin transport. Annu Rev Physiol, 60, 221–242. Shak S and Goldstein IM (1985) Leukotriene B4 omega-hydroxylase in human polymorphonuclear leukocytes. Partial purification and identification as a cytochrome P-450. J Clin Invest, 76, 1218–1228. Shi WF, Hoganson CWF, Espe MF et al (2000) Electron paramagnetic resonance and electron nuclear double resonance spectroscopic identification and characterization of the tyrosyl radicals in prostaglandin H synthase 1. Biochemistry, 39, 4112–4121.

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Shimizu T, Radmark O and Samuelsson B (1984) Enzyme with dual lipoxygenase activities catalyzes leukotriene A4 synthesis from arachidonic acid. Proc Natl Acad Sci USA, 81, 689–693. Shirley MA and Murphy RC (1990) Metabolism of leukotriene B4 in isolated rat hepatocytes: Involvement of 2,4-dienoyl CoA reductase in leukotriene B4 metabolism. J Biol Chem, 265, 16288–16295. Skoog MT, Nichols JS, Harrison BL and Wiseman JS (1988) Specificity of an HPETE peroxidase from rat PMN. Prostaglandins, 36, 373–384. Smith WL, DeWitt DL and Garavito RM (2000) Cyclooxygenases: structural, cellular and molecular biology. Annu Rev Biochem, 69, 145– 182. Smith WL and Lands WE (1972) Oxygenation of polyunsaturated fatty acids during prostaglandin biosynthesis by sheep vesicular gland. Biochemistry, 11, 3276–3285. Spearman ME, Prough RA, Estabrook RW et al (1985) Novel glutathione conjugates formed from epoxyeicosatrienoic acids (EETs). Arch Biochem Biophys, 242, 225–230. Song I, Ball TM and Smith WL (2001) Different suicide inactivation processes for the peroxidase and cyclooxygenase activities of prostaglandin endoperoxide H synthase-1. Biochem Biophys Res Commun, 289, 869–875. Stene DO and Murphy RC (1988) Metabolism of leukotriene E4 in isolated rat hepatocytes. Identification of b-oxidation products of sulfidopeptide leukotrienes. J Biol Chem, 263, 2773–2778. Surette ME and Chilton FH (1998) The distribution and metabolism of arachidonate-containing phospholipids in cellular nuclei. Biochem J, 330, 915–921. Sutyak J, Austen KF and Soberman RJ (1989) Identification of an aldehyde dehydrogenase in the microsomes of human polymorphonuclear leukocytes that metabolizes 20-aldehyde leukotriene B4. J Biol Chem, 264, 14818–14823. Thunnissen MM, Nordlund P and Haeggstrom JZ (2001) Crystal structure of human leukotriene A4 hydrolase, a bifunctional enzyme in inflammation. Nature Struct Biol, 8, 131–135.

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2 Control of Eicosanoid Production by Cellular and Secreted Phospholipase A2 Marise Andreani1, Jean-Luc Olivier2 and Gilbert Be´re´ziat1 1University

Pierre and Marie Curie, Paris; and 2Faculty of Medicine, Nancy, France

Phospholipases A2 (PLA2) are a structurally diverse group of enzymes that hydrolyse the fatty acid which esterifies the sn-2 position of glycerophospholipids (Figure 2.1), leading to the formation of a free fatty acid and a lysoderivative. The presence of various PLA2 enzymes in mammalian cells provides multiple differentially regulated pathways for the important process of fatty acid turnover and lipid mediator synthesis. VARIOUS CLASSES OF PHOSPHOLIPASES A2 The mammalian PLA2 enzymes consist of three unrelated families of enzymes: secretory PLA2s (sPLA2s), cellular PLA2s and PLA2s which hydrolyse the platelet-activating factor (PAF) or oxidized phospholipids. Cellular Phospholipases A2 Cellular PLA2s comprise at least five categories of enzymes which differ considerably from each other. The best characterized are cytosolic PLA2s (cPLA2s) (Figure 2.2). Three different enzymes have been cloned to date: cPLA2a cPLA2b and cPLA2g. cPLA2a has a molecular weight of 85 kDa (749 amino acids), is widely distributed and its membrane translocation is achieved by a calcium-binding domain (CalB) (Leslie 1997). This enzyme possesses multiple enzymatic activities exhibiting, in addition to phospholipase A2 activity, intense lysophospholipase activity and weak transacylase activity. It can also hydrolyse fatty esters of 7hydroxycoumarin. The lysophospholipase activity provides an efficient mechanism to prevent an untimely increase of deleterious cytotoxic lysophospholipids. Mutagenesis studies have demonstrated two important amino acid residues for the catalytic process. Serine 228, which is located in a region of sequence homology with the phospholipase B from Penicillum notatum that aligns the lipase consensus sequence (Gly–Leu–Ser–Gly–Gly), is involved in the esterase process in the same way as the serine of the catalytic site of trypsin. This suggests that serine 228 [S228] covalently reacts with the substrate to form an acyl-enzyme intermediate. In addition, cysteine 331 [C331] is the residue that is targeted by sulphydryl-modifying reagents leading to a loss of PLA2 activity, which suggests that it is located near the active site where its integrity is required for correct hydrolysis. However, when [S228] is replaced by an alanine [S228A], low phospholipase activity persists, suggesting that [C331] can replace serine as the active nucleophil The Eicosanoids. Edited by Peter Curtis-Prior. &2004 John Wiley & Sons, Ltd: ISBN 0 471 48984 0

site, albeit with reduced catalytic activity. Arginine 200 [R200] and aspartic acid 549 [D549] are also essential for cPLA2 activity, but the network of electron/proton charges which allows [S228] and/or [C331] activation is not understood. Several serine residues are phosphorylated in cPLA2: 437, 454, 505 and 727. While most studies have emphasized the major role of serine 505 [S505] phosphorylation for enzyme activation, other studies also suggest the participation of serine 727 [S727]. At the present time, cPLA2a is the only characterized cellular phospholipase clearly involved in agonist-induced release of lipid mediators. cPLA2b and cPLA2g have recently been cloned (Underwood et al 1998) but only cPLA2g has been characterized. This enzyme has a molecular weight of 60 kDa (541 amino acids) and is calciumindependent, since it lacks the N-terminal CalB domain. cPLA2g is closely related to cPLA2a and contains the potential critical amino acids for the active site with the exception of cysteine [C331], but lacks the key elements that regulate cPLA2a activity (serine phosphorylation sites and calcium ion-induced membrane translocation). Its membrane anchoring is obtained by a prenylation motif at the C-terminus and a myristoylation site at the Nterminus (Figure 2.2). This expression is restricted to heart and skeletal muscles and it is suggested that this enzyme participates in ischaemia-induced injury of heart muscle. Another calcium-independent PLA2 (iPLA2) has been cloned in CHO cells and macrophages (Balboa et al 1997; Tang et al 1997) (Figure 2.3). The CHO enzyme is an 85 kDa protein present in the form of a multimeric complex of 270–350 kDa. The full-length cDNA encodes a 752 amino acid cytoplasmic protein with one lipase motif (GXS465XG) and eight ankyrin repeats, which are required for enzymatic activity. It lacks extensive homologies with other phospholipases and was named iPLA2. The macrophage enzyme has marked homology with CHO iPLA2. These enzymes hydrolyse choline phospholipids at the same rate as ethanolamine phospholipids, but at a higher rate than phospholipids with negatively charged head groups (PI and PA). They exhibit a rather broad specificity towards phospholipid substrates and an additional intrinsic lysophospholipase activity. No known consensus sequences for post-translational modification, such as phosphorylation sites, are present. This indicates that this enzyme is most probably involved in the housekeeping action of remodelling phospholipids. Phospholipases A2 specific for plasmalogens have been described. One has been isolated from bovine brain. It is localized in cytosol and does not require Ca2+ for its activity. It has a molecular weight of 39 kDa and is strongly inhibited by

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Figure 2.1 Various sites of phospholipid cleavage by phospholipases

Figure 2.2 Protein domains of the two cytosolic phospholipases A2 indicating the important amino acid residues involved in the catalytic process (R, arginine; D, aspartic acid; C, cysteine; S, serine), the calcium binding domain (CalB), which is involved in calcium-mediated membrane translocation of the enzyme, and the sites for prenylation and myristoylation that might be involved in enzyme activation. Serine505 is the main residue in which phosphorylation by Map-kinase induces enzyme activation

Figure 2.3 Calcium-insensitive cellular phospholipases A2 showing the serine of the active site and the ankyrin domain

PLA2 CONTROL OF EICOSANOID PRODUCTION glycosaminoglycans, gangliosides and sialoglycoproteins. The interactions between plasmalogen-selective PLA2 and glycoconjugates may be involved in the regulation of enzymatic activity. Plasmalogen rather than monocyl phospholipids is also the preferred substrate for the cardiac PLA2 isoform activated during ischaemia. The diacyl metabolite, lysophosphatidylcholine, is arrhythmogenic, but the effects of the plasmalogen metabolite, lysoplasmenylcholine (LPLC), are essentially unknown (Caldwell and Baumgarten 1998).

Secreted Phospholipases A2 The mammalian sPLA2s constitute a homogeneous family of enzymes closely related to snake venom sPLA2s (Figure 2.4). They share an ancestral precursor and have retained several very stable characteristics: a low molecular weight (13–16 kDa); several disulphide bridges (4–8); the need for millimolar concentrations of calcium ions and a strongly conserved active site. This site comprises the presence of a nearly invariable calcium binding loop, whose role is to orientate the ester bond of the phospholipid substrate at the active site, and a network of electronic/protonic charges (Figure 2.5) that activates the histidine residue, which is always present at the active centre. This network involves one aspartic acid and two tyrosine residues which have been highly conserved through evolution.

Figure 2.4 Phylogeny of the secreted phospholipases A2

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The pancreatic enzymes belong to type IB sPLA2 and contain seven disulphide bridges and the characteristic ‘‘elapidae loop’’. Enzymes secreted during the inflammatory processes belong mostly to type IIA and type V sPLA2. Type IIA sPLA2 enzymes also possess seven disulphide bridges, but one is in a different position than in type I sPLA2. It lacks the ‘‘elapidae loop’’ and presents a characteristic extended C-terminus. It is the most important of the inflammatory enzymes (Kramer et al 1989; Seilhamer et al 1989). Type V sPLA2 possesses the six disulphide bridges common to type IB sPLA2 and type IIA sPLA2 but it lacks the ‘‘elapidae loop’’ and the C-terminal extension. This enzyme is mainly expressed and secreted in the heart and in macrophages in response to inflammatory cytokines (Tischfield 1997). Type X sPLA2 possesses eight disulphide bridges and differs from other sPLA2s by the presence of a propeptide and more acidic properties (Cupillard et al 1997).

Platelet-activating Factor Acetylhydrolases Platelet-activating factor acetylhydrolases are structurally diverse isoenzymes that catalyse the hydrolysis of the acyl group at the second position of glycerol in unusual phospholipids (Stafforini et al 1997). They have a marked specificity for phospholipids with a short acyl chain at the sn-2 position. Their main substrate is platelet-activating factor (PAF) and they are consequently

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Figure 2.5 Catalytic site of secreted phospholipase A2 showing the calcium ion which coordinates the carbonyl of the fatty acid esterifying the sn-1 position of the substrate, the phosphate group of the substrate, and two amino acid residues of the enzyme. Therefore, the calcium ion orientates the substrate in order to present the sn-2 ester bond to the reactive histidine residue, which is stimulated by an ionic network involving two tyrosines and one aspartic acid

considered to be signal terminators, but they are also able to hydrolyse phospholipids with sn-2 acyl chains up to nine methylene groups, especially when they carry a carbonyl group at the o-end of the acyl chain. These enzymes were first studied as secreted proteins, found in mammalian plasma. Further activities were identified in the cytosolic fraction of various mammalian tissues and in human blood cells. The plasma (pPAH) and intracellular (iPAH) forms of PAF acetylhydrolases are encoded by individual genes. The first 17 codons of the cDNA of the secreted isoform pPAH encode for a hydrophobic peptide that targets the protein to the secretory pathway; this sequence is followed by a propeptide sequence of 23 residues which is not present in the enzyme purified from plasma with a molecular weight of 44 kDa. The active site contains a catalytic triad composed of serine, histine and aspartic acid; the serine residue is a component of a small region of homology domain with other lipases: glycine–X–serine273–X– glycine (GXSXG). Two intracellular isoforms have been characterized. They possess the minimal lipase motif GXSXG (or valine). The first isoform, iPAH-I, shares a high degree of identity of the amino acid sequence (41%) with the plasma form. It is present mainly in liver and kidneys. The second intracellular isoform, iPAH-II, is composed of three subunits with molecular weights of 45, 30 and 29 kDa (a, b and g). Only the a and b subunits possess lipolytic activity; the g subunit is considered to be a regulatory component. This isoform is totally specific for PAF hydrolysis and its function is to precisely modulate cellular PAF levels.

PHOSPHOLIPASES A2 AND CELL PHYSIOLOGY Phospholipases A2 play a role in a very wide range of physiological functions. PLA2 enzymes participate in the digestion of dietary lipids and in phospholipid acyl turnover. They are involved in several pathophysiological situations, such as bacterial infection, sepsis and other inflammatory disorders. They are also

involved in major cellular functions, such as cell signalling, cell growth, apoptosis, endocytosis/exocytosis, cell migration and adhesion and the regulation of gene expression. These enzymes are involved in three major types of functions at the molecular level: (a) they participate in the metabolic fate of phospholipids; (b) they have a housekeeping role for lipoprotein and membrane repair; and (c) they are key enzymes for the control of bioactive mediator synthesis.

Phospholipases A2 and the Metabolic Fate of Phospholipids (Figure 2.6) The neosynthesis of all phospholipids shares a common pathway from glycerol-3-phosphate and acyl-CoA to phosphatidic acid (PA). Thereafter, two different pathways lead to aminophospholipids [phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS)] and other phopholipids [phosphatidylinositol (PI), phosphatidylinositoldiphosphate (PIP2), phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG or cardiolipid)] (Figure 2.6). This explains why the fatty acid profile of neosynthesized phospholipids is broadly the same and reflects the repertoire of cellular acyl-CoAs, which is itself greatly influenced by dietary conditions. The only specificity achieved by de novo synthesis is the presence of a saturated or monounsaturated chain in the sn-1 position of the glycerol backbone and the presence of a mono-unsaturated and poly-unsaturated chain in the sn-2 position. When considering the actual composition of cellular phospholipids, a vast number of studies have emphasized the specific fatty acid profile of each class of phospholipids. Furthermore, most mammals exhibit subcellular pools of phospholipids which remained rich in poly-unsaturated fatty acids when animals were submitted to drastic nutritional conditions of essential poly-unsaturated fatty acid deficiency. This, clearly, cannot be achieved by de novo synthesis, but requires several remodelling pathways, including either the polar head group or the fatty acids. Polar head groups are interchanged between phospholipids by several enzymes: base-exchange

PLA2 CONTROL OF EICOSANOID PRODUCTION

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Figure 2.6 De novo pathway of phospholipid synthesis (top) and fatty acid remodelling (bottom) by deacylation/reacylation using various transacylating enzymes

enzymes, PS decarboxylase, PE transmethylases, etc. (Figure 2.6). Fatty acid composition is modified by several pathways. A deacylation step catalysed by either PLA1 or PLA2 produced lysoderivatives. These lysoderivatives are then reacylated by (a) acyl-CoA:lysophospholipidacyl-transferases which exhibit lysophospholipid and acyl-CoA specificity and differential expression in various cell types, and by (b) phospholipid:lysophospholipid acyl-transferases, enzymes very specific for poly-unsaturated fatty acid transfer, which are mainly found in platelets, muscle cells and cells involved in the immuno-inflammatory process (Yamashita et

al 1997). Acyl-CoAs are either produced by acyl-CoA synthases, which are specific for a set of fatty acids (saturated, mono- or diunsaturated fatty acids and poly-unsaturated fatty acids) or by acyl-CoA:lysophospholipid acyl-transferase acting in reverse. In the lung, a particulate acyl-transferase synthesizes the characteristic dipalmitoyl-lecithin of surfactant by a CoA-independent transfer of palmitic acid from one sn-1-palmitoyl-lysolecithin to another. It has been reported that iPLA2 contributes to the remodelling of membrane phospholipids. In support of this proposal, iPLA2 overexpression leads to an increase in basal fatty

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acid release. Of particular interest was the finding that arachidonic acid released by iPLA2 was not metabolized to prostaglandin E2 (PGE2) (Balsinde et al 1998). Phospholipases A2 Housekeeping Enzymes for Lipoprotein and Membrane Repair Oxidative stress appears to be a major pathophysiological disturbance at work during the early atherosclerotic process (Livrea et al 1998). The phospholipids in LDL particles are known to undergo oxidation to produce oxidized phospholipids, which become substrates for PAF acetylhydrolases. These oxidized phospholipids could be detrimental in two ways; they mimic PAF to support sustained inflammation but could also covalently modify the apolipoprotein, resulting in unregulated cholesterolester accumulation by macrophages. In contrast, the products of oxidized phospholipid hydrolysis, lysoPC and fragments of fatty acids, are water-soluble and can be further metabolized into nontoxic products. Consequently, LDL-associated PAF acetylhydrolase can prevent modification of LDL into atherogenic particles. This protective effect might also be at work intracellularly, e.g. in erythrocytes and other cells subject to oxidative stress to ensure membrane phospholipid integrity, which is highly disturbed by the presence of oxidized phospholipids. Phospholipases A2 and Control of Lipid Mediator Production When activated by extracellular stimuli, cPLA2 triggers rapid hydrolysis of membrane phospholipids, leading to the formation of free fatty acids, mainly arachidonic acid, linoleic acid or oleic acid. This is the rate-limiting step for eicosanoid production (Figure 2.7). Arachidonic acid is the precursor of the vast family of eicosanoids which comprises two subfamilies, the cyclooxygenase family (prostaglandins and thromboxanes) and the lipoxygenase family [leukotrienes and hydroxyeicosatetranoic acids (HETEs)]. Linoleic acid gives rise to hydroxyoctadecanoic acids (HODEs) via the lipoxygenase pathway. The other cPLA2 product, lysophospholipid, might be a lipid mediator (lysophosphatidic acid or lysophosphatidylcholine) or the precusor of PAF. The production of these various mediators is tissue- and stimulation-dependent and this is achieved through the differential expression of the various enzymes involved in lipid mediator synthesis, and through the preferential coupling between cPLA2 and lipoxygenase or cyclo-oxygenase and the subcellular localization of these enzymes. Role of cPLA2 in Eicosanoid Synthesis Activation of cPLA2 is dependent on at least two mechanisms: phosphorylation of cPLA2 by various kinases and its translocation to membrane phospholipids by the calcium-binding domain (CalB) following an increase of intracellular Ca2+, which allows access of this cytosolic enzyme to its membrane substrate (Leslie 1997). The pathway by which cPLA2 is activated by extracellular stimuli, or whether this activation occurs at specific cellular sites, has not been elucidated. To date, various intracellular sites of translocation have been described for cPLA2 according to the cell type and stimuli used. They include nuclear and endoplasmic reticulum membranes, where they would co-localize with cyclooxygenases or lipoxygenases. Plasma membrane has also been proposed as a possible site for translocation of cPLA2, but early studies proposing a direct interaction of cPLA2 with heterotrimeric G proteins driven by serpentine membrane receptors have not been confirmed. Recent data reporting an interaction of

cPLA2 with receptor-activated Jak-kinases, or its activation by phosphatidyl inositol 4,5-biphosphate, have confirmed the possibility that translocation to the plasma membrane might indeed constitute the actual activation process. Using chimeric cPLA2 bearing the plasma membrane targeting signal of the Lck protein kinase, we have shown that increased expression of this chimeric cPLA2 in CHO cells gave a stronger response to epinephrine, Ca2+ ionophore or both than increased expression of the wildtype enzyme (Klapisz et al 1999). We also recently demonstrated that, in epithelial cells bearing the DF508 cystic fibrosis mutation, alteration of the cystic fibrosis transmembrane regulator (CFTR) molecule alters the functional coupling between certain receptors and cPLA2, leading to increased eicosanoid production. This might increase the inflammatory state at least in some circumstances (Berguerand et al 1997). cPLA2 has one consensus site for Map-kinase and phosphorylation of this serine (S505) by Map-kinases occurs in vivo and in vitro (Figure 2.2). This phosphorylation, which induces a characteristic electrophoretic mobility shift, is essential for receptor-mediated cPLA2 activation, since the S505A mutant can no longer be activated. Other kinases, such as protein kinases C (PKC) and A, can phosphorylate cPLA2 in vitro, but this does not result in a significant increase in enzyme activity. However, PKC might indirectly activate cPLA2 in vivo by activating other kinases. In some cell systems (neutrophils, platelets), TNFa or thrombin activate cPLA2 independently of Map-kinase activation, but via a proline-directed kinase which also phosphorylates S505. Although phosphorylation is important for cPLA2 activation in certain cells, it is not sufficient for complete activation. We recently demonstrated, in CHO cells overexpressing adrenergic receptors, that Ca2+ also participates in the activating process itself. This dual activation of cPLA2 by adrenergic receptors was also evidenced in rat cardiomyocytes and has been implicated in the contractile effect of b2-adrenergic receptors (Pavoine et al 1999). However, there is evidence to suggest alternative pathways without increasing Ca2+, e.g. in macrophages and neutrophils stimulated by the phorbol ester PMA or the phosphatase inhibitor okadaic acid. In this case, phosphorylation of S727 in addition to S505 phosphorylation might dispense with the Ca2+ increase. The actual protein kinase involved is not known but might be a basotrophic kinase. It is now clear that cPLA2 is a major element of the signalling cascade. However, this enzyme might not be as ubiquitous as may appear at first glance, e.g. its expression is confined in murine brain grey matter to astrocytes at the pial surface of the brain, and was found in white matter in addition to fibrous astrocytes, in interfascicular oligodendrocytes (Lautens et al 1998). In contrast, mRNA cPLA2-immunoreactivity was undetectable in neurons of murine brain. A direct role of cPLA2 in muscarinic signalling must be ruled out, but an indirect regulation of neurotransmission may be provided via the activity of cPLA2 in glial cells. Role of sPLA2 in Eicosanoid Synthesis Type II sPLA2 has been previously shown to hydrolyse phospholipids in the membrane of Escherichia coli, after the action of bacterial permeability-increasing protein (BPI), suggesting that it might participate in antimicrobial defence. Only indirect evidence has been published to indicate that type II sPLA2 can hydrolyse mammalian cell phospholipids in vivo to generate lipid mediators. In several cell systems, treatment by inflammatory cytokines strongly increases prostaglandin and type II sPLA2 synthesis (vascular smooth muscle cells, articular chondrocytes, mesangial cells, macrophages). This enzyme accumulates in the Golgi apparatus associated with proteoglycans. Part of the

Figure 2.7 Biosynthetic pathways of oxygenated fatty acids. The cyclooxygenase pathways (right) and the various lipoxygenase pathways (left)

PLA2 CONTROL OF EICOSANOID PRODUCTION 23

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BIOSYNTHESIS AND METABOLISM

enzyme may remain intracellular, but the majority is secreted and found associated with proteoglycans on the external cell surface, where phospholipid hydrolysis can occur. Some studies have shown that treatment of cell cultures with neutralizing anti-type II sPLA2 or anti-type V sPLA2 antibodies or antisense cDNA corresponding to the mRNA of these enzymes decreased prostaglandin synthesis. As type II sPLA2 and type V sPLA2 are not synthesized as inactive proenzymes, mechanisms must exist in vivo to protect membranes of the secretory system from untimely hydrolysis. Furthermore, numerous studies have shown that secreted enzymes cannot hydrolyse phospholipids of the plasma membrane of intact cells. It is thought that activation of the enzyme might be the result of membrane ‘‘scrambling’’ or synergistic activation of sphingomyelinase. We have also demonstrated that sphingomyelin, which is located in the external leaflet of plasma membranes, inhibits phospholipid hydrolysis by type II sPLA2 and that cholesterol, which strongly complexes with sphingomyelin, relieves this inhibitory effect (Koumanov et al 1997). Furthermore, when cells are activated by inflammatory cytokines, membrane sphingomyelin is degraded, and phospholipid asymmetry and lipid packing are known to be widely modified under these circumstances. This may have a critical role in modulating the activity of type II sPLA2 acting on biological membranes. Type II sPLA2 activity is also regulated by polypeptide factors such as phospholipase activating peptides (PLAPs) or annexins. Annexins are known to inhibit type II sPLA2 activity via so-called substrate depletion inhibition (Comera et al 1990). Annexin is strongly bound to acidic phospholipids; large amounts of annexin are supposed to mask phospholipid substrates from the action of type II sPLA2. However, we demonstrated that its action is more complex, since at low concentrations, which more probably mimic the intracellular situation, annexins increased enzyme activity. It is known that annexin binding to the cell surface induces disorders on both sides of the membrane. In addition, annexin VI participates in severing of the stalk during the budding process of endocytosis. It is therefore tempting to implicate type II sPLA2, which is present in secretory vesicles or at the surface of the plasma membrane, in events leading to exocytosis, endocytosis and to the intracellular production of lipid mediators, which often occurs during these processes. In the kidney, uteroglobin was shown to inhibit the type II sPLA2 enzyme secreted by mesangial cells and consequently decreases the level of lysophosphatidic acid (Mukherjee et al 1998). Specific receptors for secreted type I and type II enzymes have been characterized and cloned (Lambeau et al 1995). These receptors belong to the family of mannose receptors which are involved in the clearance of plasma glycoproteins. It has been suggested that its function is to eliminate the overproduction of secreted PLA2. However, some data suggest that the binding of type I sPLA2 might induce intracellular signals (Figure 2.8). For example, in a human astrocytoma cell line, type II PLA2 was found able to activate the cascade of Map-kinases and increase cPLA2 activity (Hernandez et al 1998). But other data have challenged this possibility and conflicting results have been reported concerning the actual involvement of PLA2-receptors in this process. REGULATION OF PHOSPHOLIPASE A2 SYNTHESIS The mechanisms controlling cPLA2 activity are mainly posttranslational. This occurs by phosphorylating events and Ca2+dependent translocation and allows rapid activation of cPLA2 activity in response to a variety of physiological stimuli. The mechanisms that control cPLA2 synthesis are less clearly understood. Although there is evidence that cPLA2 mRNA levels may

be subject to modest stimulation in some cells by mediators such as TNFa, glucocorticoids, macrophage colony stimulatory factor, epidermal growth factors, interferon-g, leukaemia inhibitory factor or interleukin-13, cPLA2 function is so important for a variety of cellular processes that it is likely that transcription of this gene is closely controlled. The TNFa effect was demonstrated to be mediated by the sphingomyelinase–ceramide pathway (Hayakawa et al 1996). Conversely, IL-4 inhibits cPLA2 gene expression in LPS-stimulated macrophages and rheumatoid synovial cells (Kuroda et al 1997). It has been demonstrated that a specific element (717,78) of the cPLA2 promoter is responsible for the low-level constitutive expression of the gene (Miyashita et al 1995). Furthermore, the presence of three ATTTA motifs in the 3’ untranslated region (3’UTR) suggests that the gene might be regulated at a post-transcriptional level by modulation of mRNA stability (Tay et al 1994). Inflammatory cytokines induce the synthesis and secretion of type II sPLA2 in various cell types. In several cell systems, particularly in rat mesangial cells, the cytokine effect is reinforced by the stimulation of receptors or drugs that increase cytoplasmic cAMP (Walker et al 1995). In contrast, peptide growth factors (TGFb, PDGF, EGF and bFGF) markedly inhibited the extracellular release of type II sPLA2 (Pruzanski et al 1998). The precise signalling events leading to type II sPLA2 gene regulation are not fully understood. In most cells, interleukin-1b and TNFa trigger their action by at least two main pathways involving specific receptors. They activate acid sphingomyelinase to initiate the ceramide cascade (Mathias et al 1993). IL-1b and TNFa induce signalling cascades which activate members of the NFkB family. In most cells, NFkB is retained in the cytoplasm by inhibitory proteins called IkBs (Beg and Baldwin 1993). IL-1b and TNFa induce IkB phosphorylation and promote its degradation by the proteosome pathway and the subsequent nuclear translocation and activation of NFkB family members (Baldwin 1996). The actual pathway by which they induce phosphorylation of IkBs is still poorly understood. Recently, cPLA2 has been implicated in IL-1b-mediated type II sPLA2 gene induction in rat fibroblasts (Kuwata et al 1998). This raises the possibility of a control of the type II sPLA2 gene by free fatty acids or their derivatives via peroxisome proliferator activated receptors (PPARs). PPARs are key players in lipid metabolism and are members of the nuclear receptor superfamily of transcription factors that regulate the pattern of gene expression in response to the binding of small molecular weight ligands (Mangelsdorf et al 1995). Three different subtypes have been described, PPARa, PPARb(d) and PPARg. Although the identities of the ligands that regulate activity in vivo remain to be established with certainty, clofibric acid, leukotriene B4 and oleic acid have been demonstrated to stimulate PPARa and 15-deoxyD12,14-prostaglandin J2 (15DPGJ2) (Forman et al 1995), 12- or 9hydroxyoctadecanoic acid (HODE) (Nagy et al 1998), and certain polyunsaturated fatty acids (Kliewer et al 1997) have been demonstrated to stimulate PPARg-dependent transcription. We demonstrate that, in rat vascular smooth muscle cells, Il-1b induces the type II sPLA2 gene via the NFkB pathway and PLA2/ PPARg pathway (Figure 2.9), but not via the ceramide pathway or the cPLA2–PPARa pathway. Concomitant activation of both the NFkB pathway and the cPLA2–PPARg pathway is necessary to achieve the transcriptional process, suggesting that they cooperate at the level of transcriptional machinery (Couturier et al 1999). In rabbit articular chondrocytes, we have demonstrated that large amounts of type II sPLA2 are secreted in response to IL-1b and that insulin-like growth factor-1 (IGF-1), which counteracts cartilage degradation in arthritis, inhibits this effect. We demonstrated that the IL-1b-induced synthesis and released of type II sPLA2 occurs in parallel with release of PGE2, and results in indirect stimulation of

PLA2 CONTROL OF EICOSANOID PRODUCTION

25

Figure 2.8 Putative effects of secreted phospholipase A2 via unspecific interaction with cellular membranes or via specific receptors

the transcription of type II sPLA2 gene, giving rise to a very stable type II sPLA2 mRNA. We also demonstrated that the inhibitory effect of IGF-1 markedly decreased the half-life of type II sPLA2 mRNA (Jacques et al 1997). Activation of the human counterpart of the type II sPLA2 gene is quite different. We have demonstrated that the NFkB pathway is not directly involved in this process, at least in HepG2 cells, chondrocytes or smooth muscle cells. NFkB must stimulate an intermediary gene, the product of which in turn participates in the induction of human type II sPLA2 gene. We found that the promoter of the human gene possesses a degenerated kB site, which binds C/EBP factors (Figure 2.10) (Couturier et al 2000). This binding relieves the basal silencing of the type II sPLA2 gene (Fan et al 1997). These factors were identified to bind and stimulate many acute-phase response genes. The C/EBP family has three main members, C/EBPa, C/EBPb and C/EBPd. The cell mRNA content was markedly increased after the incubation of hepatocytes with IL-6 or during an experimental systemic inflammatory process. In addition, C/EBPb and C/EBPd are also regulated by phosphorylation. The difference that we observed between humans and rodents in the regulation of the type II sPLA2 gene might be explained by differences in the promoter region of human and rat genes, which strongly diverge. Additional experiments are currently being performed in our laboratory to define the molecular basis for these differences. This might be of importance, since type II sPLA2 is thought to play an important role in human inflammatory disease and might disfavour the rat model in the search for new classes of therapeutic agents.

CONCLUSION It is now evident that phospholipases A2 are very important enzymes in cell physiology. Both cellular and secreted PLA2

Figure 2.9 IL-1-mediated signalling pathways inducing the rat type IIA sPLA2 gene in vascular smooth muscle cells. One of the pathways involves the IkB–NF-kB system, which is activated by proteosome degradation of IkB. The second pathway involves MAP-kinase-mediated phosphorylation of the cytosolic phospholipase A2 and activation of PPAR/RXR by oxygenated fatty acids. The third pathway, via Jun kinases (JNK/SAPK), remains hypothetical

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Figure 2.10 IL-1-mediated activation of the human type IIA sPLA2 gene. The IkB–NF-kB system is replaced by activation of C/EBP dimers via phosphorylation

participate in the housekeeping of cell membrane and lipoprotein integrity. They are also involved in either the immediate control of lipid mediator production after stimulation of membrane receptors by their cognate agonists (cPLA2) or the delayed production of lipid mediators after cell stimulation (type IIA or type V sPLA2). This therefore constitutes a putative target for pharmacological agents. To date, despite tremendous work conducted by pharmaceutical companies, and with the remarkable exception of quinacrine derivatives (which, however, were not initially intended to be PLA2 inhibitors), no PLA2 inhibitors can be used in human pathology. The inhibitors that are available on the market are not very specific and must be used with caution in experiments. This is probably due to the physical properties of their substrates. One additional way to search for new drugs in the future will be to identify the signalling pathways more precisely, controlling their gene expression in order to obtain efficient inhibitors of PLA2 synthesis.

REFERENCES Balboa MA, Balsinde J, Jones SS and Dennis EA (1997) Identity between the Ca2+-independent phospholipase A2 enzymes from P388D1 macrophages and Chinese hamster ovary cells. J Biol Chem, 272, 8576–8580. Baldwin AS Jr (1996) The NF-kB and IkB proteins: new discoveries and insights. Ann Rev Immunol, 14, 649–681. Balsinde J, Balboa MA and Dennis EA (1998) Functional coupling between secretory phospholipase A2 and cyclo-oxygenase-2 and its regulation by cytosolic group IV phospholipase A2. Proc Natl Acad Sci USA, 95, 7951–7956.

Beg AA and Baldwin AS Jr (1993) The IkB proteins: multifunctional regulators of Rel/NF-kB transcription factors. Genes Dev, 7, 2064– 2070. Berguerand M, Klapisz E, Thomas G et al (1997) Differential stimulation of cytosolic phospholipase A2 by bradykinin in human cystic fibrosis cell lines. Am J Resp Cell Mol Biol, 17, 481–490. Caldwell RA and Baumgarten CM (1998) Plasmalogen-derived lysolipid induces a depolarizing cation current in rabbit ventricular myocytes. Circulat Res, 83, 533–540. Comera C, Rothhut B and Russo-Marie MF (1990) Identification and characterization of phospholipase A2 inhibitory proteins in human mononuclear cells. Eur J Biochem, 188, 139–146. Couturier C, Antonio V, Brouillet A et al (2000) Protein kinase Adependent stimulation of rat type II secreted phospholipase A2 gene transcription involves C/EBP-beta and -delta in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol, 20, 2559–2565. Couturier C, Brouillet A, Couriaud C et al (1999) Interleukin 1b induces type II secreted phospholipase A2 gene in vascular smooth muscle cells by a NFkB- and cytosolic phospholipase A2-mediated process. J Biol Chem, 274, 23085–23093. Cupillard L, Koumanov K, Mattei MG et al (1997) Cloning, chromosomal mapping and expression of a novel secretory phospholipase A2. J Biol Chem, 272, 15745–15752. Fan Q, Paradon M, Salvat C et al (1997) C/EBP factor suppression of inhibition of type II secreted phospholipase A2 promoter in HepG2 cells: possible role of single-strand binding proteins. Mol Cell Biol, 17, 4238–4248. Forman BM, Tontonoz P, Chen J et al (1995) 15-Deoxy-delta12–14prostaglandin J2 is a ligand for the adipocyte determination factor PPARg. Cell, 83, 803–812. Hayakawa M, Jayadev S, Tsujimoto M et al (1996) Role of ceramide in the stimulation of the transcription of cytosolic phospholipase A2 and cyclooxygenase 2. Biochem Biophys Res Commun, 220, 681–686. Hernandez M, Burillo SL, Crespo SM and Nieto ML (1998) Secretory phospholipase A2 activates the cascade of mitogen activated protein kinases and cytosolic phospholipase A2 in the human astrocytoma cell line 1321N1. J Biol Chem, 273, 606–612. Jacques C, Bereziat G, Humbert L et al (1997) Posttranscriptional effect of insulin-like growth factor-I on interleukin-1b-induced type II-secreted phospholipase A2 gene expression in rabbit articular chondrocytes. J Clin Invest, 99, 1864–1872. Klapisz E, Ziari M, Wendum D et al (1999) N- and C-terminal plasma membrane anchoring have opposite effects on cytosolic phospholipase A2 activation. Eur J Biochem, 265, 957–966. Kliewer SA, Sundseth SS, Jones SA et al (1997) Fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferator-activated receptors alpha and gamma. Proc Natl Acad Sci USA, 94, 4318–4323. Kramer RH, Hession C, Johansen B et al (1989) Structure and properties of a human non pancreatic phospholipase A2. J Biol Chem, 264, 5768– 5775. Koumanov K, Wolf C and Bereziat G (1997) Modulation of human type II secretory phospholipase A2 by sphingomyelin and annexin VI. Biochem J, 326, 227–233. Kuroda A, Sugiyama E, Taki H et al (1997) Interleukin-4 inhibits the gene expression and biosynthesis of cytosolic phospholipase A2 in lipopolysaccharide-stimulated U937 macrophage cell line and freshly prepared adherent rheumatoid synovial cells. Biochem Biophys Res Commun, 230, 40–43. Kuwata H, Nakatani Y, Murakami M and Kudo I (1998) Cytosolic phospholipase A2 is required for cytokine-induced expression of type IIA secretory phospholipase A2 that mediates optimal cyclooxygenase2-dependent delayed prostaglandin E2 generation in rat 3Y1 fibroblasts. J Biol Chem, 273, 1733–1740. Lambeau G, Ancian P, Nicolas JP et al (1995) Structural elements of secretory phospholipase A2 involved in the binding to M-type receptors. J Biol Chem, 270, 5534–5540. Lautens LL, Chiou XG, Sharp JD et al (1998) Cytosolic phospholipase A2 (cPLA2) distribution in murine brain and functional studies indicate that cPLA2 does not participate in muscarinic receptor-mediated signaling in neurons. Brain Res, 809, 18–30. Leslie CC (1997) Properties and regulation of cytosolic phospholipase A2. J Biol Chem, 272, 16709–16712.

PLA2 CONTROL OF EICOSANOID PRODUCTION Livrea MA, Tesoriere L, Maggio A et al (1998) Oxidative modification of low-density lipoprotein and atherogenetic risk in b-thalassemia. Blood, 92, 3936–3942. Mangelsdorf DJ, Thummel C, Beato M et al (1995) The nuclear receptor superfamily: the second decade. Cell, 83, 835–839. Mathias S, Younes A, Kan CC et al (1993) Activation of the sphingomyelin signaling pathway in intact EL4 cells and in a cellfree system by IL-1b. Science, 259, 519–522. Miyashita A, Crystal RG and Hay GG (1995) Identification of a 27 bp 5’flanking region element responsible for the low level constitutive expression of the human cytosolic phospholipase A2 gene. Nucleic Acids Res, 23, 293–301. Mukherjee AB, Kundu GC, Mandal AK et al (1998) Uteroglobin: physiological role in normal glomerular function uncovered by targeted disruption of the uteroglobin gene in mice. Am J Kidney Dis, 32, 1106–1120. Nagy L, Tontonoz P, Alvarez, JG et al (1998) Oxydized LDL regulates macrophage gene expression through ligand activation of PPARg. Cell, 93, 229–240. Pavoine C, Magne S, Sauvadet A and Pecker F (1999) Evidence for a b2adrenergic/arachidonic acid pathway in ventricular cardiomyocytes. Regulation by the b1-adrenergic/cAMP pathway. J Biol Chem, 274, 628–637. Pruzanski W, Stefanski E, Vadas P et al (1998) Regulation of the cellular expression of secretory and cytosolic phospholipases A2 and cyclooxygenase-2 by peptide growth factors. Biochem Biophys Res Commun, 1403, 47–56.

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Seilhamer JJ, Pruzanski W, Vadas P et al (1989) Cloning and recombinant expression of phospholipase A2 present in rheumatoid arthritic synovial fluid. J Biol Chem, 264, 5335–5338. Stafforini DM, McIntyre TM, Zimmerman GA and Prescott SM (1997) Platelet-activating factor acetylhydrolases. J Biol Chem, 272, 17895– 17898. Tang J, Kritz RW, Wolfman N et al (1997) A novel cytosolic calciumindependent phospholipase A2 contains eight ankyrin motifs. J Biol Chem, 272, 8567–8575. Tay A, Maxwell P, Li ZG et al (1994) Cytosolic phospholipase A2 gene expression in rat mesangial cells is regulated post-transcriptionally. Biochem J, 304, 417–422. Tischfield JA (1997) A reassessment of the low molecular weight phospholipase A2 gene family in mammals. J Biol Chem, 272, 17247– 17250. Underwood KW, Song C, Kriz RW et al (1998) A novel calciumindependent phospholipase A2; cPLA2-g that is prenylated and contains homology to cPLA2. J Biol Chem, 373, 21926–21932. Walker G, Kunz D, Pignat W et al (1995) Pyrrolidone dithiocarbamate differently affects cytokine- and cAMP-induced expression of group II phospholipase A2 in rat renal mesangial cells. FEBS Lett, 364, 218– 222. Yamashita A, Sugiura T and Waku K (1997) Acyltransferases and transacylases involved in fatty acid remodeling of phospholipids and metabolism of bioactive lipids in mammalian cells. J Biochem (Tokyo), 122, 1–16.

3 Mechanisms of PGH Synthase-1 (COX-1) Activity and Role of Radical States Carol Deby and Ginette Deby-Dupont University of Lie`ge, Lie`ge, Belgium

BRIEF OVERVIEW The first works in the research on prostaglandin endoperoxide H (PGH) synthase (PGHS) were performed simultaneously by Van Dorp et al (1964) and Bergstro¨m et al (1964), who described a sequence of reactions starting when a proteic extract of sheep seminal vesicles was incubated with arachidonic acid. Further research established that three polyunsaturated fatty acids (chain length of 20 carbon atoms; Figure 3.1) incubated with seminal vesicle extracts were oxidized by two molecules of oxygen. These three precursors became cyclic compounds, passing through free radical states, finally producing the prostaglandin PGH, the direct precursor of the various biologically active prostanoids (prostaglandins, prostacyclin, thromboxanes) and their numerous metabolites. Further data demonstrated the complexity of the peroxide and endoperoxide rearrangements, leading first to endoperoxide G and second to endoperoxide H, highly unstable and quickly metabolized by a cascade of reactions into more stable compounds, the prostanoids. At the origin of the synthesis of these cyclic compounds derived from arachidonic acid and the two other 20-carbon chain unsaturated fatty acids, there are two common root compounds, endoperoxide G (PGG) and endoperoxide H (PGH), deriving from the reduction of PGG. A single enzyme, displaying two enzymatic activities (cyclooxygenasic and peroxidasic), produces the root compounds: this enzyme is referred to as prostaglandin endoperoxide H synthase (PGHS). The main pathways of arachidonic acid peroxidation by PGHS are presented in (Figure 3.2). The first step is a double dioxygenase or cyclooxygenase reaction (bis-dioxygenation; Ogino et al 1978), binding two oxygen molecules on the polyunsaturated fatty acid precursor. A cyclic molecule is produced with both 9,11endoperoxide and 15-hydroperoxyl groups (Figure 3.2), called endoperoxide G or PGG (Samuelsson 1972; Nugteren and Hazelhof 1973). The following reaction summarizes this first step: C20:3, C20:4, C20:5+2O2
!PGG1,2,3 (bis-dioxygenation) In the second step, the 15-hydroperoxide group of PGG is reduced to hydroxyl by the peroxidase activity of PGHS, forming the endoperoxide H or PGH. This reaction follows the classical scheme, using a hydrogen donor DH2: PGG+DH2
!PGH+H2O+D

(D is often a free radical)

From PGG and PGH, several prostanoids can be produced; their nature is determined by the enzymes of the considered tissue The Eicosanoids. Edited by Peter Curtis-Prior. &2004 John Wiley & Sons, Ltd: ISBN 0 471 48984 0

and according to the biochemical circumstances, e.g. from endoperoxides there is a generation of thromboxane A2 in platelets, while in endothelial cells, prostacyclin (PGI2) seems to be predominant. PGD appears to be relatively abundant in the rat spleen (Christ-Hazelhof and Nugteren 1979), as shown by in vitro experiments in the presence of albumin (Christ-Hazelhof et al 1976; Hamberg and Fredholm 1976) or glutathione-S-transferase (Christ-Hazelhof et al 1976). Until the beginning of the 1990s, it was generally admitted that only one gene existed for PGH synthase (Yokoyama and Tanabe 1989), but it was recognized that this enzyme, originally named cyclooxygenase (COX), was found in tissues in the form of two isoenzymes, COX-1 and COX-2. It is now demonstrated that these two isoforms are encoded by two different genes (Kraemer et al 1992). COX-1 is the classical constitutive (‘‘housekeeping’’) enzyme, and COX-2 is the inducible isoform. Whereas these isoenzymes differ substantially with respect to their expression and biology, they possess similar structures and express the same two catalytic activities. This chapter deals only with PGHS-1 (COX-1). A general review of COX-2 properties was published by Herschman (1996) and can be found in recent reviews with a comparison of the molecular biology (biosynthetic and signalling pathways) of the two enzymes (Smith et al 2000). The general enzymatic mechanisms described for COX-1 are applicable to COX-2.

STRUCTURE OF PGH SYNTHASE-1 (EC 1.14.99.1) PGH synthase-1 (or cyclooxygenase-1: COX-1) is a bis-dioxygenase displaying both cyclooxygenase and peroxidase activities (O’Brien and Rahimtula 1976; Miyamoto et al 1976), thus catalysing the two following reactions where AA is arachidonic acid, C20:4 (but this fatty acid can be replaced by C20:3 or C20:5): 1. AA+2O2
!PGG2 (cyclooxygenase reaction) 2. PGG2
!PGH2 (peroxidase reaction) This enzyme can be separated from the protein fraction converting PGH into PGE (Miyamoto et al 1974, 1976). PGHS-1 is a membrane-bound haemoglycoprotein containing mannose and Nacetyl-glucosamine (van der Ouderaa et al 1977). Its 576 amino acid sequence has been completely elucidated (Smith et al 1996). A dimeric structure has been proposed, with both dimers structurally and functionally similar (Roth et al 1980; van der Ouderaa et al 1977; Xiao et al 1998). The monomer is a single polypeptide chain of 72 kDa with three structural domains: an epidermal

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Figure 3.1 The three polyunsaturated fatty acids, precursors of the prostanoids, and their corresponding derived PGHs. I, eicosatrienoic (o-6) or homo-g-linolenic acid C20-3; Ia, PGH-1; II, eicosatetraenoic (o-6) or arachidonic acid C20-4; IIa, PGH-2; III, eicosapentaenoic (o-3) acid C20-5; IIIa, PGH-3

growth factor domain at the N-terminus; a neighbouring membrane-binding domain; and a large globular catalytic domain at the C-terminus (closely similar to the catalytic domain of neutrophil myeloperoxidase) with a hydrophobic channel (Smith et al 2000). Each subunit contains a haem (van der Ouderaa et al 1977; Roth et al 1980; Nugteren et al 1981). The enzyme can be solubilized and purified in non-ionic detergents (Miyamoto et al 1976; van der Ouderaa et al 1977; Hemler and Lands 1976). During purification, the apoenzyme can be separated from its haem and becomes completely inactive. The reconstitution of the enzymic activity can be driven by haemin addition (Miyamoto et al 1974). The distribution of PGHS-1 in the cell was studied by Morita et al (1995a, 1995b), using quantitative confocal fluorescence imaging microscopy. The enzyme was found equally distributed in the endoplasmic reticulum and perinuclear membranes using immunological techniques, but the activities were mainly localized in the perinuclear region. It is not our purpose to give details on the techniques and methods of purification of PGHS; however, it must be underlined that, according to Roth et al (1980), PGHS-1 can be purified to near homogeneity in an active form containing a single subunit. The combination of classical protein chemistry, UV-visible spectroscopy (Lambeir et al 1985; Dietz et al 1988), electron spin resonance (Karthein et al 1987, 1988; DeGray et al 1988, 1992; Tsai et al 1992, 1994a) and X-ray crystallography (Picot et al 1994) permits us to imagine the PGHS as a spheroid (Marnett et al 1999) (Figure 3.3).

The active site of the enzyme is formed by a hydrophobic channel opening into the outside of the protein, accessible for dioxygen molecules and presenting specific sites that intervene in the binding and stereospecific oxidation of arachidonic acid. Three amino acid residues are especially important: Arg120, which serves as counter-ion for the carboxylic function of the substrate and is the anchoring base of the substrate (Picot and Garavito 1994; Bhattacharyya et al 1996); Tyr 385, which plays the main role in the oxidation of the fatty acid; and Ser 530, of which the acetylation blocks the dioxygenase activity (Loll et al 1995). The substrate carboxylate is positioned adjacent to Arg 120, which plays a crucial role in binding arachidonate and arylalkanoic acid-type inhibitors (Kulmacz et al 1994) and the 13-pro-S hydrogen is placed near the phenolic hydroxyl of Tyr 385. The importance of the Arg 120 residue in PGHS-1 is recognized for its interaction with arachidonic acid and non-steroidal antiinflammatory drugs (NSAIDs) containing a free carboxylic acid moiety (Mancini et al 1995). The o-end of the fatty acid is inserted into a channel at the top of the cyclooxygenase active site that eventually leads to the surface of the protein. This end of arachidonate straddles the a-helix containing Ser 530, the site that is acetylated by aspirin. Acetylation blocks the arachidonate binding to PGHS (Shimokawa and Smith 1992; Barnett et al 1994). PGHS appears to have two high-affinity binding sites for metalloporphyrins. The two sites have slightly different affinities for haem. The synthase dimer is capable of cyclooxygenase catalysis when the site with the highest affinity is occupied by the haem. The two subunits of the enzyme are thus not completely identical (Kulmacz and Lands 1984). Haem can be replaced by manganese protoporphyrin, giving a system where the enzyme only catalyses the bis-dioxygenation producing prostaglandin G1 (Ogino et al 1978; Tsai et al 1998). NSAIDs compete directly with arachidonate for binding to the cyclooxygenase site and inhibit cyclooxygenase activity, but have little effect on peroxidase activity (Capdevila et al 1995; Kulmacz and Lands 1985a). THE TWO MAIN MECHANISMS OF PGH SYNTHASE ACTIVITY Two Distinct Sites The two activities of PGHS (bis-dioxygenasic and peroxidasic) are displayed on the same enzyme molecule on two distinct sites, physically and functionally separated, that are closely localized on the protein chain (Marshall and Kulmacz 1988). Tyr 385 is located between the haem- and arachidonate-binding sites, well positioned to serve a role in coupling the two enzyme activities (Picot and Garavito 1994). The existence of these distinct sites was first demonstrated by using acetyl salicylic acid (ASA), which blocks the dioxygenase reaction but does not affect the peroxidase activity. Aspirin acetylates a single serine residue of an active site on the enxyme (Roth et al 1975, 1981; van der Ouderaa et al 1977, 1980; Hemler et al 1978), related to the cyclooxygenase function, without modifications of the peroxidase activity (Miyamoto et al 1976; van der Ouderaa et al 1977). On the other hand, replacement of the haem by manganese protoporphyrin maintains only the cyclooxygenase but not the peroxidase activity (Ogino et al 1978). After treatment with ASA, PGHS is always able to transform exogenous PGG2 into PGH2. The peroxidase reaction acts not only on PGG2 but also on other peroxides. Mechanism of PGG Formation: Cyclooxygenasic Function This function is believed to involve a free radical state, initiated by the abstraction of a hydrogen atom at carbon 13 on arachidonic

PGH SYNTHASE-1 ACTIVITY AND RADICAL STATES

31

Figure 3.2 Chemical steps in the conversion of arachidonic acid into PGG2. The enzyme removes the 13-pro-S-hydrogen (I), which generates a pentadienyl radical with maximal electron density at carbons 11 and 15 (II). The trapping of the radical at carbon 11 with O2 produces a peroxyl radical, which adds to carbon 9, generating an endoperoxide and a carbon-centred radical at carbon 8 (III). The carbon 8 radical adds to the double bond at carbon 12, generating the bicyclic peroxide and an allylic radical with maximal electron density at carbons 13 and 15 (IV). The trapping of the carbon radical at carbon 15 with O2 generates a peroxyl radical (V) which, in the presence of an electron donor (D.H), is reduced to PGG2 (VI)

acid (Hamberg and Samuelsson 1967). The reality of the radical state, carbon-centred, of arachidonic acid was evidenced by Mason et al (1980). Since the end of the 1980s, it has been demonstrated that the hydrogen atom abstraction was conducted by Tyr 385 in PGHS-1 (see below). At this state, an oxygen molecule could react with carbon atom 11 to form a peroxyl radical, which could abstract a hydrogen atom to form a hydroperoxide (Schreiber et al 1986). From the work of Dietz et al (1988), it appears clearly that the hydrogen abstraction is done by the tyrosyl radical, Tyr. 385. Until 1994, the role of Tyr. was hardly debated (Kulmacz et al 1994). But recent papers have afforded numerous arguments in favour of the importance of the Tyr. 385 step (Tsai et al 1994a, 1999). Since the regeneration of Tyr. 385 is dependent on peroxidasic activity, the cyclooxygenasic function closely depends on the presence of a sufficient hydroperoxide level. A balance must thus exist between the two activities that will regulate the global synthesis of PGH. Kulmacz et al (1994) realized a computerized model which can simulate PGHS activity. However, in in vivo conditions, outcoming hydroperoxides such as hydrogen peroxide (H2O2) are able deeply to modify the expected results (see below). Other factors can also modify the reactions, such as antioxidants and the presence of carbonates (from CO2). Thus, in vitro experiences are often very difficult to reproduce. The presence of glutathione (GSH) peroxidase

results in a strong PGHS inhibition (Hemler et al 1978) because this enzyme reduces inorganic and organic hydroperoxides. Mechanism of PGH Formation: Peroxidasic Function Analogies between PGH Synthase and the Classical Haem Peroxidases The first analogy between the PGHS and compound I (Cd I) of horseradish peroxidase (HRP), the typical model of peroxidases, was given by Nastainczyk et al (1984). They observed the classical spectral characteristics of Cd I during the kinetic study of PGHS, i.e. the formation of a ferryl-oxo complex (FeO)3+ of the haem, analogous to the formation of Cd I of horseradish peroxidase. These results were confimed by Lambeir et al (1985), who calculated that Cd I of PGH synthase is quickly converted to compound II. Confirmations were afforded by the team of Marnett (Markey et al 1987). Co-factors are strongly required as electron donors since, in their absence, arachidonic acid irreversibly inactivates PGHS (Markey et al 1987), but until recently these biological reducers were unknown (Zenser et al 1999). Many naturally occurring compounds have been assayed as electron donors, such as NADPH, NADH, glutathione, methionine, tryptophan,

32

BIOSYNTHESIS AND METABOLISM

Figure 3.3 Schematic representation of the monomer of PGH synthase-1. For details see text

epinephrine, ascorbic acid, lipoic acid and uric acid (Markey et al 1987; Zenser et al 1999). However, in some cases, some of these substances could act as free radical scavengers (see section on regulation, below). Different molecules can play the role of electron donor, each itself becoming a free radical. A good model is given by various oestrogens (Freyberger and Degen 1989). Glutathione seems to slow the rate of PGHS activity (Freyberger and Degen 1989). In the resting (native) state, the haem of PGHS is in the ferric high-spin state and the iron atom is at oxidation degree III (Karthein et al 1987). On the porphyrin macrocycle, there are numerous sequences of the carbon chain containing conjugated double bonds, a favourable structure for the resonance of a free electron. During peroxidase activity, an electron of one of these sequences can be removed at the same time as an electron of the ferric atom. This phenomenon occurs by the reaction of the haem with a hydroperoxide molecule (i.e. H2O2, cumene peroxide, lipid peroxide).

The Cycle of Oxidoreduction of the Haem in PGHS (Figure 3.4) In the presence of a peroxide (R–OOH), the haem is able to reduce the –OOH function by giving two electrons. One of these electrons originates from the porphyrin macrocycle. The departure of this electron causes the formation of a radical state, with resonance by way of the conjugated double bonds of the macrocycle. This electron delocalization is represented in Figure 3.4 by dotted lines. The electron loss is associated with gain of a positive charge (+ on Figure 3.4). The second electron comes from the haem iron, which is thus oxidized to the ferryl state (FeIV) associated with an oxygen atom. This structure is analogous to the Cd I first described for horseradish peroxidase. The radical structure on the porphyric ring and the positive charge explain the name given to Cd I, pcation radical. During the activity of PGHS, it is clear that the

peroxide used will be PGG2, but other peroxides can trigger PGHS activity. H2O2 is one of these possible triggering peroxides. We shall see further what kind of peroxides are used to form Cd I. Recently, Marnett et al (1999) underlined the role played by peroxynitrite (see below: triggering). At the end of the 1980s, Karthein et al (1987, 1988) demonstrated by ESR and crystallographic studies that Cd I is transformed into Cd II by a long-range electron transfer (LRET) from a tyrosyl residue of the protein chain (Tyr 385), an observation confirmed by Shimokawa et al (1990). This electron given by the tyrosyl residue is coupled to the free electron of the porphyrin macrocycle, which returns to a non-radical state, losing its positive charge. The radical state thus becomes localized in a stereospecific site that has been described above. Facing the tyrosyl radical is carbon 13 of arachidonic acid. A hydrogen atom is abstracted from arachidonic acid at this carbon 13, forming an arachidonyl radical, which is able successively to trap two exogenous dioxygen molecules (present in the protein channel), following a mechanism previously described (see above). Finally, Cd II is reduced to the ferric state by mechanisms generating free radicals. Effectively, two molecules of hydrogen donor are needed to reduce the oxygen atom of the ferryl structure and FeIV into FeIII. These side-reactions associated with prostaglandin synthesis are described in a separate part of this chapter. How is the radical located on Tyr 385 reduced for a next dioxygenase cycle? The final step in each round of arachidonic acid oxygenation is the reduction of the peroxyl radical precursor of PGG2 by Tyr 385, which regenerates the Tyr 385 radical for the next round of cyclooxygenase catalysis (Smith et al 1992). This leads to multiple turnovers per activation event and allows the accumulation of PGG2 (Seibert et al 1994; Xie and Herschman 1995). To be able to initiate a novel cycle, Cd II must be reduced to the resting form (FeIII). It is one of the steps where an electron donor (DH) is required. By donation of an H., DH can become a free radical D. (for the central role of Tyr 385, see Figure 3.5). The interaction of haem with PGHS is stoichiometric (Tsai et al 1997). Only one subunit needs to be bound to a haem to form a catalytically active synthase dimer, and an excess of haem is an inhibitor (Kulmacz and Lands 1984). Preincubation of the enzyme with haematin or haemoglobin results in the loss of enzyme activity. The enzyme inactivation is prevented by tryptophan or various other aromatic compounds (Ogino et al 1978). Coupling of the Two Mechanisms of PGHS Activity Two mechanisms are proposed to account for the combination of the cyclooxygenase and peroxidase activities of PGHS. Dietz et al (1988) proposed a branched-chain process, hypothesizing that the cyclooxygenase reaction propagates independently of peroxidase activity. Bakovic and Dunford (1994) proposed tightly coupled mechanisms; for these authors, the peroxidase cycle participates totally in cyclooxygenase propagation. Wei et al (1995) consider the branched-chain mechanism to be more probable than the tightly coupled mechanism. NATURE OF THE AGENTS TRIGGERING PEROXIDASE ACTIVITY Requirement for a Hydroperoxide ROOH Hemler and co-workers (Hemler et al 1979; Hemler and Lands 1980) observed that the addition of purified arachidonic acid to a preparation of cyclooxygenase did not consume oxygen during a more or less long time period that depends on the degree of purity of arachidonic acid (lag time). The addition of trace amounts of

PGH SYNTHASE-1 ACTIVITY AND RADICAL STATES peroxidized arachidonic acid suppressed this lag time. Other peroxides, such as peroxidized linoleic acid, can play the same triggering role; tert-butyl-hydroperoxide (O’Brien and Rahimtula 1976) and arachidonate peroxides (Narayanan and Harrington 1980) have a significant enhancing effect. The efficient triggering action of hydroperoxides is evidenced when GSH peroxidase is added to the enzyme system: PGHS is blocked. This triggering phenomenon is now easily explained by the need to oxidize the ferric haem into its ferryl state (see Figure 3.4). A source of hydroperoxides is cytochrome P-450BM-3 (Capdevila et al 1996). The Problem of H2O2 The triggering effect of hydroperoxides may be simply obtained by H2O2. The stimulation of PGHS by optimal H2O2 concentrations was observed at the end of the 1970s (Hemler et al 1979; Deby and Deby-Dupont 1980). However, the direct role of H2O2, as cyclooxygenase trigger, was minimized by Hemler et al (1979): the action of H2O2 would be too weak and would be prevented by catalase. Later on, a complete inactivation of PGHS by H2O2 with bleaching of the haem Soret absorbance peak was found when using 20 moles H2O2/mole PGHS haem (Tsai et al 1992). But more recently the triggering effect of H2O2 has again been contested (Hajjar et al 1995). The latest publications on the subject admit that H2O2 will play an activating role at optimal concentrations (Bakovic and Dunford 1996a). The formation of Cd I, using H2O2 as substrate, was studied as a function of pH and temperature. The protonated form of H2O2 preferentially reacts with the unprotonated form of the enzyme over the pH range of 3.5–9.1, suggesting the importance of acid–base catalysis for Cd I formation (Bakovic and Dunford 1996b). Many other observations have confirmed the role of hydrogen peroxide. Ascorbate activates cyclooxygenase through the generation of H2O2 (Polgar and Taylor 1980). GSH peroxidase reduces H2O2 as well as other hydroperoxides, and is an inhibitor of PGHS activity (Hemler et al 1978). Activated leukocytes produce H2O2, which is partially released outside these cells (Badwey and Karnovsky 1980). It was also observed that activated leukocytes stimulated cyclooxygenase activity in endothelial cells (Rampart et al 1981). It is thus logical to hypothesize that H2O2 acts as a messenger from leukocytes to neighbouring cells and platelets. The intracellular sources of H2O2 are numerous, since H2O2 is formed in mitochondria and by numerous enzymatic systems (e.g. cytosolic xanthine oxidase, NADPH-oxidase). It has recently been demonstrated that endothelial cells possess a functional leukocyte-type NADPHoxidase (Meyer et al 1999) and that this enzyme (or a parent form) is present in vascular smooth muscle cells, fibroblasts and myocytes (Griendling et al 2000). It is therefore highly probable that H2O2 effectively interacts in vivo with PGHS activity. However, if many data demonstrated that H2O2 can play an efficient role during PGHS activity and that H2O2 and O.2generating systems are stimulating on prostanoid production, it also appears that an optimal concentration of H2O2 is needed to reach PGHS stimulation, H2O2 becoming an inhibitor when used above this optimal concentration (Figure 3.6) (Deby 1988; Deby and Deby-Dupont 1981). The favourable role of an optimal concentration of peroxide has long remained not clearly understood. This is now explained by the mechanism of action of PGHS, which uses peroxide to break a covalent bond on the haemic structure of the enzyme, starting the chain reaction that will lead to arachidonic acid transformation. PGHS-1 would have a higher threshold for peroxide than PGHS-2 (Kulmacz and Wang 1995; Capdevila et al 1996) and a negative allosteric regulation by low arachidonic acid concentrations (Swinney et al 1997).

33

Role of Nitric Oxide and Peroxynitrite The Role of NO . The effect of nitric oxide (NO.) on PGHS remains controversial. For some authors, NO. is an inhibitor (Kanner et al 1992; Picot et al 1994; Curtis et al 1996), without effect (Tsai et al 1994a, 1994b) or a significant activator (Corbett et al 1993; Franchi et al 1994; Kelner and Uglik 1994; Riutta et al 1994). Direct activation of PGHS-1 by nitric oxide or NO. donors has been reported (Salvemini et al 1993, 1995a, 1995b; Hajjar et al 1995). However, the effect of NO. is still discussed, as results reporting activation of PGHS by NO. have proved to be difficult to reproduce, even with enzymes from the same sources (Capdevila et al 1995; Kulmacz and Wang 1995). The biochemical basis for direct cyclooxygenase activation by NO. has yet to be demonstrated convincingly. NO has only a very weak affinity for the ferric haem in PGHS-1, and PGHS–haem ligands generally inhibit, rather than activate, cyclooxygenase activity (Kulmacz and Lands 1985b). If NO. binds tightly to ferrous PGHS-1 haem (Capdevila et al 1995), the ferrous state is rapidly oxidized in air and is not observed during catalysis (Masferrer et al 1994). Published data of a PGHS activation by NO. or derived nitrosothiols report the use of high concentrations of NO., implausible when compared to the low micromolar levels expected in vivo. These properties make it very unlikely that NO. is a significant PGHS haem ligand at the low micromolar levels expected in vivo. Nitrosothiol formation on PGHS cysteine residues has been proposed as a mechanism for cyclooxygenase activation by NO. (Hajjar et al 1995), but the published data yield an implausibly high stoichiometry of PGHS nitrosothiol formed vs. NO. added, indicating that the issue needs to be re-examined (Kulmacz 1998). The Role of ONOO7 Taking into account a regulatory regime of PGHS activity by peroxides, it is conceivable that peroxynitrite (ONOO7), the coupling product of NO. and superoxide anion (Beckman et al 1990), is a PGHS activator (Landino et al 1996; Kulmacz 1998). The indirect cyclooxygenase activation via ONOO
requires appreciable levels of O2.
(Landino et al 1996), which is not produced during catalysis by purified PGHS unless particular cosubstrates, such as NADH or NADPH, are added (Kukreja et al 1986). The team of Marnett recently published that peroxynitrite (ONOO7) is an excellent oxidant of the haemic FeIII (Figure 3.7) (Gunther et al 1997; Goodwin et al 1998, 1999). GSH peroxidase is unable to inhibit this step as it does when ROOHs are used as triggers. This triggering role of ONOO7 can occur in some cells, producing simultaneously NO. and O.2
(endothelial cells, leukocytes). When ONOO7 is the triggering agent, the PGHS activation is inhibited by superoxide dismutase, which prevents the formation of ONOO7 from NO. and O.2
(Marnett et al 1999). These data provide a biochemical link between NO. biosynthesis and prostaglandin biosynthesis, and may explain the observations that NO synthase inhibitors reduce prostaglandin biosynthesis in inflammatory lesions in vivo (Marnett et al 1999). ASSOCIATED OXIDATIVE MECHANISMS Co-oxidation Marnett et al (1975) first described co-oxygenation as the oxidation of compounds by the arachidonic acid–PGH synthase

34

BIOSYNTHESIS AND METABOLISM

Figure 3.4 Mechanisms of PGHS-1 peroxidasic function. DH2, electron donor (co-factor) which can produce a radical species; grey ellipsoid, schematic view of the apo-protein, containing Tyr–OH (Tyrosine 385). ROOH is a hydroperoxide able to oxidize the haem. A, formation of the intermediate I (Cd I); B, formation of the intermediate II, after a long-range electron transfer (LRET), reducing the macrocycle radical, and forming a tyrosyl radical, at Tyr 385; C, abstraction of H. at carbon 13 of the eicosapolyenoic acid with formation of a lipidic radical and reduction of the tyrosyl radical, giving Cd II; D, reduction of Cd II to the resting enzyme by an electron donor, DH2, which becomes a radical (D.)

system. This phenomenon has been now termed co-oxidation. A wide variety of chemicals are co-oxidized during the reduction of PGG2 to PGH2. The best knowledge of the mechanisms involved in prostaglandin biosynthesis now provides a clearer understanding of the co-oxidation processes, which appear as important as those occurring in the cytochrome P-450 system. The co-oxidation occurs when the reduction of the ferryl state FeIV of the haem to the resting state, FeIII, requires the oxidation of an electron donor, DH (see Figure 3.4). A good example is given by oxidation of p-coumaric acid by PGHS and H2O2 (Bakovic and Dunford 1993): coumaric acid serves as a reducing substrate for PGHS through two one-electron oxidation steps. The electron donor DH can become a free radical, D., which initiates radical reactions, as concluded by Eling et al (1990), reviewing the results published by different research teams. For these authors, the common point in the different oxidative mechanisms intervening in co-oxidation (epoxidation, peroxidation, etc.) was the formation of free radicals. PGHS, as well as other peroxidases (horseradish peroxidase, thyroid peroxidase, myeloperoxidase), is able to oxidize various compounds and to transform some of these into toxic derivatives. Dix et al (1986) described a hydroperoxidedependent epoxidation of an aromatic molecule derived from

anthracene. Baumann et al (1983) explained the weak effect of cyclooxygenase inhibitors of several NSAIDs (phenylbutazone, aminopyrine) by a co-oxidation, transforming these molecules into less active metabolites, while other, more active NSAIDs were found to be less oxidized by PGHS activity (diclofenac, indomethacin). Electron spin resonance (ESR) techniques using spin-traps indicated that free radical intermediates are formed during the metabolism of certain drugs by PGHS. Phenytoin is oxidized into a toxic compound by PGHS, with a free radical step, as demonstrated by ESR (Kubow and Wells 1989). Samokyszyn et al (1995) and Freyaldenhoven et al (1996) published interesting results, obtained by ESR, on the generation of free radical species during the co-oxidation of all-transand 13-cis-retinoic acid.

Singlet Oxygen Generation Marnett et al (1974) early on observed that PGH synthase activity was associated with an ultra-weak red chemiluminescence, which was attributed to the formation of singlet oxygen. Cadenas et al (1983) later confirmed Marnett’s hypothesis.

PGH SYNTHASE-1 ACTIVITY AND RADICAL STATES

35

Figure 3.5 The cycles of oxido-reduction, and the central role of Tyr 385. DH, electron donor; grey ellipsoid, schematic view of the enzyme with tyrosine 385 and the haem; AA, arachidonic acid

Co-oxidation and Cancer The relationship between PGHS activity, generating free radicals and oxidation products, and carcinogenesis has been actively studied since the 1970s. The first compound to be studied was a-benzopyrene, a polycyclic aromatic hydrocarbon, which was easily oxidized into carcinogenic epoxides (Huberman et al 1976). Further studies demonstrated the carcinogenic role of arachidonic acid oxidation (Eling et al 1990). The presumably carcinogenic food antioxidant 2(3)-tert-butyl-4-hydroxyanisole (BHA) is oxidized into various metabolites, particularly 2-tert-butyl (1,4) hydroquinone (TBHQ) and its quinone, TBQ. ESR analyses showed that PGHS activity resulted in a substantially accelerated metabolism of TBHQ into TBQ, which was accompanied by the formation of superoxide anion, hydroxyl radical and H2O2. Both acetylsalicylic acid and indomethacin induced a significant decrease in TBQ excretion into urine. Co-oxidation by PGHS of the BHA metabolite TBHQ into TBQ, yielding reactive oxygen species, might therefore be responsible for the carcinogenic and toxic responses elicited by this antioxidant (Schilderman et al 1993). NSAIDs such as aspirin are considered to be anticancerous agents. Colorectal cancer prevention by aspirin is demonstrated by a recent epidemiological study (Giovannucci 1999). In studies on animals also, colorectal carcinogenesis induced by azoxymethane was inhibited by aspirin (Li et al 1999). NATURAL REGULATION OF PGHS-1 PGHS-1 has a cyclooxygenase turnover number of +3500 moles arachidonate/min/mole dimer (Kulmacz et al 1994; Barnett et al 1994), similar to that of PGHS-2 (Smith et al 2000). The formation of prostaglandins by PGHS-1 can be limited by the availability of the fatty acid substrate, by the concentration of

triggering hydroperoxides, by cellular and cytoplasmic factors and by the self-catalysed inactivation (suicide inactivation) of PGHS. Control of the Bioavailability of the Precursors PGHS-1 function requires exogenous arachidonic acid (Reddy and Herschman 1994) and seems to have a sigmoidal dependence on this free fatty acid (allosteric activation). At this level, a control can be exerted on the release of the free fatty acids and on their transport to the active site of the enzyme. The intracellular concentration of arachidonic acid available for prostanoid synthesis seems to equilibrate with the extracellular pool of the free fatty acids. The concentration of arachidonic acid is itself a regulator of PGHS, since arachidonic acid in excess, leading to increased PGHS activity, will also induce the simultaneous production of free radicals and inactivation of PGH synthase (Gonchar et al 1999). The release of free fatty acids appears to be controlled by hormonal and adrenergic stimulations, but also by inflammatory, immunological and even mechanical mechanisms. These actions may be explained by the activity of several enzymes, phospholipases A2, cholesterol esterase and triglyceride lipases. Phospholipases exist in different forms, the cytosolic (cPLA2) and secretory (sPLA2; two isoforms) phospholipases and a Ca2+independent phospholipase (iPLA2). Phospholipase A2 activity is under hormonal control and is stimulated by bradykinin, catecholamines, angiotensin II or histamine. Calcium mobilization acts on cytosolic and secretory phospholipase stimulation by a non-receptor pathway. It also appears that the cytosolic phospholipase A2 is activated by tumour necrosis factor-a (TNFa): this cytokine produces an activation of the mitogenactivated protein kinase cascade, which is followed by the phosphorylation of phospholipase A2 and the release of arachidonic

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BIOSYNTHESIS AND METABOLISM

acid (Hernandez et al 1999). From recent work on the interrelations between the phospholipases and PGHS activities, it appears that PGHS-1 and -2 use cPLA2 and sPLA2 but that iPLA2, which is the dominant phospholipase involved in arachidonic acid release, serves in cell membrane remodelling but does not induce PGHS activity (Murakami et al 1999; Fitzpatrick and Soberman 2001). The role of glucocorticoids still remains a matter of discussion. From much reported data, it appears that glucocorticoids may initially stimulate phospholipase A2 activity, while inhibiting it at a later phase. This biphasic behaviour may be attributed to different concentrations of a PLA2-modulating protein, possibly lipocortin, that accumulates during exposure to glucocorticoids (Prajgrod and Danon 1992). The glucocorticoids thus act as inhibitors by an indirect pathway: they induce the synthesis of the proteic phospholipase inhibitors, lipocortin and its related polypeptides, macrocortin and renocortin (Douglas et al 1992). Glucocorticoids and lipocortin-1 (lipocortin peptide 2-26) may also act by inhibiting the expression of phospholipase A2 and PGHS, but this last effect has only been described for PGHS-2 (Masferrer and Seibert 1994; Masferrer et al 1994; Minghetti et al 1999; Ferreira et al 1997). Cholesterol esterase is subject to hormonal control. Its activity of free fatty acid release from cholesteryl esters may be partially controlled by the activity of lecithin cholesterol acyl transferase (LCAT), which can use the free fatty acids carried by HDL for cholesterol esterification. The activation of the triglyceride lipases that release free fatty acids from the triglyceride reserves is dependent on the activation of adenyl cyclase, a mechanism that may be important in the kidney, where the triglycerides are rich in arachidonic acid. The transport of free fatty acids in plasma is mainly regulated by albumin, with a molar ratio of free fatty acid to albumin not exceeding 4. However, free fatty acids are also carried by other plasma compounds, cyclodextrin, haptoglobin and lipoproteins. Heparin, by releasing lipoprotein lipase, induces a lysis of LDL triglycerides, increasing the plasma concentrations of free fatty acids. Heparin also weakens the bond between free fatty acids and albumin, making them more available for PGHS activity (Deby 1988).

Availability of the Triggering Agent (ROOH or ONOO7) Hydroperoxide dependence is now a firmly established fact (Chen et al 1999). At the end of the 1970s we demonstrated the enhancer role of an optimal concentration of H2O2 on seminal vesicle homogenates (Deby and Deby-Dupont 1980; see Figure 3.6). Catalase, which specifically destroys H2O2, decreases prostaglandin synthesis in activated leukocytes, while U937 cells incubated with labelled arachidonic acid show a strong enhancement of prostaglandin formation when H2O2 is added to the system (Marshall et al 1987). An important intracellular inhibitor is GSH peroxidase, acting through its peroxidasic activity, which eliminates H2O2 and other lipoperoxides necessary for the triggering of PGHS (see above). The addition of GSH-peroxidase to the PGHS–arachidonic acid solution significantly decreases the synthesis of PGH2 (Marshall et al 1987), a fact that is explained by the destruction of both lipid and hydrogen peroxides. GSH peroxidase may also be inactivated by scavenging PGG2, the hydroperoxide derived from PGHS activity that could further activate other PGHS molecules and involve a chain reaction (Marshall et al 1987). Margalit et al (1998) observed that elevated GSH levels inhibit prostanoid production in in vivo models, providing evidence for the role of GSH in the regulation of prostanoid biosynthesis.

Figure 3.6 Effects of increasing concentrations of hydrogen peroxide (H2O2) on the biosynthesis of prostanoids (PGs) by PGHS-1 (bull seminal vesicles). The rate of prostanoid formation is expressed as a percentage of the control value (absence of H2O2). Role of the addition of catalase or superoxide dismutase (SOD) on the PG biosynthesis: 1078–1077 M H2O2 (arrow I), no effect of catalase or SOD; 1077–1076 M H2O2 (arrow II), no effect of catalase, increasing effect of SOD; 1076–1075 M H2O2 (arrow III), decreasing effect of catalase, no effect or slightly inhibiting effect of SOD; 1075–1072 M H2O2 (arrow IV), increasing effect of catalase and decreasing effect of SOD

Various physiological effects that are produced by hydrogen peroxide are thus mediated by cyclooxygenase activation and are diminished or abolished by NSAIDs, e.g. tracheal smooth muscle contractions in guinea-pig (Rhoden and Barnes 1989; Gao and Vanhoutte 1993) and human airway smooth muscle tone enhancement (Rabe et al 1995). Human leukocytes produce activator hydroperoxides in quantities sufficient to enhance prostaglandin synthesis in cells (Marshall and Lands 1986). The activators appeared to be partly H2O2 and partly a lipid hydroperoxide. These effects are completely inhibited by glutathione peroxidase (0.5 U/ml) and partially by catalase (500 U/ml) (Marshall et al 1987). Low levels of H2O2 enhance arachidonic acid-induced platelet aggregation by cyclooxygenase activation, which is inhibited by aspirin (Pratico et al 1991). H2O2 alone does not induce aggregation. H2O2 thus plays a role in the activation of cyclooxygenase by providing an adequate peroxide tone (Hecker et al 1991). The role of antioxidants on PGHS activity could thus be largely explained in this perspective of a regulation role of lipoperoxides. By lowering the lipoperoxide levels, antioxidants such as atocopherol (vitamin E) could inhibit PGHS activity. But the results reported in the literature are divergent, and the role of tocopherol on PGHS remains a matter of discussion. Panganamala et al (1977) found no effects of DL-a-tocopherol, although Seeger et al (1988) observed that inhibition was obtained using Trolox, a water-soluble derivative of tocopherol. However, strong antioxidants such as a-naphthol, guaiacol, NDGA and propyl gallate are inhibitors (Panganamala et al 1977).

Regulation by Cell and Plasma Factors In vivo Experiments Intravascular injection of high doses of arachidonate is normally necessary to obtain physiological events (fall of blood pressure and

PGH SYNTHASE-1 ACTIVITY AND RADICAL STATES

37

Figure 3.7 Role of peroxynitrite (ONOO7) in the formation of the p-cation radical on PGH synthase

myocardial symptoms) that appear to be linked to the production of prostanoids. However, heparinization, short-duration hypoxia and low amounts of plasmatic free haemoglobin increase the hypotensive effect of arachidonic acid injection, that hypotensive effect being accompanied by significant rises of plasma prostanoid concentration (Deby and Deby-Dupont 1981; Deby et al 1981). These observations demonstrate that control mechanisms of PGHS are normally present in vivo. A control is also exerted at the level of the availability of the precursors of prostanoids, the free fatty acids. In man, plasma levels of unesterified arachidonic acid are normally low (a few mg/ml), but important increases of this free fatty acid were measured in pathological and stress conditions (Deby-Dupont et al 1983).

Effects of Blood Components Plasma and cells of different mammalian species have inhibitory properties on PGHS, attributed mainly to albumin (Heinsohn et al 1987) but also to other proteins such as haptoglobin and lipoproteins (LDL). Inhibitors of PGHS are present in tissue extracts (e.g. stomach, placental extracts) but are often attributed to the cells present in these tissues. The inhibitory properties of plasma proteins still remain under discussion, as it seems difficult to explain an intracellular inhibition of PGHS by intravascular proteins that cannot enter the cells. An action on cell membrane receptors would be an explanation for these inhibitory phenomena. PGHS inhibitors are present in cells such as platelets, where the inhibitory capacity would be due to b-thromboglobulin. Numerous stimulating factors of PGHS are present in blood, one of them being free haemoglobin. Another important stimulating factor of blood is heparin, which acts by releasing arachidonic acid from its albumin-binding sites and by inhibiting GSH-peroxidase and LCAT activities. A stimulating effect on PGHS is also attributed to hypoxia, by way of plasmatic uric acid increase. The stimulating activity of this purine can be explained by its free radical scavenging properties (Deby et al 1981; Deby 1988).

Self-catalysed Inactivation: the Suicidal Character of PGH Synthase The phenomenon of autoinactivation was first described during the 1970s (Smith and Lands 1972; Egan et al 1976; Ohki et al 1979). One molecule of PGHS can only run a limited number of cycles, thus transforming a limited number of substrate molecules. This number was estimated, in conditions in vitro, at 103 moles PGH produced per molecule of PGHS (Marshall et al 1987). Studies performed with human endothelial cells overexpressing PGHS-1 allowed us to quantify the kinetics of PGHS-1 turnover and indicated that approximately 30% of PGHS-1 was degraded during each catalysis-induced autoinactivation, so that the extent of prostanoid synthesis would be governed by the level of PGHS-1 mass (Sanduja et al 1994). Egan et al (1976) proposed that the inactivating factors were oxygen-centred radicals. The free radical species are thus responsible for enzyme inactivation but, as the enzymatic activity of PGHS produces free radicals at different steps of its activity, this free radical production leads to the autoinactivation of the enzyme. This autoinactivation explains the role played by antilipoperoxidants and radical scavengers on prostanoid production, which were contradictorily described as inhibitors or stimulators of PGHS. The presence of free radical scavengers such as phenols (Egan et al 1976, 1978), lipoic acid (Marnett and Wilcox 1977), uric acid (Deby et al 1981) and antioxidants (tocopherol, NDGA) prolongs enzyme activity more or less considerably according to their efficiency (affinity coefficient for radicals, concentrations, etc.). Antilipoperoxidant and radical scavenging activities are often associated. A free radical scavenger may thus protect PGHS from autoinactivation while inactivating the lipoperoxides needed to trigger the enzymatic activity. However, the active site of PGHS being hydrophobic, its access is only possible to lipophilic molecules or to hydroxyl radicals (.OH) or small alkoxyl radicals (RO.). Lipophilic antioxidants will thus protect the enzyme from inactivation by free radicals, but will also reach the active site and affect the peroxide concentration, while hydrophilic antioxidants will only inactivate free radicals in the aqueous phase without altering the lipoperoxide activity in the lipophilic phase. Substances which exert a scavenging activity only against hydroxyl

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and oxy-ferryl radicals, without inhibition of lipoperoxidation, thus protect PGHS and significantly enhance prostanoid synthesis. A typical example is given by uric acid, which traps .OH and oxoferryl radicals but is inefficient against peroxidation cycles (Deby 1988). Uric acid was demonstrated to enhance prostaglandin synthesis in vitro (Deby et al 1981) and in vivo (Bourgain et al 1982). A new explanation of the self-inactivation of the enzyme has recently been proposed, based on kinetic considerations: there is an unbalanced process which paralyses the activity of PGHS. But why does the self-inactivated enzyme remain definitively in this state? Some authors prefer the oxidative theory, where strong oxidants, such as free radicals and singlet oxygen, would destroy the active sites. But the main impact of oxidative agents seems to be exerted on the haem. Deep haem spectral changes were observed during suicidal inactivation (Tsai et al 1992). Recently, Wu et al (1999) explained the haem spectral changes: the Cd II is first converted in a faster step (0.5–2/s) into a new compound, intermediate III, which undergoes a subsequent slower (0.01–0.05/ s) transition to a terminal species. These results were obtained by the study of the kinetics of formation of intermediate I (Cd I) and intermediate II during reaction of PGHS. The ability of compounds to protect against hydroperoxide-induced inactivation correlates directly with their ability to act as reducing substrates. Hydroquinone, an excellent reducing substrate, protected against hydroperoxide-induced inactivation when present in less than threefold molar excess over hydroperoxide (Markey et al 1987). The presence of a highly efficient hydroperoxide-reducing activity appears absolutely essential for the protection of the cyclooxygenase capacity of PGHS. We may conclude that the mechanisms of control of PGHS are complex, and remain incompletely elucidated. This control is modulated in an interdependent manner by both the peroxide level and the supply of arachidonate. Moreover, it is clear that there are differences but also resemblances in the regulatory mechanisms that apply to PGHS-1 or to PGHS-2, this latter being induced within the context of inflammatory response, resulting in an exacerbated production of prostanoids. There is thus a need to control the two PGHSs separately, which can only be performed by acting on the activity of the enzyme without affecting the bioavailability of the prostanoid precursor (Fitzpatrick and Soberman 2001).

In experiments on macrophages, Masferrer et al (1994) observed that both PGHS-1 message and PGHS-1 protein levels were unaffected either by adrenalectomy or by dexamethasone administration, unlike PGHS-2. The expression of PGHS-1 appears to be stable, and mRNA for PGHS-1 is found in most cells at relatively stable concentrations, so that PGHS-1 is considered as a constitutive (‘‘housekeeping’’) enzyme supplying the low prostanoid levels indispensable for basal functions of the cells and the organisms (Smith et al 1996; Kulmacz 1998). PGHS1 and -2 are encoded by separate genes, located on different chromosomes. In contrast to PGHS-2, PGHS-1 expression is not induced in inflammatory situations. The two PGHSs are often coexpressed in the same cells but seem to function independently, channelling prostanoids to the nucleus and to the extracellular milieu, respectively. PGHS-1, like PGHS-2, is attached to the membrane, anchored to one leaflet of the lipid bilayer through the hydrophobic surfaces of amphipathic helices, and not through transmembrane motifs. PGHS-1 is especially inhibited by aspirin, acting via the inhibition of platelet thromboxane A2 formation, lowering the relative risk for mortality from cardiovascular disease. It thus seems that PGHS-1 activity is not importantly regulated at the level of its gene expression. PGHS-1 is constitutively present, expressed at constant levels throughout the cell cycle, in most tissues but not within all cells of a tissue. However, it is an oversimplification to consider that PGHS-1 is only constitutive, as PGHS-1 levels have been demonstrated to change during development, to be downregulated in endothelial cells and upregulated in mast cells under the influence of specific factors (Smith et al 1996). The role of glucocorticoids on PGHS-1 regulation remains a matter of discussion. These hormones have an inhibitory effect on PGI2 production in endothelial and mesangial cells, this effect being generally attributed to their activity on phospholipase A2 and thus to the availability of arachidonic acid. An effect of glucocorticoids on PGHS expression has been described only for COX-2 (Masferrer et al 1994; Minghetti et al 1999; Ferreira et al 1997). However, in 1999, it was reported that the glucocorticoids downregulated PGHS-1 expression in foetal pulmonary artery endothelial cells, as demonstrated by the decrease of COX-1 mRNA expression in these cells when they are treated by dexamethasone (Jun et al 1999). Glucocorticoids may downregulate COX-1 expression by acting on the glucocorticoid receptor and on gene transcription.

BIOSYNTHESIS OF PGHS-1 The two PGHSs serve separate physiological functions, as demonstrated by studies with knockout mice for either PHGS-1 or PGHS-2 (Langenbach et al 1999), and have segregated biosynthetic pathways (Smith et al 2000). PHGS-1 is expressed constitutively and assumes the regulated production of prostanoids that are needed for normal physiological functions, such as haemostasis. The subcellular location of PGHS-1 appears similar to that of PGHS-2 although the latter appears more concentrated in the nuclear envelope (Morita et al 1995a). PGHS-1 can be detected in most cells, but is preferentially expressed at high levels in endothelial cells, monocytes, platelets, renal collecting tubules and seminal vesicles (Smith and DeWitt 1996). The gene for PGHS-1 is approximately 22 kilobase pairs and contains 11 exons (Kraemer et al 1992). Typical of developmentally regulated ‘‘housekeeping’’ genes, the PGHS-1 gene lacks a TATA box. PGHS-1 expression does not vary greatly in adult animals, so that study on its transcriptional regulation is difficult. Virtually nothing is known about the details of the regulation of PGHS-1 gene expression, contrary to the large amount of data that has accumulated for PGHS-2 expression (Smith and Dewitt 1996; Smith et al 1996, 2000).

PHARMACOLOGICAL REGULATION A major method of PGHS control is the use of NSAIDs, which act through their ability to inhibit cyclooxygenase activity, reducing the synthesis of prostaglandins, prostacyclins and thromboxane (Vane 1987). Acetyl salicylic acid (aspirin) remains the model of these NSAIDs. The mechanism of acetylation of PGHS active site has been the subject of numerous studies (DeWitt et al 1990; Shimokawa and Smith 1992; Loll et al 1995). At low doses (40 mg/day), aspirin selectively inhibits production of thromboxane A2 without affecting prostacyclin. This is explained by an irreversible acetylation of PGHS and the impossibility for platelets to regenerate PGHS, while the systemic plasma aspirin concentration is likely to be too low to affect prostacyclin synthesis. However, the inhibition activity of most of the NSAIDs does not distinguish between PGHS-1 and PGHS-2. Attempts are under way to produce NSAIDs with a specific inhibition activity on PGHS-2 (Smith et al 2000; Fitzpatrick and Soberman 2001). Callan et al (1996) presented a kinetic model for the comparison of NSAID efficiencies.

PGH SYNTHASE-1 ACTIVITY AND RADICAL STATES CONCLUSIONS Since the first description of its activity in the beginning of the 1960s, PGHS has been isolated to a high degree of purity. Its amino acid sequence has been elucidated and its gene identified. PGHS-1 could be distinguished from its parent enzyme, PGHS-2, and the specific characteristics of PGHS-1 as a ‘‘housekeeping enzyme’’, not induced by an inflammatory response, could be defined. Because of its isolation and in vitro studies of pure PGHS, the molecular mechanisms of its cyclooxygenasic and peroxidasic activities could be described, underlining its connections with haemic peroxidases. PGHS belongs to the family of ‘‘radical proteins’’ and is the source of an important free radical production, which leads to an autoinactivation of the enzyme. However, a major problem remains the pharmacological regulation of its activity. As the two PGHS enzymes are free radicalgenerating enzymes which may be responsible for radical chain reactions and potentially dangerous co-oxidations of neighbouring molecules, it would be useful to inhibit the two enzymes separately by free radical scavengers. However, this remains an unsolved problem. REFERENCES Badwey JA and Karnovsky ML (1980) Active oxygen species and the functions of phagocytic leukocytes. Annu Rev Biochem, 149, 695–726. Bakovic M and Dunford HB (1993) Kinetics of the oxidation of pcoumaric acid by prostaglandin H synthase and hydrogen peroxide. Biochemistry, 32, 833–840. Bakovic M and Dunford HB (1994) Intimate relation between cyclooxygenase and peroxidase activities of prostaglandin H synthase. Peroxidase reaction of ferulic acid and its influence on the reaction of arachidonic acid. Biochemistry, 33, 6475–6482. Bakovic M and Dunford HB (1996a) Reactions of prostaglandin endoperoxide synthase and its compound I with hydroperoxides. J Biol Chem, 271, 2048–2056. Bakovic M and Dunford HB (1996b) pH and temperature dependence of the rate of compound I formation from the reaction of prostaglandin endoperoxide synthase with hydrogen peroxide. Biochem Cell Biol, 74, 117–124. Barnett J, Chow J, Ives D et al (1994) Purification, characterization and selective inhibition of human prostaglandin G/H synthase 1 and 2 expressed in the baculovirus system. Biochim Biophys Acta, 1209, 130– 139. Baumann J, von Bruchhausen F and Wurm G (1983) Decreasing inhibitory potency of prostaglandin synthetase inhibitors during their cooxidative metabolism. Studies on amino-phenols, pyrazolon derivatives and 1,3-diphenylisobenzofuran. Pharmacology, 27, 267–280. Beckman JS, Beckman TW, Chen J et al (1990) Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA, 87, 1620–1624. Bergstro¨m S, Danielsson H and Samuelsson B (1964) The enzymatic formation of prostaglandin E from arachidonic acid. Biochim Biophys Acta, 90, 207–219. Bhattacharyya DK, Lecomte M, Rieke CJ et al (1996) Involvement of arginine 120, glutamate 524, and tyrosine 355 in the binding of arachidonate and 2-phenylpropionic acid inhibitors to the cyclooxygenase active site of ovine prostaglandin endoperoxide H synthase-1. J Biol Chem, 271, 2179–2184. Bourgain RH, Deby C, Deby-Dupont G and Andries R (1982) Enhancement of arterial thromboformation by uric acid, a free radical scavenger. Biochem Pharmacol, 31, 3011–3013. Cadenas E, Sies H, Nastainczyk W and Ullrich V (1983) Singlet oxygen formation detected by low-level chemiluminescence during enzymatic reduction of prostaglandin G2 to H2. Hoppe Seylers Z Physiol Chem, 364, 519–528. Callan OH, On-Yee S and Swinney DC (1996) The kinetic factors that determine the affinity and selectivity for slow binding inhibition of human prostaglandin H synthase 1 and 2 by indomethacin and flurbiprofen. J Biol Chem, 271, 3548–3554.

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4 Regulation and Function of Prostaglandin Synthase 2/Cyclooxygenase II Harvey R. Herschman David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

The prostaglandins (PGs) play central roles in acute and chronic inflammatory responses. Pharmacological inhibition studies of prostanoid synthesis have provided enormous insight into the physiological and pathophysiological roles of the prostanoids. Vane (1971) reported that aspirin and other nonsteroidal antiinflammatory drugs (NSAIDs) exert their biological effects by blocking prostaglandin synthesis. This landmark experiment implicated prostanoids in thermoregulation, pain perception and inflammatory responses, because of the antipyretic, analgesic and antiinflammatory effects of NSAIDs, and in blood clotting, because of a major aspirin side effect— increased clotting time. Prostanoids and the leukotrienes are derived from C20 fatty acids, principally arachidonic acid. Following appropriate stimulation, cells activate a phospholipase which releases free arachidonic acid from membrane phosphoglycerolipid stores. Prostaglandin synthase, or cyclooxygenase, converts arachidonic acid to prostaglandin H2 (PGH2), the common precursor to all prostanoids. Prostaglandin synthase catalyses two concerted reactions. Initially, arachidonic acid is cyclized to PGG2 by a bis-oxygenation. PGG2 is converted by a hydroperoxidase reaction, at a distinct enzyme site, to PGH2. PGH2 serves as substrate for production of the PGE2, PGF2a, PGD2, PGI2, TXA2, etc. Cell-type-specific prostaglandin production is the consequence of cell-type-specific expression of distinct prostaglandin synthases—PGE2 synthase, PGD2 synthase, etc.

THE DOGMA WITH RESPECT TO INDUCED PROSTAGLANDIN SYNTHESIS Until the mid-1980s it was thought that: (a) the rate-limiting step in prostaglandin synthesis following an inflammatory insult is phospholipase activation to release arachidonic acid from membrane stores, and (b) constitutive cyclooxygenase in tissues is sufficient to convert released arachidonic acid to PGH2. Whiteley and Needleman (1984) demonstrated that PGE2 production induced with conditioned medium in human fibroblasts could be blocked by either cycloheximide or actinomycin D, suggesting that protein translation and mRNA transcription were necessary. Moreover, an increase in microsomal cyclooxygenase Vmax suggested that ligand stimulation induced an increase in cyclooxygenase activity. Similarly, it was found that platelet derived growth factor (PDGF)-induced PGE2 accumulation in murine fibroblasts was blocked by cycloheximide (Habenicht et al 1985) and epidermal growth factor (EGF)-induced PGE2 production in murine osteoblast-like MC3T3E1 cells was blocked by cycloheximide and actinomycin D (Yokota et al 1986). Moreover, if constitutive cyclooxygenase in 3T3 cells is inactivated by aspirin treatment, PDGF-induced PGE2 synthesis is hardly affected, suggesting that pre-existing cyclooxygenase does not play a significant role in mitogen-induced prostaglandin production (Habenicht et al 1985).

EVIDENCE FOR INCREASED CYCLOOXYGENASE EXPRESSION IN RESPONSE TO LIGAND STIMULATION PURIFICATION, CHARACTERIZATION AND CLONING OF SHEEP SEMINAL VESICLE CYCLOOXYGENASE Sheep seminal vesicles were the primary source for cyclooxygenase purification and biochemical characterization (Hemler et al 1976; Miyamoto et al 1976). Cyclooxygenase, a homodimer of two 70 kDa subunits, is found in the endoplasmic reticulum and nuclear membrane. Oligonucleotide probes, prepared from amino acid sequence data, were used to clone sheep seminal vesicle cyclooxygenase (DeWitt et al 1988; Merlie et al 1988; Yokayama et al 1988). Ovine cyclooxygenase encodes a 576 amino acid protein. Human and murine sheep seminal vesicle cyclooxygenase orthologues were later cloned. The Eicosanoids. Edited by Peter Curtis-Prior. &2004 John Wiley & Sons, Ltd: ISBN 0 471 48984 0

PGI2 induction in endothelial cells by interleukin-2 (IL-2) is inhibited by actinomycin D and cycloheximide (Frasier-Scott et al 1988). Using an affinity-purified antibody to sheep seminal vesicle cyclooxygenase, a substantial increase in immunoreactive material was observed in IL-2-treated cells. Similarly, ligand-stimulated MC3T3-E1 murine osteoblast-like cells demonstrated an increase in microsomal cyclooxygenase activity which could be immunoprecipitated by antisera to sheep seminal vesicle cyclooxygenase. Raz et al (1988) used antisera to sheep seminal vesicle cyclooxygenase to demonstrate that IL-1 treatment of human fibroblasts stimulated incorporation of radioactive methionine into immunoprecipitable cyclooxygenase. This immunoprecipitated cyclooxygenase was reported to be similar to sheep seminal vesicle cyclooxygenase, based on N-terminal amino acid sequencing and

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endogycosidase H treatment. Ras et al (1989) concluded that IL1-induced PGE2 production in human dermal fibroblasts is the consequence of ligand-induced cyclooxygenase synthesis. Following luteinizing hormone (LH) treatment of rats, granulosa cells produce large amounts of immunoreactive cyclooxygenase (Wong et al 1989). Although LH-stimulated cyclooxygenase accumulation was blocked by a transcriptional inhibitor, cyclooxygenase message was not increased in LHtreated cells. Wong et al (1989) concluded that this LH-induced increase in cyclooxygenase did not result from increased transcription of the cyclooxygenase gene. Endotoxin/lipopolysaccharide (LPS) stimulation of human monocytes also led to increased microsomal cyclooxygenase activity and immunoprecipitable cyclooxygenase. LPS-induced PGE2 production, elevated microsomal cyclooxygenase activity and increased immunoprecipitable cyclooxygenase protein were all inhibited by dexamethasone, a potent inhibitor of prostaglandin production (Fu et al 1990). These authors suggested that monocytes ‘‘. . . may contain two pools of cyclooxygenase, each with a differential sensitivity to endotoxin or dexamethasone’’. Masferrer et al (1990) demonstrated that peritoneal macrophages isolated from endotoxin-treated mice had increased levels of cyclooxygenase activity and—when incubated with radioactive amino acids in culture—had increased immunoprecipitable cyclooxygenase. Both induced cyclooxygenase activity and immunoprecipitable radioactive cyclooxygenase protein were blocked by dexamethasone. Chicken embryo fibroblasts expressing the v-src oncogene also produce elevated levels of immunoprecipitable cyclooxygenase (Han et al 1990). Thus, by 1990, it was clear that mitogens (PDGF, EGF), inflammatory agents (LPS), hormones (LH), cytokines (IL-1) and oncogenes (v-src) could induce transcription- and translation-dependent accumulation of cyclooxygenase activity and immunoprecipitable cyclooxygenase. EVIDENCE FOR A SECOND CYCLOOXYGENASE GENE Rosen et al (1989) demonstrated that anti-sheep seminal vesicle cyclooxygenase antibodies could precipitate a 12-fold increase in immunoprecipitable, radioactively labelled protein from serumstimulated sheep tracheal epithelial cells in culture. They probed northern blots from serum-stimulated and control cells with a cDNA probe for the 2.8 kb cyclooxygenase message. At high stringency, they saw no increase in this message. At lower stringency, they observed a 4.0 cross-reacting RNA species present in serum-treated cells, absent in untreated cells. They suggested that the 4.0 mRNA ‘‘may be derived from a distinct, cyclooxygenase-related gene’’ and ‘‘. . . may encode for a protein with cyclooxygenase activity’’. The following year, Masferrer et al (1990) suggested that two cyclooxygenase genes may encode two cyclooxygenase proteins, one regulated by glucocorticoids. CLONING THE COX-2 cDNA Despite circumstantial evidence for an inducible cyclooxygenase in hormone-, cytokine-, growth factor-, mitogen- and oncogenetreated cells, identification of the inducible cyclooxygenase did not result from a directed search for a second cyclooxygenase in stimulated cells. Each laboratory that reported the cloning of a second, inducible cyclooxygenase in 1991 was searching for genes that modulate growth-factor or oncogene-stimulated proliferative responses. Simmons et al (1989) identified cDNAs induced in chicken embryo fibroblasts in response to v-src expression or serum

stimulation. Xie et al (1991) sequenced one of these genes, CEF147, whose open reading frame encoded a protein with 59% homology to sheep seminal vesicle cyclooxygenase. They suggested that this gene, which they named miPHSch for ‘‘mitogeninducible PGSchicken’’, might be a new cyclooxygenase and pointed out the similar size of the CEF-147 message and the 4.1 kb message described by Rosen et al (1989). However, they could not be certain of their suggestion because, at the time their paper was published, the chicken orthologue of sheep seminal vesicle cyclooxygenase had not been cloned. Consequently, they could not be certain whether miPHSch was a distinct, inducible cyclooxygenase or the chicken orthologue of the previously cloned ovine/human/murine cyclooxygenase. Our laboratory was interested in identifying ‘‘immediate early’’ or ‘‘primary response’’ genes that mediate the transition from G0arrested cells to proliferating cells in response to mitogenic stimulation. We used differential screening of a cDNA library prepared from mitogen-treated cells to identify cDNAs for mitogen-stimulated genes. The library was screened with probes prepared from mRNAs isolated from G0 cells and from tetradecanoyl phorbol acetate (TPA)-treated cells to identify TPA-induced sequences, or TIS genes (Lim et al 1987). In 1991, we reported (Kujubu et al 1991) that the sequence of the TIS10 ‘‘immediate early’’ gene had striking sequence homology to the previously cloned murine cyclooxygenase cDNA. In contrast to the studies with miPHSch, we could conclude unambiguously that the 2.8 kb murine cyclooxygenase message and the 4.0 TIS10 message encoded highly related proteins that were derived from distinct genes. The TIS10/inducible cyclooxygenase cDNA was induced by EGF, forskolin and serum, in addition to TPA, in murine fibroblasts. The constitutive cyclooxygenase gene encoding the 2.8 kb message is now called COX-1 and the inducible cyclooxygenase gene encoding the 4.0 kb message is called COX-2. COX-2 mRNA has multiple AUUUA sequences (Xie et al 1991; Kujubu et al 1991), associated with rapid mRNA degradation. O’Banion et al (1991) described a v-src- and serum-inducible cyclooxygenase immunoprecipitable with antiserum to sheep seminal vesicle cyclooxygenase in murine fibroblasts. Induction was suppressed by glucocorticoids. Low-stringency hybridization identified a glucocorticoid-sensitive 4.0 kb message that crossreacted with the 2.8 kb probe encoding ovine seminal vesicle cyclooxygenase present in the serum-treated fibroblasts. They concluded, based on ‘‘stringency analysis and preliminary sequence data’’, that this RNA ‘‘arises from a gene distinct from that transcribed into the previously cloned 2.8 kb PGHS cDNA’’ and demonstrated that the product of in vitro translation of the 4.0 kb message could be immunoprecipitated by antiserum to sheep seminal vesicle cyclooxygenase. O’Banion et al (1991) concluded that their data ‘‘suggests that two distinct and differentially regulated cyclooxygenase species exist’’. In 1992, O’Banion et al (1992) also reported the sequence of the murine COX-2 cDNA. Using sequence data from the murine COX-2 cDNA, Hla and Neilson (1992) and Jones et al (1993) cloned the human COX-2 cDNA and demonstrated its induction in various cell types. The COX-2 cDNA was later cloned from chicken, rat, dog, guinea-pig, sheep, rabbit, mink, horse, cow and trout. THE COX-2 AND COX-1 GENES COX-1 is constitutively expressed in most cells. In contrast, COX2 is inducible by a wide range of inducers, in a variety of cell types (Herschman 1991). ‘‘Immediate early genes’’ are usually small compared to other genes (Herschman 1996). As we thought might be the case, the murine COX-2 gene is indeed smaller than the COX-1 gene. The murine COX-2 gene is found in an 8 kb DNA segment (Fletcher et al 1992), even though murine COX-2 mRNA

PROSTAGLANDIN SYNTHASE 2/CYCLOOXYGENASE II is 4.0 kb and murine COX-1 mRNA, encoded from a 22 kb gene (Yokoyama and Tanabe 1989), is only 2.8 kb. The COX-2 gene has only 10 exons and 9 introns; a COX-1 exon coding an N-terminal hydrophobic leader sequence is not present in the COX-2 gene. The remaining exons of the COX-1 and COX-2 genes and the splice sites at the exon–intron junctions are similar for COX-1 and COX-2, with the exception of their final 30 /C-terminal exons. The last, longer exon of the COX-2 gene encodes the longer 30 untranslated region of the COX-2 message. The major differences between the COX-2 and COX-2 transcription units are the absence of the first exon found in COX-1 and the consistently smaller introns in the COX-2 gene, as compared to the COX-1 gene. COX-1 and COX-2 map to distinct chromosomes, both in mouse and human. The sequences upstream of the transcriptional start sites of the COX-1 and COX-2 genes bear no resemblance to one another. In contrast, the upstream regulatory regions of the murine and human COX-2 genes have substantial sequence similarity, sharing a number of putative transcription factor binding sites. THE COX-1 AND COX-2 PROTEINS The COX-1 protein is 602 amino acids; the COX-2 protein is 604 amino acids. COX-1 and COX-2 share about 80% amino acid identity/similarity, differing most extensively at their amino- and carboxyl-terminal ends. COX-1 has a 17 amino acid hydrophobic sequence at its N-terminal that is not shared by COX-2. Conversely, COX-2 has an 18 amino acid insertion, located near its carboxyl terminal, that is not found in COX-1. COX-1 and COX-2 are membrane-bound homodimers, found in the endoplasmic reticulum and nuclear membrane. COX-1 (Picot et al 1994) and COX-2 (Kurumbail et al 1996; Luong et al 1996) share remarkable similarities in tertiary structure. Both are bound to the membrane by amphipathic helices that form monotopic membrane attachment sites. The helices comprising the COX-1 and COX-2 membrane binding sites contain the greatest divergence in their sequences, with the exception of the unique N-terminal and C-terminal peptide insertions. The cyclooxygenase and peroxidase active sites are located at distinct regions of the proteins. Arachidonate gains access to the COX-1 and COX-2 cyclooxygenase sites by a hydrophobic channel adjacent to the membrane binding sites. The COX-2 active site contains a ‘‘side pocket’’ not present in the COX-1 active site. The serine 530 residue that is the site of COX-1 aspirin acetylation is conserved at serine 516 in COX-2, and is also subject to irreversible aspirin acetylation, leading to COX-2 inhibition. COX-1 tyrosine 385, essential for cyclooxygenase activity, is conserved as tyrosine 371 in COX-2, and is also essential for activity. The sequences required for haem binding (TIWLREHNRV and RGLGF) are also conserved in COX-1 and COX-2. For additional information about COX-1 and COX-2 membrane association, substrate and inhibitor accessibility to the COX-1 and COX-2 active sites, COX-1 and COX-2 kinetic parameters and mechanisms of catalysis by COX-1 and COX-2, readers are referred to reviews that emphasize COX-1 and COX-2 biochemistry, enzymology and pharmacology (e.g. Smith et al 2000). WHY ARE TRADITIONAL NSAIDs EFFECTIVE INHIBITORS OF BOTH COX-1 AND COX-2? COX inhibitors were first identified by their ability to inhibit prostaglandin production in vitro by COX-1 purified from sheep seminal vesicles. Active compounds were then tested for efficacy in

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in vivo inflammation models. Candidates effective in inhibiting inflammation and demonstrating appropriate pharmacokinetic and pharmacodynamic properties were then brought to clinical trial. Since the animal model, in retrospect, required COX-2 inhibition, compounds that ‘‘passed’’ both the in vitro and in vivo preclinical tests were, by definition, both COX-1 and COX-2 inhibitors. Lead compounds that could preferentially inhibit COX-2 were identified only when COX-2 was used to screen chemical libraries for inhibitors that could distinguish between COX-1 and COX-2. Subsequent refinement led to development of the blockbuster drugs Celebrex and Vioxx, with additional COX-2 pharmacological agents on the horizon. THE COX-1/COX-2 PARADOX: WHY ARE THERE TWO CYCLOOXGENASES? Until the discovery of COX-2, the dogma for prostanoid synthesis suggested that the rate-limiting step in ligand-stimulated prostaglandin synthesis was the activation of phospholipase to release arachidonic acid from membrane stores. Constitutive cyclooxygenase activity in cells—now known as COX-1—was thought to be present in excess, and available to convert the stimulus-released arachidonic acid to PGH2. With the discovery of an inducible cyclooxygenase, COX-2, the question arose as to why there are two cyclooxygenases. If constitutive cyclooxygenase is available to convert arachidonate to PGH2, what is the role of an inducible cyclooxygenase? Moreover, if arachidonate released in cells following ligand stimulation is available to COX-1 and COX-2, why do COX-2-specific inhibitors such as Celebrex and Vioxx work? How can the COX-2-specific inhibitors prevent stimulusinduced prostaglandin synthesis if COX-1 is available to convert arachidonate acid to PGH2? To determine whether arachidonic acid availability is functionally restricted to distinct COX isozymes, Reddy and Herschman (1994) examined the production of prostaglandins in activated cells unable to synthesize COX-2. Antisense oligonucleotides (ASOs) directed against COX-2 mRNA were used to block COX-2 protein production in both mitogen-activated fibroblasts and endotoxin-activated macrophages. In both cases, ligandstimulated PGE2 production was blocked by ASO inhibition of COX-2 synthesis. When the fibroblasts or macrophages activated in the presence of COX-2 ASO were provided with exogenous arachidonic acid, they were able to synthesize PGE2. The data suggest: (a) that COX-1 present in fibroblasts and macrophages is active—it can convert exogenously supplied arachidonate to PGH2; and (b) that COX-1 present in fibroblasts and macrophages is unavailable to convert endogenous arachidonic acid, released following ligand stimulation, to PGH2. Endogenous arachidonic acid released from membrane stores following ligandstimulated phospholipase activation is not available to COX-1, but is available to COX-2. Using retrovirally expressed COX-1 and COX-2, Chulada et al (1996) reached fundamentally the same conclusions. When the first COX-2 inhibitor, NS-398, became available, we examined its ability to inhibit prostaglandin production from endogenously and exogenously supplied arachidonic acid, following mitogen stimulation of fibroblasts. NS-398 blocked PGE2 production from endogenous arachidonic acid following PDGF treatment, but had no effect on COX-1-dependent PGE2 synthesis when the cells were supplied with exogenous arachidonic acid (Herschman 1996). Endogenous arachidonic acid released in response to ligand stimulation can be converted to PGH2 by COX-2, but not by COX-1, under intracellular conditions. The simplest explanation for segregated access to endogenous arachidonic acid for COX-1 and COX-2 would be a difference in their intracellular location. Although initially thought to be the

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case (Regier et al 1993), immunofluorescence analyses (Reddy and Herschman 1994; Spencer et al 1998) and immunoelectron microscopy studies (Spencer et al 1998) suggest that the subcellular localizations of COX-1 and COX-2 are indistinguishable. In most cell types (fibroblasts, endothelial cells, macrophages, epithelial cells, osteoblasts) ligand-stimulated prostaglandin synthesis is relatively slow and is dependent on COX-2 gene expression. In contrast, when mast cells are activated by IgE receptor aggregation, PGD2 expression occurs in a biphasic fashion; there is a rapid ‘‘early’’ burst of PGD2 synthesis, followed by a ‘‘delayed’’ phase of PGD2 production (Kawata et al 1995; Murakami et al 1995). Aspirin inhibition, glucocorticoid inhibition and COX-1- and COX-2-specific inhibitors have been employed to demonstrate that the ‘‘early’’ PGD2 production phase is due to pre-existing, constitutive COX-1, while ‘‘delayed’’ PGD2 production is due to induced COX-2. In this special case of prostaglandin production, the temporal separation of COX-1 and COX-2 expression contributes to distinctions in the production of PGD2. COX-1 AND COX-2 USE ARACHIDONIC ACID WITH DIFFERING EFFICIENCIES AT LOW SUBSTRATE CONCENTRATIONS COX-2 is more efficient at low arachidonic acid concentrations (below 0.5 mM), while COX-1 is more efficient at higher arachidonic acid concentrations (above 0.25 mM) (Swinney et al 1997). COX-1 shows positive cooperation with arachidonic acid as substrate, in contrast to COX-2 (Swinney et al 1997; Chen et al 1999). Swinney et al (1997) suggest that COX-1 positive cooperation plays a major role in COX-1’s inability to utilize endogenous arachidonic acid at the concentrations released intracellularly by ligand-stimulated phospholipases. The haem groups within the COX-1 and COX-2 peroxidase sites require a lipid peroxide-dependent ‘‘initiation’’ reaction. The oxidized haem at the peroxidase side then oxidizes a tyrosine residue in the COX cyclooxygenase site. This oxidized tyrosine removes a hydrogen atom from arachidonate, forming an arachidonic acid radical that reacts with molecular oxygen to form PGG2. PGG2 is, itself, a lipid peroxide. Chen et al (1999) postulate that PGG2 can participate in a feedback loop to activate the cyclooxygenase activity of latent COX molecules. The required concentration for initiator peroxide is about 10-fold lower for COX-2 than for COX-1. Kulmacz and Wang (1995) suggest that the reduced requirement for initiator can explain the ability of COX-2 to preferentially utilize low concentrations of arachidonate as substrate in cells. COUPLING OF COX-1 AND COX-2 TO UPSTREAM PHOSPHOLIPASES AND DOWNSTREAM PROSTAGLANDIN/THROMBOXANE/ PROSTACYCLIN SYNTHASES Prostanoid production requires three enzymatic reactions: activation of phospholipase to release arachidonic acid from glycerophospholipids, conversion of arachidonic acid to PGG2 and then to PGH2 by COX-1 or COX-2, and conversion of PGH2 to the various prostanoids by tissue-specific prostaglandin/ thromboxane/prostacyclin synthases. In activated mast cells, we found that a secretory phospholipase (Reddy and Herschman 1996), subsequently identified as type V sPLA2 (Reddy et al 1997), provides arachidonate to COX-1 and a cytoplasmic phospholipase provides arachidonate to COX-2. Although these results were initially controversial (Bingham et al 1996), studies with cPLA2 knockout mice confirmed the requirement of cPLA2 for the

delayed, COX-2-dependent PGD2 synthesis in mast cells (Fujishima et al 1999) and transfection studies demonstrated the role of type V sPLA2 in providing arachidonate to COX-1 for ‘‘early’’ PGD2 synthesis (Enomoto et al 2000). In the past several years, a profusion of cytoplasmic and secreted phospholipases has been described, and an extensive, confusing and contradictory literature has developed concerning the identities of phospholipases that provide arachidonic acid to COX-1 and COX-2. The upshot of these studies is that both cPLA2 and several secretory phospholipases can supply arachidonic acid to both COX-1 and COX-2. The coupling of specific phospholipases and cyclooxygenases is dependent on cell type, ligand and relative enzyme concentrations. The field is currently an area of intense research, conflicting studies and relatively tentative conclusions. Previously, the rate-limiting step in prostanoid production was thought to be phospholipase activation. With the discovery of COX-2, the model shifted to COX-2 as a subsequent limiting factor for ligand-induced prostanoid production. The conversion of PGH2 to the final prostanoid product was not thought to be subject to transient, ligand-dependent events. However, Matsumoto et al (1997) demonstrated that the qualitative nature of prostanoids produced in rat peritoneal macrophages changed, depending on the stimulating ligand. Cells activated with a calcium ionophore, to stimulate COX-1-dependent prostanoid synthesis, produce TXA2 and PGD2. In contrast, when stimulated with endotoxin, these cells produce PGE2 and PGI2. When exogenous PGH2 was provided (to bypass COX-2 synthesis), LPS-induced PGE2 synthesis required transcription- and translation-dependent production of PGE2 synthase (PGES). The data suggested that (a) PGES is induced by LPS and (b) induced PGES is coupled to induced COX-2. Similarly, unstimulated rat peritoneal macrophages produce TXA2 and PGD2 predominantly but, when stimulated with endotoxin, produce primarily PGI2 and PGE2 (Brock et al 1999). However, these authors did not observe an increase in PGES levels following endotoxin stimulation. They suggested that COX-1 and COX-2 might be able to preferentially and differentially couple with alternative prostanoid synthases in cells, thereby qualitatively changing the spectrum of the prostanoids produced. Similar results have also been observed in endothelial cells (Caughey et al 2001). Unstimulated human umbilical vein endothelial cells (HUVEC) produce TXA2. Following IL-1b treatment, thromboxane levels increase two-fold. In contrast, IL-1b-treated HUVECs produce vastly increased levels of PGI2 (54-fold) and PGE2 (84-fold) from endogenous arachidonic acid. Increased PGI2 and PGE2 production relative to TXA2 also occurs if the IL-1b-treated cells are tested for prostanoid synthesis with exogenous PGH2 as substrate, suggesting that the levels of PGES and PGI2 synthase must be elevated in IL-1b-treated HUVECs. A new chapter in our understanding of the nature of prostaglandin synthesis was opened when Jakobsson et al (1999) described the cloning of an inducible, membrane-bound PGES, mPGES, from IL-1b-treated cells. Murakami et al (2000) also cloned mPGES and demonstrated that it is inducible by inflammatory signals in macrophages and osteoblasts. In a companion paper, Tanioka et al (2000) cloned a constitutive, cytoplasmic cPGES isozyme. These papers showed that mPGES is functionally coupled to COX-2 and cPGES is functionally coupled to COX-1, when cells use endogenous arachidonic acid. However, if arachidonic acid is artificially elevated in cells, this strict coupling breaks down; mPGES can use COX-2-derived PGH2 to produce PGE2 (Murakami et al 2000). Like COX-2, mPGES induction can be inhibited by glucocorticoids (Stichtenoth et al 2001). MPGES has emerged as an interesting candidate for pharmacological inhibition, since it modulates PGE2 production in an inflammatory context.

PROSTAGLANDIN SYNTHASE 2/CYCLOOXYGENASE II Using co-transfection studies to characterize the coupling of other prostanoid synthases with COX-1 and COX-2, Ueno et al (2001) found that thromboxane synthase and PGI2 synthase, like mPGES, preferentially couple with COX-2. However, the functional linkage of COX-1 and COX-2 with the prostanoid synthases was strongly modulated by the levels of arachidonic acid available to the enzymes in cells. An even more interesting ‘‘twist’’ to the qualitative changes in prostanoid production was provided by Sun et al (2001). Unstimulated RAW264.7 macrophages produce small amounts of TXA2 and PGE2. When stimulated with endotoxin or by crosslinking of ab1 integrin, COX-2 levels and PGES levels are elevated, and both TXA2 and PGES message synthesis is substantially induced. In contrast, if the hyaluronan receptor (CD44) is cross-linked, COX-2 mRNA levels are induced but PGES mRNA levels are not elevated. Moreover, the production of TXA2 is substantially increased in these cells, reaching levels obtained by LPS treatment of ab1 integrin cross-linking, but no elevation in PGE2 production occurs. Thus, the nature of the inducer determines the qualitative distribution of prostaglandin production by differentially regulating COX-2 and terminal prostaglandin/thromboxane/prostacyline synthase gene expression. Clearly, the coupling of the myriad phospholipases, the COX-1 and COX-2 enzymes and the terminal prostanoid synthases to produce prostanoids following ligand stimulation will continue to provide fertile ground for research for some time to come.

CANCER AND CYCLOOXYGENASES We have known for many years that low-dose aspirin reduces colon cancer morbidity and mortality. A substantial literature now exists demonstrating elevation of COX-2 gene expression both in colon tumours and in epithelial cells adjacent to the sites of tumour initiation, suggesting that COX-2 misregulation plays a role in colon cancer initiation and progression. COX-2-specific inhibitors are extremely effective at inhibiting induction of colon tumours in experimental animals (Kawamori et al 1998) and reduce the frequency and size of colon polyps in familial adenomatous polyposis (FAP) patients (Steinbach et al 2000). Mice heterozygous for a mutation in the multiple intestinal neoplasia (min) gene, the orthologue of the human FAP gene, develop multiple intestinal tract neoplasms. When crossed to COX-2 null mice, the frequency of intestinal tract tumours was dramatically decreased (Oshima et al 1996). Pharmacological studies suggest that antiangiogenic effects may play a major role in the efficacy of COX-2 inhibitor-based prevention of colon and lung cancer progression (Masferrer et al 2000). These data lead to the suggestion that elevated COX-2 expression plays a role in colon tumour initiation, vascularization and progression. However, when min (+) mice were crossed to COX-1 null mice a similar decrease in intestinal tumours occurs (Chulada et al 2000), suggesting that both COX isoforms play roles in colon tumour development. Because of the interest generated in ectopic COX-2 overexpression in colon cancer, COX-2 expression has been examined in a variety of tumours. Elevated COX-2 has been described in bladder, cervix, oesophageal, gastric, glioblastoma, head and neck, hepatocellular, lung, mammary, mesothelioma, ovarian, pancreatic, phaeochromocytoma, prostate and skin (melanoma) tumours, in addition to colorectal tumours. The role of COX-2 in tumours and the efficacy of COX-2 inhibitors as adjuvant chemotherapy are now major investigative areas.

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REGULATION OF THE COX-2 GENE Within a few years of its discovery, a variety of stimuli, including growth factors, cytokines, fluid shear, free-radical generators, hormones, inflammatory stimuli, ischaemia reperfusion, neurotransmitters, oncogenes, radiation and cell stressors, were shown to induce COX-2. COX-2 can be induced in amnion cells, astrocytes, decidual cells, dendritic cells, endometrial cells, endothelial cells, epithelial cells, fibroblasts, granulosa cells, lymphocytes, macrophages, mast cells, mesangial cells, mesothelial cells, microglia, neurons, neutrophils, osteoblasts, smooth muscle cells, synovial cells and others I have surely left out. The main issues considered in stimulus-generated transcriptional gene activation are: (a) the cytoplasmic signalling pathway that transduces the initial signalling event (e.g. receptor occupation) to the nucleus; (b) the transcription factors, activated by the signalling event, that initiate target gene transcription; and (c) the cis-acting regulatory elements of the target gene that control transcription. In addition to regulation by transcriptional activation, gene expression can be controlled by message splicing, message movement from nucleus to cytoplasm, message stability and message translational efficiency. COX-2 gene regulation appears to vary widely among cell types and among inducing agents.

Cis-Acting Sites of the COX-2 Gene Because the same COX-2 gene exists in all cells, it is easiest to first consider cis-acting COX-2 regulatory sites. Signalling pathways and transcription factors must converge on some subset(s) of cisacting COX-2 regulatory sites. Murine and human COX-2 genes share a number of putative transcription factor binding sequences, including an E-Box, a cyclic AMP response element (CRE), NFIL6/C-EBP sites, an NF-kB binding site, etc., in the first kilobase of DNA 50 of the transcription start site. Transient transfection assays, with constructs in which luciferase reporter gene transcription is driven either by the COX-2 promoter region or by promoter regions containing mutations of putative regulatory elements, demonstrated that the CRE plays the major role in v-src, serum and PGDF COX-2 gene stimulation in murine fibroblasts (Xie and Herschman 1995, 1996). The CRE of the murine and human COX-2 gene also plays a role in COX-2 induction of endotoxin-stimulated macrophages (Wadleigh et al 2000), activated mast cells (Reddy et al 2000), UV-induced keratinocytes (Gorgoni et al 2001), TNFa (Potter et al 2000; Hansen et al 2000) and IL-1 (Wang and Tai 1999) stimulated amnion cells, hormone and phorbol ester treated osteoblasts (Okada et al 2000; Wadleigh and Herschman 1999), and both IL-1 (Kirtikara et al 2000) and endotoxin (Inoue et al 1995) treated endothelial cells. Interestingly, the rat COX-2 gene has no CRE. Reporter gene experiments suggest that an E-Box plays a major role in stimulus-induced COX-2 gene expression in rat cells (Morris and Richards 1996). Sirois and Richards (1993) first suggested that the COX-2 NFIL-6 site(s) might play a role in regulating COX-2 expression. They demonstrated that an NFIL-6 mutation blocks hormonal COX-2 induction in rat granulosa cells. Mutational studies suggest that COX-2 NFIL-6 sites play a role in TNFa-stimulated osteoblasts (Yamamoto et al 1995; Wadleigh and Herschman 1999), TNFa-treated osteoblasts (Yamamoto et al 1995), amnion cells (Hansen et al 2000) and pancreatic islet cells (Sorli et al 1998), fluid shear-stressed osteoblasts (Ogasawara et al 2001), IL1b-treated chondrocytes (Thomas et al 2000), amnion (Potter et al 2000) and endothelial cells (Kirtikara et al 2000), endotoxintreated vascular endothelial cells (Inoue et al 1995) and

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macrophages (Wadleigh et al 2000; Mestre et al 2001), and activated mast cells (Reddy et al 2000). The most controversial of the COX-2 cis-acting prospective regulatory sites is the NF-kB site. We found that mutation of the NF-kB site does not inhibit induction of COX-2 promoter/ luciferase reporter genes in hormone-stimulated osteoblasts (Wadleigh and Herschman 1999), activated mast cells (Reddy et al 2000) or endotoxin-stimulated macrophages (Wadleigh et al 2000). Mutation of the human NF-kB site does not suppress COX-2 promoter activity in response to IL-1 (Sorli et al 1998). In the rat, which has no CRE in the COX-2 gene, mutation of COX2 NF-kB sites also did not significantly reduce promoter activity (Chen et al 1999). In contrast, several laboratories have reported that mutation of the human COX-2 NF-kB site attenuated induction from the COX-2 promoter (Wang and Tai 1998; Schmedtje et al 1997; Allport et al 2000). An extensive literature suggests that activation of NF-kB transcription factors plays a role in induced COX-2 gene expression, but in many cases the affected COX-2 cis-acting site has not been examined. The role of the COX-2 NF-kB site in stimulus-induced gene expression appears to be quite context-sensitive. Transcription Factors We initially assumed that the cyclic AMP binding protein, CREB, would be the transcription factor modulating COX-2 expression at the CRE in mitogen- and oncogene-stimulated fibroblasts. Gel shift experiments demonstrated a strong interaction between the COX-2 CRE and CREB (Xie et al 1994). However, cotransfection of a plasmid expressing wild-type CREB, along with a COX-2 promoter/luciferase reporter gene, suppressed vsrc-induced luciferase expression (Xie and Herschman 1995). The simplest interpretation is that CREB acts as a ‘‘dominant negative’’, binding at the COX-2 CRE but not acting as a functional transcription factor. ‘‘Supershift’’ experiments with anti-Jun antibody demonstrated that c-Jun also binds to the COX-2 CRE (Xie and Herschman 1995). In contrast to the inhibition with overexpressed CREB, c-Jun overexpression enhanced v-src stimulated expression from the COX-2 promoter. We also replaced the CRE in the COX-2 promoter/luciferase reporter gene with the DNA binding domain of the gal4 promoter, and tested the ability of GAL4-CREB, GAL4-ATF2 and GAL4-JUN chimeric proteins to stimulate luciferase production. Only GAL4-JUN was active; GAL4-CREB and GAL-4ATF2 were inactive. We concluded that c-JUN, not CREB, plays the major role in mediating gene expression at the COX-2 CRE following v-src stimulation in fibroblasts. CREB co-expression with the COX-2 promoter/luciferase reporter gene also suppresses luciferase activity in mitogen-stimulated fibroblasts (Xie and Herschman 1996), hormone-stimulated osteoblasts (Wadleigh and Herschman 1999), activated mast cells (Reddy et al 2000) and endotoxin-treated macrophages (Wadleigh et al 2000). In contrast, c-JUN overexpression enhances COX-2 expression in all these contexts, leading us to conclude that, in most cell contexts, c-JUN is likely to be the transcription factor modulating CREmediated COX-2 gene expression in response to ligand/oncogene stimulation. Caivano and Cohen (2000) suggest that CREB is the transcription factor active at the COX-2 CRE in endotoxin-stimulated RAW264 macrophages. Their conclusion is based primarily on pharmacological inhibition of signalling kinases upstream of transcription factor activation and the observation that endotoxin-stimulated CREB phosphorylation correlates with COX-2 gene expression. However, endotoxin treatment of these same cells also stimulates transient phosphorylation of c-JUN (Wadleigh et al 2000). Moreover, despite activation of CREB

phosphorylation by forskolin in RAW264 cells, COX-2 gene expression is not enhanced (Caviano and Cohen 2000). The correlation of CREB phosphorylation and COX-2 expression and their coordinate pharmacological inhibition following stimulation also lead to the conclusion that CREB mediates lysophosphatidylcholine induction of COX-2 in vascular endothelial cells (Rikitake et al 2001) and UV-induced COX-2 expression in human keratinocytes (Tang et al 2001). Based only on increased CREB phosphorylation, Ogasawara et al (2001) concluded that CREB mediates fluid shear-stressed COX-2 expression in osteoblasts. Inoue et al (1995) were the first to report that C/EBPd overexpression enhances induced expression from the COX-2 promoter, in this case in endotoxin- and phorbol ester-treated vascular endothelial cells. Similar co-transfection experiments suggested that C/EBP proteins, binding at COX-2 NFIL6 sites, play a role in IL-1b-treated chrondrocytes (Thomas et al 2000), hormone-treated osteoblasts (Wadleigh and Herschman 1999; Harrison et al 2000), endotoxin-stimulated macrophages (Wadleigh et al 2000) and activated mast cells (Reddy et al 2000). COX-2 induction is impaired in endotoxin-treated macrophages prepared from C/EBPb7/7 mice (Gorgoni et al 2001). In contrast, COX-2 induction is not diminished in serum, TNFa, IL1b or v-src treated fibroblasts from these C/EBPb7/7 mice (Gorgoni et al 2001) or in epidermal cells of TPA-treated C/ EBPb7/7 mice (Wang et al 2001). These experiments demonstrate both the complexity and cell-type specificity of COX-2 gene regulation and the power that genetically manipulated mice bring to the analysis of gene expression. The role of NFkB transcription factors is among the most controversial aspects of COX-2 gene expression. p65 reduction by antisense in human rheumatoid synovial fibroblasts is reported to reduce IL-1b induction of COX-2 (Roshak et al 1996; Crofford et al 1997). Nucleotide decoy experiments sequestering p65 suggest that hypoxia-induced COX-2 expression in human endothelial cells also requires p65 (Schmedtje et al 1997). Co-transfection of p65 and p50 expression vectors synergistically stimulate expression from a COX-2 reporter gene (Newton et al 1997), also suggesting a role for NF-kB-mediated COX-2 gene expression. However, when NF-kB activation is blocked in murine macrophages with a dominant negative I-kB, both basal and endotoxininduced expression from a COX-2 promoter/luciferase reporter gene are elevated, while expression from an NF-kB reporter plasmid is strongly inhibited (Wadleigh et al 2000). Again, distinctions in cell type and ligand specificity for COX-2 induction are likely to account for much of the apparently contradictory literature concerning NFkB in COX-2 induction. Signalling Pathways Because of our initial observations regarding the c-JUN transcription factor in activating COX-2 in fibroblasts (Xie and Herschman 1995, 1996) and subsequent studies in osteoblasts (Wadleigh and Herschman 1999), macrophages (Wadleigh et al 2000) and mast cells (Reddy et al 2000), we have concentrated on signalling pathways that activate the RAS–MEKK1–JNKK– JNK–JUN pathway. Using primarily co-transfection strategies with dominant-negative and activated constructs of RAS and the various signalling kinases, we demonstrated that this pathway plays a role in mitogen- and oncogene-activated COX-2 expression in fibroblasts (Xie and Herschman 1995, 1996), in hormone-induced COX-2 expression in osteoblasts (Wadleigh and Herschman 1999) and in COX-2 expression in activated mast cells (Reddy et al 2000). Using similar approaches, we also find that the RAS–RAF–MAPK–ERK pathway plays a role in COX-2 induction in these cells. Since our original reports on the role of

PROSTAGLANDIN SYNTHASE 2/CYCLOOXYGENASE II RAS-dependent activation of the COX-2 gene, many studies implicating ERK 1/2, p38 and JNK have been reported, using cotransfection and pharmacological inhibitor approaches (Herschman 2002). Specific signalling pathways appear to have differing degrees of importance in COX-2 induction, depending on the cell type and inducer examined. In contrast to fibroblasts, osteoblasts and mast cells, DN-RAS co-transfection does not block endotoxin activation of a COX-2 reporter gene in macrophages (Wadleigh et al 2000). The ability of DN-JNK and DN-MEKK1 to block endotoxin-stimulated expression from the COX-2 promoter in macrophages (Wadleigh et al 2000) suggests that c-JUN is also a major COX-2 transcriptional activator in these cells. TLR4 is the major endotoxin receptor. Evolutionary conserved signalling intermediate in toll pathways (ECSIT) is an adaptor protein that connects toll/tumour necrosis factor receptor-associated factor (TRAF60) activation to MEKK1, leading to c-JUN activation (Kopp et al 1999). Co-expression of DN-ECSIT blocks endotoxin-induced reporter gene expression from the COX-2 promoter (Wadleigh et al 2000), suggesting that ECSIT couples occupation of TLR4 by endotoxin to the MEKK1–JNK–c-JUN pathway to activate COX-2 expression. A complete pathway from TLR4 receptor ligand binding to COX-2 gene activation has been proposed (Wadleigh et al 2000). Caivano et al (2001) suggest that endotoxin/LTR4 activation stimulates the classical MAPK pathway and the p38-mediated pathway to phosphorylate MSK kinases that initially activate the COX-2 gene via CREB phosphorylation in macrophages. They do not describe the pathway(s) by which TLR4 activation is coupled to MAPK– SAPK–MSK to phosphorylate CREB. COX-2 MESSAGE STABILITY Ristimaki et al (1994) suggested that COX-2 mRNA stabilization might play a role in COX-2 elevation following cell stimulation. Research on this topic has focused on two main areas: (a) the mechanisms that alter COX-2 mRNA stability in stimulated cells and (b) the sequences in the COX-2 mRNA that regulate message stability. Several laboratories have demonstrated that ligand-dependent COX-2 mRNA stabilization can be blocked with p38 kinase inhibitors (Ridley et al 1998; Dean et al 1999; Matsuura et al 1999; Jang et al 2000; Fiebich et al 2000). Lasa et al (2001) suggest that the ability of glucocorticoids to suppress COX-2 induction is, in part, the result of glucocorticoid inhibition of p38 activation, thus blocking ligand-induced, p38-dependent stabilization of COX-2 message. Lasa et al (2000) propose that the target of p38 for COX2 message stabilization is MAPKAP2, but the downstream target of this latter kinase has not been identified. Sheng et al (2001) suggest that RAS-mediated increases in COX-2 in transformed cells may be regulated by AKT/PKB-induced stabilization of COX-2 message, as well as increased COX-2 transcription. Attempts to identify the regions of COX-2 mRNA that modulate its stability in response to ligand stimulation have used chimeric reporter genes that fuse regions of COX-2 message to stable transcripts (Dixon et al 2000; Lasa et al 2000). Both groups agree that the 123 nucleotides immediately 50 to the translation termination site play a critical role in COX-2 message regulation. This region of the COX-2 30 untranslated message is rich in AUUUA sequences that confer instability to cytokine messages. While several cytokine instability regions share the stabilizing response to p38 activation with the COX-2 sequence, this property is not universal to all AUUUA-rich regions (Lasa et al 2000). Both groups identified proteins that can bind to the region of the COX-2 message associated with regulated stability, but no causal relationships have been established. The character-

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ization of critical sequences responsible for COX-2 message stabilization, the proteins that mediate this phenomenon and the signalling pathways that translate receptor occupancy into changes in COX-2 message stability should be a fascinating chapter in the story of COX-2 regulation. ACKNOWLEDGEMENTS Thanks to all the members of my laboratory, past and present, who have contributed to the studies from our group described in this review. I also thank my many colleagues in the field of COX-2 biology who have provided advice, reagents and information. My apologies to those whose work I have not cited, due either to lack of space or to oversights by me in reviewing the literature. Supported by Grant AI50495, the UCLA Asthma and Allergic Disease Center supported by the NIAID and NIEHS and Grant CA84572 from the National Cancer Institute. REFERENCES Allport VC, Slater DM, Newton R and Bennett PR (2000) NF-kB and AP-1 are required for cyclo-oxygenase 2 gene expression in amnion epithelial cell line (WISH). Mol Hum Reprod, 6, 561–565. Bingham CO III, Murakani M, Fujishama H et al (1996) A heparinsensitive phospholipase A2 and prostaglandin endoperoxide synthase-2 are functionally linked in the delayed phase of prostaglandin D2 generation in mouse bone marrow-derived mast cells. J Biol Chem, 271, 25936–25944. Brock TG, McNish RW and Peters-Golden M (1999) Arachidonic acid is preferentially metabolized by cyclooxygenase-2 to prostacyclin and prostaglandin E2. J Biol Chem, 274, 11660–11666. Caivano M and Cohen P (2000) Role of mitogen-activated protein kinase cascades in mediating lipopolysaccharide-stimulated induction of cyclooxygenase-2 and IL-1b in RAW264 macrophages. J Immunol, 164, 3018–3025. Caivano M, Gorgoni B, Cohen P and Poli V (2001) The induction of cyclooxygenase-2 mRNA in macrophages is biphasic and requires both CCAAT enhancer-binding protein b (C/EBPb) and C/EBP D transcription factors. J Biol Chem, 276, 48693–48701. Caughey GE, Cleland LG, Penglis PS et al (2001) Roles of cyclooxygenase (COX)-1 and COX-2 in prostanoid production by human endothelial cells: selective upregulation of prostacyclin synthesis by COX-2. J Immunol, 167, 2831–2838. Chen G, Wood EG, Wang SH and Warner TD (1999) Expression of cyclooxygenase-2 in rat vascular smooth muscle cells is unrelated to nuclear factor-kB activation. Life Sci, 64, 1231–1242. Chen W, Pawelek TR and Kulmacz RJ (1999) Hydroperoxide dependence and cooperative cyclooxygenase kinetics in prostaglandin H synthase-1 and -2. J Biol Chem, 274, 20301–20306. Chulada PC, Loftin CD, Winn VD et al (1996) Relative activities of retrovirally expressed murine prostaglandin synthase-1 and -2 depend on source of arachidonic acid. Arch Biochem Biophys, 330, 301–313. Chulada PC, Thompson MB, Mahler JF et al (2000) Genetic disruption of Ptgs-1, as well as Ptgs-2, reduces intestinal tumorigenesis in min mice. Cancer Res, 60, 4705–4708. Crofford LJ, Tan B, McCarthy CJ and Hla T (1997) Involvement of nuclear factor kB in the regulation of cyclooxygenase-2 expression by interleukin-1 in rheumatoid synoviocytes. Arthrit Rheumat, 40, 226– 236. Dean JL, Brook M, Clark AR and Saklatvala J (1999) p38 mitogenactivated protein kinase regulates cyclooxygenase-2 mRNA stability and transcription in lipopolysaccharide-treated human monocytes. J Biol Chem, 274, 264–269. DeWitt DL and Smith WL (1988) Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence. Proc Natl Acad Sci USA, 85, 1412– 1416. Dixon DA, Kaplan CD, McIntyre TM et al (2000) Post-transcriptional control of cyclooxygenase-2 gene expression. J Biol Chem, 275, 11750– 11757.

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5 Mammalian Lipoxygenases Shozo Yamamoto1, Hiroshi Suzuki2, Natsuo Ueda3, Yoshitaka Takahashi4 and Tanihiro Yoshimoto4 1Kyoto

Women’s University, Kyoto; 2Tokushima University School of Medicine, Tokushima; 3Kagawa University School of Medicine, Kagawa; and 4Kanazawa University Graduate School of Medicine, Kanazawa, Japan

LIPOXYGENASE ENZYMES Lipoxygenase enzymes recognize the 1-cis,4-cis-pentadiene structure of unsaturated fatty acids such as arachidonic, linoleic and linolenic acids, and incorporate one molecule of oxygen into the polyenoic acids to produce corresponding hydroperoxy acids. The enzymes belong to the group of dioxygenases. In the 1960s the enzyme was found in soybean, and referred to as lipoxidase at that time; it was believed that there was no lipoxidase in animal tissues (Tappel 1963). Along with the discovery of bioactive thromboxane in 1974, however, an enzyme which oxygenated the position 12 of arachidonic acid was found in human platelets (Hamberg and Samuelsson 1974) and in animal platelets (Nugteren 1975). The enzyme was referred to as arachidonate 12-lipoxygenase, and it was clearly demonstrated that lipoxygenase enzymes were also present in animals. Furthermore, a protein that was isolated from rabbit reticulocytes and inhibited mitochondrial respiration was found to be a lipoxygenase (Rapoport et al 1979). The reticulocyte enzyme was later identified as 15-lipoxygenase upon reaction with arachidonic acid (Bryant et al 1982). Upon the discovery of bioactive leukotrienes, 5-lipoxygenase was found in rabbit polymorphonuclear leukocytes (Borgeat et al 1976). The 8lipoxygenase activity was detected in mouse skin (Gschwendt et al 1986). Fatty acid cyclooxygenase was also shown to be a lipoxygenase in terms of its mechanism of arachidonate oxygenation (Hamberg and Samuelsson 1967b). Figure 5.1 summarizes the oxygenation reactions catalysed by these lipoxygenases. The oxygenation site of the lipoxygenase substrate is different from enzyme to enzyme. Each enzyme is named on the basis of the oxygenation site upon reaction with arachidonic acid, which has 20 carbon atoms; thus, ‘‘arachidonate X-lipoxygenase’’ oxygenates position X of arachidonic acid as substrate, counted starting from its carboxylic carbon. For reference citation in this chapter, review articles are cited in most cases. If necessary, the readers are advised to see the original articles cited in these review articles.

REACTION MECHANISM OF SOYBEAN LIPOXYGENASE In addition to the regiospecific oxygenation by lipoxygenase enzyme, as described above, the stereospecific oxygenation initiated by stereoselective hydrogen abstraction is characteristic of lipoxygenase. This was first shown by an isotope tracer experiment of soybean lipoxygenase using [13DR-3H] and [13LSThe Eicosanoids. Edited by Peter Curtis-Prior. &2004 John Wiley & Sons, Ltd: ISBN 0 471 48984 0

3

H] 8,11,14-eicosatrienoic acid (Hamberg and Samuelsson 1967a). As illustrated in Figure 5.2, one of the two hydrogen atoms (13proS hydrogen) of the methylene group close to the oxygenation site is stereoselectively eliminated, followed by migration of a double bond and formation of a conjugated diene. The resultant radical is attacked by an oxygen molecule, and the final product is a stereospecific hydroperoxy acid, i.e. 15S-hydroperoxy-8-cis,11cis,13-trans-eicosatrienoic acid. Soybean lipoxygenase contains an equimolar amount of iron (Pistorius and Axelrod 1974), and three histidine residues and C-terminal isoleucine are considered to be the ligands, as demonstrated by site-directed mutagenesis (Steczko and Axelrod 1992) and X-ray crystallography (Boyington et al 1993).

ARACHIDONATE 5-LIPOXYGENASE 5-Lipoxygenase initiates the biosynthesis of leukotrienes in leukocytes, and plays important pathophysiological roles in allergy and inflammation (Ford-Hutchinson et al 1994). The enzyme abstracts proS hydrogen from the carbon 7, and oxygenates the carbon 5 of not only arachidonic acid but also 5,8,11-eicosatrienoic acid and 5,8,11,14,17-eicosapentaenoic acid, and produces 5S-hydroperoxy-6-trans,8-cis,11-cis,14-cis-eicosatetraenoic acid (5S-HPETE) from arachidonic acid. However, the enzyme is much less active with linoleic and linolenic acids (Yamamoto 1992). For leukotriene synthesis, the 5S-HPETE produced is further converted to leukotriene A4 with 5,6-epoxide by liberation of one molecule of water (Figure 5.3). Several lines of enzymological and molecular biological evidence indicate that leukotriene A4 synthesis from 5S-HPETE is attributed to the 5-lipoxygenase enzyme. This reaction is initiated by abstraction of proR hydrogen from position 10. Thus, 5-lipoxygenase is a bifunctional enzyme, with 5-oxygenase activity and leukotriene A4 synthase activity (Yamamoto 1991, 1992; Ford-Hutchinson et al 1994). 5-Lipoxygenase contains iron, which is essential for catalytic activity (Ford-Hutchinson et al 1994). Ca2+ is required for 5lipoxygenase activity, and Ca2+-dependent activity is stimulated by the addition of ATP (Yamamoto 1991; Ford-Hutchinson et al 1994; Ra˚dmark 2000). The intracellular function of 5-lipoxygenase is assisted by an 18 kDa arachidonate-binding protein, which is referred to as five-lipoxygenase activating protein (FLAP) and facilitates the supply of arachidonic acid to the enzyme (FordHutchinson et al 1994). Earlier, 5-lipoxygenase was known as a soluble cytosolic protein, and shown to become membrane-bound upon increase of intracellular calcium concentration. Recent

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Figure 5.2 Selective hydrogen abstraction and oxygenation by soybean lipoxygenase

Figure 5.1 Various mammalian lipoxygenases. PG, prostaglandin; HPETE, hydroperoxyeicosatetranoic acid

investigations have demonstrated the occurrence of a large part of the enzyme inside the nucleus of various types of cell (Ra˚dmark 2000; Funk and Chen 2000). Furthermore, involvement of the SH3 binding motif was suggested in the translocation of 5lipoxygenase, and a two-hybrid method has demonstrated the binding of the enzyme to cytoskeletal proteins such as a-actinin and actin (Ra˚dmark 2000). 5-Lipoxygenase cDNAs of various animals have been cloned, and the primary structures of 673- or 674-amino acid enzymes have been deduced. The 5-lipoxygenase gene consists of 14 exons, and is located on human chromosome 10q 11.2 and on murine chromosome 6. The promoter region is rich in GC boxes, and has neither TATA nor CCAAT boxes (Ra˚dmark 1995; Funk 1993, 1996). The 5-lipoxygenase-deficient mice grow normally, and are resistant to the shock induced by platelet-activating factor (Funk 1996; Austin and Funk 1999; Funk and Chen 2000).

Figure 5.3 5-Lipoxygenase catalysis. hydroperoxyeicosatetraenoic acid

LT,

leukotriene;

HPETE,

ARACHIDONATE 12-LIPOXYGENASE As described above, 12-lipoxygenase was first found in platelets, and later in leukocytes and various other animal cells (Yamamoto 1992; Yoshimoto and Yamamoto 1995; Yamamoto et al 1997). According to our earlier observations, there are two types of 12lipoxygenase. The enzymes of human and bovine platelets are almost inactive with linoleic and linolenic acids, while the enzymes of porcine and bovine leukocytes are active with these C18 fatty acids as well as C20 fatty acids such as arachidonic acid (Yamamoto 1992). Thus, platelet and leukocyte 12-lipoxygenases are distinguishable in terms of substrate specificity. The 12-lipoxygenase reaction is initiated by abstraction of proS hydrogen at position 10 of arachidonic acid, followed by the migration of a double bond and the formation of 8-cis,10-trans conjugated diene, and 12S-hydroperoxy-5-cis,8-cis,10-trans,14cis-eicosatetraenoic acid (12S-HPETE) is produced (Yamamoto 1992). Unlike 5-lipoxygenase, the 12S-HPETE produced is not

subjected to conversion to an epoxide of leukotriene type. In addition to 12S-HPETE, as shown in Figure 5.4, leukocyte 12lipoxygenase produces 15S-hydroperoxy-5-cis,8-cis,11-cis,13trans-eicosatetraenoic acid (15S-HPETE) from arachidonic acid in an amount of about 10% of the total products, whereas platelet enzyme produces 15-HPETE in a quantity of only 1–2% of 12HPETE (Yamamoto et al 1997). The 15S-HPETE produced is converted to 14,15-epoxy acid with a conjugated triene of leukotriene type by leukocyte 12-lipoxygenase at a significant rate. In this reaction, proS hydrogen is removed from carbon 10. Furthermore, 15S-HPETE is subjected to 8S-oxygenation and 14R-oxygenation by the same enzyme, and dihydroperoxyeicosatetraenoic acids (diHPETE) are produced (Figure 5.4). Such multifunctional activities of 12-lipoxygenase have been reviewed previously (Yamamoto 1991, 1992; Yamamoto et al 1997).

MAMMALIAN LIPOXYGENASES

55

Figure 5.4 12-Lipoxygenase catalysis. LT, leukotriene; HX, hepoxilin; HPETE, hydroperoxyeicosatetraenoic acid; diHPETE, dihydroperoxyeicosatetraenoic acid.

cDNAs and genomic DNAs of 12-lipoxygenase have been cloned from various animal species (Funk 1993; Yoshimoto and Yamamoto 1995; Funk 1996; Ku¨hn and Thiele 1999; Brash 1999). In terms of the deduced amino acid sequence and the intron size of the enzyme gene, leukocyte 12-lipoxygenases are closer to reticulocyte 15-lipoxygenases than to platelet 12-lipoxygenases. On the basis of these findings, together with the different antigenicities and substrate specificities, we proposed a classification of two isozyme types (platelet type and leukocyte type) for 12-lipoxygenases (Yamamoto et al 1997). As shown in Table 5.1, the platelet type and the leukocyte type are also found in tissues other than platelets and leukocytes, respectively. Tissue distribution of 12-lipoxygenase isozymes has been listed in detail in the review article by Yoshimoto and Yamamoto (1995). Recently, 12lipoxygenases with lower homologies for platelet and leukocyte enzymes have been isolated from human and murine skin and referred to as the ‘‘epidermis type’’ (Ku¨hn and Thiele 1999). As for the regulation of enzyme gene expression, 12-lipoxygenase is not a rapidly inducible enzyme, in contrast to cyclooxygenase-2. The gene structures of various 12-lipoxygenases have been described and discussed in the studies by Yoshimoto

and Yamamoto (1995) and Funk (1996). The expression of human platelet enzyme is negatively regulated by NFkB (Arakawa et al 1995). The mRNA of the platelet-type enzyme in human epidermoid carcinoma cells was induced by epidermal growth factor after a 10 h lag period (Chang et al 1993), and the interaction of c-Jun and Sp1 was shown to be involved in this induction (Chen and Chang 2000). The protein level of leukocytetype enzyme in porcine aortic smooth muscle cells is increased around 6 h after the addition of platelet-derived growth factor, and remains elevated for up to 24 h (Natarajan et al 1996). It was reported that translocation of human platelet 12-lipoxygenase from cytosol to membrane was required for the activation of the enzyme (Ozeki et al 1999). A clinical case with myeloproliferative disease showed a decreased expression of platelet 12-lipoxygenase protein and mRNA (Matsuda et al 1993). The biological functions of 12-lipoxygenase remain unestablished, although many studies have reported various biological activities of 12-HPETE or 12-HETE (Yamamoto et al 1997). Mice lacking leukocyte-type 12-lipoxygenase grow normally and are fertile, with no macroscopic abnormality of the external or internal organs (Funk 1996). The gene disruption resulted in a

Table 5.1 12-Lipoxygenase isozymes Properties Substrate specificity Arachidonic acid Linoleic and linolenic acids Phospholipids Reaction products Amino acid identity with 15-lipoxygenase* Exon-intron structure compared with 15-lipoxygenase* Distribution Human Porcine Bovine Canine Rat Mouse *Rabbit reticulocyte 15-lipoxygenase.

Leukocyte type

Platelet type

Epidermis type

Active Active Active 12-HPETE415-HPETE Higher Similar

Active Almost inactive Almost inactive 12-HPETE4 415-HPETE Lower Different

Active – – – Lower Different

Adrenal Leukocyte, pituitary Leukocyte, trachea, cornea Leukocyte, brain Leukocyte, pineal gland, aorta, lung, pancreas, spleen Leukocyte, pituitary, pineal gland, kidney

Platelet, skin, uterine cervix

Skin

Platelet Platelet Platelet Platelet, skin

Skin

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Figure 5.5 Lipoxin biosynthesis. LT, leukotriene; LX, lipoxin; HPETE, hydroperoxyeicosatetraenoic acid

reduced capacity of the macrophages to oxidize low-density lipoprotein (LDL) (Austin and Funk 1999). Furthermore, the 12lipoxygenase-lacking mice showed diminished atherosclerosis (Cyrus et al 1999). The involvement of LDL receptor-related protein (LRP) in macrophages was suggested in the oxidation process of LDL by macrophage 12-lipoxygenase (Xu et al 2001). The gene disruption of murine platelet-type 12-lipoxygenase did not enhance thromboxane formation, but the platelets were hyperresponsive to ADP-induced aggregation (Austin and Funk 1999). 12-Lipoxygenase is involved in the biosyntheses of two groups of bioactive eicosanoids. As shown in Figure 5.4, hepoxilins A3 and B3 with hydroxyl and epoxy groups are produced from 12HPETE. These compounds show various biological activities related to the release of intracellular calcium and the opening of potassium channels (Pace-Asciak 1994, 1995). Recently it was established that 12-lipoxygenase increased membrane excitability by inhibiting M-type potassium channels without affecting any other ion channels (Takahashi et al 1999). Figure 5.5 shows the biosynthesis of lipoxins A4 and B4 with a conjugated tetraene and 3 hydroxyl groups. These compounds may function as endogenous braking signals in host defence, inflammation and hypersensitivity. Platelet 12-lipoxygenase is involved in the transcellular synthesis of lipoxins from arachidonic acid via leukotriene A4 (Serhan 1994; Brady and Serhan 1996). In the 1980s the lesional skin of psoriasis patients was reported to contain an increased amount of 12R-HETE rather than 12SHETE. The 12R-HETE production was attributed presumably to cytochrome P-450 (Woollard 1986). Recently, cDNA of a new 12lipoxygenase was cloned from human skin, and the recombinant enzyme produced 12R-HPETE rather than 12S-HPETE. Thus, the enzyme was referred to as 12R-lipoxygenase (Boeglin et al

1998). cDNA of the same enzyme was also cloned from a human B cell line, CCL-156, and its gene was located on chromosome 17p13. A mouse orthologue was also cloned (Sun et al 1998). ARACHIDONATE 15-LIPOXYGENASE The 15-lipoxygenase reaction is initiated by abstraction of proS hydrogen from position 13 of arachidonic acid, followed by the formation of 11-cis,13-trans conjugated diene, and 15S-HPETE is produced. As demonstrated with the rabbit reticulocyte enzyme (Bryant et al 1985), 15-lipoxygenase is also a multi-functional enzyme. The same enzyme transforms 15S-HPETE to 14,15epoxy acid with a conjugated triene (14,15-leukotriene A4) as illustrated in Figure 5.6. In this reaction, proS hydrogen is abstracted from position 10 (Yamamoto 1992). In addition to 14,15-leukotriene A4 production, the enzyme oxygenates position 5 or 8 of 15S-HPETE, and produces 5S,15S- and 8S,15SdiHPETEs. 15-Lipoxygenase of reticulocytes has also 12-lipoxygenase activity, and 12S-HPETE is produced in one-ninth the amount of 15S-HPETE (Ku¨hn et al 1990). Linoleic and linolenic acids as substrates of the enzyme are more active than arachidonic acid (Ku¨hn et al 1990). Recently, a new 15-lipoxygenase was found and its cDNA was cloned from human hair root. The enzyme was also expressed in normal prostate epithelium, but the expression level was reduced in prostate adenocarcinoma (Shappell et al 1999). The primary structure of the 676 amino acid enzyme has 40% identity to the known 15-lipoxygenase. With the recombinant enzyme, linoleic acid as substrate was less active than arachidonic acid (Brash et al 1997). As mentioned above, 15-lipoxygenase has a broad substrate specificity in terms of the carbon chain length of substrate

MAMMALIAN LIPOXYGENASES

57

Figure 5.6 15-Lipoxygenase catalysis. LT, leukotriene; HPETE, hydroperoxyeicosatetraenoic acid; diHPETE, dihydroperoxyeicosatetraenoic acid

(Yamamoto 1992). Furthermore, the rabbit reticulocyte enzyme is also active with arachidonic acid esterified in phosphatidylcholine, and 12S- and 15S-oxygenated products are found (Murray and Brash 1988). cDNA cloning for 15-lipoxygenases of rabbit reticulocytes, human reticulocytes and human airway epithelium deduced the primary structures of 661 or 662 amino acid enzymes (Ku¨hn and Thiele 1995; Funk 1996). The gene of rabbit 15-lipoxygenase is composed of 14 exons, and its promoter is characteristic of a house-keeping enzyme (Ku¨hn and Thiele 1995). The expression of the enzyme gene is upregulated by interleukin-4 or -13, and suppressed by interferon-g (Ku¨hn and Thiele 1995; Funk 1996). Translation of the mRNA encoding rabbit reticulocyte 15lipoxygenase is inhibited by a 48 kDa protein which binds to a repeated sequence in the 30 -untranslated region (Ostareck-Lederer et al 1994). In connection with 15-lipoxygenase physiology, various biological activities have been reported for 15-HPETE and 15-HETE (FordHutchinson 1991; Ku¨hn and Thiele 1995). A currently active research project with 15-lipoxygenase is its possible role in the pathogenesis of atherosclerosis. As mentioned above, 15-lipoxygenase oxygenates esterified unsaturated fatty acids contained in membranes and lipoproteins (Murray and Brash 1988; Parthasarathy et al 1989; Ku¨hn and Brash 1990). On the basis of these findings and further experimental results, a possible role of 15-lipoxygenase is the oxygenation of LDL, which triggers the production of an atherogenic form of lipoprotein (Steinberg et al 1989; Ku¨hn et al 1994; Harats et al 1995).

ARACHIDONATE 8-LIPOXYGENASE An earlier work reported that an application of phorbol ester to mouse back skin resulted in a production of 8-hydroxy-5-cis,9trans,11-cis,14-cis-eicosatetraenoic acid (8-HETE) from arachidonic acid by a cell-free epidermal preparation (Gschwendt et al 1986; Fu¨rstenberger et al 1991). Later, the metabolite was identified as 8S-HETE (Hughes and Brash 1991). After attempts for a long time to characterize the 8-lipoxygenase, its cDNA encoding 667 amino acids was cloned from the mouse epidermal

library (Jisaka et al 1997; Krieg et al 1998). The amino acid sequence has 78% identity to the new human skin 15-lipoxygenase mentioned above and about 40% identity to mouse 5-lipoxygenase and mouse 12-lipoxygenases of three types. The recombinant enzyme oxygenates arachidonic acid exclusively to 8S-HPETE, but linoleic acid is a poor substrate and is converted to 9Shydroperoxy derivative (Jisaka et al 1997). The enzyme also converts 5S-HPETE to leukotriene A4 at a rate comparable to that of 5-lipoxygenase (Qiao et al 1999). 8S-HETE is one of the ligands for nuclear peroxisome proliferator-activated receptor-a (Brash 1999), although many other compounds, including leukotriene B4 and non-steroidal antiinflammatory drugs (NSAIDs), can activate this receptor (Lehman et al 1997; Funk 2001).

MOLECULAR EVOLUTION OF LIPOXYGENASES Since the time when 5-, 12- and 15-lipoxygenases and cyclooxygenase were subjected to computer-assisted sequence comparison by Sigal (1991) and Toh et al (1992), many versions of the lipoxygenase phylogenetic tree have been presented (Funk 1996; Ku¨hn and Thiele 1999; Brash 1999; Kikuno et al 1999) and the tree has grown bigger and bigger. According to these analyses, plant lipoxygenases are distantly related to mammalian lipoxygenases, but the C-terminal portion containing all the ligand residues for the catalytically essential iron (Boyington et al 1993) is well conserved between soybean lipoxygenase and mammalian lipoxygenases. In terms of the amino acid sequence identity, 5lipoxygenase is distinct from other mammalian lipoxygenases. As we demonstrated previously (Yamamoto 1992), the platelet type and the leukocyte type of 12-lipoxygenase are distinguishable. Moreover, in the phylogenetic tree there is a closer relationship between leukocyte 12-lipoxygenases and reticulocyte 15-lipoxygenases rather than platelet 12-lipoxygenase. This finding agrees with their similar catalytic activities, described above. Therefore, 15-lipoxygenase of the reticulocyte type and 12-lipoxygenase of the leukocyte type in combination are referred to as 12/15lipoxygenase by some investigators (Funk 1996; Ku¨hn and Thiele 1999). However, this terminology may be misleading and it should

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be noted that the major product of leukocyte 12-lipoxygenase is 12-HPETE, and that of reticulocyte 15-lipoxygenase is 15HPETE. As mentioned above, epidermis-type 12-lipoxygenase was proposed as the third isozyme type (Ku¨hn and Thiele 1999). CONCLUSION Various mammalian lipoxygenases have been investigated extensively and intensively, and their molecular and catalytic properties are now known in detail. However, except for the leukotrieneproducing 5-lipoxygenase, their physiological and pathological roles have not yet been established. Ku¨hn and Thiele (1999) have stated: ‘‘many sequence data but little information on biological significance’’. To establish the biological significance of lipoxygenases, we must demonstrate specific biological activity associated with a lipoxygenase metabolite of arachidonic acid and other polyunsaturated fatty acids, with stereoselective structure produced in various (not one single) animal species. REFERENCES Arakawa T, Nakamura M, Yoshimoto T and Yamamoto S (1995) The transcriptional regulation of human arachidonate 12-lipoxygenase gene by NFkB/Rel. FEBS Lett, 363, 105–110. Austin SC and Funk CD (1999) Insight into prostaglandin, leukotriene, and other eicosanoid functions using mice with targeted gene disruption. Prostagland Other Lipid Mediators, 58, 231–252. Boeglin WE, Kim RB and Brash AR (1998) A 12R-lipoxygenase in human skin: mechanistic evidence, molecular cloning, and expression. Proc Natl Acad Sci USA, 95, 6744–6749. Borgeat P, Hamberg M and Samuelsson B (1976) Transformation of arachidonic acid and homo-g-linolenic acid by rabbit polymorphonuclear leukocytes. Monohydroxy acids from novel lipoxygenases. J Biol Chem, 251, 7816–7820. Boyington JC, Gaffney BJ and Amzel LM (1993) The three-dimensional structure of an arachidonic acid 15-lipoxygenase. Science, 260, 1482– 1486. Brady HR and Serhan CN (1996) Lipoxins: putative braking signals in host defense, inflammation and hypersensitivity. Curr Opin Nephrol Hypertens, 5, 20–27. Brash AR (1999) Lipoxygenases: occurrence, functions, catalysis, and acquisition of substrate. J Biol Chem, 274, 23679–23682. Brash AR, Boeglin WE and Chang MS (1997) Discovery of a second 15Slipoxygenase in humans. Proc Natl Acad Sci USA, 94, 6148–6152. Bryant RW, Bailey JM, Schewe T and Rapoport SM (1982) Positional specificity of a reticulocyte lipoxygenase. Conversion of arachidonic acid to 15S-hydroperoxy-eicosatetraenoic acid. J Biol Chem, 257, 6050–6055. Bryant RW, Schewe T, Rapoport SM and Bailey JM (1985) Leukotriene formation by a purified reticulocyte lipoxygenase enzyme. Conversion of arachidonic acid and 15-hydroperoxyeicosatetraenoic acid to 14,15leukotriene A4. J Biol Chem, 260, 3548–3555. Chang W-C, Liu Y-W, Ning C-C et al (1993) Induction of arachidonate 12-lipoxygenase mRNA by epidermal growth factor in A431 cells. J Biol Chem, 268, 18734–18739. Chen B-K and Chang WC (2000) Functional interaction between c-Jun and promoter factor Sp1 in epidermal growth factor-induced gene expression of human 12(S)-lipoxygenase. Proc Natl Acad Sci USA, 97, 10406–10411. Cyrus T, Witztum JL, Rader DJ et al (1999) Disruption of the 12/15lipoxygenase gene dimisishes atherosclerosis in apo E-deficient mice. J Clin Invest, 103, 1597–1604. Ford-Hutchinson AW (1991) Arachidonate 15-lipoxygenase; characteristics and potential biological significance. Eicosanoids, 4, 65–74. Ford-Hutchinson AW, Gresser M and Young RN (1994) 5-Lipoxygenase. Annu Rev Biochem, 63, 383–417. Funk CD (1993) Molecular biology in the eicosanoid field. Prog Nucleic Acid Res Mol Biol, 45, 67–98.

Funk CD (1996) The molecular biology of mammalian lipoxygenases and the quest for eicosanoid functions using lipoxygenase-deficient mice. Biochim Biophys Acta, 1304, 65–84. Funk CD (2001) Prostaglandins and leukotrienes: advances in eicosanoid biology. Science, 294, 1871–1875. Funk CD and Chen X-S (2000) 5-Lipoxygenase and leukotrienes. Transgenic mouse and nuclear targeting studies. Am J Respir Crit Care Med, 161, S120–S124. Fu¨rstenberger G, Hagedorn H, Jacobi T et al (1991) Characterization of an 8-lipoxygenase activity induced by the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate in mouse skin in vivo. J Biol Chem, 266, 15738–15745. Gschwendt M, Fu¨rstenberger G, Kittstein W et al (1986) Generation of the arachidonic acid metabolite 8-HETE by extracts of mouse skin treated with phorbol ester in vivo; identification by 1H n.m.r. and GC– MS spectroscopy. Carcinogenesis, 7, 449–455. Hamberg M and Samuelsson B (1967a) On the specificity of the oxygenation of unsaturated fatty acids catalyzed by soybean lipoxidase. J Biol Chem, 242, 5329–5335. Hamberg M and Samuelsson B (1967b) On the mechanism of the biosynthesis of prostaglandins E1 and F1a. J Biol Chem, 242, 5336– 5343. Hamberg M and Samuelsson B (1974) Prostaglandin endoperoxides. Novel transformations of arachidonic acid in human platelets. Proc Natl Acad Sci USA, 71, 3400–3404. Harats D, Mulkins MA and Sigal E (1995) A possible role for 15lipoxygenase in atherogenesis. Trends Cardiovasc Med, 5, 29–36. Hughes MA and Brash AR (1991) Investigation of the mechanism of biosynthesis of 8-hydroxyeicosatetraenoic acid in mouse skin. Biochim Biophys Acta, 1081, 347–354. Jisaka M, Kim RB, Boeglin WE et al (1997) Molecular cloning and functional expression of a phorbol ester-inducible 8S-lipoxygenase from mouse skin. J Biol Chem, 272, 24410–24416. Kikuno R, Daiyasu H and Toh H (1999) Molecular evolution of proteins involved in the arachidonic acid cascade. In Comprehensive Natural Products Chemistry, vol 1: Polyketides and Other Secondary Metabolites Including Fatty Acids and Their Derivatives, Sankawa U (ed.) Elsevier, Amsterdam, 273–284. Krieg P, Kinzig A, Heidt M et al (1998) cDNA cloning of a 8-lipoxygenase and a novel epidermis-type lipoxygenase from phorbol ester-treated mouse skin. Biochim Biophys Acta, 1391, 7–12. Ku¨hn H and Brash AR (1990) Occurrence of lipoxygenase products in membranes of rabbit reticulocytes. Evidence for a role of the reticulocyte lipoxygenase in the maturation of red cells. J Biol Chem, 265, 1454–1458. Ku¨hn H and Thiele B-J (1995) Arachidonate 15-lipoxygenase. J Lipid Mediators Cell Signalling, 12, 157–170. Ku¨hn H and Thiele BJ (1999) The diversity of the lipoxygenase family. Many sequence data but little information on biological significance. FEBS Lett, 449, 7–11. Ku¨hn H, Sprecher H and Brash AR (1990) On singular or dual positional specificity of lipoxygenase. The number of chiral products varies with alignment of methylene groups at the active site of the enzyme. J Biol Chem, 265, 16300–16305. Ku¨hn H, Belkner J, Zaiss S et al (1994) Involvement of 15-lipoxygenase in early stages of atherogenesis. J Exp Med, 179, 1903–1911. Lehmann JM, Lenhard JM, Oliver BB et al (1997) Peroxisome proliferator-activated receptors a and g are activated by indomethacin and other non-steroidal anti-inflammatory drugs. J Biol Chem, 272, 3406–3410. Matsuda S, Murakami J, Yamamoto Y et al (1993) Decreased messenger RNA of arachidonate 12-lipoxygenase in platelets of patients with myeloproliferative disorders. Biochim Biophys Acta, 1180, 243–249. Murray JJ and Brash AR (1988) Rabbit reticulocyte lipoxygenase catalyzes specific 12(S) and 15(S) oxygenation of arachidonoylphosphatidylcholine. Arch Biochem Biophys, 265, 514–523. Natarajan R, Bai W, Rangarajan V et al (1996) Platelet-derived growth factor BB mediated regulation of 12-lipoxygenase in porcine aortic smooth muscle cells. J Cell Physiol, 169, 391–400. Nugteren DH (1975) Arachidonate lipoxygenase in blood platelets. Biochim Biophys Acta, 380, 299–307. Ostareck-Lederer A, Ostareck DH, Standart N and Thiele BJ (1994) Translation of 15-lipoxygenase mRNA is inhibited by a protein that

MAMMALIAN LIPOXYGENASES binds to a repeated sequence in the 30 untranslated region. EMBO J, 13, 1476–1481. Ozeki Y, Nagamura Y, Ito H et al (1999) An anti-platelet agent, OPC29030, inhibits translocation of 12-lipoxygenase and 12hydroxyeicosatetraenoic acid production in human platelets. Br J Pharmacol, 128, 1699–1704. Pace-Asciak CR (1994) Hepoxilins: a review on their cellular actions. Biochim Biophys Acta, 1215, 1–8. Pace-Asciak CR, Reynaud D and Demin PM (1995) Hepoxilins: a review on their enzymatic formation, metabolism and chemical synthesis. Lipids, 30, 107–114. Parthasarathy S, Wieland E and Steinberg D (1989) A role for endothelial cell lipoxygenase in the oxidative modification of low density lipoprotein. Proc Natl Acad Sci USA, 86, 1046–1050. Pistorius EK and Axelrod B (1974) Iron, an essential component of lipoxygenase. J Biol Chem, 249, 3183–3186. Qiao N, Takahashi Y, Takamatsu H and Yoshimoto T (1999) Leukotriene A synthase activity of purified mouse skin arachidonate 8-lipoxygenase expressed in Escherichia coli. Biochim Biophys Acta, 1438, 131–139. Ra˚dmark O (1995) Arachidonate 5-lipoxygenase. J Lipid Mediators Cell Signalling, 12, 171–184. Ra˚dmark O (2000) The molecular biology and regulation of 5lipoxygenase. Am J Respir Crit Care Med, 161, S11–S15. Rapoport SM, Schewe T, Wiesner R et al (1979) The lipoxygenase of reticulocytes. Purification, characterization and biological dynamics of the lipoxygenase; its identity with the respiratory inhibitors of the reticulocyte. Eur J Biochem, 96, 545–561. Serhan CN (1994) Lipoxin biosynthesis and its impact in inflammatory and vascular events. Biochim Biophys Acta, 1212, 1–25. Shappell SB, Boeglin WE, Olson SJ et al (1999) 15-Lipoxygenase-2 (15LOX-2) is expressed in benign prostatic epithelium and reduced in prostate adenocarcinoma. Am J Pathol, 155, 235–245. Sigal E (1991) The molecular biology of mammalian arachidonic acid metabolism. Am J Physiol, 260, L13–L28.

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Steczko J and Axelrod B (1992) Identification of the iron-binding histidine residues in soybean lipoxygenase L-1. Biochem Biophys Res Commun, 186, 686–689. Steinberg D, Parthasarathy S, Carew TE et al (1989) Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med, 320, 915–924. Sun D, McDonnell M, Chen X-S et al (1998) Human 12(R)-lipoxygenase and the mouse ortholog. Molecular cloning, expression, and gene chromosomal assignment. J Biol Chem, 273, 33540–33547. Takahashi Y, Kawajiri H, Yoshimoto T et al (1999) 12-Lipoxygenase overexpression in rodent NG108-15 cells enhances membrane excitability by inhibiting M-type K+ channels. J Physiol, 521.3, 567– 574. Tappel AL (1963) Lipoxidase. In The Enzymes, vol 8, Boyer PD, Lardy H and Myrba¨ck K (eds). Academic Press, New York, 275–283. Toh H, Yokoyama C, Tanabe T et al (1992) Molecular evolution of cyclooxygenase and lipoxygenase. Prostaglandins, 44, 291–315. Woollard PM (1986) Stereochemical difference between 12-hydroxy5,8,10,14-eicosatetraenoic acid in platelets and psoriatic lesions. Biochem Biophys Res Commun, 136, 169–176. Xu W, Takahashi Y, Sakashita T et al (2001) Low density lipoprotein receptor-related protein is required for macrophage-mediated oxidation of low density lipoprotein by 12/15-lipoxygenase. J Biol Chem, 276, 35454–36459. Yamamoto S (1991) ‘‘Enzymatic’’ lipid peroxidation: reactions of mammalian lipoxygenases. Free Rad Biol Med, 10, 149–159. Yamamoto S (1992) Mammalian lipoxygenases: molecular structures and functions. Biochim Biophys Acta, 1128, 117–131. Yamamoto S, Suzuki H and Ueda N (1997) Arachidonate 12lipoxygenases. Prog Lipid Res, 36, 23–41. Yoshimoto T and Yamamoto S (1995) Arachidonate 12-lipoxygenase. J Lipid Mediators Cell Signalling, 12, 195–212.

6 Biosynthesis and Biological Effects of 5-oxo-ETE and Other Oxoeicosatetraenoic Acids William S. Powell McGill University, Montreal, QC, Canada

INTRODUCTION The eicosanoids comprise a large number of 20-carbon fatty acids, many of which contain oxo groups. Oxoeicosanoids can be formed in various ways, including the PG synthase-catalysed rearrangement of PGH2 to form PGE2 and PGD2, the nonenzymatic degradation of hydroperoxyeicosanoids, which is enhanced by the presence of haem compounds, and the dehydrogenase-catalysed oxidation of hydroxyeicosanoids. This chapter will deal with the formation of oxoeicosatetraenoic acids (oxo-ETEs) from hydroxyeicosatetraenoic acids (HETEs) by specific dehydrogenases and the biological activities of the resulting products. 15-Hydroxyprostaglandin dehydrogenases, the first such eicosanoid-metabolizing enzymes to be discovered, convert prostanoids and other 15-hydroxyeicosanoids to their biologically inactive 15-oxo metabolites. 12-Hydroxyeicosanoid dehydrogenases convert LTB4, 12-HETE and similar compounds to 12-oxoeicosanoids, whereas 5-hydroxyeicosanoid dehydrogenase (5h-dh) converts 5-HETE and other 5-hydroxyeicosanoids to their 5-oxo metabolites. The latter pathways will be the major focus of this chapter, as they give rise to the potent inflammatory mediator, 5-oxo-ETE.

15-OXOEICOSANOIDS Formation of 15-Oxoeicosanoids The 15-hydroxy fatty acid precursors of 15-oxoeicosanoids can be synthesized by both cyclooxygenases and lipoxygenases. The major initial step in the metabolism of prostaglandins is oxidation of the 15-hydroxyl group by a cytosolic 15-hydroxyprostaglandin dehydrogenase (15h-PG dh), resulting, in most cases, in their biological inactivation (Okita and Okita 1996). These enzymes (referred to here collectively as 15h-PG dh) are found in a wide variety of tissues, most notably the lung (Anggard et al 1971). In addition to prostaglandins, 15h-PG dh oxidizes a variety of other eicosanoids containing a hydroxyl group in the 15-position. The 15-lipoxygenase (15-LO) products 15-HETE, 5,15-diHETE and 8,15-diHETE (Bergholte et al 1987) as well as LXA4 (Serhan et al 1993) are all excellent substrates, being converted to their 15-oxo metabolites. This enzyme also metabolizes shorter chain fatty acids with hydroxyl groups in the o6-position, including the cyclooxygenase product 12-HHTrE (Liu et al 1985) and the lipoxygenase product 13-hydroxy-9,11-octadecadienoic acid (13HODE) (Agins et al 1987) to 12-oxo-5,8,11-heptadecatrienoic The Eicosanoids. Edited by Peter Curtis-Prior. &2004 John Wiley & Sons, Ltd: ISBN 0 471 48984 0

acid and 13-oxo-9,11-octadecadienoic acid (13-oxo-ODE), respectively. Other dehydrogenases also metabolize o6-hydroxy fatty acids. An NAD+-dependent dehydrogenase in rat liver cytosol converts 13-HODE to 13-oxo-ODE (Bronstein and Bull 1997). This enzyme appears to be relatively specific, as the only other substrates found to be metabolized to an appreciable extent were 9-HODE and 15-HETE. In contrast to both the above enzyme and 15h-PG dh, which are found in the cytosol, a microsomal 15-HETE dehydrogenase has been reported in mouse liver (Sok et al 1988). Metabolism of 15-Oxoeicosanoids As with 15-oxoprostaglandins, uncyclized 15-oxoeicosanoids are metabolized by D13-prostaglandin reductase. 5-Hydroxy-15-oxoETE is converted to 5-hydroxy-15-oxo-13,14-dihydro-ETE by a NADPH-dependent cytosolic reductase in human neutrophils (Berhane et al 1998). LXA4 is converted to an analogous dihydro metabolite by monocytes, following initial oxidation of the 15hydroxyl group (Serhan et al 1993). It is likely that 15-oxo-ETE would be metabolized in a similar fashion, but this would be accompanied by the loss of the UV-absorbing chromophore, which makes this reaction more difficult to monitor by HPLC. 12-OXOEICOSANOIDS Formation of 12-oxo eicosanoids 12-Oxoeicosanoids are formed from 12-hydroxy or 12-hydroperoxy precursors, which in turn are synthesized by 12-lipoxygenases. Several such enzymes exist, including the highly specific platelet 12-LO and the less specific leukocyte 12-LO, which both convert arachidonic acid to 12S-HETE (Brash 1999). In addition, a 12Rlipoxygenase has recently been identified in human skin (Boeglin et al 1998). 12-Oxo-ETE can be formed both enzymatically from 12S-HETE or 12R-HETE or non-enzymatically by decomposition of 12-HPETE. In contrast to 15h-PG dh, 12-hydroxyeicosanoid dehydrogenases have been characterized only relatively recently. The first clue for the enzymatic formation of 12-oxoeicosanoids came from the identification of 10,11-dihydro metabolites of 12HETE and LTB4 in incubations with leukocytes (Powell 1987; Wainwright et al 1990) and corneal microsomes (Murphy et al 1988). Porcine leukocytes convert 12S-HETE to 10,11-dihydro

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and 10,11-dihydro-12-oxo metabolites. Initial mass spectrometric studies suggested that the 10,11-dihydro metabolite was formed from deuterium-labelled 12-HETE with partial retention of the deuterium at C12, suggesting direct reduction of the 10,11-double bond by an olefin reductase. However, subsequent experiments clearly showed that the initial step in the formation of this compound was oxidation of the 12-hydroxyl group of 12-HETE to give 12-oxo-5,8,10,14-eicosatetraenoic acid (12-oxo-ETE) (Wainwright and Powell 1991). Unlike cytosolic 15h-PG dh, 12h-dh resides in the microsomal fraction of neutrophils and requires NAD+ as a co-factor (Wainwright and Powell 1991). This enzyme also converts LTB4 to its 12-oxo metabolite. It appears to be relatively specific for a hydroxyl group in the 12-position, but may also exhibit some activity towards 15-HETE and 13-HODE (Wainwright et al 1990) Interestingly, 12h-dh may not be stereospecific, as porcine neutrophil microsomes convert both 12S-HETE and 12R-HETE to 12-oxo-ETE at similar rates. Furthermore, 12S-HETE and LTB4, which have opposite configurations at C12, are equally good substrates for this enzyme (Wainwright and Powell 1991). On the other hand, it is possible that this apparent lack of specificity could be due to the presence of multiple 12h-dhs in neutrophils. Corneal microsomes appear to contain a similar dehydrogenase that oxidizes 12R-HETE (Nishimura et al 1991). The neutrophil 12h-dh is quite distinct from LTB4 12hydroxy-dehydrogenase, which oxidizes the 12R-hydroxyl group of LTB4 and is found in the kidney (Yokomizo et al 1993). The kidney enzyme differs from the neutrophil dehydrogenase in that it is found in the cytosol, requires NADP+ as a co-factor, does not metabolize 12-HETE (Yokomizo et al 1993) and also has 15-oxoPG D13 reductase activity (Ensor et al 1998). 12-Oxo-ETE can also be formed non-enzymatically from 12HPETE by cells that contain 12-LO. This reaction has been investigated in platelets, and is thought to occur via the haemcatalysed loss of water from the hydroperoxide (Fruteau de Laclos et al 1987). 12-oxo-ETE has also been identified as a metabolite of arachidonic acid in nervous tissue from the marine mollusc Aplysia californica and, as in platelets, was thought to have arisen from 12-HPETE (Piomelli et al 1988). Metabolism of 12-oxo-ETE As with 12-oxo-LTB4, 12-oxo-ETE is metabolized by a D10reductase to 10,11-dihydro-12-oxo-ETE (i.e. 12-oxo-5,8,14-eicosatrienoic acid; 12-oxo-ETrE), which is further metabolized to 10,11-dihydro-12-HETE (i.e. 12-hydroxy-5,8,14-eicosatrienoic acid; 12-HETrE). The major product of metabolism of 12S-HETE by porcine neutrophils is 12R-HETrE. Although the 12-hydroxyl group of the product is in the R configuration because of the priority rules, it has the same stereochemistry as the 12S-hydroxyl group of the substrate. 12S-HETrE is also formed from 12S-HETE, but much more slowly than 12R-HETrE. The 12h-dh/D10-reductase pathway has also been found in other types of cells and tissues. Dihydro metabolites of 12-HETE, presumably formed by the metabolism of 12-oxo-ETE, are formed by this or a related process in corneal microsomes (Nishimura et al 1991). Using LTB4 as a substrate, several groups have demonstrated this or a related pathway to exist in a variety of cell types, including mesangial cells, T lymphocytes, macrophages (Kaever et al 1987; Scho¨nfeld et al 1988), monocytes (Fauler et al 1989) and keratinocytes (Wheelan et al 1993), as well as in the lung (Kumlin et al 1990). 12-Oxo-ETE can be directly reduced to 12-HETE by 12ketoreductases in microsomal fractions from rat liver (Falgueyret et al 1988), skin (Falgueyret et al 1990) and leukocytes (Falgueyret et al 1990). In the case of liver microsomes, a mixture of 12R- and

12S-HETE was formed, whereas the skin and leucocyte enzymes converted 12-oxo-ETE stereospecifically to 12S-HETE. It is not known whether or not the 12-ketoreductase and the 12-hydroxydehydrogenase activities are due to the same enzyme. Biological Activity of Metabolites of 12-HETE Formed by the 12h-dh Pathway 12R-HETE, which has the same configuration at C12 as LTB4, stimulates neutrophils by activating the LTB4 receptor (Cunningham and Woollard 1987). Although it induces the same maximal response as LTB4, it is about 250 times less potent in mobilizing calcium in these cells (Table 6.1). 12S-HETE is about 20-fold less potent than 12R-HETE in activating neutrophils. Both 12SHETE and 12R-HETE can be metabolized by the 12h-dh/D10reductase pathway (Wainwright and Powell 1991). However, neither the initial product, 12-oxo-ETE, nor its metabolite 10,11dihydro-12-oxo-ETE (i.e. 12-oxo-ETrE) has appreciable agonist activity in neutrophils (Powell et al 1995c). Reduction of the keto group restores biological activity, with 12S-HETrE and 12RHETrE exhibiting about one-third the potency of the corresponding 12-HETE (Table 6.1). Although the 12h-dh/D10-reductase pathway serves to biologically inactivate 12R-HETE and LTB4 with respect to neutrophils, it may have other functions in other cells. As noted above, 12-oxoETE is formed in Aplysia neural tissue and has been shown to elicit changes in membrane potential in neurons from this species (Piomelli et al 1988). The dihydro metabolite of 12-oxo-ETE, 12R-HETrE, which is formed in the cornea (Murphy et al 1988) and blood vessels, may have an important role as a proinflammatory agent in these tissues (Ramboer et al 1992). This compound has been reported to have vasodilatory effects (Murphy et al 1988) and it is a highly potent angiogenic agent, both in vivo in the rabbit eye (Masferrer et al 1991) and in vitro (Stoltz et al 1996b). This effect may be mediated by a specific receptor in endothelial cells (Stoltz and Schwartzman 1997). 12-HETrE may act through NF-kB in microvessel endothelial cells (Stoltz et al 1996a) and has recently been reported to stimulate VEGF production by these cells (Mezentsev et al 2002). 5-OXOEICOSANOIDS Formation of 5-Oxoeicosanoids Synthesis by 5h-dh As with 12-HETE and 15-HETE, 5-hydroxyeicosanoids are metabolized by a dehydrogenase pathway. Evidence for the existence of such a pathway initially came from studies showing that neutrophils convert 6-trans isomers of LTB4 to dihydro

Table 6.1 EC50 values for the effects of 12R-HETE and 12S-HETE and their metabolites formed by the 12h-dh/D10-reductase pathway on calcium mobilization in human neutrophils Compound LTB4 12R-HETE 12S-HETrE 12S-HETE 12R-HETrE 12-oxo-ETE 12-oxo-ETrE

EC50 (nM) 0.75 190 530 2000 6000 4 410 000 4 410 000

5-OXO-ETE AND OTHER OXO-ETES

Figure 6.1 Relative rates of conversion of different eicosanoids to oxometabolites by human neutrophil microsomes in the presence of NADP+ (1 mM)

Figure 6.2 Regulation of 5-oxo-ETE synthesis in human neutrophils. By stimulating NADPH oxidase, PKC increases the intracellular levels of NADP+, the co-factor required for the synthesis of 5-oxo-ETE by 5h-dh. The effects of PMA are inhibited by Ssp (staurosporine) or DPI (diphenylene iodonium), or by heating neutrophils at 468C for 9 min. NADPH oxidase can be bypassed by addition of PMS (phenazine methosulphate), which directly converts NADPH to NADP+

Figure 6.3 Metabolism of 5-oxo-ETE

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metabolites (Powell 1986; Powell and Gravelle 1988). Deuteriumlabelling experiments indicated that the formation of these metabolites requires prior oxidation of the 5-hydroxyl group. Interestingly, this reaction is quite specific for 6-trans metabolites of LTB4, as LTB4 itself is not a substrate (Powell et al 1992). It seemed unlikely that the major substrate for 5h-dh would be 6trans isomers of LTB4, as these compounds are formed nonenzymatically and have little biological activity. The most likely candidate for a biologically relevant substrate for this enzyme was 5S-HETE, which has a 5S-hydroxyl group and a 6-trans double bond in common with 6-trans-LTB4. Examination of the substrate specificity for 5h-dh revealed that this enzyme is highly specific for eicosanoids containing a 5S-hydroxyl group followed by a 6-trans double bond, and that 5S-HETE was indeed the best substrate (Figure 6.1). In contrast to 12-hydroxyeicosanoids, which are rapidly metabolized by 12h-dh in unstimulated porcine and rat neutrophils, 5-hydroxyeicosanoids are oxidized to only a small extent by 5h-dh unactivated human neutrophils. Instead, 5-HETE undergoes rapid hydroxylation to 5,20-diHETE (O’Flaherty et al 1986; Powell et al 1994). Activation of neutrophils with PMA or opsonized zymosan results in dramatically enhanced synthesis of 5-oxo-ETE. This effect appears to be mediated by protein kinase C (PKC) stimulation of NADPH oxidase, as it can be blocked by the PKC inhibitor staurosporine and by inhibition of the oxidase, either by gentle heating or by treatment with diphenylene iodonium (DPI) (Figure 6.2) (Powell et al 1994). The stimulatory effect of NADPH oxidase on 5-oxo-ETE production is due to elevation of intracellular levels of NADP+, the co-factor required for its synthesis. This effect can be bypassed by artificially raising intracellular NADP+ levels by addition of phenazine methosulphate, which non-enzymatically converts NADPH to NADP+ (Powell et al 1994). These results suggest that the release of 5-oxoETE is highly regulated, and requires not only activation of PLA2 and 5-LO, but also the adjustment of intracellular co-factor levels to provide an adequate level of NADP+ to permit oxidation of 5-HETE. Other blood cells, including monocytes (Zhang et al 1996), lymphocytes (Zhang et al 1996), eosinophils (Powell et al 1995a) and platelets (Powell et al 1999b), also contain 5h-dh. Of these,

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As with other oxoeicosanoids, 5-oxo-ETE can be generated nonenzymatically from 5-HPETE. 5-oxo-ETE, probably formed by this mechanism, was detected following incubation of supernatants from MC-9 cells with arachidonic acid (Bryant et al 1986). Treatment of red blood cell ghosts with t-butyl hydroperoxide led to the formation of 5-oxo-ETE esterified to phospholipids, which was detected by liquid chromatography–mass spectrometry (Hall and Murphy 1998).

that eosinophils convert arachidonic acid to a lipid chemoattractant that was not identical to LTB4 and had a UV spectrum (Morita et al 1990) similar to that of 5-oxo-15-HETE, which we isolated after incubation of 15-HETE with neutrophil microsomes in the presence of NADP+ (Powell et al 1992). Schro¨der’s group went on to show that 5-oxo-15-HETE, which they synthesized by incubation of arachidonic acid with soybean lipoxygenase, is a potent eosinophil chemoattractant, suggesting that this is the substance responsible for the eosinophil-derived lipid chemoattractant activity that they previously detected. The finding that 5-HETE is converted to 5-oxo-ETE by neutrophils can also explain earlier work by O’Flaherty’s group showing that 5-HETE is a weak neutrophil agonist that acts independently of receptors for other lipid mediators (O’Flaherty 1985; O’Flaherty et al 1988). The relatively low potency of 5-HETE suggested that it may be acting through a receptor for another unknown lipid mediator. From subsequent work (discussed below), it would appear that the biological activity of 5-HETE is due to its interaction with the receptor for the more potent 5oxo-ETE, which is a potent activator of eosinophils and neutrophils and also has effects on other cells including monocytes and epithelial cells.

Metabolism of 5-oxo-ETE

Neutrophils

5-oxo-ETE is metabolized by a variety of pathways, including o-oxidation and reduction of both olefinic and keto groups, as well as by oxidation by lipoxygenases (Figure 6.3). The major pathway for the metabolism of 5-oxo-ETE in neutrophils is ohydroxylation to its 20-hydroxy metabolite (Powell et al 1996), which is presumably catalysed by LTB4 20-hydroxylase. This enzyme is present in large amounts in neutrophils, but is absent from monocytes and other blood cells (Powell 1984). Mouse macrophages, which lack the 20-hydroxylase enzyme, convert 5-oxo-ETE to 18- and 19-hydroxylated products (Hevko et al 2001). Neutrophils also contain a cytosolic D6-reductase that converts 5-oxo-ETE to its 6,7-dihydro metabolite (i.e. 5-oxo-ETrE). This enzyme, which requires NADPH as a co-factor, is clearly distinct from PG D13-reductase because of its requirement for calcium and calmodulin, suggesting that its activity may be tightly regulated (Berhane et al 1998). 5-oxo-ETE can also be converted back to its precursor, 5S-HETE, probably by 5h-dh acting in the reverse direction (Powell et al 1992), and this is the major pathway for the metabolism of 5-oxo-ETE by unstimulated platelets (Powell et al 1999b). In contrast, stimulation of platelets with thrombin, which results in activation of 12-LO, results in the conversion of 5-oxoETE to its 12-hydroxy metabolite, 5-oxo-12-HETE. 5-oxo-ETE is also a substrate for 15-LO, which converts it to 5-oxo-15S-HETE. This may be a significant reaction in eosinophils, which possess high 15-LO activity (Turk et al 1982). 5-oxo-ETE can be converted to a glutathione conjugate, 5-oxo7-glutathionyl-8,11,14-eicosatrienoic acid (FOG7), by mouse macrophages (Bowers et al 2000). This compound, which is biologically active, appears to be formed by LTC4 synthase in these cells (Hevko and Murphy 2002). Other glutathione transferases also convert 5-oxo-ETE to similar products, but in this case they are not biologically active, suggesting that they are inactive isomers of FOG7 (Hevko and Murphy 2002).

5-oxo-ETE is nearly 100 times more potent than its precursor, 5SHETE, in stimulating neutrophil migration and calcium mobilization (Powell et al 1993). It also induces a variety of other responses in neutrophils, including actin polymerization, surface expression of the adhesion molecules CD11b and CD11c, adherence and aggregation (Norgauer et al 1996; O’Flaherty et al 1994; Powell et al 1997). Although 5-oxo-ETE by itself has only modest effects on degranulation and the oxidative burst in neutrophils, it is a strong inducer of these responses in the presence of both platelet-activating factor (PAF) and tumour necrosis factor-a (TNFa) (O’Flaherty et al 1994). Pretreatment of neutrophils with either granulocyte-macrophage colony stimulating factor (GM-CSF) or G-CSF also strongly enhances these responses (O’Flaherty et al 1994). 5-oxo-ETE has been shown to activate certain kinases involved in intracellular signalling in neutrophils. It stimulates both phosphatidylinositol 3-kinase (Norgauer et al 1996) and MAP kinases (ERK-1 and ERK-2) (O’Flaherty et al 1996a) in these cells. It also stimulates phosphorylation of cPLA2 in neutrophils and the release of arachidonic acid, the latter effect being potentiated by pretreatment with low concentrations of GMCSF (O’Flaherty et al 1996a). The 5-oxo-ETE metabolite FOG7 is also capable of activating both neutrophils and eosinophils (Bowers et al 2000). This substance is a chemoattractant for these cells, and induces actin polymerization. However, unlike 5-oxo-ETE, it does not elicit a calcium mobilization response. The nature of the receptor mediating the response to FOG7 is unclear, and may well be the cys-LT1 receptor rather than the 5-oxo-ETE receptor. The effects of 5-oxo-ETE on neutrophils are very similar to those of LTB4. However, in all of the responses tested, LTB4 is about 10–50 times more potent than 5-oxo-ETE, although the maximal responses to the two eicosanoids are similar. As LTB4 and 5-oxo-ETE are likely to be formed concomitantly and in comparable amounts, this raises the question of the physiological relevance of 5-oxo-ETE as a neutrophil agonist. In normal circumstances, it would seem likely that LTB4 is more important than 5-oxo-ETE in regulating neutrophil activity. However, it is possible that in certain pathological situations 5-oxo-ETE may also play a role. For example, the latter compound could be involved in persistent inflammation, in which case neutrophils

monocytes are the most active, producing levels of 5-oxo-ETE comparable to or greater than those of LTB4. Although platelet microsomes rapidly convert 5-HETE to 5-oxo-ETE in the presence of NADP+, intact platelets produce little 5-oxo-ETE, but instead catalyse the reverse reaction (i.e. formation of 5HETE from 5-oxo-ETE) and could thus be involved in the biological inactivation of 5-oxo-ETE. As with neutrophils, conversion of 5-HETE to 5-oxo-ETE by intact platelets is dramatically enhanced by increasing intracellular NADP+ levels by addition of phenazine methosulphate (Powell et al 1999b). Non-enzymatic Synthesis of 5-oxo-ETE

Biological Activity of 5-Oxoeicosanoids The high degree of specificity of 5h-dh suggested that the product of this reaction may have some biological significance. This concept was supported by the finding by Schro¨der’s group

5-OXO-ETE AND OTHER OXO-ETES

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may become desensitized to LTB4 (Marleau et al 1993). Furthermore, 5-oxo-ETE appears to be metabolized more slowly than LTB4, which is very rapidly converted to o-oxidation products by LTB4 20-hydroxylase in neutrophils (Powell 1984).

C5a, LTB4 and fMLP (O’Flaherty et al 1996b). 5-oxo-ETE also induces phosphorylation of MAP kinases (ERK-1 and ERK-2) in eosinophils, and is much more potent in stimulating this activity in eosinophils than in neutrophils (O’Flaherty et al 1996b).

Eosinophils

Monocytes

In contrast to neutrophils, human eosinophils undergo a much stronger chemotactic response to 5-oxo-ETE than to LTB4, which has only weak effects on the migration of these cells (Powell et al 1995a). The maximal chemotactic response elicited by 5-oxo-ETE is considerably greater than that induced by other lipid mediators, including PAF (O’Flaherty et al 1996b; Powell et al 1995a) and PGD2 (Monneret et al 2001), and is also greater than that of the potent eosinophil-specific chemokine eotaxin (Powell et al 2001). At low concentrations (1–10 nM), 5-oxo-ETE also induces a stronger response than PAF and other lipid mediators, with the exception of PGD2, which has about the same effect on eosinophil migration in this concentration range (Monneret et al 2001). However, in this concentration range eotaxin induces a somewhat stronger response than 5-oxo-ETE (Powell et al 2001). Eotaxin and 5-oxo-ETE appear to have a synergistic effect on eosinophils, as low concentrations of this chemokine induce a leftward shift in the concentration–response curve for 5-oxo-ETE (Powell et al 2001). 5-oxo-15-HETE has a similar effect to 5-oxo-ETE on eosinophil migration (Schwenk and Schro¨der 1995) but is somewhat less potent (Powell et al 1995a). 5-oxo-ETE is also a highly potent stimulator of eosinophil migration through a Matrigel matrix, an effect that may be mediated by proteinase release (Guilbert et al 1999). 5-oxo-ETE is also active in vivo, as it induces infiltration of eosinophils into the lungs of Brown Norway rats following intratracheal administration (Stamatiou et al 1998). This effect is dependent on 5-oxo-ETE itself, as it could not be blocked by antagonists to either PAF or LTB4 and was not shared by cysteinyl leukotrienes. However, it could be blocked by administration of antibodies against either of the adhesion molecules VLA-4 (CD49d) or LFA-1 (CD11a). We have also recently shown that intradermal injection of 5-oxo-ETE induces infiltration of eosinophils into human skin. However, this effect is not specific for eosinophils, as an increase in neutrophils was also observed (Muro et al 2003). In addition to its effects on eosinophil migration, 5-oxo-ETE can also promote eosinophil survival, an effect not shared by the eosinophil chemokines eotaxin and RANTES (regulated upon activation, normal T cell expressed and secreted) (Stamatiou et al 2001). However, unlike its other effects on eosinophils, this effect is indirect, as it was nearly completely dependent on the presence of small numbers of contaminating monocytes. This study suggests that 5-oxo-ETE can stimulate monocytes to release a survival factor for eosinophils (Stamatiou et al 2001). 5-oxo-ETE also induces a variety of rapid responses in eosinophils, including calcium mobilization, actin polymerization, surface expression of CD11b, and shedding of L-selectin (Czech et al 1997; Powell et al 1999a). Interestingly, LTB4 also induces most of these responses in eosinophils, with a potency similar to that of 5-oxo-ETE, in spite of the fact that it is a very weak chemoattractant for human eosinophils. However, LTB4 has only a weak effect on actin polymerization compared to 5-oxoETE, suggesting that this response may correlate more closely than other responses, such as calcium mobilization, to cell migration (Powell et al 1999a). 5-oxo-ETE displays only very weak degranulating activity for eosinophils, but its potency is markedly increased following priming with GM-CSF. Low concentrations of 5-oxo-ETE also dramatically enhance degranulation responses to a variety of other mediators, including PAF,

5-oxo-ETE also induces a chemotactic response in human monocytes (Sozzani et al 1996). Although not as potent as fMLP or MCP-1, it strongly enhances the chemotactic response to both MCP-1 and MCP-3. 5-oxo-ETE also stimulates actin polymerization, but has no effect on intracellular calcium levels in these cells (Sozzani et al 1996). Epithelial cells Guinea-pig jejunal crypt epithelial cells undergo a reduction in cell volume following treatment with 5-oxo-ETE (MacLeod et al 1999). This effect is selective for crypt cells, as villus cells do not respond to this substance. 5-oxo-ETE is much more potent than a variety of other mediators, including LTD4, bradykinin, vasoactive intestinal peptide and 5-HETE. In contrast, LTB4 has no effect on the volumes of these cells. The effect of 5-oxo-ETE is blocked by inhibitors of chloride and potassium channels, suggesting that it is due to activation of ion channels, possibly through activation of protein kinase C. Prostate Cancer Cells The 5-lipoxygenase inhibitors MK-886 and AA861 were found to be strong inducers of apoptosis in PC3 and LNCaP prostate cancer cells (Ghosh and Myers 1997, 1998). This effect was reversed by 5-oxo-ETE and, to a somewhat lesser extent, by 5HETE. In contrast, LTB4 was inactive. These cell lines were reported to constitutively synthesize immunoreactive 5-HETE from endogenous substrate. However, the production of 5-oxoETE itself was not measured directly. Thus, 5-oxo-ETE may be an endogenous survival factor for prostate tumour cells, and inhibition of its synthesis may induce cell death (Ghosh and Myers 1998). The 5-oxo-ETE Receptor The actions of 5-oxo-ETE are mediated by a unique G proteincoupled receptor. 5-oxo-ETE is specifically bound by high affinity binding sites on neutrophils (O’Flaherty et al 1998). Structure– activity studies indicate that this receptor is highly selective for 5oxo-ETE. Biological responses to this eicosanoid are subject to homologous, but not heterologous desensitization, and are not blocked by selective antagonists of other chemoattractant receptors. Structure–Activity Relationships The structural requirements for 5-oxo-ETE-induced calcium mobilization in neutrophils have been investigated in considerable detail (Powell et al 1996). This response was found to be highly selective for 5-oxo-ETE, as minor structural modifications resulted in dramatic losses in biological activity. Interestingly, nearly all of the metabolites of 5-oxo-ETE have much lower biological activities than 5-oxo-ETE itself, with the possible exception of 5-oxo-15-HETE (Figure 6.4). Methylation of the

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BIOSYNTHESIS AND METABOLISM responses, indicating that its actions are not mediated by the BLT1 receptor (O’Flaherty et al 1993; Powell et al 1993). Similarly, both LTB4 and PAF antagonists had no effect on 5-oxo-ETE-induced eosinophil infiltration into rat lungs, but strongly inhibited eosinophil infiltration induced by LTB4 and PAF (Stamatiou et al 1998).

Coupling to G Proteins The effects of 5-oxo-ETE on leukocytes can be blocked by the Gi protein inhibitor pertussis toxin, suggesting that it acts via a G protein-coupled receptor (Norgauer et al 1996; O’Flaherty et al 2000; Powell et al 1996). In agreement with this, 5-oxo-ETE stimulates GTP/GDP exchange in membrane fractions from neutrophils (O’Flaherty et al 2000). Pertussis toxin can also inhibit the effects of other chemoattractants, but to a lesser extent than those of 5-oxo-ETE, which are completely blocked by this substance (O’Flaherty et al 2000). This suggests that 5-oxo-ETE has an absolute requirement for Gi proteins, whereas other chemoattractants can also signal though other G proteins such as Gq and G12/13. Figure 6.4 Effects of structural modifications of 5-oxo-ETE on biological activity (calcium mobilization). The values were calculated by dividing the EC50 value for 5-oxo-ETE (4 nM) by that for the 5-oxo-ETE analogue, and multiplying by 100

Cloning and expression of the 5-oxo-ETE receptor

carboxyl group results in a 20-fold reduction in potency, whereas reduction of the oxo group to a hydroxyl group, as in its precursor, 5-HETE, results in a nearly 100-fold loss in potency. Reduction of the 6,7-double bond (Berhane et al 1998) results in an even greater loss in potency, whereas isomerization of the 8,9double bond from the cis to the trans configuration results in an approximately six-fold loss in potency (O’Flaherty et al 1994; Powell et al 1996). The metabolite of 5-oxo-ETE formed by the 12-lipoxygenase pathway, 5-oxo-12-HETE, has virtually no calcium-mobilizing activity in neutrophils (Powell et al 1999b). In contrast, the product formed by the 15-lipoxygenase pathway, 5-oxo-15-HETE, has about one-tenth the potency of 5-oxo-ETE (Powell et al 1993; Schwenk and Schro¨der 1995). 5-oxo-EPE, the product analogous to 5-oxo-ETE formed from 5,8,11,14,17eicosapentaenoic acid, has about one-tenth the potency of 5oxo-ETE (Powell et al 1995b). Finally, metabolism of 5-oxo-ETE by the o-oxidation pathway, to 20-hydroxy-5-oxo-ETE, results in an almost 100-fold loss in potency (Powell et al 1996).

CONCLUSIONS

Desensitization to 5-oxo-ETE Desensitization studies strongly support the existence of a specific 5-oxo-ETE receptor. Preincubation of neutrophils (O’Flaherty et al 1993; Powell et al 1993), eosinophils (Powell et al 1999a; Schwenk and Schro¨der 1995) or monocytes (Sozzani et al 1996) with 5-oxo-ETE resulted in complete desensitization to subsequent addition of this substance but did not block responses to other chemoattractants, including LTB4 and PAF. Similarly, initial treatment of leukocytes with other chemoattractants did not prevent 5-oxo-ETE-induced cell activation. Effects of Antagonists on Other Chemoattractant Receptors The effects of 5-oxo-ETE on neutrophils are not prevented by concentrations of LTB4 antagonists that block LTB4-induced

The receptor for 5-oxo-ETE has recently been cloned and expressed by two groups independently, each using a bioinformatics approach (Hosoi et al 2002; Jones et al 2003). In agreement with prior pharmacological data, the cloned receptor is highly selective for 5-oxo-ETE and is coupled to Gi. 5-oxo-ETE induced migration and inhibited forskolin-stimulated cAMP formation in cell transfected with this receptor. The 5-oxo-ETE receptor is very highly expressed in human eosinophils, with smaller amounts being present in neutrophils and macrophages.

In the cases of many eicosanoids, in particular prostaglandins and LTB4, oxidation of one of the hydroxyl groups to a keto group is an important step in their biological inactivation. However, this is not true for 5-HETE and 12-HETE, which can be converted by dehydrogenases to products with potent biological activities. Oxidation of 5S-HETE by 5h-dh results in the formation of 5oxo-ETE, a potent chemoattractant for human eosinophils that may be an important mediator in asthma. The formation of this substance is controlled not only by substrate levels, but also by the availability of the co-factor of the enzyme, NADP+. The biological actions of 5-oxo-ETE are mediated by a highly specific Gi protein-coupled receptor. 12-HETE can also be oxidized to biologically active products. In this case, the most potent product (12-HHTrE) is formed by the further metabolism of the initial oxidation product, 12-oxo-ETE, by a D10-reductase to give an 11,12-dihydro product that is a potent angiogenic agent. Thus, both of these HETE dehydrogenase pathways can give rise to potent biological products that may be important mediators in inflammatory diseases.

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O’Flaherty JT, Wykle R, Redman J et al (1986) Metabolism of 5hydroxyeicosatetraenoate by human neutrophils: production of a novel o-oxidized derivative. J Immunol, 137, 3277–3283. Okita RT and Okita JR (1996) Prostaglandin-metabolizing enzymes during pregnancy: characterization of NAD(+)-dependent prostaglandin dehydrogenase, carbonyl reductase, and cytochrome P450-dependent prostaglandin o-hydroxylase. Crit Rev Biochem Mol Biol, 31, 101–126. Piomelli D, Feinmark SJ, Shapiro E and Schwartz JH (1988) Formation and biological actions of 12-ketoeicosatetraenoic acid in the nervous system of Aplysia. J Biol Chem, 263, 16591–16596. Powell WS (1984) Properties of leukotriene B4 20-hydroxylase from polymorphonuclear leukocytes. J Biol Chem, 259, 3082–3089. Powell WS (1986) Novel pathway for the metabolism of 6-transleukotriene B4 by human polymorphonuclear leukocytes. Biochem Biophys Res Commun, 136, 707–712. Powell WS (1987) Conversion of leukotriene B4 to dihydro- and 19hydroxy metabolites by rat polymorphonuclear leukocytes. Biochem Biophys Res Commun, 145, 991–998. Powell WS, Ahmed S, Gravel S and Rokach J (2001) Eotaxin and RANTES enhance 5-oxo-6,8,11,14-eicosatetraenoic acid-induced eosinophil chemotaxis. J Allergy Clin Immunol, 107, 272–278. Powell WS, Chung D and Gravel S (1995a) 5-oxo-6,8,11,14Eicosatetraenoic acid is a potent stimulator of human eosinophil migration. J Immunol, 154, 4123–4132. Powell WS, Gravel S and Gravelle F (1995b) Formation of a 5-oxo metabolite of 5,8,11,14,17-eicosapentaenoic acid and its effects on human neutrophils and eosinophils. J Lipid Res, 36, 2590–2598. Powell WS, Gravel S and Halwani F (1999a) 5-oxo-6,8,11,14Eicosatetraenoic acid is a potent stimulator of L-selectin shedding, surface expression of CD11b, actin polymerization, and calcium mobilization in human eosinophils. Am J Resp Cell Mol Biol, 20, 163–170. Powell WS, Gravel S, Halwani F et al (1997) Effects of 5-oxo-6,8,11,14eicosatetraenoic acid on expression of CD11b, actin polymerization and adherence in human neutrophils. J Immunol, 159, 2952–2959. Powell WS, Gravel S, Khanapure SP and Rokach J (1999b) Biological inactivation of 5-oxo-6,8,11,14-eicosatetraenoic acid by human platelets. Blood, 93, 1086–1096. Powell WS, Gravel S, MacLeod RJ et al (1993) Stimulation of human neutrophils by 5-oxo-6,8,11,14-eicosatetraenoic acid by a mechanism independent of the leukotriene B4 receptor. J Biol Chem, 268, 9280– 9286. Powell WS and Gravelle F (1988) Metabolism of 6-trans isomers of leukotriene B4 to dihydro products by human polymorphonuclear leukocytes. J Biol Chem, 263, 2170–2177. Powell WS, Gravelle F and Gravel S (1992) Metabolism of 5(S)-hydroxy6,8,11,14-eicosatetraenoic acid and other 5(S)-hydroxyeicosanoids by a specific dehydrogenase in human polymorphonuclear leukocytes. J Biol Chem, 267, 19233–19241. Powell WS, Gravelle F and Gravel S (1994) Phorbol myristate acetate stimulates the formation of 5-oxo-6,8,11,14-eicosatetraenoic acid by human neutrophils by activating NADPH oxidase. J Biol Chem, 269, 25373–25380. Powell WS, Hashefi M, Falck JR et al (1995c) Effects of oxo and dihydro metabolites of 12-hydroxy-5,8,10,14-eicosatetraenoic acid on chemotaxis and cytosolic calcium levels in human neutrophils. J Leukocyte Biol, 57, 257–263.

Powell WS, MacLeod RJ, Gravel S et al (1996) Metabolism and biologic effects of 5-oxoeicosanoids on human neutrophils. J Immunol, 156, 336–342. Ramboer I, Blin P, Lacape G, Daret D et al (1992) Effects of monohydroxylated fatty acids on arterial smooth muscle cell properties. Kidney Int, 41, S67–S72. Scho¨nfeld W, Schlu¨ter B, Hilger R and Ko¨nig W (1988) Leukotriene generation and metabolism in isolated human lung macrophages. Immunology, 65, 529–536. Schwenk U and Schro¨der JM (1995) 5-oxo-Eicosanoids are potent eosinophil chemotactic factors—functional characterization and structural requirements. J Biol Chem, 270, 15029–15036. Serhan CN, Fiore S, Brezinski DA and Lynch S (1993) Lipoxin A4 metabolism by differentiated HL-60 cells and human monocytes: conversion to novel 15-oxo and dihydro products. Biochemistry, 32, 6313–6319. Sok DE, Kang JB and Shin HD (1988) 15-Hydroxyeicosatetraenoic acid dehydrogenase activity in microsomal fraction of mouse liver homogenate. Biochem Biophys Res Commun, 156, 524–529. Sozzani S, Zhou D, Locati M et al (1996) Stimulating properties of 5-oxoeicosanoids for human monocytes: synergism with monocyte chemotactic protein-1 and -3. J Immunol, 157, 4664–4671. Stamatiou P, Chan CC, Monneret G et al (2001) 5-oxo-6,8,11,14Eicosatetraenoic acid (5-oxo-ETE) stimulates eosinophil survival. Am J Resp Crit Care Med, 163, A321. Stamatiou P, Hamid Q, Taha R et al (1998) 5-oxo-ETE induces pulmonary eosinophilia in an integrin-dependent manner in brown Norway rats. J Clin Invest, 102, 2165–2172. Stoltz RA, Abraham NG and Laniado-Schwartzman M (1996a) The role of NF-kB in the angiogenic response of coronary microvessel endothelial cells. Proc Natl Acad Sci USA, 93, 2832–2837. Stoltz RA, Conners MS, Gerritsen ME et al (1996b) Direct stimulation of limbal microvessel endothelial cell proliferation and capillary formation in vitro by a corneal-derived eicosanoid. Am J Pathol, 148, 129–139. Stoltz RA and Schwartzman ML (1997) High affinity binding sites for 12(R)-hydroxyeicosatrienoic acid [12(R)-HETrE] in microvessel endothelial cells. J Ocul Pharmacol Ther, 13, 191–199. Turk J, Maas RL, Brash AR et al (1982) Arachidonic acid 15-lipoxygenase products from human eosinophils. J Biol Chem, 257, 7068–7076. Wainwright S, Falck JR, Yadagiri P and Powell WS (1990) Metabolism of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid and other hydroxylated fatty acids by the reductase pathway in porcine polymorphonuclear leukocytes. Biochemistry, 29, 10126–10135. Wainwright SL and Powell WS (1991) Mechanism for the formation of dihydro metabolites of 12-hydroxyeicosanoids. Conversion of leukotriene B4 and 12-hydroxy-5, 8,10,14-eicosatetraenoic acid to 12oxo intermediates. J Biol Chem, 266, 20899–20906. Wheelan P, Zirrolli JA, Morelli JG and Murphy RC (1993) Metabolism of leukotriene B4 by cultured human keratinocytes. Formation of glutathione conjugates and dihydro metabolites. J Biol Chem, 268, 25439–25448. Yokomizo T, Izumi T, Takahashi T et al (1993) Enzymatic inactivation of leukotriene B4 by a novel enzyme found in the porcine kidney. Purification and properties of leukotriene B4 12-hydroxydehydrogenase. J Biol Chem, 268, 18128–18135. Zhang Y, Styhler A and Powell WS (1996) Synthesis of 5-oxo-6,8,11,14eicosatetraenoic acid by human monocytes and lymphocytes. J Leukocyte Biol, 59, 847–854.

7 Synthetic Eicosanoids Norrie H. Wilson Division of Biomedical Sciences, University of Edinburgh, Edinburgh, UK

Polyunsaturated fatty acids (PUFAs) with 20 carbon atoms, present in the tissues of living organisms, can be mobilized and oxidatively metabolized to form a vast range of highly biologically active substances which mediate many important functions. Oxidative processes arise in the living organism from chemical activity due to cyclooxygenase (COX), lipoxygenase (LOX), cytochrome P450 enzyme systems and aerial/free radical-initiated oxidation. Under usual pathophysiological conditions most of these important mediators are formed in very low quantities and the ability to obtain reasonable amounts of them by synthetic organic chemistry has been crucial to advances in this research field. The biosynthetic pathways are illustrated in Figure 7.1. Virtually all eicosanoids are readily available by chemical synthesis. However, the precursor PUFAs are usually obtained from natural biological sources. Chemical synthesis of these substances is normally only employed to obtain isotopically labelled versions for tracer and analytical studies (Adlof 1999).

INTRODUCTION TO PROSTANOIDS The prostaglandins (PGs) are formed from three precursor polyunsaturated fatty acids, as shown in Figure 7.2. In mammals the 1- and 2-series PGs are most important and the latter seems to predominate in man. The 3-series precursor, timnodonic acid, in the literature often called eicosapentaenoic acid or EPA, occurs mainly in fish and marine animals. Of the three precursor fatty acids, timnodonic is regarded as the poorest substrate for COX. Usually, therefore, a diminished PG biosynthesis results from fish oil-supplemented diets. In most of this review PG synthesis is focused on the 2-series molecules, as they appear to be most important in mammals, including humans. The shorthand notation for the precursor fatty acids is shown and reflects the requirement of double bonds at 8,11,14 (n-6, n-9, n-12), since these structural features are necessary to allow prostaglandins to form. Virtually every cell type in the mammal has the capacity to produce PGs and the biology of these eicosanoids forms one of the major topics of this treatise. The salient details of the rest of the PG biosynthesis pathway are shown in Figure 7.3 for the 2-series. The 1- and 3-series precursors are transformed similarly, producing analogous PGs. The many synthetic pathways for the production of the natural eicosanoids have been modified for the creation of huge numbers of analogues. This has allowed more selective agonist or antagonist versions to be elaborated, which in turn has increased our understanding of the various roles for these substances in the organism, as well as fostering the development of useful pharmaceutical products. The Eicosanoids. Edited by Peter Curtis-Prior. &2004 John Wiley & Sons, Ltd: ISBN 0 471 48984 0

THROMBOXANE A2 AND PROSTAGLANDIN I2— PROSTANOIDS WITH OPPOSING BIOLOGICAL ACTIONS Thromboxane A2 (TXA2) is the main prostanoid metabolite of arachidonic acid in blood platelets, where it mediates aggregation and is one of the initiators of the clotting process. TXA2 also constricts vascular smooth muscle (Armstrong and Wilson 1995). These important pathophysiological actions are limited by the, pH-dependent, short half-life of TXA2 in aqueous media. The action of TXA2 is also offset by the functional antagonism of other mediators, such as nitric oxide and particularly prostaglandin I2 (PGI2; also called prostacyclin and, in the early literature before its structure was fully known, PGX). TXA2 adds water in a general acid–base-catalysed reaction to form the biologically inactive TXB2 under all pH conditions. This consideration, and clever trapping experiments where methanol or ethanol is added across the oxetane ring instead of water, led to the proposed structure shown in Figure 7.4 (Hamberg et al 1975; see also Figure 7.16 for the total synthesis of TXA2). PGI2 is formed in the endothelial cells lining the blood vessel walls, and this PG is the most powerful platelet antiaggregating agent known (Moncada et al 1976). PGI2 is unstable in acid solution, since the structure contains an enol ether group (Johnson et al 1977, 1978). It is likely that the carboxyl group, as a neighbouring acid group, can participate in the hydrolytic decay to accelerate the formation of 6-keto-PGF1a by proton delivery to the b-carbon of the enol ether via a seven-membered transition state (see Figure 7.5). PGI2, in contrast to TXA2, is stable in alkaline solution and therefore can be stored conveniently as the sodium salt. Figures 7.4 and 7.5 show the hydrolytic inactivation of TXA2 and PGI2 and also illustrate the hemiacetal and hemiketal nature of TXB2 and 6-keto-PGF1a, respectively. The ring–chain tautomerism of these species is also shown and is analogous to that found in glucose and fructose structures, respectively. In each case the open chain carbonyl form is in very low concentration, but they can both be made to react as carbonyl compounds, forming typical derivatives such as oximes and hydrazones (this ring–chain tautomerism is the reason for the undefined stereochemistry of the hydroxyls attached to the anomeric carbons, in the ring form of these molecules).

PROSTAGLANDINS E2, F2a AND D2 These primary PGs are important biologically in pain, fever, reproduction mechanisms and many other biological functions.

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Figure 7.1 Eicosanoid biosynthetic pathways

PGE2 and PGF2a are inactivated mainly, via the circulation, by the lung 15-dehydrogenase, where the 15-hydroxyl is oxidized to a carbonyl group. Thereafter, a series of reductions of the 13,14 double bond and b-oxidations truncating the a-chain produce biologically deactivated species which are excreted in the urine. PGD2 is not a good substrate for the 15-dehydrogenase, presumably because it would lead to the formation of the fluxional 15-carbonyl species which exists in an enol form resistant to further oxidation (Jones and Wilson 1978). Instead, PGD is

reduced to the PGF ring structure, which is then metabolized (Barrow et al 1984). THE SECONDARY PROSTAGLANDINS— PROSTAGLANDINS A, B, C AND J The secondary prostaglandins, PGA, PGB (Pike et al 1969), PGC (Jones et al 1972) and PGJ (Fitzpatrick and Wynalda 1983;

Figure 7.2 The three series of prostaglandins showing the numbering system and the primary product PGGx

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Figure 7.3 The derivation of the primary prostaglandins

Mahmud et al 1984; Narumiya and Fukushima 1985) are derived from the primary PGs by dehydration (Figure 7.6). These substances perhaps arise during extraction and analytical procedures and it is uncertain whether they are physiologically formed. They have varying degrees of biological activity but are in general less potent than the primary PGs. The secondary PGs can be made from the primary PGs as shown (Figure 7.6). In the case of PGC and PGJ, the conditions for the isomerization or dehydration need to be very mild, and involve the use of plasma or albumin (see references cited). However, enzymes that appear to perform these transformations have been characterized (Polet and Levine 1975). A PGA2 source is in a coral, Plexaura homomalla, where it occurs as the methyl ester, 15R-acetate. The biosynthetic origin of this PG is completely different from that in mammals

(Weinheimer and Spraggins 1969). Procedures for conversion of PGA2 to primary PGs are known (Bundy et al 1971). This route is now of lesser importance with the development of efficient total syntheses.

SYNTHETIC ROUTES TO PROSTAGLANDINS A vast number of synthetic routes to the prostanoids exist and the reader is guided to several specialist texts on the subject (Bindra and Bindra 1977; Roberts and Scheinmann 1982; Pike and Morton 1985). Only the most versatile and useful approaches are discussed in this review, with indications of how other analogues were elaborated from the basic methods.

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Figure 7.4 The decay of TXA2 to the biologically inactive TXB2. The ring–chain tautomers of TXB2

Figure 7.5 The hydrolytic inactivation of PGI2. The ring–chain tautomers of 6-keto-PGF1a

THE COREY SYNTHESIS AND RELATED METHODS This versatile route can generate a large range of PGs, including all the primary PGs and their analogues, for research purposes and pharmaceutical development. The starting materials are available by bulk production methods. The route is illustrated in Figures 7.7 and 7.8 for PGF2a and PGE2. It was possible to perform the alkylation of cyclopentadiene 1 with benzylchloromethylether to give 2 under mild conditions (Corey et al 1971a) to avoid moving the olefinic bonds such that the correct product was obtained on adding the ketene equivalent a-chloroacrylonitrile or a-chloroacryloyl chloride (Corey et al 1971b). Hydrolysis of the

initial Diels–Alder adduct then gave ketone 3, which underwent Baeyer–Villiger oxidation to give a lactone which was hydrolysed to the hydroxy acid 4. This acid 4 was optically resolved by crystallization of the (+)-amphetamine diastereoisomeric salts (Corey et al 1971c). The resolved 4 was then relactonized to 5 by iodolactonization on the olefinic bond, and then removal of the iodo group by reduction, and adjustment of the hydroxyl protecting groups yielded the key intermediate, the Corey lactone 7 (Corey and Suggs 1975). Conversion of the Corey lactone primary alcohol to the aldehyde 8 (Figure 7.8) originally employed a chromic oxidation (Corey et al 1971c); however, even this simple step gave very low

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Figure 7.6 The secondary prostaglandins

yields and subsequently the preferred method was shown to be the Pfitzner–Moffat oxidation (Bindra and Bindra 1977, p. 200; Corey and Kim 1972, 1973). The o-chain was then added by the Horner– Emmons version of the Wittig reaction, yielding the ketone 9. Reduction of this ketone was originally performed by zinc borohydride, and then the two hydroxy epimers were separated by chromatography. The yield of the naturally occurring 15Sepimer 10 can be enhanced by inversion of the 15R-isomer. Alternatively, particularly in a batch process, the unwanted epimer can be reoxidized to the ketone and added to the next batch for reduction. Later investigations using chiral borane reductants showed that the yield of 10 could be made 490% (Corey et al 1972b, 1987). The hydroxyl groups were protected as the tetrahydropyranyl (THP) ethers, and this was followed by di-isobutylaluminium hydride (DIBAL) reduction of the lactone to the lactol (hydroxyaldehyde) 11. The Wittig reaction then allowed addition of the a-chain with a very high proportion of the cis-olefin 12. Only a very small amount of trans-olefin is formed using this procedure, using dimesylsodium (formed from dissolution of sodium hydride in dimethylsulphoxide at 658C) in DMSO as the base. The removal of the THP groups gave PGF2a. This synthesis allows PGE2 to be obtained also by chromic oxidation

of the unprotected hydroxyl in 12 to carbonyl. The final step of the sequence, the removal of the remaining THP groups to produce PGE2, must be done under mild conditions to avoid dehydration to PGA2 (see above). The required synthons for the a- and o-chains are readily obtained, as shown in Figure 7.9. These reactions are readily extended to the preparation of a large range of analogues. The ‘‘Corey synthesis’’ is versatile and can be adapted to produce the 1-series PGs by reduction of the a-chain olefinic bond (Corey et al 1971b). By employing the appropriate ochain precursor, the 3-series compounds can also be prepared (Corey et al 1971d). A further modification where the a-chain is added first, in order that a large variety of o-chain 1-series analogues could be conveniently obtained, has been explored (Schaaf and Corey 1972). A huge literature exists embellishing the Corey route. For example, there are many alternatives to the ketene equivalents discussed in relation to the 4+2 cycloaddition to cyclopentadiene (Figure 7.7) (Ranganathan et al 1977). Perhaps the most convenient, and inexpensive, synthesis of the Corey lactone is that of To¨mo¨sko¨zi et al 1976, using a highly regioselective Prins reaction (Figure 7.10) on the lactone 16, which in turn can be conveniently prepared in large amounts from 2+2 cycloaddition of dichloroketene to

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Figure 7.7 The Corey lactone synthesis: a key intermediate. Reagents: i, Base/Tl2SO4; ii, BnOCH2Cl; iii, a-chloroacrylonitrile/Cu(BF4)2; iv, KOH/ DMSO/H2O; v, MCPBA; vi, NaOH/H2O; vii, CO2; viii, (+)-amphetamine salt resolution; ix, H+/H2O; x, KI3/H2O; xi, PhBzCl/pyridine; xii, Bu3SnH; xiii, H2, Pd/C, H+

cyclopentadiene (To¨mo¨sko¨zi et al 1976). The Prins reaction is interesting in this system, as it is unusually high-yielding and regiospecific. Other reactions for selective reactions at the olefinic bond in this bicyclic system are most effective on the more tightly constrained ketone 15, which can also lead to several sophisticated PG syntheses (Newton and Roberts 1980). The lactone hydroxyaldehyde 18 can be taken on into the ochain addition or alternatively the intermediate dihydroxy compound (not shown) can be selectively protected at the secondary hydroxyl before the generation of the aldehyde group. Large-scale synthesis of vast numbers of analogues in which the two side-chains can be totally differentiated was developed by the ICI Group (Brown et al 1978), using acetoxyfulvene 19 as the starting point to control the 4+2 cycloaddition of ketene equivalent. The modified route, allowing the inverse addition of the two side-chains, is depicted in Figure 7.11 (Mallion and Walker 1975). These syntheses have some advantages, since they do not use the highly toxic thallous sulphate or benzylchloromethylether as in the Corey synthesis (Figure 7.7). The rest of the process from 25 is very similar to the Corey route. The dimethoxyacetal protecting group was removed by gentle treatment with acid and water before the o-chain was deployed.

THE CONJUGATE ADDITION APPROACH All the PG syntheses discussed so far suffer from the fairly large number of steps involved. Several groups have developed more convergent processes for PG production utilizing conjugate additions. The method involves the 1,4-addition, usually of an organocopper o-chain synthon, to a suitable cyclopentenone. There

are several procedures from which PGE1, and PGE2 were successfully prepared. A typical reaction is illustrated in Figure 7.12. The components themselves for this type of synthesis require several steps in their preparation. Nevertheless the synthesis can be made very efficient. Examples are provided by Sih et al (1973, 1975a, 1975b) and Heather et al (1973). Suitable cyclopentenone precursors such as 26 have been made by several methods described in these papers. The o-chain components, such as 27, are easily available by addition of acyl chloride to acetylene (Price and Pappalardo 1961) in very high yield (Figure 7.13). The chlorine can then be replaced by the more active iodo group for use in the conjugate addition after reduction of the ketone to the alcohol(s). The o-synthon has been enantiomerically resolved by making the phthalate half-ester of the iodovinyl alcohol and purifying the diastereoisomeric (7)-a-methylbenzylamine salts by crystallization (Kluge et al 1972). The conjugate addition of the protected resolved alcohol to the unresolved cyclopentadienone then gave rise to a pair of diastereoisomers, which were separated by chromatography, thereby achieving a chiral synthesis from that point. The reader is directed to the monographs cited for other examples. Advantagous modifications of this type of synthesis include the use of organo-aluminium (Bernady et al 1979) and organo-zirconium species (Schwartz et al 1980) o-chain synthons. The conjugate addition methods can be made even more efficient and a great deal of painstaking research has been done to find reaction conditions allowing the initially formed enolate anion generated from the addition of the o-chain to be trapped using an a-chain synthon (Figure 7.14). This means that the two side-chains can be deployed in a ‘‘one-pot’’ reaction. Initial experiments succeeded in the synthesis of 11-deoxy-PG analogues (e.g. Patterson and Fried 1974) but the method failed in the preparation of the fully functionalized PG.

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Figure 7.8 Corey route to prostaglandins E2 and F2a. Reagents: i, DCC/pyridinium trifluoroacetate/DMSO; ii, NaH/DMSO/(MeO)2POCH2COC5H11; iii, chiral borane/tBuLi; iv, HOH/K2CO3/MeOH; v, DHP/H+; vi, Dibal[H]; vii, DMSO/Ph3PCHCH2CH2CH2COO7; viii, CrO3/PhH/H2O; ix, AcOH/H2O

The use of more electrophilic a-chain synthons (see 32, 33; Figure 7.15), in tandem with modification of the intermediate enolate with triphenyltin chloride and very carefully controlled reaction conditions, successfully generated the desired product which was transformed into a PG. This ‘‘one-pot’’, threecomponent coupling step gave 475% yields (Suzuki et al 1988). Removal of protecting groups completed the synthesis of PGE2. The most elaborate but successful process, devised by Noyori and his colleagues, required the modification of the organolithium ochain precursor with dimethyl zinc. This presumably stabilizes the intermediate enolate, thereby allowing time for the trapping to occur (Suzuki et al 1990). There was advantage also in the use of the stannane 35 for the salt-free generation of the organolithium 36. Synthon 33 was also used in this procedure and gives rise to 5,6-didehydro-PGs which can be cyclized to the important carbacyclins (see section on PGI2 synthesis, below). Methods for the preparation of suitable cyclopentenones, such as 29, are well documented and the enolate

trapping synthesis has been made even more efficient by the use of enantiomerically resolved versions (Harre et al 1982). The Synthesis of the Labile Prostanoids PGH, PGG and TXA These rather unstable molecules were able to be isolated from biological systems by excluding the co-factors required for the conversion to other PGs (Nugteren and Hazelhof 1973; Hamberg and Samuelsson 1973). The COX enzyme used was from ram seminal vesicles (RSV), the traditional source, and the original biological research was performed using this approach. This method is probably still a very convenient way of obtaining the endoperoxides. Nevertheless, both PGG2 and PGH2 have been chemically synthesized, providing proof of their structure and a supply of these unstable species. The pathway for PGH2 43 is illustrated in Figure 7.16, starting from PGF2a (Porter et al

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Figure 7.9 Synthons for the a- and o-chains

Figure 7.10 Corey lactone via Kovac’s regiospecific Prins reaction. Reagents: i, CCl3COCl/Et3N; ii, Zn/AcOH; iii, H2O2/AcOH/08C; iv, Prins reaction (HCHO)n/AcOH/H2SO4/708C; v, H+/MeOH; vi, Pfitzner–Moffat [O]

1978, 1979a). PGG2 was obtained by a similar route (Porter et al 1980), in which all three labile oxygen functions were added simultaneously by displacement. Of particular note is the use of 2-chloro-3-ethyloxazolium tetrafluoroborate, the Mukaiyama reagent, in the SN2 (substitution nucleophilic 2nd order kinetics) displacement reaction to introduce the bromo groups. This lowtemperature displacement reaction avoids randomizing of the stereochemistry of the leaving groups, which was initially a problem when toluenesulphonate was employed as the leaving group. Other special features of these syntheses include the following. Enzymatic ester hydrolysis, the penultimate step, was required to avoid damage to the bromo groups. Hydrogen peroxide 98% and freshly prepared silver trifluoroacetate were also necessary to optimize the yield of PGH2 (17% in the final step). In spite of the difficulties, both chemical and COX enzyme methods are viable for the production of PG endoperoxides.

An even greater synthetic challenge is TXA2. The ring structure of this eicosanoid is attacked by water and has a half-life of about 40 s in aqueous media. In consequence, TXA2 must be generated quickly and used immediately. Most biological investigations have used TX synthase enzyme prepared from microsomes of animal blood platelets treated with PGH2. It is important to appreciate that PGH2 has a great deal of TXA-like bioactivity and, since TXA2 is normally generated from it, the detection of enhanced biological activity and the subsequent decay of the activity engendered in the solution thus obtained testified to the presence of this labile substance (Needleman et al 1976; Yoshimoto et al 1977). Indeed, experiments of this type led to the discovery and elucidation of the hitherto unknown ring structure of TXA2 (Hamberg et al 1975). It was realized that there was no reason why TXA2 could not be isolated and stored at low temperatures in a dry non-protic organic solvent. Some 10 years after the original description of TXA2, a chemical synthesis was finally devised by a

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Figure 7.11 The acetoxyfulvene approach. Reagents: i, NaOEt/HCOOEt; ii, a-chloroacrylonitrile; iii, H2O/H+; iv, (MeO)3CH/H+; v, NaOH/DMSO; vi, H2O2/OH7; vii, KI3/H2O; viii, Bu3SnH; ix, DHP/H+

Figure 7.12 Conjugate addition of the o-chain

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Figure 7.13 The o-chain synthon preparation

Figure 7.14 The three-component coupling reaction

Figure 7.15 Synthons for the three-component coupling

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Figure 7.16 Synthesis of PGH2. Reagents: i, n-butylboronic acid/dimethoxypropane; ii, TBDMSCl/imidazole/DMF; iii, H2O2/H2O; iv, 2-chloro-3ethylbenzoxazolium tetrafluoroborate/Et4N+Br7/CH2Cl2/7158C; v, H+/H2O; vi, porcine lipase ester hydrolysis; vii, H2O2 98%/CF3COOAg/Et2O

group of American chemists (Bagwat et al 1985a, 1985b). The strategy depended on conversion of TXB2, for which there are several syntheses, back to TXA2. The route is shown in Figure 7.17. Interesting aspects are the use of the large ring lactone formed between the 15-hydroxyl and the carboxylic acid as a hydroxyl/carboxyl protecting group, which also prevents sidereactions at the 13,14-double bond, particularly during the free radical debromination. The formation of the oxetane ring, by a modified Mitsunobu reaction, required the presence of the bromine atom in the precursor 48. The beautiful rationale for this was the realization that in the chloral (trichloroacetaldehyde) the carbonyl reacts spontaneously with alcohols to form the acetal, and even in water the energetically favoured form of the chloral is the hydrate (gem-dihydroxy compound). This contrasts with acetaldehyde itself, where the carbonyl form is preferred. Previous attempts to make an oxetane, by dehydrative cyclization across a larger ring, have been unsuccessful and led to alkene formation. A rare, successful example of oxetane formation was that of a 1,3-dihydroxy steroid, where the fairly rigid geometry of the fused ring system did not allow alkene formation (Clayton et al 1957; see also Shibasaki et al 1980). At least one halogen atom adjacent to the putative carbonyl group was shown to be required for successful ring closure (47–49) to the oxetane. The synthetic material 49, after lactone hydrolysis in dry tetrahydrofuran with potassium trimethylsiloxide, was able to be stored dry as the solid potassium salt, which could be quickly dissolved in buffer for biological use as required. The chemical and biological properties of the synthetic TXA2 50 were identical to the biologically derived material.

STABLE MIMETIC ANALOGUES OF PGH2 AND TXA2 TXA2 suffers from instability in the aqueous media in which biological experiments are conducted. Although the aforementioned methods of obtaining TXA2 are theoretically interesting and prove the structure of this important mediator, it is extremely difficult to use the natural substance in routine investigations, where a constant concentration of pharmacologically active compound is desirable for making quantitative measurements. Since TXA2 has such potentially pathological effects, a stable mimetic was required in the search for blocking drugs for use as pharmacological tools or which could have clinical potential. The compound generally accepted as the standard TXA2 mimetic is an Upjohn analogue, U-46619 55, the so-called 9a,11a-epoxymethano-PGH2. This compound has the desired selectivity and potency on the TXA2 receptors (TP receptors) and should be more correctly regarded as a stable carba-analogue of PGH2, and named as 9a-carba-PGH2. Most analogues having this general shape, including PGH2 itself, are excellent TXA2 mimetics. However, PGH2 itself is unstable, and is converted into the large range of other PGs, thus rendering it unsuitable for biological investigations. For background and leading references to this vast and interesting field about the characterization and classification of TP and other PG receptors, see Coleman and Humphrey (1993). U46619 55 is an important research PG due to its usefulness in the discovery and testing of TXA2 agonists and antagonists (Coleman and Humphrey, 1993). The elegant synthesis of

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Figure 7.17. Synthesis of TXA2. Reagents: i, Ph3P/dipyridyl disulphide; ii, 2-chloro-1-methylpyridinium iodide/-H2O; iii, NBS/NaHCO3/Et2O/H2O; iv, (MeO)3P/ethyl azodicarboxylate; v, R3SnH; vi, saponification with either NaOD/D2O/MeOD or Me3SiOK/THF

U46619 is shown in Figure 7.18, starting from suitably protected PGE2. Very mild methylenation of the PGE2 by sulphoximine anion addition, followed by reductive elimination, gave the exocyclic olefin 51, thereby avoiding the dehydration of the PGE ring to PGB. Highly selective hydroboration of the exocyclic alkene with the sterically constrained 9-borabicyclononane (9-BBN) led to the alcohol 53, from which was prepared the methane sulphonate (mesylate) ester 54. After removing the protecting silyl groups, the generation of the alkoxide anion at the 11-position caused elimination of the mesyloxy group and cyclization to the desired structure. This final treatment with excess base was extended in order to hydrolyse the carboxylic ester group to 55, the free acid (Bundy 1975). A more elaborate synthesis of U46619 from more basic raw materials was performed later by Trost et al (1978). In a similar cyclization, the other epoxymethano-cogener, U44069 (9b-carba-PGH2) 56, has also been synthesized by the Upjohn company (Bundy 1975). U-44069 is less potent and in some biological systems acts as a partial agonist, producing slightly less than the maximum response of the biological system. Other molecules employed as TXA2 mimetics include SQ 26655 57, EP 171 58 and thia-carba-TXA2 59 (named for convenience as STA2) (Figure 7.19). Further reviews and leading references are available in Tymkewycz et al (1991) and Wilson and Jones (1985). These TXA2 mimetics are available from Cayman Chemicals Inc., a company specializing in the production and marketing of all eicosanoids.

THE SYNTHESIS OF PGI2 (PROSTACYCLIN) AND ITS STABLE ANALOGUES There are a number of variations of the chemical syntheses of the antiplatelet and vasodilatory PGI2, all employing virtually the same approach from PGF2a. The typical reaction sequence is shown in Figure 7.20. Iodination of the methyl ester of PGF2a in the presence of mild base gave reaction of the 9-position hydroxyl with the 5,6-double bond, yielding the two isomers 60 and 61. These products underwent dehydroiodination with diazabicyclononane (DBN) to the desired Z-alkene 62. Hydrolysis of the ester with base gave the final product as the stable sodium salt of PGI2 63 (Johnson et al 1977, 1978). This synthesis showed that a transaddition, followed by a trans-elimination, both normally expected in this type of reaction, would give rise to the Z-geometry of the newly created olefinic bond. The structure of PGI2 was thereby confirmed. Instead of iodine, other similar cyclization methods employing bromine, phenylselenyl chloride or mercuric acetate have been shown to give the desired product (see also Corey et al 1977; 1978; To¨mo¨sko¨zi et al 1977, 1978). The Upjohn chemists have also shown that PGI2, along with other olefinic isomers, can be isolated by heating the biologically deactivated hydration metabolite, 6-keto-PGF1a, in benzene, with Dean and Stark water removal (Johnson et al 1978). PGI2 has also been prepared by cyclization of the 5,6-didehydroPGF2a analogue, obtainable by conjugate addition of the a-chain 33 in the three-component coupling reaction (Figures 7.14 and 7.15). The ring formation step was achieved, in high yield, using a palladium chloride complex, followed by depalladation with

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Figure 7.18 Synthesis of U-46619. Reagents: i, N-methylphenylsuphonimidoylmethylmagnesium chloride/THF/7788C; ii, Al/Hg, AcOH
H2O; iii, Ph3SiCl (TPSCl); iv, 9-BBN, H2O2/OH7; v, MsCl/Et3N; vi, H3PO4/H2O/THF; vii, KOH/MeOH/H2O

Figure 7.19 Structures of other selected TX mimetics

ammonium formate or, with less stereochemical control, by mercuric trifluoroacetate followed by reductive demercuration with borohydride (Suzuki et al 1988). The use of PGI2 as a therapeutic agent is inhibited by its instability to acid. The associated administration of the alkaline sodium salt and the high rate of metabolism in mammals is a further impediment. Accordingly, medicinal chemists have prepared a vast array of PGI2 congeners, mostly based on the 6acarba-PGI2, usually named carbacyclin for convenience, 69 (Figure 7.21). It was also hoped that it would be possible to

find analogues with selectivity for the IP receptors on platelets, as opposed to those mediating hypotensive vascular activity, since the use of PGI2 to restore circulation in peripheral vascular disease, etc., requires the patient to be maintained in a supine posture to prevent loss of conciousness, due to low blood pressure. The reader is referred to the textbooks cited earlier for the huge array of synthetic efforts in this field and to more recent treatises by Nickolson et al (1985) and Collins and Djuric (1993). An example is outlined, in Figure 7.21, of an asymmetric synthesis of

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Figure 7.20 Synthesis of PGI2. Reagents: i, KI/I2/Na2CO3/H2O/08C; ii, DBN/benzene/408C; iii, NaOH/H2O/MeOH

carbacyclin (Aristoff 1981), starting from a chiral Corey pathway intermediate (compounds 10, 11; Figure 7.8). The a-chain addition to 68 produced both Z- and E-alkenes. Studies to define the conditions to enhance formation of the desired isomer were performed by the Schering group (Rehwinkel et al 1988; Westermann et al 1992). Care is required in the nomenclature of these analogues, since the Z-isomer designation in PGI2 becomes the E-isomer, for designation of the naturally occurring geometry in carbacyclin, due to the alteration in precedence of groups around the exocyclic alkene. The two carbacyclin analogues having altered o-chains, Iloprost 70 (Skuballa and Vorbru¨ggen 1983; note that in the early literature Iloprost was called Ciloprost) and Cicaprost 71 (Skuballa et al 1986) (Figure 7.22), were synthesized and showed interesting structure–activity relationships (SAR). Cicaprost, which has a highly defined quite rigid structure from C-4 to C15 (PG numbering) due to the acetylenic bond and the relatively inflexible cis ring junction, is specific to IP receptors. However, Iloprost has considerable PGE-like actions (Sheldrick et al 1988) as well as PGI activity, like carbacyclin itself and PGI2. For further useful SAR discussion, see Nickolson et al (1985) and Wise and Jones (1996). Further investigation of PGI cogeners has turned up good evidence that there may be sub-types of the IP receptor. Radioligand binding of the isocarbacyclin analogue 73 (Suzuki et al 1996) showed specificity for binding sites in most rat

brain areas. Cicaprost (the most selective IP agonist) displayed much lower binding, except in selected other CNS regions. Isocarbacyclin 72 bound efficiently to both types of brain site and is potent as an antiaggregatory analogue on platelets. Cicaprost is highly potent on platelet inhibition, where compound 73 is very weak, and so the platelet receptor has been called IP1 and the preponderant rat CNS receptor IP2 (Takechi et al 1996). OVERVIEW OF PG SYNTHESIS AND BIOLOGY The breakthrough in understanding the receptors for PGs arose about 1980 with the discovery of TP receptor antagonists using the stable TX-mimetics (see section on PGI2 synthesis, above) (Coleman and Humphrey 1993). It was then possible to quantify the often considerable amount of TX activity in other PGs. The activity of the other PG receptors could then be observed uncontaminated by TP effects, and new PG analogues could be investigated for potentially dangerous TP agonist actions. For example, the introduction of the 16,16-dimethyl o-chain, a standard strategy to attentuate the standard 15-dehydrogenase deactivating metabolism of most PGs, also increases the TP agonist activity. PGE2 has approximately 200-fold less activity as a TX mimetic than U46619, whereas 16,16-dimethyl-PGE2 has only about 15-fold less activity. Even more amazing TP agonism

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Figure 7.21. Asymmetric synthesis of carbacyclin. Reagents: i, LiCH2PO(OMe)2; ii, CrO3/pyridine; iii, K2CO3/18-crown-6/PhMe; iv, HCOO7HNEt3+/ PdC; v, a-chain Wittig reaction; vi, remove protecting groups

is produced by the introduction of a 16-p-fluorophenoxy-o-chain, as in EP 171. The increase in TP effects is dramatically large. The ring structure to which the 16-p-fluorophenoxy group is attached is of minor importance because of the large increase in TP effects (Jones et al 1987 and references therein). It is important to appreciate that most PGs can interact at least to some degree with several PG receptors and that selective antagonism of a particular receptor is the most useful criterion for classification. Currently, for prostanoid receptors there are good antagonists for TP, some EP and DP receptors. The search for powerful selective antagonists to the other PG receptors continues. For an introduction to eicosanoid receptors, see Wise and Jones (2000) and Coleman and Humphrey (1993). ISOPROSTANES These molecular species are derived from PUFAs by adventitious autoxidation. They comprise a large range of structures having features similar to PGs formed enzymatically by COX. A subgroup, the neuroprostane class, has been identified, derived from the very highly unsaturated fatty acids, particularly docosahexenoic acid, that are found in abundance in the brain. The classes of isoprostanes (iPGs) formed from arachidonic acid are shown in Figure 7.23; the nomenclature is that of Rokach et al (1997). The initial peroxide structures formed are reduced or isomerized to more stable compounds having hydroxyl or

carbonyl groups like the PGs. Note that Types I and II (not shown in Figure 7.23) constitute isoprostanes derived from fatty acids with even more degrees of unsaturation, such as timnodonic acid. Type III are close isomers of the naturally occurring enzymatically produced PGs; however, the non-enzymatic reactions give rise to a large number of racemic diasteroisomers. Many of these species have been identified in the body and it is apparent that the non-enzymatic oxidation favours formation of cisdeployed side-chains, leading to 8-iso- or 12-iso-PGs of the F, D and E ring types. Most chemical synthesis of PGs is designed to be asymmetric to reflect the natural COX activity. Since the isoprostanes are not enzymatically produced and are in consequence racemic mixtures of diastereoisomers (Morrow et al 1990), any chemically synthesized isoprostane will not reflect the natural mixture of stereoisomers. In fact, the chemical syntheses of isoprostanes will normally generate a limited number of stereoisomers. This creates a problem in ELISA and radioimmunoassay methods, where the crucial, selective enzyme or antibody detector is sensitive to a single enantiomer. Such an assay method will not reflect the full isoprostane synthesis. However, these methods can be validated to some degree by GC–MS, particularly as more of the possible isomers become available by synthetic methods. Already predominant isomers have been unambiguously made and it has been suggested that measurements, using some of these as standards, can be used as markers of the oxidative stress levels in an organism (Proudfoot et al 1999).

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Figure 7.22 Structure of important PGI analogues

Rokach’s synthesis of the two lactones 78 and 80, derived from glucose, is indicated in Figure 7.24 (Hwang et al 1994). Wittigtype reactions identical or similar to those employed in the elaboration of the side-chains in the natural PGs have been used to obtain a large range of iPGs. Very few of the iPG stereoisomers have been examined for overt biological actions. Examples include 8-iso-PGF2a, which is available as a pure enantiomer and is recognized to be a highly active TP agonist (Kromer and Tippins 1996). Another example, 8-iso-PGE2, has also been shown to be formed by non-enzymatic autoxidation and has also been synthesized chemically and investigated for biological activity. This cogener has been shown to have strong TP activity on smooth muscle of the vasculature but only causes platelet aggregation at higher dose levels. It also inhibited the aggregation due to the standard powerful TXA2 mimetics (Longmire et al 1994). These authors speculate that this may be due to a unique receptor for iPGE. One complicating factor may be that the iPGE can activate IP receptors, thereby making the platelet response capricious. Alternatively, this iPGE can epimerize readily at the 8-position, adjacent to the ring carbonyl group, via the enol form, to give PGE2 (Figure 7.25). The equilibrium position for these epimers is 90:10 in favour of the natural PGE2 over 8-iso-PGE2 (Daniels et al 1968). How fast this epimerization occurs is pH-dependent and is normally done with very mild base in the case of PGE to minimize PGB formation. In principle, any protic system can catalyse this and it is interesting to speculate whether this can occur while the iPG is still attached to the lipid pool. Similarly, 8-iso-PGD2 can give rise to some nat-PGD2 by equilibration of the suitable diastereoisomer. In an organism suffering a high level of oxidative stress, cell membrane function is likely to be affected by the presence of these polar lipids and a sudden large release of these potentially bioactive substances could have catastrophic repercussions. For an overview of this interesting subject, see Rokach et al 1997.

INTRODUCTION TO LIPOXYGENASE PRODUCTS Distinct from the COX systems are the lipoxygenase (LOX) enzymes, which oxidize PUFA to hydroperoxyeicosatetraenoic acid (HPETE) compounds as an initial product. These are ubiquitous in both plants and animals. The HPETE initial product is usually reduced to the hydroxyeicosatetraenoic acid (HETE) but can also go on to more exotic epoxy species, as discussed later. The function of these metabolic pathways is not fully understood, but in the mammalian system research has shown that these pathways are involved in a vast array of crucial functions, including inflammation, control of the immune system, programmed cell death (apoptosis), fat metabolism and transcriptional control within the cell nucleus. The energy balance within an organism at a deep level appears to be modulated by PUFA, such as docosahecaenoic acid and arachidonic acid as well as oxidation products derived from them, and these substances have been implicated as ligands for peroxisome proliferator-activator receptors (PPARs) in the cell nucleus (for reviews of this area, see Desvergne and Wahli 1999; Kliewer et al 1999; Willson et al 2000). Different cell types have their own unique range of lipoxygenases and, in the case of polyunsaturated eicosanoic acids, nearly every possible position for oxidation has been observed. The LOX enzyme system is therefore categorized by the position at which the oxygen is introduced. In man the main lipoxygenases are 5-, 12- and 15- and these occur in leukocytes and lymphocytes, platelets, lung and several other tissue types (Maclouf 1996). A general review of lipoxygenases is available (Brash 1999). Dihomo-g-linolenic acid, the 1-series PG precursor, has no 5,6double bond, and therefore cannot undergo 5-lipoxygenation, whereas arachidonic and timnodonic acids, with their higher degrees of unsaturation, can form products of 5-LOX. All these acids are susceptible to the actions of LOX enzymes at other

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Figure 7.23 Isoprostanes derived from arachidonic acid

Figure 7.24 Rokach synthesis of lactones leading to iPGs. Reagents: i, acetone/H3PO4, ZnCl2/208C; ii, NaH/THF/08C then CS2/208C then MeI/208C; iii, Bu3SnH/toluene/1118C; iv, H2O/AcOH/208C; v, thiocarbonyldiimidazole/dichloroethane/838C; vi, H+/H2O/THF/668C; vii, methyl (triphenylphosphoranylidene)acetate/base/THF; viii, Bu3SnH/AIBN/C6H6/808C; ix, TBDMSCl/imidazole/DMF/608C

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Figure 7.25 The equilibration of 8-iso-PGE2 and its enantiomer to the more stable trans side-chain isomers

suitable positions. Experiments to alter diets, and hence perturb the lipid pools of the body, and the blocking of pathways such as the COX systems by aspirin-like drugs, which may lead to more lipoxygenase products, are under investigation (DeWitt 1996). It appears, therefore, that even when no biological role for a given metabolite is initially apparent, further research usually discovers a role, either a binding site (enzyme modulation) or a receptor (cell signalling), with important consequences for the organism. The contribution of synthetic chemistry in providing these mediators as standards for analytical work and pharmacological study cannot be overestimated.

HETEs, HPETEs, LEUCOTRIENES AND LIPOXINS An important lipoxygenase pathway in humans is the one involving 5-LOX (Samuelsson 1983). This metabolic route leads to the physiologically important leukotrienes (LTs) and is depicted in Figure 7.26. LTA4 is very unstable and adds water to form LTB4 or glutathione to form LTC4. These reactions are mediated by hydrolase or glutathione-S-transferase enzymes, respectively. The glutathione in the latter case is added to the epoxide by nucleophilic attack of the cysteine thiol group. The enzymatic routes are still used to obtain these esoteric compounds but chemical synthesis is also available and was required to prove the stereochemistry. The lipoxygenases mostly produce the S-enantiomer of the hydroperoxide and subsequent enzymic reactions are all stereospecific. Low-temperature HPLC is the key to obtaining pure material for biological study, particularly of the labile hydroperoxides. All the highly unsaturated lipoxygenase products suffer from rapid aerial autoxidation/polymerization.

LTB4 is important as a major chemotactic agent (Samuelsson et al 1987; Ford-Hutchinson 1990). The ketone derived from oxidation of 5-HETE (5-oxo-ETE) has similar actions to LTB4, but it acts by a different mechanism and is less potent (Powell et al 1995). LTC4 and the other peptido-LTs (Figure 7.26) have been identified with the slow-reacting substance of anaphylaxis (SRSA). This is released along with histamine in allergic responses and promotes hyper-reactivity of airways. The 12- and 15-lipoxygenase pathways, shown in Figures 7.27 and 7.28, appear to be more important than was first realized. Initially it was considered that the formation of these very polar lipids constituted methods for deactivation and elimination of very bioactive substances from the body, and this may still be true in some cases. However, it is now realized that virtually all of these products have some role to play in the signalling systems of the organism, e.g. 12-HPETE and LTB4 have been proposed to be endogenous ligands for the vanilloid (VR1) receptor which mediates the burning pain sensation caused either by heat or acid (Hwang et al 2000). These lipoxygenase routes also lead to the hepoxilins and lipoxins. These substances are examples of subtle mediators of important functions (see Lumin et al 1992; Reynaud et al 1995; Yoshimoto and Yamamoto 1995; Bratt et al 1995). The following section on the synthesis of the oxylipins is designed as an introduction to their chemistry. CHEMICAL SYNTHESIS OF LIPOXYGENASE-DERIVED EICOSANOIDS Chemical synthesis of many lipoxygenase products has been performed using standard aliphatic olefinic and acetylenic chemistry. Excellent monographs on the subject are available

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Figure 7.26 The 5-lipoxygenase biosynthetic pathway

(Corey and Cheng 1989; Scheinmann and Ackroyd 1984; Pike and Morton 1985). In many cases these syntheses were done to prove the stereochemistry of molecules of biological prominence. Practical routes, mostly starting from the precursor fatty acids, are discussed as methods of obtaining these exotic substances for research.

SYNTHESIS OF HYDROPEROXY- AND HYDROXY-DERIVATIVES OF EICOSAPOLYENOIC ACIDS (HPETEs AND HETEs) Enzymatic methods have the advantage that the starting material is the readily available PUFA and even very labile products are easy to obtain under mild conditions by HPLC, since the reaction often forms a UV chromophore. The enzymic products are also normally chiral. Apart from the asymmetric aspect, the enzymatic systems can be imitated chemically by use of an excited state of dioxygen, singlet oxygen, which is conveniently generated from ground state dioxygen by dye sensitization. Irradiation of a solution of methylene blue dye with light of red wavelength causes excitation of the dye to the singlet electronic state, which is then transferred to oxygen dissolved in the solvent. Air can often be

used as the source of oxygen and is much less hazardous than pure oxygen gas. The singlet oxygen thus formed reacts with suitable substrates in the solution, in this case the methylene-interrupted polyunsaturated fatty acid. The ‘‘ene’’ reaction, thus engendered, produced HPETE products where the shifted double bond became trans. This general rule, that double bonds which move during a reaction become trans, only seems to be violated in the enzymic addition of water to LTA4 to form LTB4. The oxygenation reaction can be run at low temperatures, such that thermally labile products can be isolated. The general reaction is shown in Figure 7.29 (Porter et al 1979b) and when applied to arachidonic acid gave all possible HPETE products. These were characterized by HPLC separation guided by UV absorbing fractions. Hydrogenation of all double bonds and reduction of the hydroperoxy group to hydroxy followed by derivatization of the hydroxy-saturated acid to methyl ester, trimethylsilyl ether (Me, TMS), followed by gas chromatography–mass spectrometry, determined the position of the oxygen function (Porter et al 1979b). Milligram quantities of the pure racemic HPETE compounds were available by this onestep process. Woollard (1986) used this method to make standard HETEs and demonstrated that 12-HETE formed in psoriatic skin was the R-enantiomer, in contrast to the usual 12S-HETE formed in platelets. He was also able to chromatographically separate the

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Figure 7.27 The 12-lipoxygenase biosynthetic pathway

two enantiomers using the diastereoisomeric dehydroabietylurethane derivatives, showing that this procedure can produce chiral material identical to that formed in biological systems. Arguably the most important synthetic approach to the lipoxygenase products is by the selective epoxidation of arachidonic acid. This can be adjusted to provide, directly, either the 5,6-epoxide 83 shown in Figure 7.30, or the 14,15-epoxide 85 shown in Figure 7.31 (Corey et al 1980a). The related HPETEs and HETEs can thus be obtained. Furthermore these 5,6- and 14,15-epoxides can be used as starting points for the transfer of the oxygen function along the chain (Figure 7.32) to give, respectively, the 8,9-epoxide 89 (Falck and Manna 1982) and the 11,12-epoxide 91 (Corey et al 1980a). The highly efficient allylic epoxidation mediated by vanadium as catalyst provided the key step in this shift of the oxirane function to the adjacent double bond position. The HPETE/HETE systems related to these new epoxides were thus able to be obtained. HPETEs can be generated chemically at low temperature from HETEs by nucleophilic displacement, as in Figures 7.30 and 7.31, and the scheme can be used to obtain either the free acid or the methyl ester. The stereochemistry of these products was determined by comparison with enzymatically produced compounds from either plant or animal sources.

LTA4, LTB4 AND LTC4 SYNTHESIS FROM 5-HPETE 5-HPETE was converted to the labile leukotriene A4 (LTA4) by a hydroperoxide-oxiranylcarbinol rearrangement (Corey and Barton 1982; Corey et al 1984). LTA4 has been converted to LTB4 by enzymatic addition of water (Maycock et al 1982). LTC4 and the other peptido-LTs have been obtained by addition of glutathione, etc. to LTA4 (see Figure 7.26) (Corey et al 1980b). Lipoxins and Hepoxilins Synthesis of these esoteric and highly unstable species has been crucial to prove the stereochemistry and make them available for biological studies. Lipoxin A4 (LxA4) was most conveniently prepared from 15HETE, which can in turn be obtained in chiral form, and in large amount, from oxidation of arachidonic acid by soya bean lipoxygenase. The 15-HETE hydroxyl group was protected, and then the carboxyl group was utilized in an iodolactonization reaction to introduce the 5-hydroxy function in an analogous method to that shown in Figure 7.30. Vicinal oxidation directed by the 5-hydroxyl then allowed addition of the final hydroxyl.

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Figure 7.28 The 15-lipoxygenase biosynthetic pathway

Figure 7.29 Formation of hydroperoxides by singlet oxygen

5S,6S,15S-Trihydroxy-7,9,11,13-eicosatetraenoic acid (LxA4) was obtained as a 1:1 mixture with the 5R,6R-diastereoisomer and was identical to biological material from human neutrophils (Corey and Su 1985a; see also Corey et al 1989). Lipoxin B4 (LxB4) was prepared analogously. The crucial step was the conversion of the hydroperoxy soya bean lipoxygenase product, 15-HPETE, to a mixture of threo- and erythroepoxyalcohols with titanium tetraisopropoxide. The threo form was then treated with base to produce the required 14,15dihydroxy compound which, after protecting as the trimethylsilyl ether, underwent the iodolactonosation process to introduce the hydroxy group at the 5-position. One of the 5-epimers was shown by HPLC retention times to be identical to natural LxB4 (Corey et al 1985b). Other elegant but longer syntheses of the lipoxins are known which further confirm the assigned structure. Most employ standard alphatic chemistry techniques, involving coupling of alkynes and/or chiral starting points to control the stereochemistry. An example is given by Gravier-Pelletier et al (1991), where the starting material is D-isoascorbic acid. A key step is a modified

Wittig-type reaction involving the use of arsonium ylide. The arsenic moiety acts as a leaving group but the lower affinity for oxygen allows the starting material carbonyl oxygen to be retained as an epoxide. Both LxA4 and LxB4 were synthesized. The synthesis of hepoxilin A3 (HxA3) (Chabert et al 1989) is depicted in Figure 7.33 as an example of the use of this approach using arsonium ylides. An interesting, almost biomimetic, synthesis of HxA3 and, by hydrolysis, the associated trioxilin A3 (TxA3) has also been described by Lumin et al (1992). THE MONOOXYGENASE PATHWAY (CYTOCHROME P450) A range of oxidized derivatives are known to be formed by the hepatic cytochrome P450 enzyme systems, which require molecular oxygen and NADPH. The enzymes are site-specific and each double bond of the polyunsaturated eicosanoic acids can be attacked, usually to form a monoepoxide (EET), with almost total

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Figure 7.30 Alternative synthesis of 5-HETE, 5-HPETE and the 5,6-epoxide of arachidonic acid. Reagents: i, KI3/K2CO3/THF/H2O/O8C; ii, DBU; iii, Et3N/MeOH; iv, LiOH then CH2N2; v, MsCl/Et3N/CH2Cl2/7658C then H2O2/71108C

Figure 7.31 The selective 14,15-epoxidation of arachidonic acid and conversion to 15-HPETE. Reagents: i, (imidazolyl)2CO/CH2 Cl2 then H2O2/lithium imidazolide then KHSO4/CH2Cl2; ii, CH2N2/Et2O; iii, Mg(NR2)2; iv, MsCl/Et3NCH2Cl2/7428C then TBDMSOOH; v, H+/H2O

enantiofacial selectivity. In the case of arachidonic acid, all epoxides are known (see previous section) and are further modified by ubiquitous epoxide hydrolases to form vicialdihydroxyeicosatrienoic acids (DHETEs). These EETs are often formed with the unsaturated acid still incorporated in the phospholipids and thus can be released by phospholipases. The consequences to membrane structure and the pathophysiology of these metabolites is still under investigation (Capdevilla et al

1992). The DHETEs and some epoxyhydroxy derivatives have mostly been characterized by GC–MS, and the DHETEs synthesized by addition of water across the various epoxides discussed in the previous section. Addition of water or glutathione to various positional isomers of LTA4 produced by non-enzymatic oxidations could, in principle, lead to a large range of isoleukotrienes and related substances. This would be analogous to the

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Figure 7.32 Synthesis of the 8,9- and 11,12-epoxides of arachidonic acid. Reagents: i, KBr/THF/H2O/AcOH; ii, ButOOH/VO(acac)2/PhH; iii, (CF3SO2)2O/pyridine/HMPT/CH2Cl2

Figure 7.33 Use of arsonium ylides in synthesis of HxA3

Figure 7.34 Structures of the endogenous cannabinoids and D9-tetrahydrocannabinol

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formation of the isoprostanes. There are some reports of these substances occurring in vivo; however, the pathophysiological significance of these reactions remains to be determined (Rokach et al 1997).

ANANDAMIDE AND ENDOGENOUS CANNABINOIDS In 1992 it was shown (Devane et al 1992) that cannabinoid receptors existed in the mammalian system and that the endogenous ligand was an eicosanoid, namely arachidonylethanolamide. Since then another ligand 2-arachidonylglycerol has been shown to be endogenous (for a review of this field, see Pertwee 1998). These natural ligands have affinity for the cannabinoid receptors similar to tetrahydrocannabinol, the main pharmacologically active constituent of the traditional marijuana plant. The chemical constitution of the endogenous and the plant ligands is enormously different, and these investigations have led to a large SAR study resulting in new agonists, antagonists and inverse agonists for these receptors. The endogenous ligands can be prepared in large amounts by simple derivatization of arachidonic acid. Review papers (Martin et al 1999; Hillard 2000) are available which discuss the chemical synthesis and SAR of anandamide and its analogues. More problems attend the discovery of 2-arachidonylglycerol as a ligand for CB receptors, since it is known that 2 acylglycerols can easily rearrange to the 1substituted compound. 1-Arachidonylglycerol is much weaker as a CB agonist than 2-arachidonylglycerol. The chemical synthesis and activity is discussed in a series of papers (Sugiura et al 1999 and references therein). Both endogenous ligands, so far discovered, are achiral substances, unlike the plant cannabinoids (phytocannabinoids), where it is known that only the naturally occurring enantiomeric substance has high biological activity. However, chiral analogues of the endogenous CB ligands have been prepared and exhibit a eudesmic effect and some have advantages since they are not metabolized as readily.

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Section Two Analytical Methods

8 Perspectives of Analytical Methods for Eicosanoids Jay Y. Westcott1 and Angelo Sala2 1National

Jewish Medical and Research Center, Denver, CO; and 2Department of Pharmacological Sciences, Milan, Italy

The methods that have been utilized to measure eicosanoids are diverse and reflect the chemistry and physiology of these bioactive lipids (Andersen et al, 1985). Early investigators utilized bioassay techniques to discover arachidonic acid metabolites in biological fluids. Later, mass spectrometry was utilized to determine the chemical structure of these lipids we now call prostaglandins and leukotrienes. Immunoassays, first radioimmunoassays and then enzyme immunoassays, became the workhorse methods for the quantification of eicosanoids in a wide variety of biological fluids, cell culture supernatants and tissues. Immunoassay technology continues to advance with the development of time-resolved fluoroimmunoassays, which can be easier, faster and more sensitive than the more traditional immunoassays. This chapter briefly discusses some general considerations for the analysis of eicosanoids before focusing on each of the four analytical techniques mentioned above. GENERAL CONSIDERATIONS Eicosanoids are hydrophobic lipids, typically being more soluble in organic solvents than in aqueous media. This hydrophobicity in some cases can be extreme, with the eicosanoid sticking to glass or plastic rather than staying soluble in an aqueous buffer. This is often most pronounced in aqueous media with very low protein, and the inclusion of even 0.1% albumin is usually enough to keep eicosanoids in solution. Stock solutions of eicosanoids are usually kept in organic solvents, such as methanol or ethanol, to help keep them in solution. Keeping them in these solvents can at times increase the stability of eicosanoids, although this is not always the case. When adding even small concentrations (0.5%) of solvents with eicosanoids to biological preparations, it is important to do a vehicle control, as these solvents can have a variety of effects on their own (Westcott and Murphy 1985). The enzymatic catabolism of eicosanoids in vivo can be very rapid. Prostaglandin (PG)E2 is quickly cleared from the circulation during a single pass through the lung (Robinson et al 1985). Peptidoleukotriene metabolizing enzymes are prevalent both on the cell surface and in fluids such as blood and urine (Kuo et al 1984). Thus, it is important to measure the correct eicosanoid in the correct biological fluid, e.g. in urine leukotriene (LT)E4 is the correct cysteinyl-leukotriene to measure, since its precursors would be metabolized to this product in either blood or urine (Westcott et al 1991). In some fluids, such as epithelial lining fluid in the lung, there could be a mix of leukotrienes and an assay system that can measure LTC4, LTD4 and LTE4 is desirable (Wenzel et al 1990). In urine there can be both the prostacyclin The Eicosanoids. Edited by Peter Curtis-Prior. &2004 John Wiley & Sons, Ltd: ISBN 0 471 48984 0

metabolite 6-keto-PGF1a (felt to be an index of kidney prostacyclin production) as well as another metabolite 2,2-dinor-6-ketoPGF1a (an index of extrarenal prostacyclin production). The urinary prostacyclin metabolite to be measured is dependent on what compartment the investigator desires to investigate. In addition to enzymatic degradation, eicosanoids are subject to non-enzymatic oxidation and hydrolysis. Arachidonic acid can be oxidized to a variety of hydroxy-acids, which can be shown to be different from the enzymatic products by being a fairly equal racemic mix rather than one specific stereoisomer (Fretland and Djuric 1989). Arachidonic acid can also oxidize to form isoprostanes, which are thought to be indicators of the oxidative stress in a system (Morrow and Roberts II 1996). Prostacyclin has a half-life of minutes in plasma before it is hydrolysed (Lucas et al 1986). The cysteinyl leukotrienes also become oxidized to a variety of products (Westcott et al 1984) that can catalyse further degradation, such that once degradation begins it can accelerate quickly. The warning here (particularly for leukotriene quantification) is to be very wary of eicosanoid standard concentrations, especially if they have been stored for 6 months or more. For any analytical technique it is important to perform some type of recovery experiment. In these experiments a spiked amount of the compound to be measured is added to various components of the system and the resultant amount recovered at the end of analysis is determined. These types of studies are often performed early on in assay development and neglected by later investigators who often take this work for granted. However, there are situations in eicosanoid analysis in which the individual investigator should perform additional assay validation. For example, when a complex system is being studied and no eicosanoid is found, it is imperative to add the eicosanoid to the system to show that it could be found if it were indeed present in the system. Metabolism or uptake of the eicosanoid could have occurred, which would have prevented finding the eicosanoid in the sample although it was indeed produced in the system. A second situation is when a sample has to undergo substantial purification or concentration prior to quantification. Since eicosanoids are both ‘‘sticky’’ and prone to degradation/metabolism, it is important to have some idea of what recovery is. In instances where multiple samples are routinely run, it is a reasonable practice to spike one sample with the eicosanoid to be measured, treat it as an unknown and run it through all the analytical steps. For example, this should be done in the quantification of urinary LTE4, where an immunoaffinity resin is used to initially bind the leukotriene followed by centrifugation to remove the resin, washing, methanol elution, evaporation and final quantification by enzyme immunoassay (Westcott et al 1998).

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BIOASSAY The bioassay represents a unique technique which is capable of detecting biological activities in samples (Kellaway and Trethewie 1940). Although today a number of sophisticated techniques are available for the immunological or physicochemical detection of eicosanoids, it is important to remember that properly mastered bioassays contributed crucial information to the development of the eicosanoid field. For example, the discoveries of prostacyclin (PGI2) and thromboxane (TXA2) were driven by their unique behaviours in bioassay systems (Piper and Vane 1969) and it is questionable whether the presence of such unstable compounds would have been recognized without bioassay techniques. This was clearly recognized by the assignment of the Nobel Prize for Physiology or Medicine to Sir John Vane in 1982 (Vane 1983). Bioassays were also utilized to elucidate the mechanism of action of non-steroidal antiinflammatory drugs (NSAIDs) and in the identification of early antagonists of slow-reacting substances of anaphylaxis (Augstein et al 1973; Vane 1971). Numerous examples exist for the use of bioassays in the quantitation of eicosanoids (Fleisch et al 1979). Before the development of specific immunoassays, bioassays were among the most accessible methods for sensitive semi-quantitative determination of biologically active compounds. A possible problem associated with the use of bioassays for quantitative analysis is that additional endogenous substances can also be present which affect the responsiveness of isolated organs to specific eicosanoids, and vice versa (Gryglewski and Korbut 1976). For example, platelet activating factor (PAF), when released from pulmonary tissues following hypersensitivity reactions, may cause a 10-fold increase in the contracting activity of cysteinyl leukotrienes on the isolated guinea-pig ileum, even at sub-nanomolar concentrations (Clancy and Hugli 1983). While this can be regarded as a significant pitfall in quantification, the occurrence of this phenomenon presents a clearer picture of the final biological relevance (in terms of the biological activity measured) linked to the production of a given metabolite. Considerable specificity can be achieved using bioassay methodology. An appropriate selection of assay organs can be set up in a cascade so that the substance to be tested sequentially superfuses a series of different isolated organ preparations. The typical pattern of biological activities (contraction, relaxation or no effect) observed in the different preparations can provide a unique fingerprint, allowing discrimination between different eicosanoids, e.g. while both PGI2 and PGE2 relax the rabbit mesenteric artery, the latter contracts the bovine coronary artery while the former relaxes it. By utilizing five or six different isolated organ preparations, it is indeed possible to achieve remarkable specificity. The specificity of very simple bioassays can also be substantially increased by using appropriate mixtures of receptor antagonists/synthesis inhibitors added to the superfusion buffer (Gilmore et al 1968). It is also common for NSAIDs to be supplemented to systems to inhibit the generation of endogenous prostaglandins that might occur in response to superfusion. There exist numerous variations on the typical isolated organ bioassay. An interesting evolution of a typical superfusion cascade was introduced by Vane (1964), in which heparinized blood taken from an artery by means of a peristaltic pump was used to superfuse an isolated organ cascade and then returned to the animal by gravity or using another pump. In another bioassay variation, increased sensitivity was achieved by a laminar flow superfusion technique (Ferreira and De Souza Costa 1976). This modification of the classical superfused organ cascade lowers the superfusion flow to 0.1–0.3 ml/min, substantially prolonging the contact time between tested substances and the isolated organs used for detection. This leads to a significant increase in

sensitivity, such that as little as 5–10 pg PGE2 or 50–100 pg cysteinyl-leukotrienes can be easily detected. The technique of bioassay represents not only ‘‘a way of biological thinking’’, as stated by Professor R. Gryglewski, but is a versatile tool in the hands of experienced pharmacologists. Bioassays can provide highly reliable quantitative and qualitative information of unique importance in the discovery of biologically active unstable products. Recent technological advances have provided additional valuable support for the use of bioassays in the analysis of eicosanoids. For example, the use of computerassisted video-microscopy provides enhanced sensitivity by detecting changes in smooth muscle size of the order of a few microns while providing recording of smooth muscle activity in conditions very similar to that observed in vivo (Wilkens et al 1992). Parallel use of analytical techniques providing structural information, such as liquid chromatography–mass spectrometry (LC–MS), with bioassay will continue to represent a powerful approach for the unequivocal identification of bioactive arachidonic acid metabolites (Subbanagounder et al 2002). Mass Spectrometry The structure elucidation of prostaglandins and their metabolites in the 1960s was one of the most successful early applications of gas chromatography–mass spectrometry (GC–MS) to the solution of biological structural problems (Bergstrom et al 1962). Less than 20 years later, MS again played a crucial role in the structural identification of the slow reacting substance of anaphylaxis (SRS-A) as a cysteine containing oxygenated arachidonic acid derivative named leukotriene C (Murphy et al 1979). The importance of MS in the field of eicosanoid research can easily be observed in the several thousand publications pertaining to MS of eicosanoids. In addition to the pivotal role of MS in the identification of prostaglandins and leukotrienes, this technique has also been widely applied to the quantitative analysis of arachidonic acid metabolites and in validating other methodologies. Over the years, MS analysis has represented the ‘‘golden standard’’ for the validation of different analytical techniques and confirming (or not) immunoassay results. For example, during the early 1970s several laboratories reported peripheral plasma levels of PGA compounds (PGA1+PGA2) measured by RIA at about 1 ng/ml and higher, suggesting that PGA might represent a circulating hormone (Attallah et al 1974; Pletka and Hickler 1974). However, no PGA metabolites could be detected (detection limit 5 pg/ml) when specific GC–MS techniques were utilized, strongly implicating the presence of interfering cross-reacting substances in the immunoassays (Green and Steffenrud 1976). The basis for MS methods in the quantitation of eicosanoids is the use of appropriate internal standards added immediately after collection of samples. These internal standards are usually stable isotope-labelled analogues of the eicosanoids to be measured, i.e. synthetic eicosanoids in which a given number of hydrogen, carbon-12 or oxygen-16 isotopes are replaced by the corresponding stable isotopes deuterium, carbon-13 or oxygen-18. After purification, derivatization and chromatographic separation, the ratio between the ions arising from stable isotope-labelled and -unlabelled molecules is measured by mass spectrometers, allowing calculation of the amount of unlabelled compound present in a given sample. These stable isotope-labelled analogues have physical/chemical properties almost identical to the unlabelled molecules and thus co-elute during purification and derivatize with similar efficiency. This appropriate use of stable isotope-labelled analogues of eicosanoids as internal standards corrects for any loss of analyte arising from less than quantitative extraction or derivatization, and also

PERSPECTIVES provides a significant carrier effect, helping to improve recovery of trace amounts of the compound of interest (Green et al 1973). Although capable of providing an excellent analytical approach for the specific and sensitive quantitation of eicosanoids, the use of MS has several problems or disadvantages. Probably the biggest drawback is that MS still requires very expensive instrumentation and highly trained personnel that are not available in all laboratories. Second, while extremely sensitive, many GC–MS applications require extensive extraction and purification of samples before analysis, in particular when complex biological matrices, such as plasma or urines, are involved. Fairly simple solid phase extraction on normal or reverse phase cartridges has been successfully adopted in a few cases, resulting in significantly improved signal:noise ratios. However, in many cases either thin layer chromatography (TLC) or high pressure liquid chromatography (HPLC) are required to attain satisfactory purification of the sample. In addition to the need to purify many samples, another major drawback for methodologies based on GC is the absolute requirement for volatility and thermal stability of the compounds to be analysed at the temperatures used for GC. This prerequisite initially precluded the use of GC–MS for the analysis of polar or thermally labile eicosanoids, such as the cysteinyl-leukotrienes. Nevertheless, catalytic desulphurization to yield the corresponding 5-hydroxyeicosatetraenoic acid (5-HETE) followed by derivatization and GC–MS was successfully used to quantitate the total amount of LT in biological samples (Murphy and Sala 1990). Different chemical reactions involving the functional groups present on eicosanoids have also been used to increase the volatility and thermal stability of eicosanoids, such as esterification of the carboxy group, methoximation of the keto group and trimethylsilylation of the hydroxy group. The rapid progress of analytical methods observed with other technologies has also occurred in the field of MS. The introduction of more sensitive instrumentation coupled with new ionization techniques has allowed significant advancement in the analysis of eicosanoids in complex matrices. Multiple-sector mass spectrometers allow for selected reaction monitoring which, together with chromatographic resolution, results in extreme selectivity while still maintaining excellent signal:noise ratios as a result of a very low background (Dawson et al 1988; Hughes et al 1988). New ionization techniques, such as electrospray ionization and negative ion/electron capture-chemical ionization (Barrow et al 1982), have created new opportunities for the analysis of compounds previously not suitable for MS as intact molecules, such as LTE4 (Wu et al 1996). A major advantage of electrospray ionization is that it allows the direct sampling of the effluent from HPLC analysis into tandem mass spectrometers (LC–MS–MS). In addition to expanding the number of eicosanoids that can be analysed by mass spectrometry, LC–MS–MS substantially decreases the required sample manipulation, resulting in a significant decrease in the total analysis time and allowing for a completely automated analytical protocol to be utilized (Kishi et al 2001). The introduction in recent years of new, lower-cost mass spectrometers with MS–MS capability is also an advantage that might allow for wider utilization of MS techniques. Enzyme Immunoassays The first reported immunoassay was a radioimmunoassay (RIA) for insulin in 1959 (Yalow and Berson 1959). This new methodology was quickly accepted and its use expanded to thousands of other large and small molecular weight compounds (including eicosanoids) in both clinical and basic research applications (Dray et al 1975). This technique was considered so important that Rosalyn Yalow was awarded the Nobel Prize for

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Physiology or Medicine in 1977 (Yalow 1978). Numerous variations of this RIA have appeared, of which enzyme immunoassays (EIA) became widespread in use after their first description in 1971 (Engvall and Perlman 1971; Van Weemen and Schuurs 1971). The use of EIAs to quantify prostaglandins and leukotrienes rapidly replaced radioimmunoassays (RIA) for the measurement of these lipids. The success of immunoassays has been due largely to the specificity of antibodies and the sensitivity of detection systems utilized. Both RIAs and EIAs for eicosanoids utilize a specific antibody for the eicosanoid to be measured, a labelled tracer eicosanoid modified so that it can be easily detected, and a method to separate what is bound to the antibody from what is not bound. A competition takes place between the labelled tracer and the eicosanoid itself (either in an unknown sample or a standard) for the small amount of specific antibody available. For RIA and the eicosanoid could be labelled with radiolabelled iodine or tritium, the separation of bound from unbound tracer taking place by absorption of the small, unbound component to added charcoal. The charcoal is removed by centrifugation and the radioactivity in the supernatant measured using a scintillation counter. In EIAs, the tracer is the eicosanoid covalently linked to an enzyme (usually acetylcholinesterase with eicosanoids). The antibody is made to adhere to a polystyrene plate (often utilizing an additional species-specific antibody), separating what is bound to it from what remains in solution. After allowing a suitable time (2–24 h) for binding to occur, what is not bound is washed away. The amount of tracer bound is then determined after adding substrate by measuring enzyme activity (the enzymatic conversion of a colourless substrate to a coloured product). Figure 8.1 shows the main steps in a simple eicosanoid EIA. The specific antibody can be produced in a rabbit, mouse, goat or other species and the same antibody will often work in both RIAs and EIAs. However, just because an antibody works in an RIA does not mean it necessarily will work in an EIA. One reason for this is that in EIAs the small molecular weight eicosanoid is linked to a much larger enzyme, such that now an antibody might completely fail to recognize it, or an antibody might recognize it much better than the unlabelled compound. In either case there is not a good ‘‘competition’’ of tracer and unlabelled eicosanoid for the antibody and the assay will not work well. Pradelles, Grassi and Maclouf first reported the use of EIAs in the quantification of prostaglandins in 1985 (Pradelles et al 1985). One unique aspect of this assay was the utilization of acetylcholinesterase as the detection enzyme instead of horseradish peroxidase (HRP) or alkaline phosphatase. Acetylcholinesterase has a variety of advantages, including great stability, allowing synthesized tracer to be frozen (unlike HRP) or lyophilized and remain usable for years (usually until the eicosanoid degrades). Acetylcholinesterase is also very active in producing product and is not inactivated during activity (like HRP). Finally, the enzyme can be linked in subunits of two to four, which effectively amplifies the activity of the enzyme complex. These advantages are especially important for competitive assays in which enzyme activity and stability are crucial for a sensitive assay. It should be stressed that commercial EIAs utilizing other enzymes are available and can work well. How well an assay works depends not only on the type of tracer utilized but, most importantly, on how specific and how avid the antibody is. Although competitive EIAs are generally utilized to quantify eicosanoids, another type of immunoassay called a sandwich enzyme-linked immunosorbent assay (ELISA) can be utilized for the quantification of larger molecular weight antigens. Two antigen-specific antibodies are utilized in sandwich ELISAs, one to trap the antigen on a surface and the other to detect the antigen. Thus, although sandwich ELISAs can be utilized to measure enzymes involved in prostaglandin synthesis, such as

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Figure 8.1 Methodology of competitive enzyme immunoassay of eicosanoids utilizing an acetylcholinesterase-labelled tracer (figure provided by Dr Kirk Maxey, Cayman Chemical, Ann Arbor, MI)

cyclooxygenase-1 and -2 (Creminon et al 1995), competitive EIAs are generally required for the eicosanoids themselves. Compared to sandwich ELISAs, competitive EIAs have increased risk of cross-reactivity problems, since only one specific antibody for an eicosanoid is utilized. Competitive immunoassays are inherently more affected by matrix effects, especially the pH and protein concentration of the buffer, and are also typically less sensitive than sandwich ELISAs. Finally, unlike sandwich ELISAs in which the antibodies are typically present in excess, competitive assays have a trade-off in which the assay can be made more sensitive by decreasing the antibody or tracer concentration but at the cost of the assay taking longer to develop. It is often unclear whether samples need to be purified prior to performing immunoassays for eicosanoids. The answer is a definitive—it depends. Typically, cell culture supernatants do not have to be purified before analysis. That having been said, the presence of serum in these samples can cause increased background in some cases (TXB2 or PGE2 can be present in serum) or can add metabolic enzymes (e.g. in the quantification of LTC4). Serum samples are usually not utilized to quantify what is in blood because of low endogenous levels and the risk for production during collection. However, blood samples can be utilized ex vivo to provide an approximation of the total metabolic capacity of blood cells (often in the presence of in vivo treatment with potential inhibitors). Urine samples require purification for some eicosanoids but not for others, so it is advisable to check with the assay vendor for the validation of data available under different conditions (purified/unpurified). Urine LTE4 is one eicosanoid that requires fairly extensive purification prior to quantification (Westcott and Taylor 1998). Other fluids, e.g. bronchoalveolar lavage fluid, do not require purification as much as concentration, so that they can be processed efficiently using solid phase extraction cartridges. Tissue homogenates are occasionally utilized to measure eicosanoids but, as with serum, it is very difficult to get endogenous eicosanoid levels in tissue; rather, one can get a synthetic capability approximation. Tissue can be homogenized in an aqueous buffer (with or without an inhibitor) or in methanol to extract eicosanoids and precipitate proteins. EIAs are the most common method utilized for the quantification of eicosanoids and most likely will continue to be for at least the next 5–10 years. There are several reasons for this. The assay is

sensitive (often in the low pg/ml range), which makes it typically more sensitive than other methods. The assay is relatively easy to perform and can be performed quickly or at least does not require much labour/time. This type of analysis does not require unique expensive equipment, although a plate reader costing as little as $3000 is required. EIAs utilize reagents that are commercially available and should provide consistency over prolonged periods. The cost of these reagents when compared to other analytical alternatives makes it reasonably priced. Although this technique is not as specific as GC–MS (often considered the gold standard for analysis), many antibodies are very specific and are quite suitable for this desired task. TIME-RESOLVED FLUOROIMMUNOASSAYS Fluoroimmunoassays represent the most recently developed variation of the RIA theme. In this version, the labelled tracer used in competitive assays (or the enzyme/biotin-labelled detection antibody in sandwich ELISAs) has been replaced with a fluorescent-labelled molecule (Soini and Hemmila 1979). This type of assay has the potential advantages of increased sensitivity, faster assay time and use of stable reagents. Fluoroimmunoassays have been utilized to measure a wide variety of compounds, including bendazac and metabolites (Staley et al 1988), cytokines (Kimura et al 2001), steroids (Fiet et al 2001) and immunoglobulin E (Yuan et al 1997), to name a few. Hundreds of published papers describing fluoroimmunoassays have appeared over the past 10 years, but only a few have been concerned with the analysis of eicosanoids. Luke and Schlegel (1990) described an early assay for PGF2a and later other assays for metabolites of PGE and PGF (Luke and Schlegel 1992). These assays utilized biotin to label the prostanoid and streptavidin to add to the fluorescent europium (Luke and Schlegel 1992). Detection sensitivities of these assays were between 0.2 and 1.2 pg/ assay which, although better than RIAs, were similar to EIAs. Fluoroimmunoassays were also utilized to quantify cytochrome P450 epoxygenase products of arachidonic acid (Nithipatikom et al 1997). This assay utilized fluorescein-labelled 14,15-dihydroxyeicosatrienoic acid as the labelled tracer to compete with the unlabelled eicosanoid. The assay sensitivity was approximately 3 pg/ml. Finally, a two-site sandwich-type immunofluorimetric

PERSPECTIVES assay was developed for PGD synthase, which was sensitive (50 pg/ml), accurate and very specific for this enzyme (Melegos et al 1996). Although the first report of the use of fluoroimmunoassays for prostaglandins was 10 years ago, there has been only minimal subsequent work reported utilizing this type of assay. There have been several reasons for this, but probably the main reason is the lack of pre-made commercial reagents. Although the tracer reagent is supposedly easy and quick to synthesize, most investigators do not want to spend this time and effort, especially if it is required to be made often. Second, this assay utilizes a specialized fluorescent reader for microplates that many laboratories do not have. Finally, this assay is not that much more sensitive than the commercial EIAs that utilize acetylcholinesterase. This is in large part due to the advantages of the acetylcholinesterase-labelled tracer compared to radioisotopes used in RIAs. There are some advantages that could drive considerable investments in fluoroimmunoassays of eicosanoids. One of the unique aspects of fluoroimmunoassays is that multiple different fluorescent tags (for different antigens) with different spectral emissions can be utilized at the same time and quantified in the same sample (Merio et al 1996; Zerwes et al 2002). For example, Zerwes et al (2002) utilized europium, samarium and terbium to label antibodies recognizing VCAM-1, E-selectin and ICAM-1 in a multiparameter screening assay. This saves time and sample volume compared to assaying each compound separately. The availability of standard methods to prepare reagents and universally used reagents could also make the assay much more user-friendly. Recently, a universal streptavidin-labelled europium reagent has been described that could be utilized in a wide range of assays that currently utilize biotin-labelled tracers and intermediates (Scorilas and Diamandis 2000). In the competitive assays of eicosanoids, biotin-linked eicosanoids would be synthesized and used with the streptavidin–europium in a fashion very similar to that described by Luke and Schlegel (1992). The biotin-labelled eicosanoids should be fairly easy to synthesize and would be quite stable.

CONCLUSION Eicosanoids play an important role in normal physiology as well as in pathophysiology. In addition to the numerous known metabolic pathways and arachidonate metabolites, new metabolic enzymes and new products will undoubtedly continue to be discovered over the coming years. In fact, a new cyclooxygenase-3 that is specifically inhibited by acetaminophen was recently reported (Chandrasekharan et al 2002). Analytical techniques, including bioassay, GC–MS, EIA and fluoroimmunoassay, will be extremely important in discovering these new products and in determining their biological roles in health and disease. While this present chapter has presented a brief introduction to these four analytical techniques commonly used in eicosanoid analysis, the following four chapters will examine these techniques in more detail.

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9 Enzyme Immunoassays of Metabolites and Enzymes Using Acetylcholinesterase as Label Christophe Cre´minon1 and The Late Jacques Maclouf 1CEA-Saclay,

2*

Gif s/Yvette Cedex; and 2U348 INSERM, Hoˆpital Lariboisie`re, Paris, France

Eicosanoids, including prostaglandins (PGs), thromboxanes (TXs), leukotrienes (LTs) and hydroxyeicosatetranoic acids (HETEs), are a class of molecules derived from 20-carbon essential fatty acids. The oxidative modifications of the arachidonic acid backbone (this compound being the main precursor in humans) can follow two different pathways, either enzymatic or non-enzymatic, leading to the production of eicosanoids or isoeicosanoids, respectively. Our knowledge of eicosanoids has grown considerably over the last 10 years. Different biosynthetic pathways have been demonstrated, viz. constitutive, inducible and transcellular. The list of non-enzymatically generated isoeicosanoids is constantly being enriched by newly identified compounds. While the physiological importance of eicosanoids has been known for many years, these recent findings have opened up new lines of research for numerous scientists of differing backgrounds. In addition to the scientific interest of these different themes, these new molecules appear to be promising targets for pharmacological purposes, due to their probable involvement in pathophysiological processes, particularly in vascular diseases. Identification and characterization of eicosanoids are generally achieved using molecular biology or mass spectrometry techniques for macromolecules (receptors or enzymes) and small active mediators (isoeicosanoids), respectively. Routine analysis of these different molecules clearly requires suitable analytical methods. Antibodies thus appear as reagents of choice due to their high specificity and affinity. These properties have been advantageously exploited for over 30 years in quantitative and specific immunoassays, in combination with labelled molecules of high specific activity. Initially developed using (125I or 3H) radiolabelled molecules (Maclouf et al 1976), these methods now tend to involve non-isotopic labelling, particularly enzymatic tracers (Hayashi et al 1983), which are at least as sensitive, more stable and subject to fewer safety-related constraints. We have been developing such systems for many years and have applied this strategy to measure PGs, LTs and TXs using acetylcholinesterase (AChE; EC 3.1.1.7) from Electrophorus electricus as a label (Pradelles et al 1985, 1990), in combination with a chromogenic non-toxic substrate for the staining step. The use of acetylcholinesterase for the labelling of molecules (haptens, antigens or antibodies) presents numerous advantageous features, such as a high specific activity, a very low non-specific binding (in spite of a 320 kDa molecular weight) and the possibility of using different chemical strategies to label molecules over a large pH range. Moreover this enzyme *Deceased, 14 July 1998 The Eicosanoids. Edited by Peter Curtis-Prior. &2004 John Wiley & Sons, Ltd: ISBN 0 471 48984 0

provides a continuous signal for more than 24 h, offering the possibility of increasing the precision and the sensitivity of the immunoassay by allowing the enzymatic reaction to proceed for longer periods. In this chapter, we will focus on findings concerning recently identified molecules (specific immunoassays for 8-epi-PGF2a, cyclooxygenases 1 and 2) and the development of a new method to assay LTC4. DEVELOPMENT OF COMPETITIVE ENZYME IMMUNOASSAY FOR 8-epi-PGF2a Background Recent observations have shown that lipid peroxides accumulate in certain diseases due to attack by free radicals produced in atherosclerosis or myocardial infarction (Patrono and FitzGerald 1997). The recent discovery of a new family of biologically active derivatives of arachidonic acid (the F2-isoprostanes), formed by a non-enzymatic mechanism catalysed by free radicals, may yield information on illnesses linked to oxidative stress. One of these compounds, 8-epi-PGF2a, acts as a powerful vasoconstrictor and its action appears to involve participation of the TXA2/PGH2 vascular receptor. We have produced polyclonal antibodies against this compound to develop a competitive enzyme immunoassay (EIA). Strategy All eicosanoids are low molecular weight substances (haptens) which must be covalently attached to a macromolecule (antigenic carrier) to elicit antibody production. Like most eicosanoids, 8epi-PGF2a has but one useful chemical group, a carboxylic function, which was activated to allow covalent coupling to KLH (immunogen for antibody production) or to AChE (enzyme tracer). This strategy previously proved to be successful for developing a competitive enzyme immunoassay for a large set of eicosanoids (Table 9.1). Rabbit polyclonal antibodies were obtained and tested using the 8-epi-PGF2a-AChE tracer. A competitive enzyme immunoassay was developed (Wang et al 1995). Assay sensitivity and specificity were analysed. The assay was applied to the measurement of 8-epi-PGF2a in biological samples and validated by correlation with other analytical methods (RIA, GC–MS).

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Table 9.1 Sensitivity of the different competitive enzyme immunoassays of eicosanoids using acetylcholinesterase as label Compound PGB2 PGD2-MO PGE2 Bicyclo-PGE2 6-Keto-PGF1a 11b-PGF2a 13,14-Dehydro-PGF2a PGF2a TXB2 2,3-Dinor-TXB2 11-Dehydro-TXB2 LTB4 LTC4 LTE4 12-HETE 15-HETE

IC50 (pg/ml) 38 80 110 10 60 55 400 70 70 200 180 30 100 140 1000 740

Results As shown in Figure 9.1A, the competitive enzyme immunoassay was very sensitive (B/B0 50% &8 pg/ml). It was also highly specific, with less than 0.1% cross-reactivity with related compounds such as 8-epi-PGE2 or PGF2a. Analysis of the same urine samples by two independent methods, EIA and GC–MS, yielded very similar results, thus validating the assay. Although no circadian variation of the compound in urine was observed (Figure 9.1B), preliminary results revealed a positive correlation between dose and the subject’s age. Urinary elimination was not modified by the treatment of volunteers with two non-steroidal antiinflammatory drugs (NSAIDs). However, the analysis of plasma samples indicated low but real production of 8-epi-PGF2a by an enzymatic mechanism (cyclooxygenase-dependent), a synthesis that was completely suppressed by aspirin.

PRODUCTION OF SPECIFIC ANTIBODIES AND DEVELOPMENT OF SPECIFIC ENZYME IMMUNOMETRIC ASSAYS FOR CYCLOOXYGENASES 1 AND 2, KEY ENZYMES IN ARACHIDONIC ACID METABOLISM Background Cyclooxygenase (COX, also known as PGH synthase; EC 1.14.99.1) is the first enzyme that intervenes in the biosynthesis of prostaglandins and thromboxanes starting with arachidonic acid. This activity therefore controls the synthesis of molecules acting as mediators in numerous cell functions. Thus an increase in prostaglandins accompanies the majority of clinical inflammation symptoms. Recent data have shown that there are two isoforms of this enzyme (Smith et al 1996), which is the target of numerous NSAIDs, such as aspirin, indomethacin and ibuprofen. The first form (COX-1) appears to be constitutively expressed and its production is only slightly affected by stimuli and hormones. This 70 kDa protein was cloned, purified and sequenced from various tissue and animal sources. More recently, a new form (COX-2) of similar molecular weight was identified as the product of an early response gene. Its expression can be induced by numerous external ligands. This novel form was cloned and its primary sequence determined for different species. COX-1 presents more than 90% between-sequence homology, whereas the homology is about 80% for COX-2. Comparison of the COX1 and COX-2 sequences in a given species shows around 60% similarity, with extensive conservation of the major residues involved in enzymatic activity. However, a significant difference is observed between these two sequences. There is a significant deletion in the amino-terminal part of COX-2 (including a large part of the COX-1 peptide signal sequence) compensated for by the insertion of an 18-residue sequence in the C-terminus (absent in COX-1). Strategy

Prospects This method could facilitate a rationally-based evaluation of antioxidant drugs in humans. Moreover, these compounds have a longer life than hydroperoxides, for example, and could therefore be used as stable markers in clinical studies of oxidizing reactions.

Detailed comparative analysis of human COX-1 and COX-2 sequences has identified different limited zones specific to each of the two isoforms. We have thus raised several polyclonal and monoclonal antibodies against synthetic peptides (Figure 9.2). Purified COX-1 (from ram seminal vesicles) was used to raise polyclonal and monoclonal antibodies, since the extensive conservation of the inter-species primary structure could lead to

Figure 9.1 (Panel A) Dose–response curve of 8-epi-PGF2a enzyme immunoassay. (Panel B) Daily variation of urinary 8-epi-PGF2a excretion analysed by competitive enzyme immunoassay in 10 healthy subjects

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Figure 9.2 Scheme of the h-COX-1 and h-COX-2 peptides used for development of specific immunometric assays. SP: signal peptide

recognition of human COX-1. Polyclonal antibodies were also raised against a 12-residue peptide specific to the human COX-1 amino-terminus. Our strategy for COX-2 was to produce monoclonal antibodies directed against the specific 18-residue insert. Polyclonal antibodies were raised against a 12-residue peptide located immediately upstream of this insert, a sequence completely modified with respect to COX-1. These different antibodies were first used and characterized in Western blot experiments. Most were also used in competition assays. Subsequently, two-site immunometric assays specific to each of the two isoenzymes were developed and used to study the production of these two forms in a model of human umbilical vein endothelial cells (HUVEC). Results We obtained two immunosera against COX-1, one raised against the first 12 residues of the human enzyme (anti-N-COX-1) and the other against the total sequence of purified ovine COX-1 (antiCOX-1). We also developed six mAbs with the same protein (Cre´minon et al 1995a). We obtained polyclonal antibodies and 10 mAbs raised against the C1 peptide of COX-2 (580–596 sequence of COX-2, specific insert of this form). Polyclonal antibodies directed against the C2 peptide (570–581 sequence) were also prepared (Cre´minon et al 1995b). Western blot experiments, designed to characterize the usefulness and specificity of these antibodies (Habib et al 1993), showed that COX-2 can be induced in HUVEC, since this isoform had been previously observed only at the transcriptional and genomic level. In HUVEC stimulated by interleukin-1a (IL-1a), or by a phorbol ester (PMA), COX enzymatic activity increased in parallel with a protein doublet detected using anti-C1 antibodies, while COX-1 immunoreactivity remained practically unchanged. Induction kinetics experiments with IL-1a and PMA revealed

different patterns of COX-2 production: faster and shorter for PMA and longer for IL-1a. We carried out immunoprecipitations with cells stimulated by IL-1a, incubated with Met 35S. Using an anti-C1 antiserum, a 35S-labelled protein doublet appeared, with a molecular weight of around 70 kDa, while the anti-COX-1 polyclonal antibodies showed only a very slight difference between the reference and stimulated cells. COX-2 immunoreactivity detected in these experiments was completely suppressed by preincubation of antibodies with the C1 peptide used in their production. We also verified that COX-1 and COX-2 immunoprecipitates exhibited COX enzymatic activity inhibited by a NSAID, flurbiprofen. Treatment of COX-1 and -2 by endoglycosidase-H revealed the glycosyl character of these enzymes (molecular weight decrease) without suppressing the doublet observed for COX-2 immunoreactivity. In parallel with the Western blot experiments, we developed immunological assays for COX-1 and COX-2. Two of the six mAbs did not recognize human COX-1 in Western blotting. All the polyclonal and monoclonal antibodies used in a competitive type assay (using AChE-labelled ram COX1 as tracer) were insufficiently sensitive, thus ruling out any assays of biological samples. We then developed two-site immunometric assays using one antibody coated onto the solid phase to ensure capture of the molecule, with staining by another AChE-labelled antibody (in a Fab’ form). We first analysed all the possible binding complementarities between these different antibodies by using ovine COX-1 or human platelet extracts (which contain only COX-1). Various combinations with good sensitivities [minimum detectable concentration (MDC) 20–60 pg/ml with ovine COX-1] were eliminated due to limited recognition of human COX-1. Polyclonal anti-COX-1 and anti-N-COX-1 antibodies were shown to be good capture antibodies, used with AChE-labelled CX-110 mAb as tracer. As shown in Figure 9.3A, the assay combining coated polyclonal anti-COX-1 antibodies and CX-110-AChE as tracer gave an MDC of 113 pg/ml with ovine COX-1, with good accuracy and good recognition of the human enzyme (parallelism with serial dilutions of the sample).

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Figure 9.3 (Panel A) Standard curve obtained using polyclonal anti-COX-1 IgGs as capture antibody and AChE-labelled mAb CX-110 as enzyme tracer, ram COX-1 as standard (full line) and serial dilutions (1/10, 1/20, 1/40, arbitrarily positioned on the abscissa) of human platelets extract (dotted line). (Panel B) Standard curve obtained using affinity-purified polyclonal anti-C2 peptide antibodies as capture antibody and AChE-labelled (anti-C1 peptide) mAb CX-329 as enzyme tracer, C3 peptide as standard (full line) and serial dilutions (1/20, 1/40, 1/80, 1/160, arbitrarily positioned on the abscissa) of PMA-stimulated HUVEC extract (dotted line)

The assay with the anti-N-COX-1 polyclonal antibody as capture antibody was less sensitive (MDC&1 ng/ml) and was set aside. This sandwich assay indicated 100 ng COX-1/mg proteins (&6.666108 platelets) in platelet extracts, a value near that reported by other authors. All the anti-C1 antibodies detected COX-2 in Western blot experiments, but showed appreciable labelling intensity fluctuations. The best results were obtained with the CX-308, -329 and -367 antibodies. The anti-C1 polyclonal and monoclonal antibodies were tested in a competition assay (with the AChE-labelled C1 peptide as tracer). The results were very disappointing, with detection thresholds of a few pmol/ml (corresponding to a minimum concentration of 70 ng/ml for the complete protein). This assay did not detect COX-2 production after stimulation of HUVECs. On the other hand, the use of anti-C2 polyclonal antibodies (total IgG fraction) as capture antibodies provided good complementarity with three AChE-labelled anti-C1 mAbs (CX-320, -329 and -394 mAbs), both with the C3 peptide as standard (human COX-2 570–595 sequence) and PMA-stimulated HUVEC samples. Assay sensitivity was greater using affinity-purified anti-C2 antibodies (Figure 9.3B). This assay was used to measure (with respect to the C3 peptide) COX-2 production in HUVECs under different conditions. The assay does not recognize COX-1 in human platelet extracts. These two assays were validated by molecular sieve chromatography experiments. The two isoforms were eluted at very high molecular weights (&300 kDa), while electrophoretic analysis of these immunoreactive peaks gave only the expected value of 70 kDa. The high molecular weight peaks very probably resulted from non-covalent associations between COXs or between COXs and other (unidentified) entities, since after a simple SDS denaturing treatment the eluted immunoreactivity was found at the expected molecular weight. Comparison of the measured reactivities of stimulated and reference cells confirmed the very high production of COX-2, in contrast with the very small changes recorded for COX-1. A combination of these two assays yielded different data for the HUVECs (Figure 9.4). An excellent correlation was seen between the parallel increase in poststimulation enzyme activity (measured by 6-keto-PGF1a production; Figure 9.4C) and COX-2 immunoreactivity (detected by both sandwich assay and Western blot; Figure 9.4A), while COX1 immunoreactivity (measured by the same techniques; Figure

9.4B) varied only slightly. We verified a dose–response effect with PMA and the sensitivity of COX-2 production (PMA-induced) to a PKC and a transcription inhibitor. Recent application of this assay to recombinant human COX-2 gave an MDC close to 250 pg/ml (unpublished results). Prospects These two assays are sensitive and high-performance tools for routine measurements of numerous samples (in comparison with Western blot techniques). However, two major problems remain: (a) the detected immunoreactivity for these two proteins corresponds to non-covalent aggregates; (b) the difficulty of obtaining suitable standards, i.e. recombinant or purified proteins. The first problem appears to be difficult to solve, since the denaturing conditions used (SDS) are generally incompatible with the formation of the antigen–antibody complex (and also with use of AChE). However, the good correlation between the Western blot results, the COX immunoassays, and the assays of PG production suggests that this aggregation does not introduce marked bias. The second problem will probably soon be resolved since various groups have prepared purified or recombinant human proteins which will be useful for calibrating the assays. These COX assays are also of potential value in examining putative interactions (Habib et al 1997) between the COX system and the NO-synthases system (also essential at the cardiovascular level). In fact, the two enzyme systems appear to share certain features and be complementary in function, and there may be interdependency in cardiovascular regulation. This example demonstrates the wide-ranging potential application of these immunoanalytical methods. ENZYME IMMUNOMETRIC ASSAY OF LTC4 Background Since the theoretical bases of competition assays were laid down in the early 1960s, several different types of immunological assays have been developed. However, two major general principles have emerged: competitive and non-competitive assays. Competitive

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Figure 9.4 Time-course of immunoreactivity of lysates from PMA-stimulated HUVEC. Cells were incubated in the absence (open blocks) or presence (full blocks) of 20 nM PMA for the indicated times. (Panel A) Immunometric analysis of COX-2 in the lysates performed using affinity-purified polyclonal anti-C2 peptide antibodies as capture antibody and AChE-labelled (anti-C1 peptide) mAb CX-329 as enzyme tracer. The inset corresponds to Western blot analysis of COX-2 synthesized by the same lysates using anti-C1 peptide mAb CX-329. (Panel B) Immunometric analysis of COX-1 in the same lysates performed using polyclonal anti-COX-1 IgGs as capture antibody and AChE-labelled mAb CX-110 as enzyme tracer. The inset corresponds to Western blot analysis of the same membranes as in Panel A (after stripping) using anti-COX-1 mAb CX-111. (Panel C) COX activity was evaluated in the same cells before lysis

assays use a labelled molecule and a limited concentration of a specific corresponding antibody. Immunoassays of small molecules (haptens, including the eicosanoids) are founded on this principle (see 8-epi-PGF2a immunoassay, above). In noncompetitive assays, the most widespread method corresponds to two-site immunometric assays (also called sandwich assays), which use two antibodies that simultaneously bind the molecule assayed. One of these antibodies (immobilized on a solid phase) captures the molecule, while the other is labelled and serves as a tracer. Both antibodies are used in excess. It should be specified that these methods, which are extensively used to assay high molecular weight or antigen molecules (45–10 kDa), are much more sensitive (see the COX-1 and COX-2 immunoassays above). It is generally admitted, however, that they are not applicable to small haptens, which cannot simultaneously bind two antibody molecules. Nonetheless, by disregarding this assumption we first demonstrated that this assay format was functional with small peptides (Cre´minon et al 1995c). However, in spite of the successful development of two-site immunometric assays for small peptides, it is clear that this procedure cannot be applied to the lowest molecular weight haptens (51000 Da). In fact, the joint and simultaneous binding of two complementary antibodies becomes impossible, due to steric hindrance alone. A new approach must therefore be envisaged if this assay format (which is more sensitive due to use of reagents in excess) is to be applied to small haptens. This is why we developed another methodology (Pradelles et al 1994) based on SPIE-IA (solid-phase immobilized epitope immunoassay; SPI-CEA patent). Theoretically, this technique

allows an immunometric format assay of molecules of any size, using just a single antibody (preferably monoclonal). This new technique is particularly suitable for haptens bearing a free amino group, since this function is generally used for conjugation with the antigenic carrier during immunogen preparation. It is assumed that this group is not directly involved in recognition by the antibodies (as supported by the observation that chemical modification of this amino group does not alter antibody binding) and is thus available for application of our new methodology. General principle This assay includes four major consecutive steps which are shown schematically in Figure 9.5. The first step is the immunological capture of the hapten by antibodies immobilized on a solid phase. After washing, the second step corresponds to covalent binding of the hapten to the antibodies. This involves the reaction of a homobifunctional reagent reactive with amino groups, thereby crosslinking the hapten to the proteins on the solid phase. After further washing, the third step (epitope release) uses a denaturing reagent to disrupt the interactions between the antibody binding sites and the hapten (still covalently bound to the antibody), thus allowing recovery of epitope accessibility. Ideally, the capture antibody is inactivated in this step. Finally, the antibody tracer addition step is an incubation with an mAb–AChE conjugate, the tracer antibody used being identical to that employed in the immunological capture step. Addition of the enzyme substrate after washing

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Figure 9.5

ANALYTICAL METHODS

General scheme of SPIE-IA

yields a typical immunometric calibration curve with a signal proportional to the amount of molecule introduced. Although the initial and final steps are common to standard immunometric assays, the originality of the procedure derives from the covalent binding and epitope release steps, which allow the use of a single antibody in a sandwich assay.

5 min reaction with 2.5% glutaraldehyde and a further reduction of remaining aldehyde active groups by treatment with 0.1 M NaBH4. Epitope release is achieved in 2 min with 0.1 M HCl before AChE-labelled anti-LTC4 mAb tracer is added for 1 h incubation. A final 30 min or 1 h revelation of AChE bound to the solid phase completes the assay.

Strategy

Results

This new procedure was tested with a monoclonal anti-LTC4 antibody (Volland et al 1994), which is not specific to LTC4 as it also cross-reacts with LTD4 and LTE4. The whole SPIE-IA takes less than 3.5 h. The capture step requires a 1 h reaction of standard or biological samples. The cross-linking step involves a

Different experimental conditions were studied for both the covalent binding and the epitope release steps, which constitute the originality of this method. After optimization, this assay gave a linear standard curve typical of immunometric assay (Figure 9.6A) and, as classically observed with immunometric assays

Figure 9.6 (Panel A) Standard curve obtained for SPIE-IA of LTC4. (Panel B) Time-course of LTC4 after addition of LTA4 to human platelets

IMMUNOASSAYS USING AChE AS LABEL involving AChE tracers, assay sensitivity and accuracy were improved when the enzymatic reaction was allowed to proceed for a longer period: MDC close to 5 pg/ml and 2 pg/ml for the 30 min and 1 h reaction, respectively. This assay is 60 times more sensitive than the competitive assay performed with the same mAb and LTC4–AChE tracer. SPIE-IA appears more specific, since the cross-reactivity with other LTs decreased (15% and 9% with LTD4 and LTE4, respectively) by comparison with the competitive assay. This probably results from steric hindrance due to the smaller size of LTE4 and LTD4, which are less accessible after covalent immobilization to the tracer antibody. The time-course of LTC4 production by platelets after addition of LTA4 was studied by SPIE-IA (Figure 9.6B) and correlated with competitive assay results. Validation was performed by HPLC fractionation experiments.

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the Hoˆpital Lariboisie`re. He was an ardent advocate of immunoanalytical methods since using them in his doctoral work at the Institut Pasteur, and of the use of acetylcholinesterase as an enzymatic marker. Jacques Maclouf was both a respected scientist and an accomplished sportsman but, rather than participate in the usual scientific ‘‘competition’’, he preferred to establish a large network of contacts with both friends and colleagues, an approach which better suited his deep human qualities. The perennial smile of this marathon-running scientist and gentleman in everyday life will be missed by all his friends and above all by his family, and our thoughts go out to his wife Nicole and their children, Be´atrice, Antoine and Guillaume. Farewell, Jacques . . .

REFERENCES Prospects This procedure appears to be limited in the case of eicosanoids to leukotrienes, which alone have a reactive amino function. However, experiments using photoactivation, a technique of great potential, are being carried out with different study models so as to extend the procedure to other chemical groups. UV and laser irradiation experiments are now under way, using various molecules. CONCLUSIONS Eicosanoid research is constantly evolving, with the identification of new molecules (or even new biosynthetic pathways, as shown for COX-2) and the reappraisal of the role of previously identified molecules. These constant changes are mainly due to the increasingly complex image emerging from the investigations of these biological systems. The cardiovascular system presents all the characteristics of this complexity in which all the cells, both circulating and constituting the vascular wall, interact dynamically and cannot be dissociated from other fundamental phenomena, such as proliferation, programmed death or cell activation. In this context, antibody affinity and specificity are undeniably very useful, and immunolocalization (Western blot, immunocytochemistry and immunohistochemistry) is most informative. Moreover, these antibodies can be used to develop routine, analytically efficient immunoassays. Lastly, the use of AChE as labelling enzyme yields inexpensive and reliable tools which will improve our understanding of numerous eicosanoids and isoeicosanoids. IN MEMORIAM Jacques Maclouf passed away on 14 July 1998. As a pharmacist, and above all as a practised and judicious scientist, he quickly understood the full potential of eicosanoids in the cardiovascular field, and this represented the main research thrust of his team at

Cre´minon C, Frobert Y, Habib A et al (1995a) Immunological studies of human constitutive cyclooxygenase (COX-1) using enzyme immunometric assay. Biochim Biophys Acta, 1254, 333–340. Cre´minon C, Habib A, Maclouf J et al (1995b) Differential measurements of constitutive (COX-1) and inducible (COX-2) cyclooxygenase expression in human umbilical vein endothelial cells using specific immunometric enzyme immunoassays. Biochim Biophys Acta, 1254, 341–348. Cre´minon C, De´ry O, Frobert Y et al (1995c) Two-site immunometric assay for substance P with increased sensitivity and specificity. Anal Chem, 67, 1617–1622. Habib A, Cre´minon C, Frobert Y et al (1993) Demonstration of an inducible cyclooxygenase in human endothelial cells using antibodies raised against the C-terminal region of cyclooxygenase-2. J Biol Chem, 268, 23448–23454. Habib A, Bernard C, Lebret M et al (1997) Regulation of the expression of cyclooxygenase-2 by nitric oxide in rat peritoneal macrophages. J Immunol, 158, 3845–3851. Hayashi Y, Ueda N, Yokota K et al (1983) Enzyme immunoassay of thromboxane B2. Biochim Biophys Acta, 750, 322–329. Maclouf J, Pradel M, Pradelles P and Dray F (1976). 125I derivatives of prostaglandins. A novel approach in prostaglandin analysis by radioimmunoassay. Biochim Biophys Acta, 431, 139–146. Patrono C and FitzGerald GA (1997) Isoprostanes: potential markers of oxidant stress in atherothrombotic disease. Arterioscler Thromb Vasc Biol, 17, 2309–2315. Pradelles P, Grassi J and Maclouf J (1985) Enzyme immunoassays of eicosanoids using acetylcholine esterase as label: an alternative to radioimmunoassay. Anal Chem, 57, 1170–1173. Pradelles P, Grassi J and Maclouf J (1990) Enzyme immunoassays of eicosanoids using acetylcholinesterase. Methods Enzymol, 187, 24–34. Pradelles P, Grassi J, Cre´minon C et al (1994) Immunometric assay of low molecular weight haptens containing primary amino groups. Anal Chem, 66, 16–22. Smith WL, Garavito RM and DeWitt DL (1996) Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. J Biol Chem, 271, 33157–33160. Volland H, Vulliez Le Normand B, Mamas S et al (1994) Enzyme immunometric assay for leukotriene C4. J Immunol Methods, 175, 97– 105. Wang Z, Ciabattoni G, Cre´minon C et al (1995) Immunological characterization of urinary 8-epi-prostaglandin F2a excretion in man. J Pharm Exp Therapeut, 275, 94–100.

10 Bioassay of Eicosanoids Robert L. Jones The Chinese University of Hong Kong, Shatin, NT, Hong Kong

Bioassay has played a fundamental role in the discovery of the eicosanoids and their development into a major research area. Goldblatt (1933) and von Euler (1934) first described the smooth muscle stimulant and vasodilator actions of acidic lipid extracts of various reproductive tissues. Many years later, Bergstro¨m’s group in Sweden isolated PGE1 and PGF1a from sheep seminal vesicles (Bergstro¨m and Sjo¨vall 1960a, 1960b; Bergstro¨m et al 1963), and the corresponding arachidonate-derived products, PGE2 and PGF2a, were soon recognized as local hormones of great biological relevance. Subsequently, the primary products of cyclooxygenase (COX) activity, PGG2 and PGH2, and their biologically active metabolites thromboxane A2 (TXA2) and prostacyclin (PGI2) were initially detected and quantified by bioassay methods. In a similar but slightly later scenario, cobra venom challenge to normal guinea-pig lung or antigen challenge to sensitized lung resulted in the release of lipid mediators with smooth muscle spasmogenic activity. The term ‘‘slow-reacting substance of anaphylaxis’’ (SRS-A) was coined for one of these principles, but its identity remained elusive for many years (Feldberg and Kellaway 1938; Kellaway and Trethewie 1940; Brocklehurst 1953; Orange and Austen 199). Eventually, the massive advances in chemical analysis techniques that occurred in the 1970s led to the identification of two peptidoleukotrienes, LTC4 and LTD4, as the main components of SRS-A, and to the identification of arachidonate 5-lipoxygenase as the initiating enzyme (Murphy et al 1979; Shimizu et al 1984, 1986; Ueda et al 1986). While LTC4 and LTD4 are powerful smooth muscle stimulants (Dahle´n et al 1981), particularly of the bronchial tree, an alternative 5-lipoxygenase pathway leads to LTB4, a nonpeptide mediator with potent chemotactic activity (see FordHutchinson, 1990). This chapter mainly deals with smooth muscle bioassays for eicosanoids, of which there are two main types. In a conventional assay, muscle tension is recorded from the assay tissue immersed in physiological salt solution in an organ bath. Standard and unknown agents are added to the bath in sequence, with wash and rest periods between each dose. This type of assay is best suited to the estimation of stable eicosanoids in either collected tissue fluid or solvent extracts/chromatography fractions reconstituted in water. In a superfusion assay, physiological salt solution drips continuously over the assay tissue and agents are added to the inflow stream over a few seconds (Gaddum 1953; Figure 10.1). In a cascade superfusion system, two or more assay tissues are arranged in a vertical bank and the effluent from the upper one drips over the next lower tissue. This arrangement may be attached to the outflow of a perfused organ (the generator in Figure 10.1), thereby affording real-time estimation of mediator release from the organ. Both stable and unstable mediators can be The Eicosanoids. Edited by Peter Curtis-Prior. &2004 John Wiley & Sons, Ltd: ISBN 0 471 48984 0

detected by this method, which was pioneered by Sir John Vane over many years (Vane 2000). CONVENTIONAL BIOASSAYS FOR EICOSANOIDS Practical Considerations The purity of standards and unknowns is a critical factor in bioassay. The earliest PGE and PGF standards were derived from incubation of precursor fatty acids with crude prostaglandin synthase obtained from sheep seminal vesicles. However, the fatty acids were not always of the highest purity, and as a graduate student in Eric Horton’s laboratory in the late 1960s, I recall our concern over the 1–2% PGE1 present in our precious few milligrams of PGE2, in the light of work showing that PGE1 and PGE2 had opposite actions on blood platelet aggregation (Kloeze 1967). We now know, of course, that PGE1 is a much more potent agonist than PGE2 at the prostacyclin (IP) receptor (see Wise and Jones 2000). Fortunately, the Upjohn Company was soon supplying researchers with pure PGE2 and PGF2a prepared by chemical synthesis. In those early days, purification of tissue fluids or homogenates involved a solvent extraction process followed by a fairly crude chromatography process. In one commonly used extraction process, the retained phases (italics) sequentially contained low– medium polarity, neutral–acidic lipids (pH 3 water:ethyl acetate), then low–medium polarity, acidic lipids (ethyl acetate:pH 8 water; pH 3 water:ethyl acetate) and then medium polarity, acidic lipids (petroleum spirit:67% ethanol) (Jouvenaz et al 1970). The Karolinska group had developed reversed-phase partition chromatography systems with an adsorbed stationary phase (as opposed to the modern bonded stationary phases) for the purification of prostaglandins (see Gre´en et al 1978). Although these systems had great separating power, they were not suitable for purifying many samples containing only nanogram quantities of prostaglandins. Consequently, most groups used adsorption chromatography on small columns of silicic acid. The material supplied by commercial manufacturers was notoriously variable in its retention and solvent flow characteristics, and much time was spent removing fines by sieving and obtaining the desired absorptivity by careful heating. Nevertheless, good separation and recovery of PGE2 and PGF2a could usually be obtained (see Salmon and Karim 1976). The low quantities of prostaglandin present in most extracts did not allow the use of a 2+2 Latin square design (see Rang and Dale 1991). A bracketing assay was the preferred method, in which two or three doses of unknown were sandwiched between low and high doses of standard. This meant that confidence limits

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Figure 10.1 Bioassay using the cascade superfusion procedure. T1, T2 and T3 represent assay tissues. The mediator generator can be an immobilized enzyme system, an isolated organ, or an intact animal

for the estimate were not usually presented. I have memories of A. S. Milton standing behind a postgraduate struggling to obtain the optimum tissue load or dose cycle and saying ‘‘It takes 10 years to become a competent bioassayist’’. Indeed, the process of increasing the steepness of the lower portion of the dose–response curve was mastered by some, whilst remaining a complete mystery to others. A ‘‘good preparation’’ would often be taken over by several workers (including ‘‘The Prof’’), well into the early hours of the morning. The rat stomach fundus, which was developed to assay 5-HT (Vane 1957), was probably the most widely used preparation for prostaglandins. It responded well to both PGE2 and PGF2a and therefore one had to have confidence in the preceding chromatographic separation. Other useful tissues included the guinea-pig ileum and the colon of the gerbil or jird (Meriones trystiami and M. libycus), which were more sensitive to PGE2 than PGF2a. The rabbit jejunum, which shows rhythmic spontaneous activity, was sometimes used to assay PGF2a. Turning to the leukotrienes, the commonest assay preparation for SRS-A has been the guinea-pig ileum, prepared as either a tube or a longitudinal strip, in the presence of a histamine H1 receptor antagonist (Kellaway and Trethewie 1940; Brocklehurst 1953); its utility has extended into studies that followed the structural elucidation of the peptidoleukotrienes and the provision of authentic standards. LTD4 is about 10 times more potent than LTC4 on guinea-pig ileum (Piper et al 1981). SRS-A was distinguished from bradykinin, PGE2 and PGF2a by its greater activity on guinea-pig ileum relative to rat uterus and gerbil colon (Orange and Austen 1969). In later studies, SRS-A in tissue extracts was assayed against an in-house laboratory standard (Conroy et al 1976). The Contribution of Conventional Bioassays to Discoveries in the Eicosanoid Field For about 10 years from 1965, conventional bioassay played a major part in establishing several physiological and pathological

roles for PGE2 and PGF2a. Starting with the pro-inflammatory actions of PGE2, Ambache and colleagues (1965) demonstrated the presence of unsaturated hydroxy fatty acids in the aqueous humour of the rabbit eye following irritation of the iris. They found that the gerbil colon had little spontaneous activity and, because of this, was a better assay preparation than the rat colon. Later workers reported increased production of PGE1, PGE2 and PGF2a in anterior uveitis in the rabbit using the rat stomach fundus as a bioassay preparation (Eakins et al 1972). Discrimination between PGE and PGF-like activity was partly based on the instability of PGE under mild alkaline conditions, while PGE1 and PGE2 were separated by thin-layer chromatography on silverimpregnated silica or paper. A considerable number of studies were performed on eicosanoid function in skin inflammation, e.g. A¨ngga˚rd et al. (1970) and Arturson et al (1973) used rat stomach fundus, gerbil colon and guinea-pig ileum to show the presence of PGE2-like material in scald and burn fluids. The group led by Malcolm Greaves was in the forefront of studies on human skin, using thin-layer chromatography (TLC) to separate E and F prostaglandins, followed by assay on the rat uterus preparation. His group showed that flucinolone, but not hydrocortisone, dramatically reduced the biosynthesis of PGE2 and PGF2a from arachidonic acid in human breast skin (Greaves and McDonald-Gibson 1972). They also incorporated tritium-labelled arachidonic acid into the incubation medium so that appropriate zones on the TLC plate could be accurately defined, and the extent of biosynthesis could also be confirmed. It is of interest that during my collaboration with Amersham Radiochemicals to produce heptatritio-PGE2 and heptatritio-PGF2a for radioimmunoassay purposes, the mass component for calculation of specific activity was determined by assay on the guinea-pig ileum and rabbit jejunum, respectively. It had to be assumed that the introduction of seven tritium atoms into a prostaglandin molecule does not alter its biological potency. One of the major effects of NSAIDs, such as aspirin, indomethacin and meclofenamic acid, is to reduce fever. In 1970, Milton and Wendlandt reported that PGE1 injected into the

BIOASSAY third ventricle of the conscious cat produced shivering and a rapid rise in body temperature. They noted that the fever was similar in character to that produced by bacterial pyrogen, except for its shorter duration. In collaboration with Feldberg’s group, cerebrospinal fluid collected from the third ventricle of cats during Shigella dysenteriae fever was assayed on the rat fundus strip preparation and found to contain elevated levels of PGE1-like activity (Feldberg et al 1973). Significantly, indomethacin, aspirin and paracetamol reduced the fever and also reduced the prostaglandin concentration to below the level of detection; this led to general acceptance of the role of prostaglandins in the generation of bacterial fever and of the inhibition of prostaglandin biosynthesis as the antipyretic mechanism of NSAIDs. A fuller description of the early work on the role of prostaglandins in fever has been written by Milton (1976). Turning to PGF2a, the paper presented by Eric Horton’s group at the New York Academy of Sciences meeting in 1970 (Horton et al 1971) illustrated the value of bioassay for detecting small quantities of a mediator of unknown structure, followed by identification of the mediator in a bulked sample by a sophisticated physicochemical method. Internal distension of the isolated uterine horn of the guinea-pig resulted in release of PGF2a-like material into the bathing medium, but there was little change in the release of PGE2, as assayed on the rat stomach strip. Extracts from distended and non-distended horns from 35 guineapigs on day 3 of the oestrous cycle were then subjected to combined gas chromatography–mass spectrometry after methyl ester–trimethysilyl ether derivatization. Ten times more PGF2a was found in the distension sample compared to the nondistension sample, and this supported the emerging hypothesis that PGF2a is a natural luteolytic agent in several animal species (see Horton and Poyser 1976). CASCADE SUPERFUSION BIOASSAYS FOR EICOSANOIDS Practical Considerations Figure 10.1 shows the basic superfusion unit and several ways of using the cascade and accessory equipment to measure mediator release and metabolism. The essence of the cascade is that the assay tissues are chosen to detect specific mediators or combinations of mediators (as a consequence of their different complements of cell surface receptors). For example, the rabbit aorta contracts to nanogram quantities of PGH2 and TXA2, while the rat stomach strip contracts to similar amounts of PGE2. Receptor antagonists can be applied to one or more of the assay tissues to improve the specificity of the detection process. For example, in many prostanoid studies, an antagonist cocktail of atropine (M receptors), mepyramine (H1 receptors), phenoxybenzamine (a1-adrenoceptors and others), propranolol (badrenoceptors) and methysergide (some 5-HT receptors) has been employed. A delay coil can be inserted into the cascade to determine the stability of the mediator under the conditions of the assay (say, pH 7.4 and 378C). Also the effluent from the generator can be split and run over two banks of assay tissues, one of which may be treated with antagonist(s). In a small number of studies, a blood-bathed system has been used. The generator is an anaesthetized animal or a blood-perfused isolated organ, such as the spleen. Blood is taken from the carotid artery of the animal or the venous outflow of the isolated organ to the top of the cascade, and instead of running to waste at the bottom it is returned to a reservoir and then to the animal or organ. It is essential to have good anticoagulation with heparin, otherwise the assay tissues become coated with a layer of fibrin and their sensitivity to mediators becomes depressed. Release of ADP and

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5-HT from aggregating platelets can also produce variation in the basal tone of the assay tissues. The whole cascade system must be thermostatically controlled to obtain reproducible responses to standards and released agents. It should be realized that the exposure profiles of assay tissues to standard agents and mediators may not be similar (as they are in conventional assays) and this may affect the accuracy of an assay. In addition, release of a mixture of mediators from the generator may lead to physiological antagonism in the assay tissues, with underestimation of the level of one or both of the mediators (see next section, final paragraph). Finally, while a highly unstable agonist may lose activity during passage down the cascade, the reverse may also occur. For example, contractile responses to LTC4 increased in size on passage over a bank of guinea-pig ileum preparations, whereas those to LTD4 remained virtually unchanged (Piper et al 1982); the explanation lay in the conversion of LTC4 to the more potent LTD4 by g-glutamyl transpeptidase present in the assay tissues. Despite these caveats, the cascade superfusion method has worked remarkably well for the bioassay of eicosanoids. The Contribution of Cascade Superfusion Bioassays to Discoveries in the Eicosanoid Field The cascade superfusion method was first used in eicosanoid research to show the release of prostaglandins from the Krebsperfused spleen of the dog during stimulation of the sympathetic nerve supply (Gilmore et al 1968). Assay bank 1 contained rat stomach strip, rat colon and chick rectum in the presence of phenoxybenzamine and propranolol; strong contractions of the first two assay tissues indicated the release of both PGE2 and PGF2a; assay bank 2 contained the same preparations continuously contracted with a combination of PGE2, angiotensin IIamide and 5-HT and was used to demonstrate catecholamine release through relaxant responses of the tissues. Contraction of the spleen induced by adrenaline is also accompanied by the release of prostaglandins (conventional bioassay) (Davies et al 1967) and in the cascade experiments the infusion of adrenaline into the chronically-denervated spleen still caused contraction of the assay tissues. Thus, it was concluded that the prostaglandins derive from activated smooth muscle cells in the spleen (or other cells with an adrenergic innervation) and not from nerves supplying the spleen. Probably the greatest utility of the cascade superfusion method in the eicosanoid field has been to detect and quantify unstable prostanoids such as PGH2, TXA2 and PGI2 (prostacyclin). Our story starts with attempts to determine the nature of ‘‘rabbit aorta-contracting substance’’ (RCS), which could be generated from lung tissue by several different stimuli and appeared to derive from enzyme action on membrane phospholipids. Gryglewski and Vane (1972) allowed arachidonic acid to interact with a crude COX preparation from dog spleen for several minutes in a silicone rubber coil (generator), and directed the effluent over rabbit aorta and rat stomach strip preparations. Arachidonate infusion alone caused weak contraction of the rabbit aorta, which was dramatically enhanced when COX was also added (Figure 10.2). PGE2 generation could also be detected using the rat stomach strip, but was complicated by a contractile response to COX alone. Addition of meclofenamic acid or indomethacin to the generator reduced the contractions of both preparations, indicating COX as the source of RCS. Further experiments showed that the contractile activity of RCS on rabbit aorta decayed considerably following passage through a delay coil, whereas activity on rat stomach strip increased. The conclusion that RCS is simply PGG2/PGH2, which decays spontaneously to give mainly PGE2, did not, however, fit all the facts; the half-lives

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Figure 10.2 Record from a cascade superfusion assay of rabbit aortacontacting substance (RCS) generated by the interaction of arachidonic acid with a crude cyclo-oxygenase (COX) enzyme preparation from dog spleen during passage through a silicone rubber coil. Modified from Gryglewski and Vane (1972) with permission of the author and publisher

of RCS (52 min) and the prostaglandin endoperoxides (ca. 5 min) were different. This led Samuelsson’s group to search for an even more unstable COX product, which they identified as thromboxane A2 (TXA2) (Hamberg et al 1975). TXA2 is several times more potent than PGH2 on the rabbit aorta (Needleman et al 1976). Rabbit coeliac or mesenteric arteries show biphasic responses, contractile progressing to relaxant, to the prostaglandin endoperoxides, while they only contract to TXA2. This profile allows the discrimination of PGH2 and TXA2, and was used to show that TXA2 is the major component of RCS released from guinea-pig lung incubated with arachidonic acid (Moncada and Vane 1977). Jones et al (1982) used the same guinea-pig lung–rabbit aorta system to show that EP 045, one of the first selective TP receptor antagonists to be synthesized, could block the contractile actions of TXA2 and U-46619 (a synthetic TP receptor agonist) to the same extent, while having no effect against angiotensin II. RCS activities generated in rabbit platelets during aggregation (Vargaftig 1977) and rabbit polymorphonuclear leukocytes during phagocytosis (Higgs et al 1976) were also shown to be due to TXA2. In following up the dual action of prostaglandin endoperoxides on rabbit coeliac and mesenteric arteries, Bunting et al (1976) detected a new natural prostanoid generated by a microsomal enzyme preparation (generator) from vascular tissue. This product, which was initially called PGX, did not contract rabbit aorta (which does not contain relaxant prostanoid receptors), had weak contractile activity on rat stomach strip and relaxed rabbit coeliac and mesenteric arteries. PGX was subsequently characterized and renamed prostacyclin, and is a potent vasodilator and inhibitor of platelet aggregation through agonist actions on IPreceptors (see Wise and Jones, 2000 for a fuller account of the discovery of prostacyclin). In the original studies, PGX did not affect the superfused rat colon, but in later experiments, purified material was reported to relax and reduce the spontaneous activity of the colon (Moncada et al 1978). The inhibitory action of prostacyclin on the colon is due to activation of IP receptors on non-adrenergic non-cholinergic (NANC) nerves, causing the release of NO and a second unidentified inhibitory transmitter (Qian et al 1995). In some preparations, spontaneous activity decays completely over several hours, and this may account for the variable action of PGX. Prostacyclin is one of three endothelium-derived relaxant factors (EDRFs) released by agents acting on the intimal surface of vascular endothelium cells, the others being nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) (see

Mombouli and Vanhoutte 1997). Endothelium-denuded vessel rings have been used in a cascade superfusion system to differentiate and assay NO and prostacyclin released from endothelium cells cultured on microcarrier beads packed into a column (generator). The underlying principle involves choosing two assay tissues, both of which relax well to NO, while only one relaxes to prostacyclin. Cocks et al (1985) used the pig and dog coronary arteries for this purpose, while Gryglewski et al (1986) used the rabbit aorta and the cow coronary artery. An example from the latter study is shown in Figure 10.3, where bradykinininduced release of both NO and prostacyclin from pig aorta endothelial cells, and the much shorter half-life of NO relative to prostacyclin, can be seen by comparing the responses of the first and second tissues of each type in the cascade. Tone in the preparations was generated by inclusion of U-46619 in the superfusate. The name ‘‘slow-reacting substance of anaphylaxis’’ (SRS-A) was derived from the very slow relaxation of the guinea-pig ileum to baseline tension following washout of the mediator from the organ bath. Thus, it was natural to attempt the bioassay of SRS-A by cascade superfusion due to the efficient washing of the assay tissues in this technique. In addition to the guinea-pig ileum, various respiratory smooth muscle preparations, such as tracheal and bronchial rings and strips of lung parenchyma, have been used on the basis of their high contractile sensitivity to the peptidoleukotrienes. The simultaneous release of RCS and peptidoleukotrienes from guinea-pig lung was measured using an initial bank of rat stomach strip/guinea-pig trachea/rabbit aorta, followed by a delay circuit, and then by a second bank of the same three preparations (Bakhle et al 1985). RCS activity decayed during the delay, whereas LT-like activity did not. The Ca2+ ionophore A23187 induced much greater release of LT-like material than either bradykinin or arachidonic acid, prompting the authors to suggest that different cell types in the lung have different complements of biosynthetic enzymes. Surprisingly, the bradykinin- and arachidonic acid-released mediators that contracted the guinea-pig trachea appeared to be COX products, but

Figure 10.3 Record from a cascade superfusion assay of both prostacyclin and NO released from pig aorta endothelial cells after challenge with bradykinin (BK). Equivalent sensitivity of the assay tissues to NO is shown by the relaxant responses to sodium nitroprusside (NP). The ring preparations were denuded of endothelium and therefore did not relax to direct application of bradykinin. Modified from Gryglewski et al (1986) with permission of the author and publisher

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Figure 10.4 Assay of LTB4 extracted and purified from 500 mg of human psoriatic skin scale using chemokinesis of human peripheral blood polymorphonuclear leukocytes. Modified from Brain et al (1984) with permission of the author and publisher

their actions were blocked by the LT receptor antagonist FPL 55712. FPL 55712 blocked authentic LTC4, but not PGE2, on guinea-pig ileum, and blocked both agonists on guinea-pig trachea, leading the authors to question the specificity of FPL 55712. However, the guinea-pig trachea contains both EP1 receptors responsible for contraction and EP2 receptors that mediate relaxation (Dong et al 1986). Consequently, just a small degree of EP1 receptor block by FPL 55712 may allow the EP2 system to become dominant, leading to a false impression of antagonist specificity.

ASSAYS FOR CHEMOTACTIC ACTIVITY Stimulation of rat neutrophils with A23187 caused the release of a lipoxygenase product with chemoattractant and aggregating activity on neutrophils (Bray et al 1980). Isolation of the product by HPLC was followed by its identification as leukotriene B4 (Ford-Hutchinson et al 1980). The agarose micro-droplet chemokinesis system employed for assay of LTB4 involves mixing neutrophils with agarose solution, placing droplets of the suspension in a 96-well micro-titre plate and allowing solidification of the agarose. Aliquots of solutions containing potential chemoattractant are then added to the wells and, after waiting for several hours, the movement of the white cells is measured microscopically (Smith and Walker 1980). Figure 10.4 shows log concentration–response curves for the LTB4 standard and LTB4-like material isolated from a bulked sample of human psoriatic skin scale (Brain et al 1984); the high sensitivity and good linearity and parallelism of this assay are readily apparent.

CONCLUDING REMARKS Classical bioassay methods are rarely used in laboratories today. Nevertheless, we should not abandon them altogether, since they are capable of detecting new biologically active natural agents, which other methods cannot.

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Gryglewski R and Vane JR (1972) The generation from arachidonic acid of rabbit aorta contracting substance (RSC) by a microsomal; enzyme preparation which also generates prostaglandins. Br J Pharmacol, 46, 449–457. Gryglewski R, Moncada S and Palmer RMJ (1986) Bioassay of prostacyclin and endothelium-derived relaxing factor (EDRF) from porcine aortic endothelial cells. Br J Pharmacol, 87, 685–694. Hamberg M, Svensson J and Samuelsson B (1975) Thromboxanes: a new group of biologically active compounds derived from prostaglandin endoperoxides. Proc Nat Acad Sci USA, 72, 2994–2998. Higgs GA, Bunting S, Moncada S and Vane JR (1976) Polymorphonuclear leucocytes produce thromboxane A2-like activity during phagocytosis. Prostaglandins, 12, 749–757. Horton EW, Thompson C, Jones R and Poyser NL (1971) Release of prostaglandins. Ann NY Acad Sci, 180, 351–361. Horton EW and Poyser NL (1976) Uterine luteolytic hormone: a physiological role for PGF2a. Physiol Rev, 56, 595–651. Jones RL, Peesapati V and Wilson NH (1982) Antagonism of the thromboxane-sensitive contractile systems of the rabbit aorta, dog saphenous vein and guinea-pig trachea. Br J Pharmacol, 76, 423–438. Jouvenaz GH, Nugteren DH, Beerthuis RK and Van Dorp DA (1970) A sensitive method for the determination of prostaglandins by gas chromatography with electron capture detection. Biochimica Biophysica Acta, 202, 231–234. Kellaway CH and Trethewie ER (1940) The liberation of slow-reacting smooth muscle-stimulating substance in anaphylaxis. Qu J Exp Physiol, 30, 121–145. Kloeze J (1967) Influence of prostaglandins on platelet adhesiveness and platelet aggregation. In Nobel Symposium II. Prostaglandins, Bergstro¨m S and Samuelsson B (eds). Interscience, New York, 241–252. Milton AS and Wendlandt S (1970) A possible role for prostaglandin E1 as a modulator for temperature regulation in the central nervous system. J Physiol, 207, 76–77. Milton AS (1976) Modern views on the pathogenesis of fever and the mode of action of pyretic drugs. Journal of Pharmacy and Pharmacology, 28, 393–399. Mombouli JV and Vanhoutte PM (1997) Endothelium-derived hyperpolarizing factor(s): updating the unknown. Trends Pharmacol Sci, 18, 252–256. Moncada S and Vane JR (1977) The discovery of prostacyclin—a fresh insight into arachidonic acid metabolism. In Biochemical Aspects of Prostaglandins and Thromboxanes, Kharasch N and Fried J (eds). Academic Press, New York, USA, 155–177. Moncada S, Ferreira SH and Vane JR (1978) Bioassay of prostaglandins and biologically active substances from arachidonic acid. Adv Prostagland Thromboxane Res, 5, 211–236. Murphy RC, Hammarstro¨m S and Samuelsson B (1979) Leukotriene C: a slow reacting substance from murine mastocytoma cells. Proc Nat Acad Sci USA, 76, 4275–4279.

Needleman P, Moncada S, Bunting S et al (1976) Identification of an enzyme in platelet microsomes which generates thromboxane A2 from prostaglandin endoperoxides. Nature, 261, 558–560. Orange RP and Austen KF (1969) Slow-reacting substances of anaphylaxis. Adv Immunol, 10, 105–144. Piper PJ, Letts LG, Samhoun MN et al (1982) Actions of leukotrienes on vascular, airway and gastrointestinal; smooth muscle. Adv Prostagland Thromboxane Leukotriene Res, 9, 169–181. Piper PJ, Samhoun MN, Tippins JR et al (1981) Pharmacological studies on pure SRS-A, and synthetic leukotrienes C4 and D4. In SRS-A and Leukotrienes, Piper PJ, (ed). Wiley, New York, 81–99. Qian YM and Jones RL (1995) Inhibition of rat colon contractility by prostacyclin (IP-) receptor agonists: involvement of NANC neurotransmission. Br J Pharmacol, 115, 163–171. Rang HP and Dale MM (1991) Measurement in pharmacology. In Pharmacology. Churchill Livingstone, Edinburgh, 49–71. Salmon JA and Karim SMM (1976) Methods for analysis of prostaglandins. In Prostaglandins: Chemical and Biochemical Aspects, Karim SMM (ed). MTP Press, Lancaster, 25–85. Shimizu T, Izumi T, Seyama Y et al (1986) Characterisation of leukotriene A4 synthase from murine mast cells: evidence for its identity as arachidonate 5-lipoxygenase. Proc Nat Acad Sci USA, 83, 4175–4179. Shimizu T, Radma˚rk O and Samuelsson B (1984) Enzyme with dual lipoxygenase activities catalyses leukotriene A4 synthesis from arachidonic acid. Proc Nat Acad Sci USA, 81, 689–693. Smith MJH and Walker JR (1980) The effect of some antirheumatic drugs on an in vitro model of human polymorphonuclear leucocyte chemokinesis. Br J Pharmacol, 69, 473–478. Ueda N, Kaneko S, Yoshimoto T and Yamamoto S (1986) Purification of arachidonate 5-lipoxygenase from porcine leukocytes and its reactivity with hydroperoxyeicosatetraenoic acids. J Biol Chem, 261, 7982–7988. Vane JR (1957) A sensitive method for the assay of 5-hydroxytryptamine. Br J Pharmacol Chemother, 12, 344–349. Vane JR (2000) The Second Gaddum Memorial Lecture: the release and fate of vaso-active hormones in the circulation. Br J Pharmacol, 131, 27–60. Vargaftig BB (1977) Carrageenan and thrombin trigger prostaglandin synthetase-independent aggregation of rabbit platelets: inhibition by phospholipase A2 inhibitors. J Pharm Pharmacol, 29, 222–228. Von Euler US (1934) Zur kenntnis der pharmakalogischen wirkungen von nativsekreten und extrekten mannlicher accessorsicher geschlechsdrusen, Naunyn Schmiedeberg’s Arch Exp Path Pharmacol, 175, 78–84. Wise H and Jones RL (2000) An introduction to prostacyclin and its receptors. Prostacyclin and Its Receptors. Kluwer Academic, New York, 1–27.

11 Gas Chromatography and Mass Spectrometry in Eicosanoid Analysis Michinao Mizugaki1, Takanori Hishinuma2, Naoto Suzuki2 and Junichi Goto2 1Tohoku

Pharmaceutical University and 2Tohoku University Hospital, Sendai, Japan

Since the discovery of the structure of prostaglandins, thromboxanes, leukotrienes, other eicosanoids and their metabolites, one of the most challenging tasks in eicosanoids research has been to define the role of eicosanoids in human health and disease. Since the measurement of eicosanoids and their specific metabolites is a useful approach to assess in vivo formation of these compounds, numerous analytical techniques have been developed for their analysis in biological samples. These include radioimmunoassay (RIA) and enzyme immunoassay (EIA), gas chromatography– mass spectrometry (GC–MS) using different ionization techniques, liquid chromatography (LC) in combination with ultraviolet (UV), fluorescence and e