Prostaglandins, Leukotrienes and Other Eicosanoids: From Biogenesis to Clinical Application

F. Marks, G. Furstenberger (Eds.)

Prostaglandins, Leukotrienes and Other Eicosanoids

F. Marks, G. Furstenberger (Eds.)

Prostaglandins, Leukotrienes and Other Eicosanoids From Biogenesis to Clinical Application

@3wI LEY-VCH Weinheim - New York Chichester Brisbane Singapore Toronto

Prof. Dr. Friedrich Marks Dr. Gerhard Furstenberger Abteilung Biochemie der gewebsspezischen Regulation (BOSOO) Deutsches Krebsforschungszentrum Im Neuenheimer Feld 280 D-69120 Heidelberg This book was carefully produced. Nevertheless, editors, authors and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertentlv be inaccurate.

Library of Congress Card No.: applied for A catalogue record for this book is available from the British Library. Deutsche Bibliothek Cataloguing-in-Publication Data: Prostaglandins, leukotrienes and other eicosanoids: from biogenesis to clinical application I Friedrich Marks; Gerhard Fiirstenberger (ed.). - Weinheim; New York; Chichester; Brisbane; Singapore; Toronto: Wiley-VCH, 1999 ISBN 3-527-29360-4

0 WILEY-VCH Verlag GmbH, D-69469 Weinheim (Federal Republic of Germany). 1999 Printed on acid-free and chlorine-free paper. All rights reserved (including those of translation in other languages). No part of this book may be reproduced in any form - by photoprinting, microfilm, or any other means - nor transmitted or translated into machine language without written per-mission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Composition: DATA SOURCE SYSTEMS, Timisoara 1900, Romania. Printing: betz druck gmbh, D-64291 Darmstadt. Bookbinding: J. Schaffer GmbH&Co. V.G., D-67269 Griinstadt. Printed in the Federal Republic of Germany.

Preface

Although discovered more than 65 and identified more than 30 year ago, eicosanoids were considered for a long time to represent biocompounds of minor importance, for instance , when compared with hormones and cytokines. As a consequence eicosanoid research was regarded as a subject for narrow specialist rather than being a major field in biomedical science. This situation has changed completely, since mainly due to the impact of modern analytical and molecular-biological technology, eicosanoids have become recognized as a physiologically and pathologically very important family of cellular signal transducers; this also underlined by two Nobel prizes. Eicosanoids and related fatty acid derivatives are produces throughout the eukaryotic kingdom by probably every organism and every cell type. Most of these compounds fulfill the functions of local mediators, also called autocoids or tissue hormones. As such they modulate the effects of all kinds of hormonal, immunological, and nervous signals as well as of environmental influences. in fact, the eicosanoid system may be understood as a device of biological signal transduction and signal processing which is placed between the environment and the systemic network of signaling on the one side and the intracellular machinery of signal processing on the other side. At the level of the molecular mechanism of prostanoid action there are indeed fluid transitions between extra- and intracellular signaling. The enormous versatility of eicosanoids is a result of the extraordinary multiplicity of polyunsaturated fatty acid metabolism that leads to innumerable bioactive compounds, which are still far from being known as a whole. This metabolic complex certainly represent one of the most fascinating examples of biochemical evolution in nature. It appears as if eicosanoids - together with other local mediators - form a highly sophisticated network of locally restricted inter- and intracellular communication which is not fixed but becomes organized 'on demand. The high degree of multiplicity and feedback interactions indicate that this machinery operates in a non-linear fashion which may help to produces 'order in chaos', that is, to carry out a characteristic performance of living matter. Considering the central role eicosanoids and related compounds play in cellular (patho)-physiology it is everything but surprising that both basic research and pharmaceutical industry undertake tremendous efforts in the development of drugs which interact with this metabolic complex. As a major milestone in this field the identification of nonsteroidal antiinflammatory drugs, such as aspirin, as inhibitors of prostanoid biosynthesis has to be mentioned. Today most investigators and clinicians agree that an in-depth elucidation of eicosanoid metabolism is a prerequisite not only for an understanding but also for an advanced treatment or prevention of some of the most serious diseases, such as atherosclerosis, cancel, Alzheimer's dementia, allergic asthma, and others. This opinion is reflected by the chapters of this book. Following an introductory

VI

Prejace

overview (Chapter I ) , Chapters 2-6 deal with the major enzymatic routes of eicosanoid biosynthesis, while Chapter 7-12 focuss on clinical aspects. The selection of topics may appear to be rather arbitrary. However, considering the enormous and rapidly enlarging extent of the field, any attempt to reach completeness would have been doomed to failure from the very beginning. Thus, the selection of the topics had to aim necessarily at exemplarity, and the goal of this book is to provide an introduction into, rather than a comprehensive review, of the field. As a compensation the authors have done their best in providing up-dated reference lists which may help the reader to follow-up special aspects in more detail and to the roots. Notwithstanding these joint intentions, every contribution reflects the authors' personal approach to handle the subject as far as style, arrangement, content, in-depth treatment, and the emphasis on special aspects are concerned. Since every chapter should stand by itself some overlapping and redundancy was inavoidable. As a whole the book offers a snapshot of one of the most fascinating subjects of current biological research and its clinical applications. The editors are aware of the fact that in such a rapidly developing fields ideas, hypotheses, conclusions, and the latest information are highly perishable goods. Nevertheless, they hope that the book contains a substantial wealth of hard facts and, thus, will serve for a reasonable period of time as a stimulation and guidance for both students and experts. Heidelberg, June 1999

Friedrich Marks

Gerhard Fiirstenberger

Contents

1

Arachidonic acid and companions: an abundant source of biological signals

................................................

1

Friedrich Murks

1.6.3 1.7 1.8

The world of PUFAs .................................................................................. The discovery of prostaglandins and related eicosanoids .......................... Mammalian eicosanoids............................................................................. Free arachidonic acid: a signaling compound? .......................................... Prostanoids ................................................................................................. HPETEs, HETEs and leukotrienes........................................................... Lipoxins ................................................................................................... 15-Epi-lipoxins......................................................................................... Hepoxilins ................................................................................................ Monooxygenase-derived eicosanoids ...................................................... Isoprostanes.............................................................................................. Anandam& ............................................................................................. Eicosanoids in invertebrates..................................................................... Eicosanoid-related signaling compounds in plants .................................. The cellular functions of eicosanoids in mammals .................................. Eicosanoids as local mediators................................................................. Specific membrane receptors mediate many biological effects of eicosanoids............................................................................... Nuclear eicosanoid receptors: a new frontier in research......................... Addendum: Methods of eicosanoid research ........................................... References................................................................................................

2

The generation of free arachidonic acid

1.1 1.2 1.3 1.3.1 1.3.2 1.3.3 1.3.4 1.3.5 1.3.6 1.3.7

1.3.8 1.3.9 1.4

1.5 1.6 1.6.1 1.6.2

...............................................

1 3 6 7 8 11 15 17 18 20 21 24 27 30 34 34 35 38 40 40

47

Peter Dieter 2.1 2.2 2.3 2.3.1 2.3.2 2.3.2.1 2.3.2.2 2.3.2.3 2.4

Introduction .............................................................................................. (Re)Incorporartionof arachidonic acid into phospholipids ..................... Phospholipases A ..................................................................................... Phospholipase A, ..................................................................................... Phospholipases A2 .................................................................................... Secretory phospholipases A2 .................................................................... Cytosolic phospholipase A2..................................................................... Calciurn-independent phospholipases A2................................................. DAG lipase and PLC or PLDPA phosphohydrolase...............................

47 48 49 49 49 51 52

56 56

VIII

Contents

2.5 2.5.1 2.5.2 2.6 2.7

Cellular models ........................................................................................ P388D1macrophages ............................................................................... Rat liver macrophages.............................................................................. Conclusions.............................................................................................. References................................................................................................

3

Cyclooxygenases Karin Miiller-Decker

3.1 3.2 3.3 3.4 3.5 3.5.1 3.5.2 3.5.3 3.5.4 3.6 3.7 3.8 3.9 3.10 3.11

Introduction .............................................................................................. 65 Cloning of cyclooxygenase isoforms ....................................................... 66 Cyclooxygenase gene structures .............................................................. 67 Regulation of cyclooxygenase isoenzyme expression ............................. 69 Cyclooxygenase proteins ......................................................................... 73 Sequence comparisons ............................................................................. 73 Post-translational modification ................................................................ 74 X-ray analysis of crystal structure............................................................ 74 Subcellular localization............................................................................ 75 Coupling of COX isoenzymes with phospholipases A2 ........................... 76 Substrate specificities............................................................................... 77 Mechanism of enzyme catalysis............................................................... 78 Biological functions of COX isoforms..................................................... 79 Isoenzyme-specific inhibitors .................................................................. 81 References ................................................................................................ 83

4

Prostanoid synthases Christian Martin and Volker Ullrich

4.1 4.2. 4.3 4.4 4.5. 4.6 4.7 4.8 4.9 4.10

Introduction .............................................................................................. Thromboxane A2 synthase ....................................................................... Prostacyclin synthase ............................................................................... Prostaglandin D syntase ........................................................................... Prostaglandin E synthase.......................................................................... Prostaglandin F synthase........................................................................ Glutathione S-transferases ..................................................................... Detection of prostaglandin synthases in various tissues ........................ Summary and outlook ............................................................................ References..............................................................................................

5

Lipoxygenases Hartmut Kiihn

109

5.1.

lntroduction............................................................................................

109

.....................................................................................

..............................................................................

.......................................................................................

57 57 57 58 59

65

89 89 91 93 97 99 100 101 102 104 104

Contents

5.2. 5.3. 5.4. 5.5 5.5.1. 5.5.2 5.6 5.6.1 5.6.2 5.6.3 5.6.4 5.6.5 5.7 5.7.1 5.7.2 5.7.3 5.7.4 5.8 5.8.1 5.8.2 5.8.3 5.8.4 5.8.4.1 5.8.4.2 5.8.4.3 5.9

IX

Lipoxygenase reaction ........................................................................... 110 Common properties of lipoxygenases .................................................... 111 Classification of lipoxygenases.............................................................. 113 Structural aspects of lipoxygenases ....................................................... 115 X-ray crystallography............................................................................. 115 Substrate alignment and determinants of positional specificity .............118 5-Lipoxygenases .................................................................................... 120 Enzymatic properties.,............................................................................ 120 5-Lipoxygenase activating protein ........................................................ 122 Molecular biology of 5-lipoxygenases................................................... 123 Tissue distribution and regulation of 5-LOX expression ....................... 123 124 Biological functions of 5-lipoxygenases................................................ 12-Lipoxygenases.................................................................................. 125 Subclassification and enzymatic properties ........................................... 125 Molecular biology of 12-lipoxygenases................................................. 126 Tissue distribution and regulation of 12-LOX expression..................... 127 Biological functions of 12-lipoxygenases.............................................. 127 Mammalian 15 -1ipoxygenases .............................................................. 129 Subclassification and enzyme properties ............................................... 129 Molecular biology of the reticulocyte-type 15-lipoxygenases...............130 Tissue distribution and regulation of 15-LOX expression ..................... 131 Biological functions of 15-lipoxygenases.............................................. 132 Structural modification of lipid-protein assemblies. Implication in cell maturation and atherogenesis................................... 132 133 Modulation of intracellular lipid signal transducers .............................. Formation of bioactive oxygenated fatty acid derivatives ..................... 134 References.............................................................................................. 134

.................143

6

Oxygenation of arachidonic Acid by cytochromes P-450 Ernst H . Oliw and Johanna Ericsson

6.1 6.2 6.3 6.3.1 6.3.2 6.3.3

Introduction............................................................................................ 143 Early work on oxidation of fatty acids by cytochromes P-450 ..............144 Oxygenation of arachidonic acid by cytochromes P-450....................... 146 Hydroxylation of o side chain ............................................................... 146 Epoxidation ............................................................................................ 147 Bisallylic hydroxylation and hydroxylation with double bond migration ................................................................................................ 149 Metabolism of epoxides ......................................................................... 152 152 Epoxide hydrolases ................................................................................ Incorporation into phospholipids ........................................................... 153 Analysis of arachidonic acid metabolites............................................... 153 153 Radioimmunoassay................................................................................ 154 GC-MS and LC-MS analyses................................................................. Steric analysis of hydroxy fatty acids .................................................... 154

6.4 6.4.1 6.4.2 6.5. 6.5.1 6.5.2 6.5.3

X

Contents

6.5.4 6.6 6.6.1 6.6.2 6.6.3 6.6.4 6.6.5 6.7 6.8

Steric analysis of epoxy fatty acids and vicinal diols ............................. Biological effects ................................................................................... Kidney .................................................................................................... Heart....................................................................................................... Vascular tree .......................................................................................... Central nervous system and the pituitary ............................................... Genital glands and endocrine organs ..................................................... Summary ................................................................................................ References .............................................................................................

7

Renal eicosanoids Margarete Goppelt-Striibe and Joachim Fader

7.1 7.1.1 7.1.2 7.1.3 7.1.4 7.1.4.1 7.1.4.2 7.1.4.3 7.1.4.4 7.1.4.4.1 7.1.4.4.2 7.1.4.4.3 7.1.5 7.1.6

Renal prostanoids ................................................................................... 169 Localization of prostanoid biosynthesis in the kidney ........................... 170 Regulation of prostaglandin synthesis in mesangial cells ...................... 172 Cyclooxygenase expression in renal inflammation................................ 173 173 Prostanoid receptors ............................................................................... TXA2 receptor ........................................................................................ 174 PGFza receptor ....................................................................................... 175 Prostacyclin (PG12)receptor .................................................................. 175 PGE? receptors ....................................................................................... 176 EP, receptors .......................................................................................... 176 EP2/EP4 receptors ................................................................................... 177 EP3receptors .......................................................................................... 177 Vasoactive effects of prostanoids: relation to hypertension................... 178 Regulation of water and electrolyte transport by prostanoids: clinical implications ............................................................................... 180 181 Cyclooxygenase inhibitors and the kidney............................................. Renal leukotrienes.................................................................................. 181 Biosynthesis ........................................................................................... 181 Biological activity of leukotrienes in the kidney.................................... 182 Renal lipoxins ........................................................................................ 183 Biosynthesis of lipoxins ......................................................................... 183 Biological activity of lipoxins in the kidney .......................................... 184 Cytochrome P-450 enzyme-generated arachidonic acid metabolites.....185 P-450 arachidonic acid o-hydroxylases ................................................. 186 Renal epoxygenases ............................................................................... 186 Functional properties of 19- and 20-HETE............................................ 187 187 Functional properties of epoxides .......................................................... Monooxygenase products in animal models of renal hypertension ....... 188 P-450-catalysed arachidonic acid metabolism in man ........................... 189 Isoprostanes............................................................................................ 190 190 Generation of isoprostanes in the kidney ............................................... Functional properties of isoprostanes..................................................... 191

7.1.7 7.2 7.2.1 7.2.3 7.3 7.3.1 7.3.2 7.4 7.4.1 7.4.2 7.4.3 7.4.4 7.4.5 7.4.6 7.5 7.5.1 7.5.2

.................................................................................

155 156 156 158 158 159 160 161 161

169

Contents

XI

7.6 7.7

Perspective ............................................................................................. References ..............................................................................................

8

The role of eicosanoids in reproduction H.P. Zahradnik. B . Wetzka and W.R.Schaler

199

8.1 8.2 8.2.1 8.2. 1 .1 8.2.1.2 8.2.2 8.2.3 8.2.4 8.3. 8.3.1 8.3.2 8.3.3 8.3.4 8.3.4.1 8.3.4.2 8.3.4.3 8.4 8.4.1 8.4.2 8.4.3 8.5

Introduction ............................................................................................ Female reproductive system ................................................................... Ovarian function .................................................................................... Follicular phase: ovulation ..................................................................... Luteal phase: luteolysis .......................................................................... The Fallopian tube ................................................................................. Menstruation .......................................................................................... Endometriosis ......................................................................................... Pregnancy ............................................................................................... Implantation ........................................................................................... Placenta .................................................................................................. Pregnancy-induced hypertension and pre-eclampsia ............................. Parturition .............................................................................................. Cervical ripening .................................................................................... Labor ...................................................................................................... Pre-term labor ........................................................................................ Male reproductive system ...................................................................... Acrosome reaction ................................................................................. Immunosuppressive actions of PGE ...................................................... Erectile dysfunction ............................................................................... References ..............................................................................................

199 199 199 200 201 202 203 207 208 208 211 214 216 218 220 222 223 224 224 224 225

9

The role of eicosanoids in inflammation and allergy Eva Wikstrom Jonsson and Sven-Erik Dahlkn

9.1 9.2 9.3

Introduction ............................................................................................ Formation of eicosanoids in allergic inflammation ................................ Biological activities and receptors with relevance for asthma and allergic inflammation ...................................................................... COX products ........................................................................................ LTB4....................................................................................................... Cysteinyl leukotrienes ............................................................................ Lipoxins ................................................................................................. Cysteinyl leukotrienes as mediators of allergen-induced airway obstruction and bronchial hyper-responsiveness .................................... Biological activity .................................................................................. Endogenous formation ........................................................................... Influence of inhibitors of leukotriene synthesis or CysLT, receptor

9.3.1 9.3.2 9.3.3 9.3.4 9.4 9.4.1 9.4.2 9.4.3

.............................................

............

192 192

233 233 234 238 238 239 241 243 243 244 244

XI1

9.4.4 9.5 9.5.1 9.5.2 9.5.3 9.5.4 9.5.5 9.6 9.7 9.7.1 9.7.2 9.7.3 9.7.4 9.7.5 9.8 9.9

Contents

antagonists on allergen-induced airway obstruction .............................. 245 Allergen-induced bronchial hyper-responsiveness ................................ 247 Cysteinyl leukotrienes as mediators of airway obstruction induced by 249 other factors ........................................................................................... Exercise .................................................................................................. 2S0 Adenosine .............................................................................................. 250 Sulfur dioxide......................................................................................... 250 250 PAF ........................................................................................................ Intolerance to aspirin and other NSAIDs ............................................... 25 1 Treatment of asthma with anti-leukotrienes........................................... 253 COX products as modulators of asthma and allergic inflammation.......254 Effects of prostaglandins and thromboxane ........................................... 254 255 Release of COX products....................................................................... Influence of inhibition of COX products on allergen-induced bronchoconstriction................................................................................ 256 COX products in airway hyper-responsiveness ..................................... 256 Anti-inflammatory and anti-asthmatic effects of PGE2 ......................... 257 Conclusions............................................................................................ 258 259 References..............................................................................................

..........................................

273

10

Prostanoids in the cardiovascular system Lukasz Partyka. Arsineh Arakil Aghajanian and Helmut Sinzinger

10.1 10.1.1 10.2 10.3 10.3.1

Introduction............................................................................................ 273 Defects in the eicosanoid system related to vascular disease .................274 Unsaturated fatty acids, vascular system and atherosclerosis ................276 Endothelium, vascular wall. platelet aggregation and eicosanoids ........278 Aspirin interaction with COX-1. Role in cardiovascular prevention. ‘Resistance to aspirin’............................................................................ 280 Eicosanoids and restenosis ..................................................................... 281 Function of eicosanoids in the heart and kidney .................................... 283 Heart muscle activity, contractility, ejection fraction ............................ 283 Myocardial infarction............................................................................. 283 Kidney, eicosanoids and cardiovascular regulation ............................... 283 Isoprostanes-novel indicators of cardiovascular disease ..................... 285 Prostaglandins as drugs in the therapy of cardiovascular disorders .......286 Mechanisms of action ............................................................................ 286 Side-effects............................................................................................. 290 Route of administration and dosage ....................................................... 291 Gene therapy-experiments with prostaglandins in cardiovascular disorders ................................................................................................. 291 292 Methods of clinical prostaglandin research ............................................ Sample collection and preparation ......................................................... 292 Bioassays................................................................................................ 293 Physicochemical methods ...................................................................... 293

10.3.2 10.4 10.4.1 10.4.2 10.4.3 10.5 10.6 10.6.1 10.6.2 10.6.3 10.6.4 10.7 10.7.1 10.7.2 10.7.3

Contents

XI11

10.7.4 10.8

Immunochemical methods ..................................................................... References..............................................................................................

11

Eicosanoids and cancer Friedrich Murks and Gerhard Fiirstenberger

11.1. 11.2 11.3

303 Carcinogenesis: a multistage process ..................................................... Immunosuppression by prostaglandins .................................................. 307 Metabolic activation of carcinogens in the course of prostaglandin synthesis ................................................................................................. 308 The generation of agressive products in the course of eicosanoid formation ................................................................................................ 308 The role of eicosanoids in tumor promotion .......................................... 309 COX inhibitors suppress tumor promotion in animal models ................309 Prevention of colorectal cancer in man by COX inhibitors ...................312 Promotion of tumor development by prostaglandins: a widespread phenomenon? ......................................................................................... 315 Mechanistic aspects................................................................................ 316 Lipoxygenase-catalyzed arachidonic acid metabolism .......................... 321 Effects of eicosanoids on tumor cell metastasis ..................................... 322 Cyclopentenone prostaglandins: a new class of anti-cancer drugs?.......324 References.............................................................................................. 324

11.4. 11.5 11.5.1 11.5.2 11S.3 11.5.4 1 1 S.5 11.6 11.7 11.8

........................................................................

294 295

303

.........

12

Synthetic eicosanoids: development and clinical applications 331 Bemd Buchmann. Ulrich Klar. Hartmut Rehwinkel and Werner Skuballa

12.1 12.2 12.3 12.4 12.5 12.6 12.7

Introduction............................................................................................ Prostaglandins ........................................................................................ Prostacyclins .......................................................................................... Thromboxane receptor antagonists ........................................................ Cystenilleukotriene antagonists ............................................................. LTB4antagonists.................................................................................... References..............................................................................................

......................................................................................................

Index

331 331 336 343 350 354 360

375

List of Contributors

Arisineh Arakil Aghajanian Institut fur Nuklearmedizin Universitat Wien Wahringer Giirtel 18-20 1009 Wien Austria Bernd Buchmann Schering AG Tnstitut fur Arzneimittelchemie MullerstraBe 170- 178 13342 Berlin Germany Sven-Erik DahlCn Institute of Environmental Medicine Karolinska Institute

xx

17177 Stockholm Sweden Peter Dieter Institut fur Physiologische Chemie Technische Universitat Dresden Karl-Marx-StraBe 3 0 1109 Dresden Germany Joachim Fauler Universitatsklinikum Carl Gustav Carus Institut fur Klin. Phhmakologie Techn. Universitat Dresden FriedlerstraBe 27 0 1307 Dresden Germany Gerhard Furstenberger Abteilung des Biochemie

gewebsspezifischen Regulation (BOSOO) Deutsches Krebsforschungszentrum Im Neuenheimer Feld 280 69 120 Heidelberg Germany Margarete Goppelt-Striibe Medizinische Klinik IV Nephrologische Forschungslaboratorien Universitat Erlangen-Nurnberg LoschgestraBe 8 9 1054 Erlangen Germany Ulrich Klar Schering AG Institut fur Arzneimittelchemie MiillerstraBe 170-178 I3342 Berlin Germany Hartmut Kiihn Institut fur Biochemie/Charitt Humboldt Universitat SchumannstraBe 20-2 1 10117 Berlin Friedrich Marks Abteilung Biochemie der gewebsspezifischen Regulation (BOSOO) Deutsches Krebsforschungszentrum Im Neuenheimer Feld 280 69 120 Heidelberg Germany Christian Martin Fakultiit fur Biologie Universitat Konstanz UniversitatsstraBe 10

XVJ

List of Contributors

78464 Konstanz Germany Karin Muller-Decker Abteilung Biochemie der gewebsspezifischen Regulation (BOSOO) Deutsches Krebsforschungszentrm Im Neuenheimer Feld 280 69 120 Heidelberg Germany Ernst H. Oliw Institute for Pharmacology Uppsala University Box 59 1, Biomedicum 75124 Uppsala Sweden Lukasz Partyka Department of Clinical Biochemistry Collegium Medicum Jagiellonian University 15a Kopernika Str. 31-501 Krak6w Poland Hartmut Rehwinkel Schering AG Institut fur Arzneimittelchemie MullerstraBe 170-178 13342 Berlin Germany Wolfgang Schafer Universitats-Frauenklinik Hugstetter Stral3e 55 79106 Freiburg Germany Helmut Sinzinger Institut fur Nuklearmedizin Universitat Wien Wlihringer Giirtel 18-20 1009 Wien Austria

Werner Skuballa Schering AG Institut fur Arzneimittelchemie MullerstraBe 170-178 13342 Berlin Germany Volker Ullrich Fakuitat fur Biologie Universitat Konstanz Universitatsstrafie 10 78464 Konstanz Germany Birgit Wetzka

Universitats-Frauenklinik Hugstetter StraBe 55 79 106 Freiburg Germany Eva Wikstrom Jonsson Institute of Environmental Medicine Karolinska Institute

xx

17177 Stockholm Sweden Hans-Peter Zahradnik Universitats-Frauenklinik Hugstetter StraBe 55 79 106 Freiburg Germany

Abbreviations

ADH AEA AMP AOD AOS APC ASA ATL bFGF CaLB CDNB CHD CHO CLASP

cox

cPLA~ CRH CSF CSF CYP DAG DART DGLA DHEA DHET(E) DMSO DRE Dsh DTT EDHF EDRF ECP EDTA EET EFA EGF EGFR EPR EPR

Anti-diuretic hormone Arachidonyl-ethanolamide Adenosine monophosphate Arterial occlusive disease Allene oxide synthase Adenomatous polyposis coli Acetyl-salicylic acid Aspirin-triggered lipoxin Basic fibroblast growth factor Calcium-dependent lipid binding 1-Chloro-2,4-dini trobenzene Coronary heart disease Chinese hamster ovary Collaborative Low-dose Aspirin Study in Pregnancy Cycloox ygenase Cytoplasmic phospholipase A2 Corticotropin-releasing hormone Cerebrospinal fluid Colony-stimulating factor Cytochrome P-450 Diacylglycerol Diet and Reinfarction Trial Dihomo-y-linoleic acid Deh ydroepiandrostendione Dihydroxyeicosatrienoic acid Dimethylsulfoxide Dioxin-responsive element Dishevelled Dithiothreitol Endothelium-derived hyperpolarizing factor Endothelium-derived relaxing factor Eosinophil cationic protein Ethylenediamine tetraacetate Epoxyeicosatrienoic acid Essential unsaturated fatty acids Epidermal growth factor Epidermal growth factor receptor Electron paramagnetic resonance Endoplasmic reticulum

XVIII

EXAFS FABP FAP FEN 1 FLAP FMRF

FPR FS H Fz GAP GC-MS GLA GM-CSF GnRH GRE GSH GSK GST GT HAT/HIT hCG HHT HETE HNPCC HODE HOT HOTE HPETE HPLC HPODE HX

IFN Ig IL

IP Ip3

IRE isoP IUGR KETE LASS LC-MS LDL LEF LH LIF

A bbrc4rrtiotz.s

Extended X-ray absorption fine structure Fatty acid-binding protein Familial adenomatous polyposis Forced expiratory volume in 1 s 5-Lipoxygenase-activating protein Phenylalanyl-methionyl-arginy1-phenylalanin PGF receptor Follicle-stimulating hormone Frizzled GTPase-activatingprotein Gas chromatography mass spectrometry y-Linoleic acid Granulocyte/monocyte colony-stimulating factor Gonadotropin-releasinghormone Glucocorticoid-responsiveelement Glutathione Glycogen synthase kinase Glutathione S-transferase Gonadotropin Heparin-associated thrornbocytopenia Human chorion gonadotropin Hydroxy-heptadecatrienoic acid Hydroxyeicosatetraenoic acid Hereditary non-polyposis colorectal carcinoma Hydroxyoctadecadienoic acid Hydroxyoctadecatrienoic acid Hydroxyoctadecatetraenoic acid Hydroperoxyeicosatetraenoicacid High-performance liquid chromatography Hydroperoxyoctadecadienoic acid Hepoxilin Interferon Immunoglobulin Interleukin Isoelectric point Inositor - 1,4,5- triphosphate Insulin-responsive element Isoprostanes Intrauterine growth retardation Ketoeicosatetraenoicacid Labile (platelet) aggregation-stimulating substance Liquid chromatography mass spectrometry Low density lipoprotein Leukocyte-enhancing factor Luteinizing hormone Leukemia inhibitory factor

Abbreviations

LPS LT LTH LX MAPkinase MCP MEK MGST Min MS NAD(H) NADP(H) NEM NGF NICI NKcell NMDA NO NOS NSAID PA PAF PAGE PAOD PC PDA PDGF PGDH PE PEFR PG PGHS PHGPx pHMB PI PIH PKC PL PLAP PLC PMNL PPAR PS PTCA PUFA PVD

XIX

Lipopol y saccharide Leukotriene Luteotropic hormone Lipoxin Mitogen-activated protein kinase Monocyte chernotactic protein MAP kinase/Erk kinase Microsornal glutathione S-transferase Multiple intestinal neoplasia Mass spechornetry Nicotinarnide adenine dinucleotide (reduced form) Nicotinarnide adenine dinucleotide phosphate (reduced form) N-Ethyl-maleirnide Nerve growth factor Negative-ion chemical ionization Natural kdler cell N-Methyl-D-aspartic acid Nitric oxide Nitric oxide synthase Non-steroidal anti-inflammatory drug Phosphatidic acid Platelet-activating factor Polyacrylamide gel electrophoresis Peripheral arterial occlusive disease Phosphatidylcholine Phytodienoic acid Platelet-derived growth factor Prostaglandin dehydrogenase Phosphatidylethanolarnine Peak expiratory flow rate Prostaglandin Prostaglandin H synthase Phospholipid hydroperoxide glutathione peroxidase p-Hydrox ymercuribenzoate Phosphatidylinositol Pregnancy-induced hypertension Protein kinase C Phospholipase Phospholipase-activating protein Phospholipase C Polyrnorphonuclear leukocyte Peroxisome proliferator-activated receptor Phosphatidylserine Percutaneous translurninal coronary angioplasty Polyunsaturated fatty acid Peripheral vascular direase

XX

Abbreviutioris

RBP RCS RT-PCR SDS SH SHR SMC sPLA~ SRS SRS-A TGF TLC TMD TNF TX UTR VLDL

Retinol-binding protein Rabbit aorta-contracting substance Reverse transcriptase polymerase chain reaction Sodium dodecyl sulfate Sulfhydryl Spontaneously hypertensive rat Smooth muscle cell Secretory phospholipase A2 Slow-reacting substance Slow-reacting substance of anaphylaxis Transforming growth factor Thin layer chromatography Transmembranedomain Tumor necrosis factor Thromboxane Untranslated region Very low density lipoprotein

Prostaglandins, Leukotrienes and Other Eicosanoids: From Biogenesis to Clinical Application Edited by F. Marks, G. Fürstenberger Copyright © WILEY-VCH Verlag GmbH, D-69469 Weinheim (Federal Republic of Germany). 1999

1 Arachidonic acid and companions: an abundant source of biological signals Friedrich Marks

1.1 The world of PUFAs Polyunsaturated fatty acids (PUFAs), such as linoleic acid, a-linolenic acid, dihomoy-linolenic acid, arachidonic acid, eicosapentaenoic acid, etc., are found throughout the eucaryotic kingdom. For men and most animals these fatty acids--or at least the precursors linoleic and a-linolenic acid-are ‘essential’, i.e. they cannot be synthesized de novo but have to be taken up in a vegetable diet (Fig. 1-1). While the major function attributed to these compounds in earlier textbooks of biochemistry was the control of membrane viscosity, it is now known that they play a key role in intercellular communication as well serving as precursors of a very large and extremely versatile family of signaling compounds formed primarily along oxidative pathways. A characteristic structural feature of the PUFAs found in living matter is that the double bonds are cis-configurated and not conjugated, i.e. are each separated by a CH2 group (1.4-cis, cis-pentadiene structure). Upon contact with air, such structures become rapidly autoxidized resulting in a bewildering variety of peroxy, hydroxy and epoxy compounds which easily undergo further reactions. Biological evolution has made use of this chemical versatility by forcing the individual reactions step by step under the control of enzymatic catalysis. As a result, cells then dispose of a very large collection of oxygenated fatty acid derivatives called ‘oxylipins’ [3] which they mainly use for signaling purposes. At least in animals, the C20 fatty acid-derived eicosanoids [4] constitute the most abundant subfamily of oxylipins with arachidonic acid being the major precursor. In cells PUFAs are normally found to be sequestered in membranes, i.e. esterified in phospholipids. They are released from this store by phospholipases which are under the control of environmental or cellular signals. Such signal-activated phospholipases [ 5 ] also regulate the formation of other signaling molecules, such as diacylglycerols, inositol-phosphates, lyso-phosphatidic acid, ceramides, etc. Most of them are released into the cytoplasm where they act as intracellular signal transducers, whereas others, such as lysophosphatidic acid, operate as mediators of intercellular communication [6]. In contrast, fatty acid-derived signaling molecules can be released into the cell’s interior as well as into the extracellular space, acting both as second messenger-like intracellular and hormone- and even pheromone-like intercellular signals. Moreover, at the receptor level they share the properties of hydrophilic hormones, i.e. they activate cellular signaling cascades by interaction with receptors at

2

1 Ar~ichidonicacid und companions: an abundunt source cf. biological s i p i d s

the cell surface, with the ability of lipophilic hormones, i.e. they penetrate the plasma membrane and modulate the activity of intracellular receptor proteins such as transcription factors. It appears, therefore, that fatty acid-derived oxylipins provide the most versatile signaling molecules in living nature. ESSENTIAL FATTY ACIDS

9

oleic acid

a - linolenic acid

linoleic acid

I

1

t

6.9-octadacadienoic acid

y-linolenic acid

stearidonic acid

i

i

I1

8.1 I-eicosadienoicacid

11

14

i

AS-DESATL‘RASE

e2x7 eez? I1

11

I4

arachidonic acid

11

(22zYooH m

C

17

5,8,11,14,174cosapentaenoic acid

ELONGASE

i

t

7.10.1 3dowsatrienoic acid

1

8.1 1,14,17-eicosatetraenoicacid

dihomo-y-linolenic acid

i

mead acid

ELONGASE

i

t O

adrenic acid

O

H

m

C

O

O

H

13

16

19

7,10,13,16,19-docosapentaenoicacid

Figure 1-1. Biosynthetic pathways for the formation of PUFAs. Linoleic and a-linolenic acids are synthesized from oleic acid in plants only (upper line) and, therefore, are essential for animals. In animal cells, these plant fatty acids undergo chain elongation with acetyl CoA at the carboxylic end (broken arrows) and desaturation at A 5 and 6. Beyond carbon atom 9 no double bonds can be introduced by animal cells, strictly limiting the interconversibility between linoleic acid- and a-linolenic acid-derived compounds. The major oxilipin precursors in vertebrates are arachidonic acid, dihomo-y-linolenic acid, eicosapentaenoic acid and linoleic acid. In invertebrates and plants, oxilipins are also synthesized from alinolenic acid and stearidonic acid. The role of other PUFAs in oxilipin biosynthesis is unknown, although some of them (i.e. mead acid and adrenic acid) have been shown to be substrates of oxilipin-forming oxygenases. As far as the nomenclature of unsaturated fatty acids is concerned, the positions of the double bonds are given as prefix numbers (beginning with the terminal carboxylic group as number 1). In another nomenclature system the position of the double bond nearest to the terminal methyl group (carrying the so-called w C-atom) is designated in order to set out the biosynthetic interrelationships of fatty acid families (for instance, the a-linolenic acid-derived w3-fatty acids and the a-linoleic acid-derived o6-fatty acids). Fish oil is particularly rich in 03-fatty acids. For more details see Willis [ I ] and Brenner [Z].

As far as evolutionary aspects are concerned, lipid mediators such as oxylipins confirm the hypothesis that the formation of signaling molecules may have developed as a ‘by-product’ of ancient metabolic pathways which were initially required for the biosynthesis and metabolism of basic biomolecules such as lipids, amino acids, peptides, nucleotides and carbohydrates.

1.2 The discovery of prostuglandins and related eicosunoids

3

1.2 The discovery of prostaglandins and related eicosanoids For a detailed treatment of this subject the reader is referred to Willis [ 11 and von Euler and Eliasson [7]. Historically, prostaglandins are the leading compounds of the eicosanoid subfamily of oxylipins. In 1930 Kurzrock and Lieb [8] discovered that seminal fluid exerted pronounced pharmacological effects on uterus preparations. Depending on whether the tissue was obtained from formerly pregnant or from sterile women it responded either by relaxation or by contraction. Analogous results obtained with extracts from the sheep vesicular gland or human seminal fluid were published a few years later by von Euler [9] and Goldblatt [ 101. Von Euler characterized the active principle as an unsaturated acidic lipid thus ruling out a hypothesis put forward by others, i.e. that acetylcholine had caused the observed effects. Since the active lipid was also initially found in extracts from the prostate von Euler [ 1I ] proposed the name ‘prostaglandin’. Only later was it realized that the prostaglandin in prostate extracts came mostly from the vesicular glands [7]. However, by that time it was already too late to change the name which had become established in the literature. Because of the inadequacy of the analytical methods available at that time it still took another 20 years until prostaglandins became identified and the whole field of research could leave the gallery of curios. In cooperation with the Bayer Company (formerly part of the IG Farben Company) in Elberfeld (Germany), von Euler and his group in Stockholm. prepared prostaglandin as barium salt from sheep vesicular glands in amounts large enough for further biological and chemical characterization. In the course of this work it soon became clear that prostaglandin represented a whole family of related factors rather than being a single compound. This finding and the chemical characterization as well as partial synthesis of prostaglandin Fza from arachidonic acid were primarily the merit of Bergstrom and Sjovall [12]. In 1957 these authors published the isolation of the first prostaglandin, i.e. PGF2a, in a crystalline form [12] and, in 1960, PGE2 was purified to homogeneity [ 131. The structures of both these prostaglandins were elucidated by Bergstrom et al. [ 141 and the absolute configuration was determined by them, in collaboration with van Doorp’s [15] group at the Unilever Research Laboratories in Vlaardingen, The Netherlands, in the early 1960s. Moreover, the biosynthesis of prostaglandin from radioactively labeled Cl0 PUFAs, such as dihomoy-linoleic acid, arachidonic acid and 5,8,11,14,17-eicosapentaenoic acid, was investigated by both groups using homogenates from sheep vesicular glands as enzymatic preparations [ 16,171. The involvement of molecular oxygen and the formation of a cyclic peroxide intermediate was demonstrated in these studies. This breakthrough in Prostaglandin research depended critically on the introduction of novel preparative and analytical methods such as thin-layer and gas chromatography and nuclear magnetic resonance and mass spectrometry. It left the way clear for the start of a steep increase in the scientific career of the prostaglandins and other eicosanoids, i.e. the number of corresponding publications began to increase exponentially El]. Nevertheless, it appears as if eicosanoid research has still not

4

I Arachidonic acid and companions: an abundunt tsource ojbiological signals

become fully integrated into the mainstream of today’s biomedical science, probably because it requires sophisticated analytical equipment which only a very limited number of laboratories possess and is not easily accessible for current methods of molecular biology. An important step forward in the development of eicosanoid research was the discovery of the prostaglandin endoperoxides PGG2 and PGH2 [ 18,191. In the beginning, these intermediates of enzymatic prostaglandin synthesis were thought to be identical with so-called rabbit aorta-contracting substance (RCS) [20] and labile (platelet) aggregation-stimulating substance (LASS) [21]. Both were found in pharmacological experiments as short-lived activities which were released, for instance, from allergenchallenged guinea-pig lung. Later on, thromboxane A2 was identified as the major component of RCS and LASS [22]. At about the same time, the Vane and his collegues. [23] postulated the existence of an endogenous thromboxane A2 antagonist, ‘PGX’, which was then characterized as PGI2 or prostacyclin. Vane[24] and another group [25] made the most important and remarkable discovery that cyclooxygenase, i.e. the enzyme controlling the initial step of prostaglandin, thromboxane and prostacyclin biosynthesis, is specifically inhibitied by non-steroidal antiinflammatory drugs such as aspirin. In 1974, while studying thromboxane and prostacyclin formation in platelets, Hamberg and Samuelsson [26] as well as Nugteren [27] observed the appearance of non-cyclic hydroxylated arachidonic acid derivatives. This finding opened a new avenue of eicosanoid research which led to the discovery of the lipoxygenase enzyme family [28] and the leukotrienes as powerful pro-inflammatory and pro-anaphylactic mediators. However, the fact that the first lipoxygenase (then called ‘lipoxidase’)had already been discovered in plants in the early 1930s should not be overlooked [29]. Eventually, lipoxygenase-catalyzed fatty acid metabolism in both animal and plant cells will move to the center of interest since it is the source of a large array of biologically hghly active substances with putatively important functions in the healthy organism and in numerous diseases. This may also hold true for eicosanoids derived from cytochrome P-450-catalyzed monooxygenation of arachidonic acid. The latter reaction, together with the cylooxygenase and lipoxygenase pathways, have been thought to represent the three major routes of eicosanoid biosynthesis. Recently, however, additional pathways, such as the formation of arachidonyl-Aethanolamide (anandamide, see Section 1.3.9), a putative neuromodulator or neurotransmitter, and the non-enzymatic biosynthesis of isoprostanes (see Section 1.3.8) have been discovered (Table 1-1). This may not be the end of the story, however. Thus, the possibility of a.large group of compounds synthesized by an interaction of the major pathways is just beginning to emerge. Moreover, there is an increasing body of evidence that, besides arachidonic acid, other unsaturated fatty acids sequestered in membrane phospholipids may give rise to a wide variety of signaling compounds which rival the eicosanoids in functional importance. Presently, the linoleic acid-derived octadecanoids are gaining increasing attention, whereas the corresponding linolenic acid derivatives are still almost entirely neglected as far as animal cells are concerned, although for plants their fundamental role in cellular signaling is fully recognized (see Sectionl.5). For the regulation of eicosanoid formation in cells, it is of central importance that,

1.2 The discovery of prostaglandins and related eicosanoids

5

in 'resting' cells, the concentrations of the free polyunsaturated precursor fatty acids are far below the K M values of the enzymes involved in the biosynthesis of eicosanoids. Instead, these fatty acids are sequestered in membrane phospholipids and are released from these stores only upon demand, i.e. through endogenous or environmental signaling (Fig. 1-3). Since eicosanoid precursor fatty acids are predominantly esterified in the sn-2 position of glycerol phospholipids, signal-activated phospholipases of the A2 type play a critical role in controlling the rate-limiting step of eicosanoid biosynthesis, although alternative pathways of fatty acid release have been identified (see Chapter 2). Table 1-1. Eicosanoid families Name Prostaglandins

Biosynthetic pathway Cyclooxygenaselprostaglandin synthases

Thromboxanes

Cyclooxygenase/Thromboxane s ynthases

Prostacyclins HPETEs and HETEs~

Leukotrienes Lipoxins 15-Epi-lipoxins Hepoxilins Trioxilins Epoxyeicosatrienoic acids Isoprostanes

CyclooxygenaseProstacyclin synthases Lipoxygenases and cytochrome P-450-dependent monooxygenases Lipoxygenase/Leukotriene synthases Lipoxygenase Acetylated cyclooxygenase-2 Lipox ygenase Lipoxygenase Cytochrome P-450-dependent monooxygenases Non-enzymatic lipid peroxidation Unclear

Major function Control of smooth muscle activity, secretory processes, immunosuppression, luteinization, etc. Platelet aggregation and aorta constriction Thromboxane antagonists Control of blood pressure, renal function, synaptic transmission and inflammation Bronchoconstriction and leukotaxis Anti-inflammatory (?) Anti-inflammatory (?) Intracellutar Ca2+release neuromodulation (?) Unknown (hepoxilin metabolites) Vasodilatation and control of renal function Signaling of oxidative stress (?)

Inhibitory neurotransmitter (?), endogenous cannabinoid receptor ligand "HPETEs, mono-hydroperoxy eicosatetraenoic acids; HETEs, mono-hydroxy eicosatetraenoic acids. Anandamide

Once released, the precursor fatty acid immediately becomes transformed into eicosanoids (the type of which depends on the pattern of biosynthetic enzymes that are expressed by a given cell) or is re-esterified. Eicosanoids are short-lived, i.e. they either hydrolyze spontaneously (such as thromboxane A2 and prostacyclin) or undergo rapid metabolic inactivation by enzymatic dehydrogenation, o-hydroxylation and fatty acid &oxidation (Fig. 1-2). As a consequence eicosanoids are not stored in cells. An exception to this rule is provided by certain invertebrates which are able to store large amounts of prostaglandins as esters or lactones (see Section 1.4).

6

k

I Aruchidonic mid and compunions: an abundunt source of biological signals

O

HO

O

H

OH

NAD+

1

15-hydroxy prostaglandin debydrogenase (PDGH)

O H HO

0

i

15-ketoprostaglandin-AI3-reduetase

0

fatty acid P-oxidation (2x)

.t C O O H

HO

0

o-hydroxylation and oxidation

- \GOOH 0

Figure 1-2. A major pathway of prostaglandin metabolism. E- and F-prostaglandins are rapidly inactivated by oxidation of the hydroxyl group at C15. This reaction is catalyzed by a ubiquitous cytosolic NAD+-dependent prostaglandin dehydrogenase (PDGH), the key enzyme of prostaglandin inactivation. The subsequent steps of prostaglandin degradation involve reduction of the A13 double bond (by a cytosolic 15-ketoprostaglandin A'3-reductase plus NADH), w-hydroxylation and oxidation and shortening of the side chains by fatty acid B-oxidation.

1.3 Mammalian eicosanoids As a concise and comprehensible introduction into the field Piomelli 1301 is highly

recommended.

1.3 Manirnaliari eicosanoids

7

1.3.1 Free arachidonic acid: a signaling compound? Eicosanoids are thought to play a modulatory role, i.e. they either promote or attenuate the reactions of cells to systemic signals, as well as collecting various cell types in an overall tissue response. Prominent examples of such integrated responses are defense reactions becoming apparent as tissue inflammation or anaphylatic reactions, wound repair and functional blood supply of organs (functional hyperemia) caused by vasodilatation. Cytoplasmic phospholipase A*, a major enzyme of eicosanoid biosynthesis (see Section 2.3.2.2.), is activated by MAPkinase (mitogen-activated protein kinase; see Lin et al. [311) through phosphorylation. In the network of cellular signal processing this lunase is located at a central point where several signaling cascades converge [32]. This clearly shows that arachidonic acid release is controlled by a wide variety of extracellular signals and activated concomitantly with many other cellular responses, let alone the fact that additional mechanisms of arachidonic acid release and probably also of phospholipase A2 activation exist (see Chapter 2 and Smith [33]). Once released from membranes free arachidonic acid is both metabolized to eicosanoids and reincorporated into phospholipids. For the latter reaction the formation of arachidonyl-CoA through an ATP- and Mg”-dependent synthase is required [34]. The CoA derivative rapidly delivers the arachidonyl residue to the sn-2 position of lysophospholipids yielding the corresponding arachidonic acid ester. This reaction is catalyzed by a specific arachidonyl-CoA-lysophospholipidtransferase. Other metabolic reactions of arachidonyl CoA, which are probably of minor importance, include the formation of diacylglycerol esters and cholesterol esters. A major question concerns the transport of arachidonic acid within cells since the enzymes catalyzing eicosanoid formation are not necessarily found at the sites of arachidonic acid release and, moreover, free arachidonate has a high tendency to stay in a membranous (i.e. lipophilic) environment rather than to diffuse into the cytosol. There is now evidence that so-called fatty acid-binding proteins (FABPs) may control the intracellular distribution of arachidonic acid [35]. Due to their structural features these ubiquitous 14-20 kDa proteins, which are tissue-specifically expressed in various isoforms, successfully compete with lipid bilayers for free fatty acids [36]. The signal-dependent release of arachidonic acid and its rapid removal from the cytosol has led to the proposal that, besides the eicosanoids, arachidonic acid itself may serve as an intracellular signaling molecule [37]. Considerable effort has been made to test this hypothesis. However, due mainly to the technical problems inherent in lipid analysis in cells, the issue has not yet been settled. Instead, much more information on the actual concentrations of free arachidonic acid in different compartments of signal-activated cells, on the role of FABPs in arachidonate signaling and on the specific interaction with putative target proteins is required. Progress in this field is hampered in particular by the lack of a simple arachidonate assay (such as an immunoassay or a chemoluminescense reaction) and the tendency of arachidonic acid (and other lipophilic compouds) to interact non-specifically with a vast number of proteins. Nevertheless, several proteins have been shown to become functionally modulated upon interaction with free arachidonic acid [30]. The most prominent example is protein kinase C [PKC], a family of at least 1 1 Ser/Thr kinases [38] which occupy a central position in cellular signal processing 1391. A characteristic property of these

8

I Arachidonic acid and companions: un ubundunt source of biologicul signals

enzymes is that their activity is strictly controlled by lipid mediators such as diacylglycerol, phospholipids, phosphatidic acid, cholesterol sulfate, sphingolipids and unsaturated fatty acids, with diacylglycerol being a bona fide cellular protein kinase C activator, whereas the physiological role of the other activators is still a matter of dispute. It has been argued that the arachidonate concentrations required for PKC activation in vitro appear to be unphysiologically high. However, the fact that PKC activation in general takes place upon contact with cellular membranes and that in this environment free arachidonic acid may temporarily approximate levels of more than 0.1 mM due to its lipophilicity should not be overlooked [30]. These concentrations are sufficient for PKC activation in vitro as well as for functional modulation of other putative target proteins of free arachidonic acid. On the other hand, based on in vitro experiments it has been proposed that unsaturated fatty acids may preferentially interact with cytosolic rather than with membrane-bound PKC [38]. Such a mechanism, which would considerably increase the intracellular range of PKC effects, requires a release of free arachidonic acid from membranes into the cytosol as perhaps promoted by FABPs. Another signaling protein, the function of which is possibly modulated by a direct interaction with free arachidonic acid, is the GTPase-activating protein (GAP) of the small G protein Ras. Activation of GAP terminates the function of Ras. As shown in vitro, arachidonic acid specifically inhibits the RasGAP activity [40,41] which would result in the activation of the signaling cascades controlled by Ras [3 11. The inactivation of RasGAP by arachidonic acid might be mediated by PKC since RasGAP is an in vitro substrate of PKC [42]. On the other hand, RasGAP has been found to directly and specifically associate with arachidonic acid-containing lipid micelles [43]. As a third example of a direct regulatory effect of arachidonic acid the modulation of ion channels may be mentioned [44]. In patch-clamp experiments with amphibian smooth muscle [45] and other tissues 1301 activation of potassium channel conductance by free arachidonate rather than by eicosanoids was found. However, this response could be evoked by a wide variety of negatively charged lipids ruling out a specific second messenger effect of arachidonic acid. Whether this argument also holds true for the activation of cation channels regulated by the N-Methyl-D-aspartictype glutamate receptor in rat brain neurons by arachidonate [46] remains an open question. Since arachidonic acid is released upon stimulation of NMDA receptors a positive feedback loop would develop, which has been speculated to participate in long-lasting synaptic potentiation (long-term potentiation, LTP) which is thought to play a role in memory storage [30,47].

1.3.2 Prostanoids For Gerrnan-speaking readers the earlier literature has been thoroughly reviewed by Schror [48]. Enzymatically generated eicosanoids with ring structures are called prostanoids. The prostanoid family includes prostaglandins and thromboxanes. The common biosynthetic precursor of each prostanoid series is an endoperoxide, prostaglandin H (PGH). PGH is produced from the corresponding fatty acid in two steps. The first step, a twofold dioxygenation, yields a labile 15-hydroperoxy-

1.3 Mummuliun eicosunoids

9

Prostanoid nomenclature Formally prostanoids are derivatives of prostanoic acid, which, however, has not been found in living cells. Instead, all biologically active prostanoids are enzymatically produced from &-polyunsaturated C2" fatty acids. Prostanoids are characterized by a A13 trans double bond, a hydroxyl group at C15 and a trans configuration of the two side chains of the cyclopentane ring. Dependin on the precursor fatty acid, additional cis double bonds are found, i.e. A5 and AF7. The total number of double bonds is given as an index number. I

Rostanoic acid

kmH e

&WH

OH

OH

OH

-H

PGE2

PGEi

(From dihomo-r -linolenic acid)

OH

W

€

l

OH

s E 3

prom arachidonicacid)

(From 5.8.1 1.I 4.1 7-erwsapentaenoic acid)

As far as the prostaglandin (PG) are concerned, the oxygen substitution of the five-membered ring determines the subfamily, which is marked by latin letters. (This nomenclature is based on the original preparative work in Bergstrom's laboratory, where E-prostaglandins were extracted with ether, F-prostaglandins with phosphate buffer (Swedish fosfat) and A- and B-prostaglandins obtained by treatment with acid or base.) Independently of this scheme, the thromboxanes (TX)are classified in an A and a B subfamily.

-

TXA

IXB

endoperoxide called prostaglandin G (PGG). In the second step, a peroxidase reaction, the 15-hydroperoxy group is reduced yielding PGH (Fig. 1 -3), whereby the cel-

I0

I Arachidonic acid und companions: an ahundunt source of biological signuls

lular source of the reduction equivalents is not known. Both reactions are catalyzed by a single enzyme, PGH synthase, which exhibits cyclooxygenase and PGG peroxidase activity (see Chapter 3 ) . Frequently, the terms prostaglandin H synthase (PGHS) and cyclooxygenase (COX)are used as synonyms. Phospholipid

>I

1 PHOSPHOLLPASE

J

arachidonicacid

I'

CYCLOOXYGENAS

prostaglandin Gz

PEROXLDASE

prostaglandin Hz

I

+'T, OH

7( Prostaglandins

Prostncyclin

Thromboxanes

Figure 1-3. Biosynthesis of prostanoids from sn-2-arachidonyl phospholipids. The rate-limiting step is the release of free arachidonic acid catalyzed by signal-activated phospholipases.

PGH is transformed into D-, E-, F- and I-type prostaglandins (the latter are also called prostacyclins) as well as into A-type thromboxanes by various specific synthases exhibiting either isomerase andor reductase activity (see Chapter 4). Prostaglandins A, B and J, which carry an a,B-unsaturated ketone structure, are probably formed from D- and E-prostaglandins by (non-enzymatic?) dehydration (see also Section 1.6.3). In some invertebrates A-prostaglandins are produced along a cyclooxygenase-independentpathway (see Section 1.4). C-type prostaglandins have not yet been found in living cells.

1.3 Mammaliun eicosanoids

11

1.3.3 HPETEs, HETEs and leukotrienes A review of earlier work on HETEs is provided by Spector et al. 1491. The most recent and most comprehensive review on leukotrienes is found in Denzlinger [50]. Mono-hydroperoxy eicosatetraenoic acids (HPETEs) are the primary products of lipoxygenase-catalyzed oxygenation of arachidonic acid. In the cell they are rapidly reduced to the corresponding monohydroxy compounds (HETEs). In mammalian cells 5-, 8-, 12- and 15-H(P)ETE have been conclusively identified (Fig. 1-4). Their formation is catalzyed by lipoxygenases, i.e. 5-, 8-, 12- and 15-lipoxygenases which have been purified and cloned (see Chapter 5 and Funk [51], Jisaka et al. [52], Krieg et al. [53], Yamamoto [54], Ford-Hutchinson et al. [55] and Yamamoto et al. [56]). These enzymes are both regio- and stereo-specific yielding mostly S-enantiomers, but sometimes also R-enantiomers. The stereoselectivity distinguishes lipoxygenasecatalzyed arachidonic acid metabolism from H(P)ETE formation through unspecific lipid peroxidation which generally results in racemic mixtures. Another important pathway of HETE fromation is controlled by cytochrome P-450-dependent monooxygenases (see Section 1.3.7 and Chapter 6). In contrast to lipoxygenase-catalyzed HETE synthesis it does not involve hydroperoxidation as an intermediate step.

Figure 1-4. Sites of oxygen insertion into arachidonic acid as catalyzed by the major mammalian lipoxygenases and structures of the hydroperoxy eicosatetraenoic acids primarily formed.

As compared with the cyclooxygenase pathway the lipoxygenase pathway of eicosanoid formation is a source of a much wider variety of products with manifold biological actions. Moreover, it seems to be the more ancient pathway. In fact, lipoxygenases have been found in invertebrates which apparently do not contain cyclooxygenases (see Section 1.4). The widespread occurrence of lipoxygenases indicates that the corresponding pathways of eicosanoid formation fulfill physiologically important functions. However, as compared with the prostanoids much less is known about the cellular roles and mechanisms of action of lipoxygenase-derived eicosanoids, except the leukotrienes.

12

I Aruchidonic acid and companions: an abundant source of biological signals

Mechanistic aspects of the lipoxygenase reaction Lipoxygenases are non-heme iron proteins which recognize the &,cis- 1,4pentadiene structure (A) of PUFAs. In the first step of the reaction a hydrogen atom is removed from the methylene group between the two double bonds (bisallylic methylene group) yielding a resonance-stabilized pentadienyl radical (B). The latter is attacked by molecular oxygen from the site opposite to hydrogen elimination (so-called antarafacial attack) and the cis,&- 1.4-pentadienestructure changes into an energetically more favorable conjugated cis,truns-1.3-pentadiene structure (C). The C-atom carrying the hydroperoxy group represents a chirality center, occumng either in an S - or an R-configuration. The whole process involves several one-electron transitions which are catalyzed by the iron ion in the catalytic center of the enzyme. The cyclooxygenase reaction is subject to the same rules and can be formally understood as consisting of two successive lipoxygenase reactions l-471. 4

FeH

Fe"

/=+!fV&..f+ 0-1 HH

H

H

H

Fez++H+ A

-B-

H Fez++ H'

C

D

Lipoxygenase-catalyzed formation of an epoxide from a hydroperoxide as required for leukotriene A4 biosynthesis is due to stereoselective dehydration energetically driven by an additional cis-trans isomerization and conjugation of a double bond.

In plant cells another pathway of epoxide formation from a-linolenic acidderived hydroperoxides is found, which is catalyzed by allene oxide synthase, a cytochrome P-450 protein. It does not involve a shift and an isomerization of double bonds (see Section 1.5). Hydroperoxides can also be isomerized to epoxy alcohols by hydroperoxide isomerases found in plants (see Section 1.5). The latter pathway resembles hepoxilin biosynthesis in animal cells (see Section 1.3.6).

Although circumstantial evidence exists for receptor-mediated cellular effects no cellular receptors for HPETEs or HETEs or the corresponding linoleic acid-derived octadecanoids have been cloned and characterized as yet. Nevertheless, some highly interesting biological effects of such substances have

1.3 Mammalian eicosanoids

13

been found in particular in invertebrates (see Section 1.4). It has to be emphazied, however, that many of these effects may result from secondary lipoxygenase products such as hepoxilins (see Section 1.3.6), lipoxins (see Section 1.3.4) and others (see Sections 1.4 and 1.5) rather than from HPETEs or HETEs. The pharmacological effects of I2-H(P)ETE have been recently reviewed in detail [49]. They include a very potent effect on the migration of aortic smooth muscle cells as well as chemotaxis of eosinophils and neutrophils indicating perhaps an involvement in inflammatory processes. Evidence for a pathophysiological function of 8-, 12- and 15-lipoxygenase-derived H(P)ETEs will be provided in Chapters 5 , 9 and 11.

Octadecanoids Most lipoxygenases and also the cyclooxygenases accept not only CZo- but also Cls-unsaturated fatty acids such as linoleic and linolenic acid as substrates. In mammalian cells peroxidation of linoleic acid yields the so-called octadecanoids, i.e. 9- and 13-hydroperoxy- and hydroxy-octadecadienoic acids (HPODEs and HODEs) resembling the HPETEs and HETEs.

OOH Linoleic acid

9-HPODE

dOH 13-HPODE

9- and 13-HODE formation is catalyzed by both lipoxygenases and cyclooxygenaes. These and other HODEs (but not the corresponding hydroperoxide precursors) are also formed by cytochrome P-450-dependent monooxygenases (see Chapter 6). The formation and effects of octadecanoids have been investigated, in particular for skin epidermis which is especially rich in linoleic acid [57-60]. There is, on the other hand, an increasing body of evidence that the linoleic acid derivatives also fulfill similar-sometimes opposite-mediator functions as the corresponding eicosanoids in other mammalian cell types. The putative (patho)physiological roles played by octadecanoids are presently under investigation (see, for instance, Tang and Honn [61] and Sections 11.5.5 and 11.6). In contrast, practically nothing is known about the formation and function of the linolenic acid-derived hydroperoxy - and hydroxy-octadecatrienoic acids in mammalian cells, while in plants and invertebrates a wide variety of lipoxygenation products of both linoleic and linolenic acid is found (see Sections 1.4 and 1.5). An important subgroup of lipoxygenase-derived eicosanoids are the leukotrienes, i.e. ‘leukocyte-derived trienes’ [50]. Leukotrienes are derivatives of 5(S)-HPETE, which is either reduced to 5-HETE or, upon 5-lipoxygenase-catalyzed elimination of

14

I Arachidonic acid and companions: an abundant source of biological signals

water, yields a 5,g-epoxy compound with 3 conjugated double bonds called leukotriene A4 or LTA4 (Fig. 1-5).Enzyme-catalyzed opening of the epoxy ring by either water or glutathione leads to 5,12-diHETE also known as LTB4 or to a glutathione conjugate termed LTC4, respectively. By successive removal of the glutamate (catalyzed by a yglutamyl transferase) and the glycine residue (catalyzed by LTD4dipeptidase) LTG is transformed into the leukotrienes LTD4 and LTE4. Analogous pathways yielding LTj and LTs species exist for dihomo-y-linolenic acid and eicosapentaenoic acid. The enzyme catalyzing the hydration of LTA4 to LTB4 is an LTA4-specific epoxyhydrolase which has been found in the cytosol of a wide variety of cell types [62). The 69 kDa zinc metallo-protein belongs to the aminopeptidase family.

I'

5-LIPOXYGENASE

Figure 1-5. Biosynthesis of leukotrienes from S(S)-HPETE.

LTG formation from LTA4 is catalyzed by LTC4 synthase, i.e. a LTA4-specific Mg*+-dependentglutathione-S-transferdse found in the endoplasmatic reticulum of

I .3 Mammalian eicosanoids

15

hemopoetic, endothelial and vascular smooth muscle cells [63]. LTC4 synthase exhibits sequence homology to the 5-lipoxygenase-activating protein (FLAP, see Section 5.6.2), a membrane protein that has been thought to be required for the proper ‘docking’ of 5-lipoxygenase at sites of arachidonic acid release [54,64], rather than to other glutathione-S-transferases. Both LTA4 hydrolase and LTC4 synthase are also found in cell types such as platelets, vascular smooth muscle and small intestinal epithelial cells which do not express 5-lipoxygenase. It is assumed that these tissues are supplied with the substrate LTA4 by other cell types, which do contain 5-lipoxygenase. This provides an interesting example of transcellular eicosanoid formation [65]. As far as (patho)physiological functions are concerned the leukotrienes are presently the best characterized of the lipoxygenase-derived eicosanoids. Three types of specific leukotriene receptors have been found in the plasma membrane of a wide variety of cell types (see Section 1.6.2). LTB4ranks among the most powerful leukotactic agents and, therefore, plays a pivotal role in inflammatory processes [66]. The major biological response evoked by the cysteinyl leukotrienes (also referred to as sulfidopeptide leukotrienes, sp-LTs), i.e. LTC4, D4 and E4, is smooth muscle contraction. In fact, this effect was discovered in 1940, when Kellaway and Trethewie [67] incubated guinea-pig ileum with perfusates from allergen-challenged guinea-pig lung. The active principle of the perfusates was called slow-reacting substance of anaphylaxis (SRS-A) since its constrictory effect was slower when compared with that of histamine, the classical mediator of anaphylactic reactions [6,68]. However, it took almost 40 years until SRS-A was identified as a mixture of LTC4, D4 and E4.[69]. These cysteinyl leukotrienes are extremely potent inducers of bronchoconstriction and are thought to provide major endogenous mediators of asthma (see Section 9.4 and Hay et al. [70]). The 5-lipoxygenase pathway of arachidonic acid metabolism is, therefore, of pivotal interest in drug development (see Chapter 12 and Brooks and Summers [71]).

1.3.4 Lipoxins Lipoxins (lipoxygenase interaction products) were discovered upon incubation of human leukocytes with the Ca2+ionophore A23187 [74] but they are now known to be produced as endogenous mediators as well. For reviews see Serhan [72,73]. Lipoxins are conjugated tetraenes which are formed from arachidonic acid by the interaction of two lipoxygenases. Each of the major lipoxygenase species in mammalian cells, i.e. 5-, 12- and 15-lipoxygenase, may participate in lipoxin synthesis (see Fig. 1-6). As an intermediate of lipoxin synthesis an unstable 15-hydroperoxy-5(6)epoxy-tetraene is found which undergoes hydration and reduction to several isomeric trihydroxy eicosatetraenoic acids including the two major lipoxins lipoxin A4 (LXA4) and lipoxin B4 (LXB4).While 5(6)-epoxydation of arachidonic acid yielding LTA4 is catalyzed by 5lipoxygenase, the 15-hydroperoxy group is introduced by either 15-lipoxygenase or platelet 12-lipoxygenase which, under these conditions, exhibits 15-lipoxygenase activity (see Section 5.8). In another pathway of lipoxin synthesis 15-HPETE may serve as a substrate for 5-lipoxygenase-catalyzed 5(6)-epoxydation [721. The lipoxygenases involved in lipoxin formation may be unequally distributed

16

1 Arachidonic acid and compunions: an ubundunt source of biological signals

between different tissues. This implies that lipoxins-similar to leukotrienes-are produced along transcellular routes, i.e. through interaction between cell types with different lipoxygenase expression patterns. For instance, LTA4 produced by Slipoxygenase in granulocytes may be delivered to platelets where it is transformed into lipoxins by platelet 12-lipoxygenase. That would mean that a major function of platelet 12-lipoxygenaseis to act as a lipoxin synthase [73,75].

OOH

5(S)-HPETE

15(S)-HPETE

LTA4

5,6-epoxy-15(S)-bydroperoxy-7,9,13-trans1 I-cis-eicosatetraenoic acid

OOH

I L X h HYDROLASE I

PLATELET 12-LOX

-

Hd

bH

Lipoxin B4

ecoo CYS

f-

I GLUTATHIONE-S- I ITRANSFERASE

I

OH

Lipoxin C4

Figure 1-6. Biosynthesis of lipoxins from S ( S ) - or I S(S)-HPETE. along a pathway resembling cystenilleokotriene metabolism L X G is transformed into LXD4 and LXEj by successive removal of the glutarnyl and glycyl residues catalyzed by a y-glutamyltransferase and a dipeptidase, respectively (not shown).

1.3 Mammalian eicosanoids

17

In pharmacological experiments lipoxins have revealed both vasoconstrictory and vasodilatory effects, depending on the organ and species. Moreover, LXA4 exhibits some leukotactic activity which in its details clearly differs from that of LTB4. Such responses would classify lipoxins as pro-inflammatory mediators. An increasing body of evidence indicates, on the other hand, that the major functions of lipoxins are antiinflammatory, i.e. that they act as endogenous antagonists of pro-inflammatory eicosanoids such as prostanoids and leukotrienes [73,76,77; see also Section 7.31. In fact, numerous pro-inflammatory responses are inhibited by lipoxins. In addition, lipoxins appear to mediate the actions of anti-inflammatory cytokines such as interleukin -4 and -13, which, vice versa, induce the 15-lipoxygenaseactivity required for lipoxin biosynthesis. The idea of leukotrienes and lipoxins antagonizing each other is supported by the finding that, under conditions of lipoxins biosynthesis, leukotriene formation is blocked. It has been postulated, therefore, that lipoxin may act as ‘chalones’, i.e. specific local inhibitors, in that they control the self-limitation of inflammatory processes [72]. To guarantee quick counter-control of inflammation, lipoxin precursors such as 15-HETE may be stored in membrane phospholipids and released upon stimulation of phospholipase A2 by pro-inflammatory signals together with arachidonic acid. Such a mechanism is implied by the unique property of 15lipoxygenase of accepting not only free but also esterified arachidonic acid as a substrate (see Section 5.8). In view of the proposed biological role of lipoxins and their potentially very important clinical implications considerable efforts are being made to elucidate the molecular mechanisms of these eicosanoids. Recently a specific LX& receptor has been cloned [73]. The amino acid sequence classifies this protein as a G protein-activating receptor exhibiting seven transmembrane domains (see Section 1.6.2). The LX& receptor has been proven to be identical to pINF 114, one of the so-called ‘orphan’ receptors the endogenous ligands of which have not been identified yet. The intracellular signal transduction pathway controlled by the LXA4 receptor is still not known.

1.3.5 15-Epi-lipoxins Besides cyclooxygenase activity, PGH synthase-2 (COX-2), as induced by cytokines or endotoxin in various epithelial and endothelial cell types (see Section 3.4), also exhibits a latent 15(R)-lipoxygenaseactivity. As shown in 1995 by Claria and Serhan I781 the latter becomes apparent when the cyclooxygenase activity is inhibited by aspirin-dependent acetylation of the enzyme. The 15(R)-HETE thus formed from arachidonic acid can be released from its cells of origin and, upon uptake by leukocytes, be transformed into two 15-epi-lipoxins & and B4 via 5-lipoxygenasecatalyzed formation of an epoxy intermediate (Fig. 1-7). In the absence of aspirin 15(R)-HETEand 15-epi-lipoxin can also be produced by cytochrome P-450-catalyzed oxygenation of arachidonic acid. In 15-lipoxins the hydroxyl group at C15 is R configurated instead of S configurated as in conventional lipoxins [73]. 15-epi-LXA4has been found to be more potent than LXAl in inhibiting granulocyte adhesion and 15-epi-LXB4 is an inhibitor of cell proliferation 1791. It has been

18

I Arachidonic acid and companions: an abutidlint .source of hiolo~yicvilsigiia1.s

suggested that, upon aspirin treatment, the pro-intlammatory pathway of prostanoid synthesis becomes switched to an anti-inflammatory pathway of 15-epi-lipoxin synthesis 1731. This idea appears particularly attractive since in inflamed and damaged tissue PGH synthase-2 becomes strongly upregulated. On the other hand, aspirintriggered 15-epi-lipoxins (ATLs) have still to be found in vivo. If so, one arrives at the interesting question of whether or not an endogenous pathway for the modulation of PGH synthase-2 activity exists which is somehow mimicked by aspirin treatment.

arachidonic acid

I

OH

1-

lS(R)-HETE

OH

J COOH

%--

&coo,

OH

15-epi-LW

HO

6H

15-epi-LXBd

Figure 1-7. Biosynthesis of 15-epi-lipoxins by aspirin-inhibited cyclooxygenase-2 (COX-2) and Slipoxygenase. In contrast to the lipoxin (Fig. 1-6) the 15-hydroxyl group is in the R configuration.

1.3.6 Hepoxilins For reviews see Pace-Asciak [80] and Pace-Asciak et al. [8 I]. Hepoxilins are mono-hydroxy epoxy eicosatrienoic acids which are generated along the IZlipoxygenase pathway. The name hepoxilin refers both to the structure of these compounds and to their property to increase glucose-induced insulin secretion in pancreatic islets (hydroxy-epoxide, insulin). Hepoxilins and their hydrolysis products, i.e. trihydroxy eicosatrienoic acids, were first isolated from human platelets in 1978-1979 [82,83J.In the early 1980s PaceAsciak et al. [84] started a still ongoing systematic investigation of the hepoxilin pathway. Two hepoxilins called HXA3 and HXB3 were shown to be generated

1.3 Mammalian eicosanoids

19

through an intramolecular rearrangement from 12-HPETE, the primary product of the 12-lipoxygenase-catalyzed oxygenation of arachidonic acid (Fig. 1-8). This rearrangment can be non-enzymatically catalyzed by ferriheme (using both 12S- and 12(R)HPETE as substrates). On the other hand, the heat sensitivity and stereo selectivity of cellular hepoxilin synthesis indicates the existence of a specific hydroperoxide isomerase or hepoxilin synthase (using exclusively 12(S)-HETE as a substrate), which is, however, still ill-defined [85].

H

O

O

-

C

c

o

o

H

12(S)-HPETE

ccooH H

O

-

G

H

0

Hepoxilin &

Hepoxilin A3

II 1

I

t

t

HEPOXILIN EPOWDE HYDROLASE]

I

Hocco H

HO

-

Trioxilin A3

O

HO

-

G

H

OH

Trioxitin Bj

Figure 1-8. Biosynthesis of hepoxilins and trioxilins from 12(S)-HPETE.

Hepoxilins are short-lived compounds which are rapidly metabolized predominantly by epoxide ring opening. In analogy to leukotriene biosynthesis this may occur either hydrolytically, being catalyzed by an epoxide hydrolase and yielding the socalled trioxilins, or by glutathione as catalyzed by glutathione-S-transferase yielding C-type hepoxilins such as hepoxilin A3-C. Hepoxilins and trioxilins have been found in a wide variety of organs, but also in lower organisms such as Aplysiu and certain algae species (see Sections 1.4and 1 S ) . Moreover, preparations from the rat pineal gland and hippocampus have been found to transform the arachidonic acid analog 4,7,10,13,16,19-docosahexaenoicacid, a constituent of these tissues, into the corresponding As-type hepoxilins and trioxilins [85].

20

1 Arachidonic acid and companions: an abundant source of hiologicol signals

Hepoxilins exhibit a series of prominent pharmacologic effects and, therefore. are presumed to represent a new class of lipid-derived signaling molecules [80,8I]. Hepoxilin-binding proteins have been found in particular in human granulocytes. Whether they act as signal-transducing receptors remains an open question, however. Since these binding proteins are located intracellularly and since hepoxilins evoke their biological effects on whole cells only when administered in a membranepenetrable form (i.e. as methylesters) they are proposed to represent intra- rather then intercellular mediators [SO]. It is remarkable that some responses are evoked by both S(S)- and 8(R)-hepoxilins, whereas others show sterospecificity for the 8R epimers only. This may indicate different mechanims of action already at the 'receptor' level. Whether or not hepoxilins activate a G-protein-regulated signaling cascade, as indirectly indicated by the pertussis toxin sensitivity of several hepoxilin effects, remains to be shown. A major pharmacologic effect of hepoxilins is the opening of intracellular Ca" stores resulting in a rise of the cytoplasmic Ca2' level [SO]. This Ca*'-mobili~ing effect may explain other cellular responses to hepoxilin such as the stimulation of glucose-induced insulin secretion in pancreatic islets, of vascular contraction and of vascular permeability. In addition, effects of hepoxilin on nerve cells have been described. These include presynaptic inhibition of Aplysia neurons, where hepoxilin A 3 (HXA3) was found to mimic the effect of 12(S)-HPETE, and hyperpolarization of hippocampal neurons in rats (see Section 1.5). Both may result from an HXA3induced opening of potassium channels. Further development in this exciting field of eicosanoid research waits for the cloning and characterization of key proteins such as hepoxilin synthase and cellular hepoxilin receptors and an elucidation of physiological hepoxilin effects.

1.3.7 Monooxygenase -derived eicosanoids For reviews see Schwartzman and McGiff [86] and Chapter 6. PUFAs such as arachidonic acid are endogenous substrates of cytochrome P-450dependent monooxygenases. Cytochrome P-450-dependent monooxygenases represent one of the largest enzyme families known. Up to now, approximately 500 isoforms have been identified. Many of them control the metabolism of xenobiotics, but they are also involved in the biotransformation of numerous endogenous compounds. Cytochrome P-450 isoforms are already found in prokaryotes. Thus, cytochrome P450-catalyzed fatty acid oxidation probably represents the most ancient pathway of oxylipin formation. A major pathway of arachidonic acid monooxygenation consists of the epoxidation of the cis double bonds yielding epoxy eicosatrienoic acids (EETs; Fig. 1-9). The cytochrome P-450 isoforms involved are therefore referred to as arachidonate epoxigenases. However, these enzymes also attack other PUFAs and even eicosanoids such as prostaglandins. Moreover, cytochrome P-450-dependent monooxygenases also catalyze the hydroxylation of such substrates at various positions. Direct hydroxylation of arachidonic acid yields numerous isomeric HETEs which differ from lipoxygenase-derived HETEs in that they are not derived from intermediate hydroperoxides (Fig. 1-9). Arachidonic acid expoxides are hydrolyzed

1.3 Mammalian eicosanoids

21

enzymatically to the corresponding a,B,-dihydroxy eicosatrienoic acids (DHETs). Arachidonate epoxides exhibit a wide variety of biological effects such as modulation of blood flow and tubular function in the kidney (see Section 7.4) and vasodilatation in other organs. However, specific cellular epoxide receptors have not yet been identified so that the mechanisms of action of these eicosanoids are largely unknown. Cytochrome P-450 derived arachidonic acid metabolites may be substrates of other enzymes of eicosanoid biosynthesis such as cyclooxygenases (see, for instance, Sections 6.6 and 7.4) dramatically increasing the number of different eicosanoid species, some of which exhibit pronounced pharmacological effects (see Section 6.6.3). For more information on the synthesis, metabolism and (patho)physiological functions of cytochrome P-450-derived eicosanoids the reader is referred to Chapter 6. a,$-dihydroxy eicoaatricnoicacids (DHETs)

tk

EPOXlDE HYDROLASE

1

cpoxidation (1)

3 1 3

I-HETE 10-HETE 13-HETE

c

biullylic hydrorylation (4)

,

4

.-_. .*

1 3

COOH

rn!

a%bydroxyhtion(2)

1 -

3 1 3

1 3

2

16-HETE 17-HETE lEHETE 19-HETE 20-HETE

2

hydroxylationwith double bond migration (3)

5-HETE EHETE

9-HETE 11-HETE If R-HETE 15R-HETE

Figure 1-9. Formation of eicosanoids by cytochrome P-450-dependent monooxygenation of arachidonic acid. The four pathways which have been identified are shown. They are catalyzed by different cytochrome P-450 isoforms. The hydroxylation with double bond migration (3) resembles the lipoxygenase reaction (see Section 1.3.3 and the scheme on page 12) in that the cis,cis-1,4-pentadiene structure attacked by oxygen isomerizes to an energetically more favorable conjugated cis,truns- 1,3-pentadiene structure. However, in contrast to the lipoxygenase reaction no hydroperoxy intermediate is formed. For the complex stereochemistry of arachidonic acid monoxygenation see Chapter 6.

1.3.8 Isoprostanes For reviews see Morrow and Roberts [87] and Rokach et aZ. [89]. Like other unsaturated fatty acids arachidonic acid is prone to non-enzymatic

22

I Amclzidonic ucid and comimrzions: an cibundtrnt soi~rceof’hiological signu1.s

autoxidation, occurring in particular in aqueous solution at physiological pH. For a long time this process was thought to represent an unphysiological or even artifactual side reaction with deleterious consequences for the cell. Only recently has it been shown that, along autoxidative pathways, biologically highly active compounds are generated in cells, which like the conventional eicosanoids may fulfill the requirements of endogenous signaling molecules. These so-called isoprostanes were discovered in human plasma and, later on, in other body fluids and tissues by Morrow er ul. 1901 in 1990. They are regio- and stereoisomers of prostaglandins, thromboxanes and B-type leukotrienes and are formed by non-enzymatic free-radical peroxidation of arachidonic acid. It is assumed [87,88] that as an initial intermediate an arachidonylperoxyl radical is produced which then undergoes further oxygenation and endocyclization followed by reduction of the peroxide to the corresponding hydroxy compounds (Fig. 1 - 1 0). The high levels of isoprostanes in body fluids and organs indicate that non-enzymatic peroxidation represents a major branch of arachidonic acid metabolism. In animal experiments isoprostane formation can be strongly induced by administration of compounds such as CC4 which stimulate lipid peroxidation in the liver [87]. Isoprostanes are predominantly found to be esterified in phospholipids indicating that phospholipid-bound rather than free arachidonic acid is a substrate of lipid peroxidation. Free isoprostanes are thought to be released from phospholipids by a phospholipase A2-type enzyme. The isoprostane family is of bewilding diversity requiring a special nomenclature [91,92]. As far as eicosanoid analysis is concerned the easy formation of isoprostaneseven at temperatures below 0 OC-represents a serious obstacle and source of error, in particular since the isoprostanes exhibit a similar chromatographic behavior as prostanoids and may even cross-react with anti-prostanoid antibodies. A steadily increasing body of evidence indicates that isoprostanes are bona j X e endogenous mediators rather than accidental by-products of lipid peroxidation. Part of this major function may be the signaling of oxidative stress [88]. Most isoprostanes have been found to exert dramatic pharmacologic effects. Thus, 8-iso-PGE2 and 8iso-PGF2, are extremely potent inducers of vascoconstriction in the glomerulus thereby reducing the renal filtration rate (see Section 7.5). Moreover, 8-iso-PGF2, evokes bronchoconstriction in experimental animals, induces the proliferation of vascular smooth muscle cells and provokes endothelin release from endothelial cells. There is pharmacologic evidence for a cellular 8-iso-prostanoid receptor which may induce the release of the second messengers inositol-l,4,5-trisphosphateand diacylglyerol. Such a mechanism would easily explain the above-mentioned biological effects of the isoprostanes, in particular of the most abundant 8-iso-PGF2,. Whether or not this receptor is identical to the thromboxane receptor or a variant thereof, as recently suggested [93,94], remains to be shown by cloning experiments. As already mentioned, the primary event in isoprostane formation is the attack of reactive oxygen species (such as hydroxyl, hydroperoxyl, peroxyl and superoxide anion radicals) on arachidonic acid yielding an arachidonyl-peroxyl radical. Reactive oxygen species are products or by-products of various enzymatic and non-enzymatic cellular reactions and are normally scavenged by antioxidants and antioxidative enzyme reactions. Under certain conditions the cell can be overloaded with reactive oxygen species. This situation, called ‘oxidative stress’, is thought to be causally

I .3 Mammalian eicosanoids

23

cH i t

(A)

I 1 12

r&--

(B)

no CWH HO

I

COOH Ho

HO

I1

HO

111

IV

Figure 1-10. Formation of isoprostanes by free radical-catalyzed (non-enzymatic) peroxidation of arachidonic acid. (A) The arachidonic acid molecule is attacked by reactive oxygen species at the C7, CIO or C13 bisallylic CH:! groups. As shown exemplarily for the pathway leading to compound I the free radicals formed by hydrogen abstraction interact with molecular oxygen at one of the four positions marked by latin ciphers and arrows (the numbers in parentheses designate the C-atoms reacting with each other to form the five-membered ring). (B) In analogy to Iipoxygenasekyclooxygenase-catalyzedoxygen insertion (see p. 12) the cis,cis-1.4-pentadiene structure to be oxygenated changes into a conjugated cis,truns-l.3pentadiene structure and a hydroperoxyl radical is formed. (C) The latter undergoes cyclization to the endoperoxide radical, which again is attacked by molecular oxygen. (D) The resulting PGGz analogs are reduced to the four F2-isoprostaglandins I, 11, I11 and N (the roman ciphers correspond to the sites of the initial oxygen insertion in arachidonic acid as shown in A) or to other isoprostanes. Oxygen insertion can also occur at C5 and C15 leading to isoprostane formation along a different pathway (dioxetane mechanism, see Rokach et al. 1891). In isoprostanes the two side chains of the five-membered ring are in the cis position rather than trans configurated as in cyclooxygenase-derived prostanoids. Compound IV is, therefore, the 8 stereoisomer (8-epi-PGFz,) of the cyclooxygenase-derived PGF2,. For more details about the complex stereochemistry of isoprostane formation see Rokach rt al. [8Y I.

24

1 Arachidonic acid and companions: an abundant source of biological signuls

involved in a number of diseases, such as atherosclerosis, cancer, Alzheimer’s dementia, chronic intoxications, etc., as well as in aging in general [87,88]. Isoprostane formation represents a reliable measure of oxidative stress and may be indicative of free radical-induced tissue damage in vivo. Moreover, determination of isoprostanes in biological material appears to be superior to other assay methods for lipid peroxidation [88]. Therefore, the measurement of isoprostanes may become of considerable practical importance, for instance for an exact evaluation of antioxidant therapies and preventive approaches for the above-mentioned diseases. However, at present the method is still hampered by the expensive analytical technique which is based mainly on mass spectrometry. Notwithstanding these problems some individual diseases previously assumed to be causally related to oxidative stress have already been shown to correlate with isoprostane formation and, therefore, may become subject to antioxidant therapy. These include injury related to heavy smoking, renal failure in patients suffering from severe liver disease, as for instance caused by acute drug poisoning (hepatorenal syndrome), and scleroderma 187,881. In animal experiments alcohol- and drug-induced liver and kidney injury as well as organophosphate poisoning were shown to correlate with isoprostane formation [87,88]. Moreover, nutrional deficiency of antioxidants such as selenium and vitamin E was found to result in a massive increase of isoprostanes in the blood plasma and tissue phospholipids of rats. Other products of non-enzymatic PUFA oxidation in dissues have been described. They include the docosahexaenoic acid-derived “neuroprostanes” and the so-called isolevuglandins[95]. The later resemble the levuglandins, i.e. y-hetoaldehydes formed by rearrangement of prostaglandin H2 [96]. Like isoprostanes neuroprostanes and isolevuglandins are thought to represent markers of oxidant injury [95]. It is hoped therefore that the systematic investigation of autoxidative PUFA metabolism will help to put the beneficial effects of antioxidants in food, which hitherto were a matter of guessing rather than of precise knowledge, on a sound scientific basis.

1.3.9 Anandamide For reviews see Berdyshev et al. 1971, Hillard and Campbell [98] and Randall and Kendall [99]. The unexpected finding that arachidonylethanolamide or anandamide is an endogenous ligand of the cannabinoid receptor has opened a new and exciting avenue of eicosanoid research. The cannabinoid receptor was identified as a cellular binding protein for A’tetrahydro-cannabinol, the active drug of marihuana, hashish, etc., in 1988 [loo]. So far, two subtypes have been cloned, named CBI and CB2 [loll. The CB, type is predominantly found in the brain, but also in a variety of other tissues, whereas the expression of the CB2 type appears to be more orless restricted to immune cells. Both receptors belong to the very large family of 7-transmembrane domain receptors (7TMD receptors) which probably originate from bacterial rhodopsin [ 1021. The major-

1.3 Mummuliun eicosanoids

25

ity of 7-TMD receptors of eucaryotic cells have been found to induce transmembrane signaling by coupling with their cytoplasmic domain to trimeric G proteins, thus effecting the major second messenger systems of the cell. Upon ligand binding the cannabinoid receptors activate Gi,” proteins. This results in the attenuation of adenylate cyclase activity and a fall in the cellular cyclic AMP (adenosine monophosphate) level. In addition, the cell membrane becomes hyperpolarized through the activation of otassium channels which in turn hinders Ca2+entry via voltage-dependent N-type Ca channels and, thus, for instance, neurotransmitter release [ 1011. Therefore, cannabinoids appear to mimic the effect of an inhibitory neurotransmitter or neuromodulator. Most cellular drug receptors identified thus far interact with endogenous ligands, such as, for example, the nicotine receptor with acetylcholine and the opiate receptor with the enkephalin and endorphin peptides. Measuring the competition of tissue extracts with a synthetic cannabinoid for CB receptor binding, Devane et al. [103], isolated an endogenous ligand from porcine brain in 1992. The factor was identified as arachidonyl-ethanolamide (AEA) and named ‘anandamide’ (from the Sanskrit word ananda for bliss). The steady-state concentration of anandamide in brain tissue is very low (around 20 pmol/g) and differs widely between different brain regions, being highest in the hippocampus [98]. It is assumed that this low tissue level results from the fact that anandamide-like other eicosanoids-is only produced on demand and is rapidly metabolized. Interestingly, the anandamide level in the brain has been found to increase considerably in the first couple of hours after death, probably as a consequence of oxygen starvation. The physiological meaning of this response is quite mysterious as is the function of the cannabinoid system in general. Anandamide is a strong activator of the CB, receptor, but only a partial agonist for the CB;! receptor, whose activation by synthetic cannabinoids is inhibited by anandamide [98]. At higher concentrations anandamide also evokes cellular responses which apparently occur independently of CB receptor activation. An important example of such a response is the inhibition of gap junctional conductance [104], in particular in the astrocyte network [1051. Whether this response is physiologically important and due to an interaction of anandamide with a non-cannabinoid receptor is still not known. The behavioral responses of animals to anandamide treatment are similar (albeit shorter) to those observed upon administration of cannabis drugs, i.e. sedation, antinociception, etc. Recently, the effects of anandamide on the cardiovascular system have gained increasing interest. There is circumstantial evidence that anandamide may be identical to (the still structurally ill-defined) endothelium-derived hyperpolarizing factor (EDHF) which supports the relaxing effect of nitric oxide and prostacyclin on vascular smooth muscle leading to a fall in blood pressure [99]. The pathway of anandamide biosynthesis is still a matter of debate [98,99]. In analogy to an enzymatic reaction discovered for saturated and monounsaturated fatty acids anandamide has been proposed to be released from N-arachidonylphosphatidylethanolamide by phospholi ase D-catalyzed hydrolysis (Fig. 1-1 1). The latter compound is derived from a Ca -dependent transacylase reaction which involves the transfer of the arachidonyl residue from I -arachidonyl-phosphatidylcholine to phosphatidylethanolamine. This reaction is thought to be rate limiting for ananda-

R

8

26

1 Arachidonic acid and companions: un ubundunt source of biological sigricils

mide biosynthesis. Both the phospholipase D- and transacylase-catalyzed reaction have been discovered in tissue extracts, in particular from the brain. The proposed mechanism is, however, somewhat at odds with the extremely low level of Narachidonyl-phosphatidylethanolamidein the brain and the fact that, in this tissue, arachidonate is almost exclusively found in the sn-2 rather than in the sn- 1 position of phospholipids. Moreover, the acyltransferase reaction requires approximately 1 m M Ca2', i.e. a Ca2' concentration which is found only in the extracellular fluid and in the cytoplasm of dying rather than of intact cells. The direct condensation of ethanolamine and arachidonic acid has been proposed as an alternative biosynthetic pathway. Such a reaction has indeed been found to occur in homogenates from various tissues; its physiological significance is, however, questionable [98].

P

H2C-0 I

R2- C- 0-CH

I

* 9

+

H2C-O-p-O-CH2-CH2.N(CH3)3

0-

1 -arachidonyl-I-phosphatidylcholinc

phosphatidylethanolamine

I

1I

TRANSACYLASE

1

lyso-phosphatidylcholine

?!

9

b C - 0-C- R1

1

R2- C- 0-CH

I

9

H$-O-?-O-CHZ-CH~-NH

0-

N-arachidonyl-I -phosphatidylethanolamide

phosphatidic acid

l

/I

I PHOSPHOLIPASE D]

i

5xxl

HO-CH2-Cb-NH

anandamide

Figure 1-11. Proposed pathway of anandamide biosynthesis.

Anandamide is rapidly degraded by hydrolysis catalyzed by a widespread microsoma1 aminohydrolase. In addition, it is rapidly taken up by cells through a mechanism which apparently resembles the reuptake of neurotransmitters by presynaptic terminals [98].

1.4 Eicosanoids in invertebrutes

27

Anandamide is also a substrate for the enzymes of eicosanoid biosynthesis such as 12- and 15-1ipoxygenases, cytochrome P-450-dependent monooxygenases and cyclooxygenases (see, for instance, Yu et al. [1061). At least 12(S)- and 15(S)-hydroxy anandamide, the lipoxygenase-derived metabolites, exhibit a similar affinity to the CB I receptor as anandamide [98]. Among the large number of other anandamide derivatives and analogs tested for CB I receptor binding, methylated anandamide, as well as the corresponding ethanolamides of docosatetraenoic acid and eicosatrienoic acid, have been found to be at least as active as anandamide [98]. Thus, anandamide may represent the leading compound of a family of structurally related endocannabinoids.

1.4 Eicosanoids in invertebrates In nature, the soft coral Plexaura homomalla was identified as possibly the richest source of prostaglandins in 1969 [107]. This finding not only gained commercial interest but gave the starting signal for an extensive search for eicosanoids and related compounds in invertebrates. Now it appears as if eicosanoids occur throughout the animal kingdom, from protozoans and nematodes to sponges, coelenterates, crustaceans, arachinoids, insects, echinoderms and molluscs up to all classes of vertebrates (for reviews see Gerwick et al. [IOS], Genvick [109], De Petrocellis and Di Marzo [ 1 101 and Stanley-Samuelson [ 1 1 11). Even in certain yeast species prostaglandin production has been shown to be of vital importance in reproductive processes (see, for instance, Coetze et al. [ 1121). The major prostaglandin species found in P. homomalla is PGA2 which, depending on the geographic subspecies, occurs either as the 15s- or 15R-enantiomer or as a mixture of both (note, that in mammals only 15s-prostaglandins are found!). Surprisingly and in contrast to vertebrate prostaglandins, the soft coral PGA2 is synthesized along a lipoxygenase rather than a cyclooxygenase pathway providing a ‘remarkable example of convergentbiochemical evolution’ [ 1081. Although the biosynthetic route is still not fully understood, there is now agreement that the lipoxygenase-catalyzed formation of 8(R)-HPETE provides a key event (other pathways proposed involve the 8R-hydroperoxidationof 1 1(R)- and 15(R)-HETEformed by the corresponding lipoxygenases). 8(R)-HPETE is then transformed into a short-lived allene-oxide intermediate which subsequently yields PGA2 along an ill-defined pathway (see Fig. 1-12). It has been proposed that, in analogy to jasmonate biosynthesis in plants (see Section 1S ) ,the cyclization of 8(R)-HPETEis catalyzed by an allene oxide synthase and an allene oxide cyclase. For more details regarding the highly complex biochemistry of prostaglandin formation in soft corals the reader is referred to corresponding reviews [108-1101. In corals the prostaglandins are stored as 15-acetoxy-I-methylesters. One important function of these compounds is the deterrence of predatory fish, many of which respond to prostaglandin intoxication by vomiting and illness and the development of adversive behavior. An analogous deterrent function has been attributed to E- and A-prostaglandins

28

I Arachidonic acid and companions: an ubundant source of biologicul signals

which are produced in large amounts along the cyclooxygenase pathway by molluscs such as the Mediterranean nudibranch Tethysfimbria [ 108,1101. These animals store the prostaglandins as 1,15-lactones (Fig. 1-13) in dorsal appendages (cerata), which are shedded upon attack by a predator. Both the lactones and the free prostaglandins formed by hydrolyzation are highly toxic to fish. In addition, there is evidence that mollusc prostaglandins and their lactones are involved in the control of smooth muscle contraction, mucus secretion and larvae hatching. Examples of other eicosanoid lactones found in invertebrates and strikingly demonstrating the versatility of arachidonic acid metabolism are shown in Fig. 1-13.

c arachidonic acid

1’

SR-LIPOXYGENASE

8R-HPETE

I’ I ‘ b :

ALLENE OXIDE SYNTHASE

ALLENE OXIDE CYCLASE

0

z

COOH

preclavulone A

OH

prostaglandin A2

Figure 1-12. Cyclooxygenase-independent biosynthesis of PGAz in P. homotncillu.

1.4 Eicosunoids in invertebrates

29

Prostaglandins, as well as a rather exotic collection of lipoxygenase-derived fatty acid metabolites, have also been found in sponges and a role of such compounds in species-specific cell aggregation has been postulated I 1 101. Prostaglandins have been shown to play an important role in insects where they are involved in various physiological processes such as egg-laying behavior (oviposition), fluid secretion by salivary glands and Malpighian tubules and hemocytic immune responses to bacterial infections [ 1 11,112,1131. Considering the evolutionary ancientness of the lipoxygenases it is not surprising that lipoxygenase-derived fatty acid metabolites are particularly abundant in invertebrates. In a few cases evidence has been provided for a physiological function of such compounds.

prostaglandin E2-1,I 5-lactone (mollusc Tethysjmbria)

halicholactone (sponge Halichondria okaabi)

didemnilactone (ascidian Dia'emnum candidurn)

apl ydilactone (sea hare Aplysia kurodnr)

Figure 1-13. Exotic eicosanoids from marine invertebrates.

A well-known example is provided by the role of the IZlipoxygenase pathway in certain types of neurons of the marine snail Aplysia californica [30,115,116]. These nerve cells are subject to presynaptic inhibition by two neuromodulators, i.e. histamine and the tetrapeptide FMRF amide, which, through activation of potassium channels, induce hyperpolarization and, thus, inhibit neurotransmitter release. Simultaneously with presynaptic inhibition, 12-lipoxygenase activity is induced in the neurons, pointing to the possibility that the corresponding eicosanoids may act as intracellular mediators of the neuromodulatory effect. In fact, treatment of such neurons with 12HPETE induced hyperpolarization, whereas 12-HETE and 5-HETE proved to be inactive. Subsequently, it was found that 12-HPETE only served as a biosynthetic precursor for the active compounds proper, which are probably 12-ketoeicosatetraenoic acid and/or hepoxilin A3. In neurons from rat hippocampus, hepoxilin A3 evoked a similar inhibitory effect as in Aplysia neurons [ 1 171. The question as

30

I Arachidonic acid and cornpanions: an ahundmt source of hiologicrrl . s i g n d s

to whether these effects reflect a physiological process and along which mechanism it would occur (hepoxilin A? acting as an intercellular signal molecule or an intracellular messenger?) have not yet been convincingly answered. 8(R)-HETE, which is produced from arachidonic acid by a stereo-specific 8(R)lipoxygenase in starfish, turned out to be a highly active and specific inducer of starfish oocyte maturation whereas the 8s enantiomer was inactive [ 1 181. It has been proposed that, in oocyte maturation, 8(R)-HETE mediates the physiological effect of the starfish hormone I-methyl-adenine[ 1 101. Together with 11(R)-HETE, 12(S)-HETE and linoleic acid-derived octadecanoids, 8(R)-HETE also seems to control the morphogenesis of freshwater polyps (Hydru) and the metamorphosis of larvae of marine Hydructiniu species [I 10,119,120]. The 12-lipoxygenase-derived trioxilin B3 and the corresponding hepoxilins B4 and B5 (see Section 1.3.6) produced by barnacles have been found to promote hatching, i.e. the release of larvae into the seawater 11211. Considering the-admittedly rather incomplete-body of data presently available Stanley-Samuelson [ 1 111 arrived at the conclusion that, in invertebrates, eicosanoids and other oxylipins are predominantly involved in the regulation of three major physiological processes, i.e. reproduction and development, ion and water transport and cellular defense. In addition, these lipid mediators play an important ecological role as pheromones and allomones, i.e. interindividual signals of attraction or deterrence. The pheromone function of eicosanoids is not restricted to invertebrates. Thus, PGF2, metabolites, in particular 15-keto PGF2a, have been found to be released by recently ovulated goldfishes and other fish species into seawater, stimulating male sexual behavior, i.e. spawning [ 1221.

1.5 Eicosanoid-related signaling compounds in plants For a comprehensive and up-dated review the reader is referred to B16e [ 1231. While arachidonic acid and eicosanoids are only found in very limited amounts in plants and their biological role is widely obscure, CISunsaturated fatty acids, such as linoleic and a-linolenic acid, are abundant. They are the preferred substrates of various plant lipoxygenases [ 124,1251 which, based on genetic studies, have been grouped into two families, i.e. lipoxygenase-1 and lipoxygenase-2. Soybean lipoxygenase-1 was, in fact, the first lipoxygenase to be discovered [29]. As one of the best characterized enzymes it serves as a model compound for both plant and animal lipoxygenases [ 1261. Soybean lipoxygenase-I catalyzes the w6-oxygenation of linoleic and linolenic acid leading to the corresponding 13(S)-hydroperoxides. Another lipoxygenase type found in potatoes catalyzes the formation of 9(S)-hydroperoxides. In addition, less specific lipoxygenase isoforms have been found which catalyze both 9(R,S) and 13(R,S) oxygenation [ 1231. The hydroperoxy compounds are intermediates in pathways leading to a wide variety of oxygenated fatty acid derivatives. In the presence of specific hydroperoxide isomerases, for instance, such hydroperoxides may be transformed into epoxy-hydroxy-octadecadienoic or -0ctadecatrienoic acids

1.5 Eicosanoid-related signaling compounds in plants

31

which structurally resemble the hepoxilins of mammalian cells [ 1271. The isomerization of linoleic acid- and linolenic acid-derived hydroperoxides to epoxy alcohols is also catalyzed by peroxygenases, i.e. an abundant family of hemoprotein enzymes in plants that bring about monooxygenation of a wide variety of compounds and epoxidation of unsaturated fatty acids with hydroperoxides as oxygen donors. Hydroperoxide isomerization occurs by intramolecular epoxidation [ 123,1271.The linoleic acid metabolites formed along these pathways exhibit antifungal efficacy [ 1231.

Nomenclature of plant oxylipins In plants CI8PUFAs are the major substrates of lipoxygenase-catalyzed oxidative metabolism leading to eicosanoid-related oxylipins. The predominant pathways of oxylipin biosynthesis in plants are controlled by lipoxygenases. Depending on the substrate fatty acid the nomenclature of the lipoxygenase-derived oxygenation products is as follows. Linoleic acid (A 9,12) + hydroxyoctadecadienoic acids, HODS (or HODEs). a-Linolenic acid (A 9,12,15) + hydroxyoctadecatrienoic acids, HOTS. Stearidonic acid (A 6,9,12,15) + hydroxyoctadecatetraenoic acids, HOTEs. For the hydroperoxy precursors the abbreviations HPOD (or HPODE), HPOT and HPOTE are used. It is indeed intriguing that lipoxygenase-derived fatty acid metabolites of plants resemble the closely related eicosanoids of animals in playing a key role in defense reactions and wound healing [ 123,128-1301. The perhaps most prominent wound hormones of plants are the jasmonates [131,132]. The pathway of jasmonate biosynthesis in plant cells [ 1331 is supposed to start with an enzyme-catalzyed release of alinolenic acid from storage or membrane lipids. This reaction, which would resemble phospholipase Az-catalyzed arachidonic acid release in animal cells, is actually the less-defined step in jasmonate biosynthesis. Free a-linolenic acid is transformed into the 13(S)-hydroperoxide by an w6-lipoxygenase. The hydroperoxide is rapidly converted to an unstable allene oxide by cytochrome P-450-dependent allene oxide synthase (AOS), an abundant plant enzyme [134]. In the presence of an allene oxide cyclase [ 1211 the allene oxide rearranges stereospecifically to 9(S), 13(S)-12-oxophytodienoic acid (12-oxo-PDA) which is structurally reminiscent of cyclopentenone prostaglandins such as PGA, B and J. Enzymatic reduction of the double bond in the cyclopentenone ring and shortening via B-oxidation of the a side chain (as well as whydroxylation) finally results in 7-iso-jasmonic acid , curcurbic acid and tuberonic acid (Fig. 1-14). These compounds are representatives of the large jasmonate family of plant hormones. This family also includes volatile derivatives such as jasmonic acid methyl ester which are thought to play a role in long-range signaling between distant parts of the plant, but-as perfume ingredients-also between men. A major function of jasmonates is to trigger the inducible chemical defense system of plants against parasites and predators. Plant cells respond to certain constituents (‘elicitors’) of such attackers by the rapid formation of jasmonates which in turn activate a series

32

I Aruchidonic ucid and companions: an abundant source qf biological signals

of genes coding for defense proteins (such as protease inhibitors) and enzymes required for the formation of toxic defense chemicals, i.e. phytoalexins and phenolic compounds [128]. In analogy to animal physiology it is possible to distinguish between local and systemic defense mechanisms.

c22?CooH a-linolcnic acid

I

w6-LIPOXYGENASE

ccoo

HOO‘

-

11

ALLENE OXIDE SYNTHASE~ COOH

11

ALLENE OXIDE CYCLASE

12-oxophytodienoic acid ( 12-0x0 PDA) 0

0

jasrnonic acid

0

7-is0 jasrnonic acid

HO

0

tuberonic acid

curcurbic acid

Figure 1-14.Biosynthesis of jasmonates.

The systemic response of plants seems to be controlled by ‘circulating’ primary wound hormones such as the 18 amino acid polypeptide systemin which is found in tomato plants [129]. Systemin has been shown to be a potent inducer of jasmonate

1.5 Eicosanoid-relatedsignaling compounds in plants

33

biosynthesis, possibly by stimulating linolenic acid release via the activation of a still ill-defined phospholipase-type enzyme in the target cells. Plant cells carrying mutations in genes which control systemin or jasmonate biosynthesis suffer from impaired pest resistance. Thus, jasmonates functionally resemble local pro-inflammatory mediators such as the eicosanoids of animals while systemin has been speculated to resemble systemic cytokines such as tumor necrosis factor a [129]. The regulatory function of jasmonates is, however, not restricted to acute emergency situations but includes other 'stress' situations which require responses such as formation of nitrogen storage proteins, seed storage, tuberization, inhibition of growth, senescence, flowering, ethylene-dependentfruit ripening, movements, etc.

stearidonic acid

a-linolenic acid

1 o6-LIPOXYGENASE 1

+

t

1lOSLIPOXYGENASE]

HOO-OH '= ' OOH

1OS-HPOTE

13s-HPOT

I

I

I HYDROPEROXIDE LYASE I

Ho -

n traumatin

0

Cymathere ether

Figure 1-15. Proposed biosynthetic pathways for the formation of the plant wound hormone traumatin (left) and the kelp flavouring cymathere ether (right).

In a natural environment microbial and fungal infection occurs mostly upon wounding. It is, therefore, intriguing that the o6-lipoxygenase-catalyzed pathway of linolenic acid metabolism in plants is strongly activated by injury and damage and that it provides-besides defense chemicals-factors which promote wound healing by triggering cell proliferation. Such plant wound hormones are derived from en-

34

I Arachidonic acid und companions: an ubimduiit .source of biologicul , s i g i i d s

zyme-catalzyed heterolytic cleavage of 13(S)-HPOTE yielding 12-oxo- I O(E)dodecenoic acid (Fig. 1-15), also called ‘traumatin’ and its derivatives [ 124,130,1351. In fact, traumatic acid, which is generated by autoxidation of traumatin and is found in wounded plant tissue, was the very first compound identified as a hormone-like substance in plants [ 1361. The hydroperoxide lyase catalyzing traumatin formation is a cytochrome P-450-dependent enzyme [ 1231. A particular rich source of oxylipins are marine algae [ 1.371. In general these compounds are formed along lipoxygenase-catalyzed pathways from polyunsatured C?,, fatty acids (in particular in red algae Rhodophyra), C I 8fatty acids (in green algae Chlorophyra) and from both (in particular in brown algae Phaeophyceae). Thus, in certain Rhodophytu species a 12(S)-lipoxygenase has been found which, together with a hydroperoxide isomerase, catalyzes the formation of 12(R), 13(S)-diHETE from arachidonic acid. For other species the synthesis of 9(R)-, 11(R)- and 13(S)HODE from linoleic acid as well as of jasmonic acid has been reported and the high amounts of PGE2, PGF?, and PGA2 found in Gracilariu and Hypnea species are potentially of commercial interest. A major pathway of oxylipin formation in green algae is the dioxygenation of ctlinolenic acid by 9- and 16-lipoxygenases. Subsequent rearrangements and cleavage of the hydroperoxides results in a wide variety of compounds the functions of which are unknown. The small acyclic and cyclic hydrocarbons, which serve as pheromones for gamete attraction of many brown algae, are formed along similar pathways, i.e. probably via cleavage of lipoxygenase-derived oxylipins [ 138,1391. To give a final example of the amazing variability of oxylipin biochemistry in plants, the formation of cymathere ether by the kelp Cymathere triplicaru may be mentioned. This unusual cyclic ether, which is structurally reminiscent of a prostaglandin, is synthesized from stearidonic acid via lipoxygenase-derived 1O( S)-HPOTE and an epoxide intermediate (Fig. 1-15; ref. [140]). Cymathere ether gives the edible kelp of the Puget Sound a cucumber-like taste. For more information the reader is referred to the detailed review of Gerwick [ 1371.

1.6 The cellular functions of eicosanoids in mammals 1.6.1 Eicosanoids as local mediators Despite their pronounced physiological effects, eicosanoids differ in several ways from classical hormones. First of all, they are synthesized by practically all tissues instead of being products of a single endocrine gland. In addition, metabolically they are extremely unstable, i.e. their area of activity is normally restricted to the immediate vicinity of their place of origin. In fact, at a concentration of I00 1250

~~

The molecular weight of both enzymes is the same and their PI values are similar. The enzymes seem to be more active in alkaline solutions as documented by their pH optimum. The main difference between the two enzymes is the dependence on glutathione (GSH). The spleen type strictly requires GSH for its catalytic properties, whereas the brain-type enzyme shows full activity with other sulfhydryl compounds, such as dithiothreitol (DTT) or 2-mercaptoethanol. In 1961 p-trace, a major protein constituent of human cerebrospinal fluid (CSF) was discovered by electrophoresis [57]. Recently Hoffmann er LII. [S8]and Urade et

98

4 Prostanoid synthases

af. [59] showed that p-trace protein is produced by cells of the choroid plexus. It has been demonstrated that a partial sequence termed p-trace protein, is almost identical to the GSH-independent PGD synthase from human brain [60,61]. Recently Hoftmann et al. [62] demonstrated the full amino acid identity, except for a leading sequence of 22 amino acids and position I54 (T instead of S). These differences result in only minimal changes in enzymatic properties. For the human p-trace protein and rat GSH-independent PGD synthase, the K, values for PGH? are 4 and 14 pM, respectively. The v,, values are 1000 and 4000 (nmol/min/mg) and optimal pH values are 11 and 9.5, respectively. The cDNA of the GSH-independent PGD synthase was sequenced and analyzed in order to find the location of the synthase in the human genome and to compare it with other proteins in the cDNA library [63,64]. White et uf. [65] found the gene of PGD synthase to be mapped on chromosome 9, bands q34.2-34.3, whose expression could be influenced by thyroid hormone [66]. The cDNA and protein sequence showed sequence similarities with lipophilic proteins belonging to the lipocalin superfamily [64,67], such as 0-lactoglobulin, a-urinary protein and a,-microglobulin [68]. As the p-trace protein is the second most abundant protein in human CSF after albumin and it is the most prominent one of presumably tissue-specific origin, the question of its function arises. Despite its high concentration in the CSF 1691, the low concentrations of PGD2 in the CSF questions its function as a PGD synthase. Recently Tanaka et al. 1701 proposed a new function of 0-trace protein as a retinoid transporter. Retinoids play an important role in regulating a variety of biological processes, such as differentiation, morphogenesis and cell proliferation. The p-trace was found to belong to the group of secretory retinoid transporters, such as retinoidbinding-protein (RBP) or P-lactoglobulin. Binding analysis of different retinoids showed strong binding of most retinoids (retinoic acids, retinals and retinols) except all-trans-retinol. The retinoid acids bind on a different site to PGHl and are therefore noncompetitive inhibitors of PGD synthase [70]. Inhibition studies were performed with a recombinant protein from Escherichia coli (JM109) in which the cDNA of PGD synthase was expressed from a plasmid [71], making use of the vector pUCl19. Site-directed mutagenesis allowed the investigation of different mutants. The core region of the amino acid sequence was found between Val30 and Gln 182, which fits the sequence of the p-trace protein. In particular the three cysteines (Cys65, Cys89 and Cys186) were exchanged separately or in combination against alanin, in order to test for catalytic properties for PGD production, folding and intermolecular disulfide bonds. Only Cys65 is necessary for catalytic activity. The other cysteines may take part in intramolecular bonding, but do not influence the folding of the monomeric protein. Functional binding studies show that Cys65 is the essential amino acid in the active site 1711 and can be inhibited by N-ethyl-maleimide (NEM) and quadrivalent seleno compounds 172,731. Oxidation of Cys65 leads to inactivation of the enzyme. Inhibition studies of the GSH-independent PGD synthase revealed that divalent and quadrivalent but not hexavalent selenium compounds can inhibit enzyme activity [74]. The reversibility of the inhibition by selenium compounds with excess of dithiothreitd points to the involvement of sulfhydryl groups. The ICSOof enzyme inhibition was approximately 5 pM. The inhibitory effect of selenium compounds can also

4.5. Prostaglandin E sythase

99

be shown by intracerebroventricularadministration of selenium chloride, which leads to serious interference with the sleep-wake rhythm of freely moving rats 148,751.

4.5. Prostaglandin E synthase Prostaglandin E synthase catalyzes the isomerization of PGH to PGE. A rich source of PGEz and PGH2 are the seminal vesicular glands of different species. Microsomes from these glands are widely used for the biosynthesis of PGH2 used in all assays testing prostaglandin synthase activities. This tissue was used in the first purification protocol for prostaglandin H-E isomerase (PGE synthase). After homogenization the main activity was found in the 105 OOOx g fraction, known as the microsomal fraction. The resulting purified or partially purified enzymes and their properties are listed in Table 4 4 . Table 4-4. Historical overview of the purification of PGE synthases. Organ and Compartment MW (kDa) species Human brain Cytosolic 24.5 25 Sheep veMembrane 43,51 sicular gland bound 120,180 19 ? Rabbit kidney Membrane bound 60-67 ( 1 0Bovine veMembrane sicular gland bound 30) ? Bovine veMembrane sicular gland bound

Km(PGH2) Optimal PH GST (mM) 147 308 44 40

Reference

activity 8 8

+ +

Ogorochi et al.[1001

150

6.2 6.5-7.5

10-20

?

Tanaka and Smith [ 1031 and Tanaka et al. [I041 Sheng et al. [77]

?

37

Moonen et a/. [78]

?

7.8

Ogino et al. [76]

?

A partially purified membrane-bound enzymes, has been obtained after several column purification steps. The enzyme requires GSH as an activating and protecting factor and has a half-life time of 30 min at 24 "C. In the tubular system of the kidney PGE2 has an effect on the sodium retention process. Therefore, the purification of PGE synthase was performed using rabbit kidney by Sheng et al. [77]. The isolation resulted in enzymes requiring GSH in the millimolar range, which could not be replaced by other agents with sulfhydryl groups (DTT, cysteine, etc.). The enzyme has a K,,, value of 10-20 pM for the conversion of PGH2 to PGE2. The reaction can be blocked by SH blocking agents, such as phydroxymercuribenzoateor NEM. The first purification of PGE synthase which achieved distinct bands on SDSpolyacrylamide gel electrophoresis was published in 1982 [78]. Two bands in the range of 60-67 kDa were the main proteins, while silver staining showed additional bands below 30 kDa. All proteins showed a clear GSH dependence of the isomerizing

100

4 Prostunoid synthases

low 30 kDa. All proteins showed a clear GSH dependence of the isomerizing activity and had an optimal activity at pH 7. Ogorochi et ul. [ 1001 improved the isolation and purification of PGE synthase by using GSH agarose chromatography followed by separation on a Mono Q column. The elution profile of the last column showed two peaks, which led to single bands on SDS-polyacrylamide gel electrophoresis of 24.5 and 25 kDa (peak 1 ) and 25 kDa (peak 2). The 25 kDa proteins are the same in both fractions and had an isoelectric point (IP) of 5.4. All proteins showed gluthathione S-transferase (GST) activity, The K,,, (PGH?) value of which could be inhibited by 1 -chloro-2,4-dinitrobenzene. peaks 1 and 2 were as high as 147 and 308 pM, respectively. Based on these findings for the two protein fractions Tanaka and Smith 1791 made an immunochemical attempt to isolate the enzymes. Proteins were used to produce monoclonal antibodies, leading to different hybrids. Immune precipitation with the three hybrids (hei-2, hei-7 and hei-26) led to different proteins. With these antibodies several proteins between 19 and 180 kDa were precipitated. The K,,, values varied between 40 and 150 pM with maximal isomerase activity in a pH range of 6.2-7.5. At least three different proteins could be isolated from one tissue, all showing isomerasc activity, but the combination of all three antibodies resulted in a precipitation of 45% maximum of the activity. This indicates that the isomerase is not characterized up to now. Surprisingly no further purification of the PGH-E isomerase has been published, except the finding that different GSTs also possess PGH-E isomerase activity. Recently Watanabe et al. [80]published the occurrence and distribution of PGE synthase in the rat (see also Table 4-6). Interestingly, they found two different types of the enzyme depending on GSH requirement. In addition, their data permitted the conclusion that non-enzymatic production of PGE2 is insignificant compared to the enzymatic pathway. So far, 20 years has not been sufficient time to characterize and sequence a PGE synthase with distinct enzyme activity. Interestingly there are several proteins which can function as an isomerase, such as the GSTs and serum albumin [3]. The nucleophilic properties of a thiol or thiolate group in a protein may probably be sufficient for the isomerization of PGHp which can explain the activity in different proteins.

4.6 Prostaglandin F synthase In 1967 Hamberg and Samuelson [81] reported the conversion of PGH2 to PGF?,,. So far three different biosynthetic pathways are known for the formation of PGF: (1) the reduction of PGH2 [82,83],(2) I 1-keto-reduction of PGD2 (84,851,(3) 9-ketoreduction of PGEp [86,87]. PGD2 1 1-ketoreductase was independently isolated from rat lung and rabbit liver by Watanabe et al. [88] and Wong [89]. Watanabe er al. 1901 also purified the reductase from bovine lung. The enzyme shows 65% similarity in its amino acid sequence to human liver aldehyde reductase. It has two different binding sites, where a reduction of PGD2 and PGH;? with K,,, values of 120 and 10 pM, respectively, occurs.

NADPH is an essential co-factor for this reaction. Another type of PGD- 1 I -keto reductase was isolated from bovine liver, with a K,,, value for PGD? of approximately 10 pM, which means a higher specificity for PGD? than PGHl [!Ill. This reductase from bovine liver shows minimal cross-reactivity to the PGF synthases. PGF synthases have a molecular weight of approximately 37 kDa and belong to the gene faniily of aldo-keto reductases [92], as a result of cDNA analysis [93]. Many aldo-kcto reductases like the human liver aldehyde reductase also catalyze the reduction of PGH2 [94] (Table 4-5) Table 4-5. PGF syntheses from three tissues Properties Source MW ( m a ) Km(PGH2) (PM) K m (PGDd (PW Optimal pH NAPDH requirement Turnover Imin-') a See Watanabe et al. [90]. n.c., not catalyzed. See Chen et al. [91]. See Hayashi et al. [ 1051.

PGF synthas2 B o v i n e lung 37 I0 100-200 6.0

PGF synthaseh Bovine liver 36 25 10 6.5

Aldehyde red ucl;iwL Human liver 36 I 00 n.c. 6.5

+

+

+

2.1

0.1

3. I

The isolation of cDNA and the expression of PGF synt,ase from bovine lung in E. coli [95] allowed structural studies. Barski and Watanabes [96] proposed a ping-pong mechanism for the reduction of PGD2. The examination of the active site by sitedirected mutagenesis did not reveal any essential cysteine but showed that ProlY5is necessary for high enzyme activity [97]. In p-crystallin, which has a high similarity in amino acid sequence (77%) to bovine lung PGF synthase, this proline is exchanged into valine resulting in decreased activity. In binding studies a covalent attachment of PGD2-p-azidophenacyl-ester into the active site was observed. Photoaffinity labeling indicates that His48,Leu'", and Ile277,are in the active site of PGD synthase [98]. Rondeau et al. [99] reported the importance of the C-terminus, the tertiary structure and the location of the NADPH binding site of the enzyme. PGF synthases are well characterized in their primary structure, their gene sequence and chemical properties, indicating a dual function of the enzyme consisting of endoperoxide cleavage and simultaneous reduction which depends on NADPH. However, GSTs have also PGH-F converting activity and contribute to the formation of PGF2,.

4.7 Glutathione S-transferase (GSTs) In 1976 Christ-Hazelhof et al. [3] reported that GST, in the presence of GSH, converts prostaglandin endoperoxides into PGF2U, PGE2 and PGD2. This group

102

4 Prostunoid synthases

purified a GST isoenzyme which mainly converted PGHl to PGF?,, production. Oiie of the earlier published PGH-E isomerases was a type of anionic GST, which lorms PGE2 as the major product [loo]. Rat spleen PGD synthase shows GST activity lS.61. A GST isoenzyme from rat liver produces a mixture of PGE? and PGF2(I, whereas an isoenzyme from sheep lung in the presence of GSH leads to a mixture of PGD2 and PGF2,. Ujihara et al. [loll systematically examined GSTs from liver and testis. GST I - 1 , GST 1 -2 and GST 2.2 show high production of PGE? and PGF?,, and only low PGD, concentrations are observed. Other GSTs (3.3, 3.4, 4.4 and 7.7) mainly yield PGE?. GST also has an intrinsic reductase activity and produces approximately equal amounts of PGF2, and PGE2 in the presence of GSH 1601. Thus GSTs have multiplc enzymatic activities with the ability to form PGE, PGD and PGF. Interestingly most of the PGE and PGD synthases, in contrast to PGF synthases, have an optimal pH higher than 7.4, which indicates that they are present in an anionic form in physiological conditions. The proposed models of the enzymatic conversion of PGH2 are based on an anionic cysteine that attacks the peroxide bond [71] or the nucleophilic carbons (C-1 1 and C-9) [ 1021. The cloning and expression of prostaglandin PGD and PGF synthases and the method of site-directed mutagenesis provide some hope that the mechanisms of these enzymatic reactions will be soon understood.

4.8 Detection of prostaglandin synthases in various tissues Prostaglandin synthases were isolated from different tissues. Specific antibodies allow immunohistochemical detection of the enzymes and reveal their location and distribution in the body. Table 4-6 summarizes the occurrence of the different enzymes, which catalyze the formation of PGE, PGD, and PGF. In the following only special tissues representative of prostaglandin production will be mentioned (Table 4-6). In the brain, mainly PGD synthase and small amounts of PGE synthase and GST are present but there is no PGF synthase. This finding supports the importance of PGE and PGD in the sleepwake cycle [75]. Immunochemical analysis showed the presence of PGF2, synthase in immune cells (histocytes and dendritic cells of the spleen) and other cells which have contact with pathogens (such as lung cells). The male reproductive tissue is a rich source of PGE synthases; therefore, this tissue was used for the first enzyme isolation procedure. In particular the deferent duct, vesicular gland and coagulation gland show high PGE synthase activity. In contrast, the reproductive system of females produces less PGE2 and no PGE synthase activity is found in the uterus. PGD synthase is found in almost all immune cells. such as macrophages, mast cells, and Langerhans cells in the skin which indicates the function of PGD in the immune response of the skin. The lung is an organ where all isomerases for PGH2 can be found.

4.8 Detection of prostunoid synthasrs in vurious tissues

103

Table 4-6. Localization of different prostaglandin synthases Tissue or organ

PGD synthase PGD synthase brain type spleen type

Brain

Glia, small neurons, spinal cord and choroid plexus 1106-1 101

Schwann cells. satellite cells and large neurons [107,1 101

GSHdependent PGH-E isomerases, membrane bound Extract 1801" - .

GSHPGF independent synthase PGH-E isomerases, membrane bound n.d 1961 . >

Tissue [SO] n.d.[80]

n.d. .[SO]

Thymus Liver

Tissue [ I 121 Kupffer cells II121

Stomach

Submucosal laver Extract .~ r801" Extract [80]"Tissue 1961 [106,112] Peyer path 11 121 Large and Extract [80]"Tissue 1961 small intestine and cecum 1801 Peyer path [ I 121 Colon cells ~131 Tissue [ 1061 n.d [I 121 Langerhans cells, macrophages, mast cells and epidermis [ 1 14,1061 Tissue [ 1061

Intestine

Colon Skeletal muscle Skin

Bone marrow Reproductive system

Adrenal gland Lung Heart Spleen Kidney

Deferent duct, testis, vesicular gland and coagulation gland [76,78,80,104]b Ovary [ 801'

Tissue 191 I

Uterus [SO]

GST activity

PGE I100,111]

PGD [3,96.106. Ill] PGD 12,561

PGD 12,561

PGD [106,111]b and PGE [ 1OO,56,2]

Tissue 1801 Tissue [lo61 Tissue [ I061 Histocytes and dendritic cells [112l Tissue [62]

Tissue [SO]

Aveolar cells PGFz, [ 31 and non-ciliated PGD 12.561 cells [3,96,97) Extract [Sol4 Tissue 1961 PGD [2,56] Extract [ Sola Cortex and Tissue 1961 medulla [ 7 7 ] Tissue 1961 n.d. in blood I961

PGD I1061

Pancreas Tissue [ 1061 Body In most body fluids fluids [ I 151 Hormone EDididvmis system li061 Measurement of prostaglandin in the presence and absence of GSH. which indicatch hynthasc aclivitj. Male. ' Female.

104

4 Prostanoid synthases

4.9 Summary and outlook Cyclooxygenase products are tissue hormones and, as such, are part of the communicating network between cells of a given tissue. The isolation of biosynthetic enzymes has allowed the production of specific antibodies for their immunochemical detection and using this technique has shed more light on the role of individual synthases in their physiology or pathophysiology. In general, the cells of the immune system use prostaglandins abundantly for communication as well as cells of the reproductive system. It is interesting that prostaglandin synthesis is upregulated upon demand and that the cyclooxygenase-2 isoenzyme plays a crucial role as an early response gene. Future work should emphasize the role of individual prostaglandins in physiological and pathophysiological conditions and the mechanisms of up- and downregulation of the enzymes involved. This should include the receptors and their pharmacology.

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Prostaglandins, Leukotrienes and Other Eicosanoids: From Biogenesis to Clinical Application Edited by F. Marks, G. Fürstenberger Copyright © WILEY-VCH Verlag GmbH, D-69469 Weinheim (Federal Republic of Germany). 1999

5 Lipoxygenases Hartmut Kiihn

5.1. Introduction Lipoxygenases constitute a family of dioxygenases which catalyze the stereospecific insertion of molecular oxygen into polyunsaturated fatty acids (PUFAs) containing an (lZ,4Z)-penta-I ,4-dienoic system [ 1-41. The primary products of the lipoxygenase reaction are hydroperoxy fatty acids containing a Z,E-conjugated diene system. Lipoxygenases occur in plants [3,4] and in the animal kingdom [ 1,2] but have not been detected so far in bacteria or yeast. However, there are several reports on the expression of lipoxygenases in lower marine organisms such as algae, sea urchin, star fish, surf clam and corals (see Section 1.4) [5-81. Most of our knowledge on lipoxygenase structure and function originates from studies on plant enzymes. Some 60 years ago a lipoxygenase was detected in dry soybeans [9]. This enzyme, which was called 'lipoxidase' at that time, was purified to homogeneity and was characterized with respect to its protein chemical and enzymatic properties [4-111. In parallel to the characterization of this lipoxygenase isoform (LOX- 1) other isozymes were detected and the pattern of isoenzymes became more and more complex with time. So far eight lipoxygenase isoenzymes have been described in soybeans [12]. In several aspects (reaction mechanism, kinetic parameters, iron content etc.) the soybean LOX-1 constitutes a suitable model for certain types of mammalian lipoxygenases. However, it should be stressed that experimental data obtained with the soybean enzyme should be interpreted with care when conclusions to mammalian physiology are drawn. For a long time it was believed that lipoxygenases were restricted to the plant kingdom and lipid peroxidation in animal tissues was due to the pseudo-lipoxygenase activity of hemoproteins. However, along with the discovery of thromboxane in 1974, the formation of (1 2S,5Z,8Z, 10E,14Z)-12-hydroxyeicosa-5,8,10,14-tetraenoicacid ( 12-HETE) was described when human thrombocytes were incubated with exogenous arachidonic acid [13]. In 1975 a lipoxygenase was detected in rabbit reticulocytes which was capable of oxidizing phospholipids and biomembranes [14]. This enzyme was purified to homogeneity in milligram amounts and was well characterized with respect to its protein chemical and enzymatic properties [15]. In 1978 a 'lipoxygenase-like enzyme' was detected in the microsomal fraction of the rat testis [ 161. The structural identification of the slow-reacting substance of anaphylaxis (SRS-A) as a mixture of the peptidoleukotrienesC4 and D4 [ 171 and the finding that arachidonate 5LOXs are involved in leukotriene biosynthesis marked a milestone in lipoxygenase

1 10

5 Lipoxygenuses

research and prompted many researchers to intensify their activities in this field. Writing a review on lipoxygenases is not an easy task because of the structural and functional diversity of this enzyme family. A MedLine search covering the last 25 years revealed 7 178 references when screened with the keyword 'lipoxygenase'. More than half of these papers (3986) were published between 1990 and 1997. Intcrestingly, the vast majority of these articles (more than 80%) are on 5-LOXs and 5-LOX inhibitors. Of the papers published between 1990 and 1997 in the lipoxygenase field. 7.1% are on I2-LOXs and 6.6% on 15-LOXs. It is of course impossible to consider all of these references for a review and I hereby apologize to all scientists whose papers will not be cited.

5.2. Lipoxygenase reaction In principle, the lipoxygenase reaction consists of three consecutive steps (Fig. 5- I ).

(1) Initial stereo-selective hydrogen abstraction from a doubly allylic Inethylene group forming a carbon-centered fatty acid radical. The hydrogen abstraction is the rate limiting step of the overall reaction as indicated by kinetic isotopic effects. Formally, hydrogen is abstracted as proton and the electron is picked up by the ferric lipoxygenase. (2) Radical rearrangement which is accompanied by a Z,E-diene conjugation. (3)Stereo-specific insertion of molecular dioxygen at C-1 or C-4 of the pentadienyl system forming a oxygen-centered fatty acid hydroperoxide radical. This radical intermediate is reduced to the corresponding anion and the enzyme is oxidixd back to a ferric form. It should be stressed that the radical intermediates remain enzyme bound; otherwise the high product specificity of most lipoxygenases could not be explained. The detailed mechanism of the lipoxygenase reaction is still a matter of discussion [3]. Recently, it was suggested that a strong base at the active site may abstract a methylene proton facilitating an electrophilic attack by the enzyme-bound ferric iron on the pentadienyl system which leads to iron reduction and to the formation of an organic radical whose unpaired electron is delocalized over the entire pentadienyl system. Alternatively, after proton abstraction the ferric iron may covalently bind to a terminal carbon atom of the pentadienyl carbanion and thus does not formally undergo reduction. In most mammalian cells the primary lipoxygenase metabolites, the hydroperoxy fatty acids, are rapidly reduced to the corresponding alcohols or converted to other secondary products such as double and triple oxygenation products [ 1 8,191, epoxy leukotrienes [20-22], short chain aldehydes [23], alkanes [ 241, etc. Intracellularly. there are two major types of glutathione peroxidases capable of reducing hydroperoxy fatty acid derivatives [25,26]. Type- 1 glutathione peroxidases preferentially reduce free hydroperoxy fatty acids and other organic and inorganic hydroperoxides, but

5.3. Common properties nf lipoxygenuses

III

only exhibit low affinity for hydroperoxy ester lipids. In contrast. type-2 glutathione peroxidases, the so-called phospholipid hydroperoxide glutathione peroxidases (PHGPx) effectively reduce esterified hydroperoxy fatty acids [27,28]. Since glutathione peroxidases are selenium-containing enzymes, selenium deficiency may lead to a decrease in the cellular capability for reducing free and esterified hydroperoxy fatty acids. Recently, it was shown that the human thioredoxin reductase [291 is also capable of reducing lS(S)-HPETE to IS@)-HETE in an reduced nicotinamide adenine dinucleotide phosphate-dependent reaction [ 301. In plants, several other metabolic pathways for metabolization of fatty acid hydroperoxides have been described: (1) the hydroperoxide lyase pathway [3 I], (2) the allene oxide synthase (AoS) pathway [32], (3) the peroxygenase pathway [33] and (41 diol formation [34].

I

activation

42-i

b product

hydrogen abstraction

dissociation

. LLqX-.FP OX-FP %

s'"p 3

oxygen oxygen losertion in.??rUon

\

LOX-F2+ ....'..._

-

iJ

step 2 radical rearrangement

/

LOX-FZ'/ ..... ...__

Figure 5-1. Schematic view of the lipoxygenase reaction. The catalytically inactive ferrous lipoxygenase is activated by hydroperoxy lipids.

5.3. Common properties of lipoxygenases Lipoxygenases are non-heme iron, non-sulfur iron-containing enzymes. They contain 1 mole of iron per mole of enzyme and the iron ligand sphere has been identified as distorted bipyramide by crystallography studies and X-ray absorption spectroscopy

112

5 Lipoxygenases

[3541]. As an exception a heme-containing 'lipoxygenase-like enzyme' has been identified in Fusarium oxysporum which was identified later on as cytochrome P-450 isoenzyme [42,431. Ferrous lipoxygenases are catalytically inactive. In order to convert them to an enzymatically active form small amounts of hydroperoxides are required. When peroxide-free fatty acids are used as substrate, the oxygenation reaction starts with a kinetic lag-phase [44,45]. The mechanism of the activation process is not entirely clear but it appears to involve oxidation of the native ferrous lipoxygenase (inactive ground state) to an activated ferric form [46,47]. Lipoxygenases require repeated activation, since a share of the activated enzyme returns to the inactive ground state 1441. It was calculated that single activation of the enzyme is sufficient for about ten catalytic cycles for the rabbit enzyme [48]; then a second activation is required. At higher concentrations, fatty acid hydroperoxides act as enzyme inactivators and most lipoxygenases undergo suicidal inactivation during fatty acid oxygenation. For the rabbit enzyme it has been shown that this inactivation is most prominent at higher temperatures (35 "C) but can be suppressed largely if the lipoxygenase reaction is carried out at 2 "C [15]. The molecular mechanism of suicidal inactivation remains unclear [49,50]. There are most probably several processes which contribute to suicidal inactivation. Detailed kinetic investigations on the lipoxygenases from soybeans [45] and rabbit reticulocytes [44] indicated that these enzymes exhibit a high affinity for fatty acids and oxygen. However, the native ferrous soybean lipoxygenase does not bind oxygen and, thus, the high affinity towards oxygen may be exhibited by the enzyme-fatty acid radical complex. The primary products of the lipoxygenase pathway, the hydroperoxy fatty acids. can be further metabolized by lipoxygenase via different routes (Fig. 5-2). (1) Double or triple oxygenation. Since 5-, 12- and 15-HETE still contain doubly allylic methylenes they constitute substrates for lipoxygenase-catalyzed oxygenation. In fact, the formation of various DiH(P)ETE and TriH(P)ETE isomers has been reported for various lipoxygenases [51,521 (2) Hydroperoxidase reaction. Under certain conditions (reduced oxygen tension or anaerobiosis) lipoxygenase catalyzes the decomposition of hydroperoxy fatty acids. This reaction, which requires the presence of a reductant in addition to the hydroperoxide substrate, is initiated by a homolytic cleavage of the hydroperoxy 5-HPETE

I

i

6y*mc

5,6-LT A4

Figure 5-2. Lipoxygenase-catalyzed conversion of hydroperopxy fatty acids. Lipoxygenases are capable of converting hydroperoxy fatty acids via three different routes. For details see text.

5.4. Classificution of lipoxygenases

1 13

bond and leads to a complex array of secondary products including keto dienes (KETEs), short chain aldehydes and alkanes (Fig. 5-3). (3) Epoxy leukotriene formation [20-221. This reaction involves a stereo-selective hydrogen abstraction from C- 10 and a homolytic cleavage of the hydroperoxy bond. Thus, lipoxygenase-catalyzedformation of epoxy leukotrienes may be regarded as concerted action of the oxygenase (hydrogen abstraction) and hydroperoxidase (peroxide bond cleavage) activity of the enzyme. non-oxygenated dimers

II HOO

1 proxy fatty acid

Lox-Fe2+\

OH-

\ L akoxy radical

a

/-

U

-

fatty acid radical

n H+

fatty acid (reductant)

LOX-Fe3+

ketodienes short chain aldehydes alkanes oxygenated dimers

Figure 5-3.Lipoxygenase-catalyzed, reductant-dependent hydroperoxidase reaction. At reduced oxygen tension or anaerobiosis lipoxygenases catalyze the decomposition of hydroperoxy fatty acids in the presence of reducing agents. The initial step of this hydroperoxidase reaction is the homolytic cleavage of the peroxide bond forming an alkoxy radical (left site). During this reaction the native ferrous lipoxygenase is oxidized to its ferric form. The alkoxy radical is subsequently decomposed forming a complex array of secondary products including KETEs, short chain aldehydes, alkanes and oxygenated fatty acid dimers. The reductant reduces the ferric enzyme back to its ferrous form and the next catalytic cycle may start. When polyenoic fatty acids are present as reducing agent, carbon-centered fatty acid radicals are formed which may stabilized by forming non-oxygenated fatty acid dimers.

5.4. Classification of lipoxygenases According to the currently used nomenclature, lipoxygenases are categorized with respect to their positional specificity of arachidonic acid oxygenation. In mammalian cells three major types of lipoxygenases have been differentiated [ 1,2]:

1 14

5 Lipoxygenuses

(1) 5-LOXs which introduce molecular oxygen at carbon 5 of the arachidonic acid backbone, (2) 12-LOXs converting arachidonic acid to (12S,5Z,8Z,1OE, 142)-12-hydroperoxyeicosa-5,8,10,14-tetraenoicacid (1 2-HPETE) and (3) 15-LOXs which catalyze the formation of 15-HPETE (Fig. 5-4). human niyeloic cells human kerutinocytes

5-LOX

LOX 12-LOX

15-LOX

leukocyte-type

platelet-type

reticulocyte-type

epidermis-type

porcine leukoqres bovine airways murine nuxrophaRes

h

human rcriculoc?rcs h u n M nurcrfiphager human airuav

human hair roofs human prosrare human cornea

w platelets

human mgacaryoqres human skin

Figure 5-4. Arachidonic acid-related classification of mammalian lipoxygenases. 12- and 1 5-LOX itre subclassified into two subgroups. Examples for the expression of the various enzyme subspecies are given in italic letters.

This nomenclature suffers from several disadvantages which became inore and more evident with the growing diversity of the lipoxygenase family. One of these disadvantages is the fact that the positional specificity of lipoxygenases is not an absolute enzyme property but depends on the structure of the substrate and on its alignment at the active site. If linoleic acid is oxidized by arachidoniate I5-LOXs. (13S, 9Z, 11E)-l3-hydroperoxyoctadeca-9,ll-dienoicacid ( 1 3-HPODE) is formed. Consequently, these enzymes should be named linoleate 13-LOXs. The same product is produced when various arachidonate 12-LOXs oxidize linoleic acid. Thus, with respect to linoleate oxygenation both lipoxygenase subtypes should be categorized in one group. If 15-HPETE is used as substrate for the soybean LOX-I, oxygen is introduced at C-5 and C-8. Thus, with 15-HPETE this arachidonate 15-LOX exhibits a 5- and 8-LOX activity. A second disadvantage of the arachidonic acid-related nomenclature is that arachidonate 12-LOXs are subdivided in two groups [2,53]. A leukocyte-type 12-LOX has been identified in porcine and murine leukocytes as well as in bovine airway epithelial cells (Fig. 5-4). With the exception of positional specificity these enzymes are very similar to the human and rabbit reticulocyte ISLOXs [2,53]. On the other hand, there is a platelet-type 12-LOX which differs strongly from the leukocyte-type enzyme with respect to its substrate specificity

5.5 Structurd aspects of 1ipox.ygenase.Y

1 I5

[2,53]. In mice the genes for both the leukocyte-type and the platelet-type 12-LOX have been identified [54]. The high degree of similarity between the leukocyte-type 12-LOX and the reticulocyte-type 15-LOX suggested that both subtypes of enzymes may be functionally related. The finding that both the human reticulocyte-type 15LOX and the murine leukocyte-type 12-LOX can be induced by interleukin 4 (IL-4) and the apparent lack of a leukocyte-type 12-LOX in rabbits and man supported this hypothesis. On the other hand, the cloning of a leukocyte-type 12-LOX in rabbits [55] and reverse transcriptase polymerase chain reaction (RT-PCR) data from human cells [56,57] may suggest the simultaneous expression of a leukocyte-type 12-LOX and a reticulocyte-type 15-LOX in rabbits and man. 15-LOXs may also be subclassified into two groups (Fig. 5-4). In addition to the reticulocyte-type 15-LOX [58], an epidermis-type 15-LOX has recently been cloned [59]. This enzyme shares only a low degree of amino acid identity with the reliculocyte-type 15-LOX and, thus, may not be functionally related to this enzyme. As alternative criterion for the classification of lipoxygenases, their enantioselectivity, has been suggested [5-81. Most lipoxygenases described so far in plants and animals are S-LOX; they selectively add an oxygen molecule to the pro-S face of the bisallyl radical. However, in lower marine organisms such as sea urchin, corals and starfish R-LOXs have been detected [5-81. In various plants lipoxygenase isoenzymes have been described which form racemic product isomers. A lipoxygenase was purified from green peas which converted linoleic acid to an almost 1 : 1 mixture of 9- and 13-HPODE and each of these positional isomers was a racemic mixture [60]. Similarly, the soybean LOX-3, the crystal structure of which has recently been solved [39] catalyzes the formation of racemic 9- and 13-HPODE. A comprehensive classification of lipoxygenases should consider functional aspects of the enzymes. However, at this stage in research little is known about the biological role of most lipoxygenases. In fact, except from the involvement of 5LOXs in the biosynthesis of inflammatory mediators (see Section 9.5) there is no well-established biological function of any lipoxygenase isoform. Thus, the arachidonic acid-related nomenclature of mammalian lipoxygenases which includes the subclassification of 12- and 15-LOXs may still be used for the next couple of years. In order to categorize the recently cloned murine 8(S)-LOX more experimental data on the properties of this enzyme are needed.

5.5 Structural aspects of lipoxygenases 5.5.1. X-ray crystallography Although soybean LOX-1 had already been crystallized in 1947 [61] it took 46 years to solve its crystal structure at 2.6 8, resolution [35]. More recently, the structure of this enzyme at 1.4 A resolution and the crystal structure of soybean LOX-3 at 2.6 A resolution where reported [36,39]. The first crystallization of a mammalian lipoxygenase was described in 1990 [62]. For this enzyme it took only 5 years to solve the

116

5 Lipoxygenuses

crystal structure [40]. Although the two soybean LOX-I structures reported so far are similar to each other, there are considerable differences with respect to the iron ligand sphere and to the number and shape of cavities (4,35,36]. Both structures indicate that the enzyme is a two-domain protein. The smaller N-terminal domain, which comprises the amino acids 1-146, constitutes an eight-stranded anti-parallel p-barrel. The larger C-terminal domain, which is relatively loosely associated to the N-terminal domain, comprises residues 147-839 and includes the non-heme iron. Although the small N-terminal domain, which structurally resembles the C-terminal co-lipase binding domain of mammalian pancreatic lipases [63], does not contribute to the putative substrate binding site it appears to be essential for enzymatic activity and was implicated in binding, transport and release of substrate and product 1361. The Cterminal domain consists of 23 helices and two anti-parallel P-sheets. Seventeen helices are approximately parallel or anti-parallel to each other and surround a 65 A long central leading helix (helix 9). Helix 18 lies parallel to this leading helix and both helices contain four of the five protein iron ligands. As indicated above the structurc of the iron ligand sphere of soybean LOX-1 has been a matter of discussion. Boyington et al. [35] described a four-coordinate iron with two adjacent unoccupied positions . Three of the four ligands are the NE-atoms of His499, His504 and His690 and, as fourth ligand, an oxygen of the C-terminal Ile839 was identified. The 6 1oxygen of Asn694 was not considered an iron ligand. In contrast, Minor et al. [36] described a six-coordinate iron with the E-nitrogens of His499, His504 and His690, a carboxylic oxygen of the C-terminal IleX39 and the 6 I oxygen of Asn694 as direct protein iron ligands. The sixth ligand position which is positioned trans to His690 at the boundary of a large internal cavity is occupied by a water molecule. This water may be displaced by the substrate fatty acid. A sixcoordinate iron ligand sphere is consistent with earlier extended X-ray absorption fine structure (EXAFS) and electron paramagnetic resonance (EPR) data [64,65]. In the first report on the 3-D structure of the soybean LOX-I two cavities in the major domain were described [35]. Cavity I, which was suggested as path for the movement of dioxygen, forms an 18 8, long hydrophobic tunnel. The second cavity, which is 40 long, is lined by the side chains of 46 mainly hydrophobic amino acids most of which are highly conserved among lipoxygenases. This cavity has two major bends. One is adjacent to the non-heme iron close to the bottom of the cavity and the second bend is at the middle of the cavity where it becomes very narrow. In the 1.4 p\ structure [36] cavity I (oxygen path) is less obvious. In fact, the 1.4 8, model does not allow for the transport of dioxygen through this cavity without substantial rearrangement of several amino acid side chains. This structure suggested that dioxygen may pass through cavity I1 or through a 20 8, pathway starting from His248 and ArgS33 at the surface of the protein, passing between Pro834 and Ile839 and ending in the vicinity of the side chain of His690. Mammalian 15-LOXs also have a two-domain structure [40]. The smaller Nterminal domain comprises 115 amino acids and is composed of eight P-barrels. The 13-barrel domain of mammalian lipoxygenases is similar in sequence, size and structure to analogous C-terminal p-barrel domains of mammalian lipases and, thus, it may play a critical role in localizing the enzyme near their substrates which may bc scquestered in biomembranes and/or lipoproteins [40]. The p-barrel domain sharcs a

5.5 Structuml aspects oj'1ipo.r~gena.sc.s

I I7

1600 A' interface with the larger C-terminal domain (Fig. 5-5) which consists of I8 helices interrupted once by a small P-sheet subdomain.

Figure 5-5. Two-domain structure of the rabbit reticulocyte 15-LOX. A preferred tryptic cleavage site in the N-terminal P-barrel and the non-heme iron are labeled.

The center of the C-terminal domain contains two long helices which contain four of the five protein iron ligands. The fifth iron ligand constitutes a carboxylic oxygen of the C-terminal isoleucine and water appears to occupy the sixth ligand position [41]. The iron ligand sphere has a fair octahedral geometry [40,41]. As for the soybean enzyme a putative substrate binding cavity was observed in mammalian 15LOXs but there are no indications for a tunnel via which oxygen may enter the active side. Thus, it was concluded that the rate of oxygen insertion is diffusion controlled and that the enzyme does not specifically position oxygen relative to the substrate fatty acid. The stereospecificity of the reaction may be determined by the forced conformation of the substrate and/or by stabilizing a certain configuration of the transition state. The substrate binding cleft of mammalian 15-LOXs appears to be a ushaped cavity which is directly accessible from the surface of the protein. Mutagenesis experiments and modeling studies on enzyme-substrate complexes suggested that the positively charged Arg403 may interact with the carboxylic group of the fatty acid

1 18

5 Lipoxygenuses

substrate [40,66]. Since mammalian S-LOXs also contain a positively charged amino acid at this position an inverse head to tail orientation of arachidonic acid 167.681 at the active site of these enzymes appears unlikely 1401.

5.5.2 Substrate alignment and determinants of positional specificity The mechanistic reasons for the positional specificity of lipoxygenases have been investigated in the past by altering the structure of the substrate [69-711 and by sitedirected mutagenesis [72-761. Experiments with a set of arachidonic acid isomers. in which the double bond system is shifted gradually towards the methyl or C-terminus of the fatty acid, suggested that the oxygenation rate and the positional specificity of 15-LOXs depend on the distance of the doubly allylic methylene from the methyl end of the fatty acid [69,70]. From these data it was concluded that the size of the substrate binding pocket may be important for the positional specificity of 12/1S-LOXs [70]. Later on, when sufficient sequence information on mammalian lipoxygenases became available, sequence alignments and mutagenesis studies indicated that the amino acids at positions 418 and 419 may function as sequence determinants for the positional specificity of 12415-LOXs [72,73]. Mutation of the space-filling Tle418 and Met419 of the human 15-LOX to smaller residues converted the enzyme to a lipoxygenase species favoring arachidonate 12oxygenation. This concept was supported by similar results on the human platelet [74) and the porcine leukocyte 12-LOXs [75] but failed to explain the mutagenesis data on the 12-LOXs of murine and rat leukocytes. When the above mentioned amino acids were mutated in the rat enzyme no alterations in the positional specificity were observed [76]. To solve this problem, chimeric lipoxygenase species were created. The results of these experiments in connection with site-directed mutagenesis studies revealed that amino acid 353 appeared to be the decisive primary determinant for thc positional specificity of 12/15-LOXs [77]. According to a computer model of the substrate binding region, the three amino acid residues at positions 353, 41 8 and 4 I9 form the bottom of the substrate binding cleft. (Fig. 5-6) and the following rules have been suggested [77]: (1) mammalian lipoxygenases containing a small amino acid at position 353 iire 12LOX (type 11) independent of the bulkiness of residues 4 18 and 4 19, (2) when a space-filling amino acid is localized at position 353, residues 418 and 419 become important for the 12 positional specificity and (3) when bulky amino acids are localized at positions 353, 418 and 419, 15lipoxygenation is favored. However, when a bulky residue at position 353 is combined with less space-filling residues at positions 4 1 8 and 4 19, arachidonic acid is oxygenated at C- 12 (12-L0X, type I). In addition to amino acids 353,418 and 419 other residues have been implicatcd in substrate binding [66]. The positively charged Arg403 may form a salt bridge with the negatively charged carboxylic group of the fatty acid substrate and Phe4 IS may interact with the substrates' double bonds via 7c-n interactions. Mutation of Phe4 I5 t o

5.5 Structuml aspects of 1ipos~griia.sr.t

1 19

an amino acid with an aliphatic side chain led to an enzyme species with a lower affinity towards fatty acid substrates [66]. 12-LOX (type4

I

\

phe353

12-LOX (type 11)

I-\

ile418 met4 19

I

HsC,

'.... ".

"

2.I...

0 '

..../

-

-

Figure 5-6. Primary determinants of the positional specificity of 12-and IS-lipoxygenases. The bottom of the substrate binding pocket is formed by the amino acids 353, 418 and 419. 12-LOX5 of type I (porcine leukocytes and human platelets) contain a large Phe353, but less space filling amino acids at positions 418 and 419. The large Phe353 forces the substrate fatty acid into a bent orientation so that its methyl terminus comes into contact with these residues. In contrast, 12-LOXs of type I1 (murine and rat macrophages) contain a small leucine at position 353 and, thus, the fatty acid does not come into contact with the residues at 418 and 419. 15-LOXs contain large amino acids at positions 353 and 418/419 which causes a substrate alignment favoring 15-lipoxygenation. The open hexagon represents the non-heme iron which is involved in hydrogen abstraction. If hydrogen is abstracted from C-10 a 12-lipoxygenation is favored whereas hydrogen removal from C- I3 leads to the formation of 15-HPETE.

So far, all studies on the enzyme-substrate interaction and on the positional specificity have been carried out with free fatty acids as substrates. On the other hand, the reticulocyte 15-LOXs are capable of oxygenating more complex substrates and there are several lines of experimental evidence suggesting that membrane phospholipids and lipoprotein cholesterol esters may be the natural substrates. Since these substrates are much more space filling than polyenoic fatty acids, it remains to be investigated how they are aligned at the active site and whether the rules established for polyenoic fatty acids are also applicable for complex substrates.

120

5 Lipoxygenases

As discussed above it is relatively easy to convert a 15-LOX to a 12-LOX and vice versa. However, all attempts failed to transform either of these lipoxygenase isoforms to a 5-LOX. When residues 418 and 419 of the human and the rabbit reticulocyte-type 15-LOX were mutated to the amino acids present in the human S-LOX inactive enzyme species resulted. When Arg403 was mutated to a leucine, the mutant was inactive too. According to the recently published X-ray data of a mammalian 15-LOX [40] the volume of the hydrophobic substrate binding pocket appears to be important for the positional specificity. Lipoxygenases exhibiting a 12-LOX activity have a substrate binding cleft which is approximately 5% larger than that of 15-LOX. The substrate binding pocket of 5-LOXs is approximately 20% larger than that of 15-LOX. If this hypothesis were correct, mutagenesis of large amino acids at the active site to less space-filling residues may convert a 15-LOX to a 5-LOX. We have carried out mutagenesis studies along this line but were not able to create a mutant enzyme which showed significant 5-LOX activity. These data suggest that space requirement may not be the most important factor for arachidonic acid 5-lipoxygenation.

5.6 5-Lipoxygenases Comprehensive review articles on mammalian 5-LOX which cover the molecular biology, enzymology and biological importance of these isoenzymes are available [78-801 and, because of space limitations, only the most important structural and functional aspects will be summarized here.

5.6.1 Enzymatic properties 5-LOXs catalyze the oxygenation of arachidonic acid to (5S,6E,8Z,1 IZ, 14Z)-5hydroperoxyeicosa-6,8,11,ICtetraenoic acid (Fig. 5-7)but also the subsequent dehydration of the peroxy intermediate forming the (5S,6S,7E,9E,I 12,142)-5,6oxidoeicosa-7,9,11,14-tetraenoicacid (leukotriene A,; LTA,). In intact leukocytes there are two metabolic pathways for LTA4 conversion:

( 1 ) the LTA4 hydrolase pathway leading to the formation of LTB4 and (2) the LTC4 synthase pathway which involves conjugation of LTA4 with glutathione forming the cysteinyl leukotriene LTC4. Subsequent cleavage of glutamic acid by a membrane-bound y-glutamyltranspeptidase produces LTDj and additional cleavage of glycine leads to the formation of LTE4 (Fig. 5-7). The human 5-LOX which was cloned in 1988 [81] is a non-heme iron-containing enzyme with a molecular weight of approximately 78 kDa which consists of 674 amino acids. The native ferrous enzyme appears to be catalytically inactive and requires activation by hydroperoxy fatty acids [78]. During the activation process the ferrous enzymeis oxidized to a ferric form, but it is still a matter of discussion whether

5.6 5-Lipoxygenases

12 I

arachidonic acid

5-LOX

SS-HPETE

I

5-LOX

LT A4

LTArWmh/

C,synthase

LT B4 no

CONHCH&OOH COOH

~glulamylhanspeptiahe C

C

LTD4

O

O

H

'-aNH? CONHCH&OOH

dipepridase

COOH

Figure 5-7. Chemical structure and biosynthesis of leukotrienes. Arachidonic acid which is liberated from the membrane phospholipids upon cell Stimulation is converted to SS-HPETE by the oxygenase activity of the 5-LOX. Then the peroxy intermediate is converted to LT&, by the LT& synthase activity of the same enzyme. Enzymatic hydrolysis (LTA4 hydrolase) of the epoxy derivative leads to the formation of LTB4. The LTC4 synthase catalyzes the conjugation of LT& with glutathione forming the peptidoleukotriene LTC4. Subsequent cleavage of glutamic acid by a membrane-bound y-glutamyltranspeptidase produces LTD4 and additional cleavage of glycine leads to the formation of LTB.

whether the ferric enzyme is catalytically active or whether it requires additional peroxide activation [78]. When compared with mammalian 12- and 15-LOXs there is an overall amino acid identity of approximately 40%.However, there are two regions of striking amino acid conservation within the entire lipoxygenase family. The first region runs from amino acid 350 to 421 in the 5-LOX and contains five conserved histidines. The second region of homology occurs between amino acids 545 and 579. Detailed functional analysis and comprehensive characterization of 5-LOXs have

122

5 Lipmygenuses

been severely hampered by the fact that the enzyme is only expressed at a low level in most mammalian cells, but also by low purification yields and by the instability of the enzyme. However, the availability of high-yield expression systems for recombinant 5-LOXs [82-S5] made more detailed investigations on the requirements and behavior of 5-LOXs possible. In contrast to other lipoxygenases the 5-LOX is activated by ATP although no ATP binding consensus sequence is apparent in the primary structure 186,871. Lower degrees of activation were observed with other adenine nucleotides and nucleoside triphosphates. Calcium in submillimolar concentrations has also been identified as a potent activator of 5-LOXs [88,89]. The mechanism by which calcium activates 5LOXs is likely by causing attachment of the enzyme to phospolipids andor biomembranes. In cell-free systems the 5-LOX associates with the membrane fraction in the presence of calcium ions and membrane translocation is reversed upon removal of calcium [90]. In intact cells membrane translocation appears to depend on the presence of the five lipoxygenase activating protein (FLAP) [91]. Phospholipids stimulate the oxygenase activity of purified 5-LOXs and phosphatidylcholine vesicles stabilize the enzyme during the purification procedure [92,93]. Although the mechanism of phospholipid activation is not well understood it was suggested that the 5-LOX reaction proceeds preferentially at the interface between the aqueous solution and the phospholipid vesicle surface. Recently, it has been shown that the mole fraction of arachidonic acid in phosphatidylcholine vesicles appears to be more important for the overall enzyme activity than the total arachidonic acid concentration in the assay system [94]. As other lipoxygenases, 5-LOXs are multifunctional enzymes which not only catalyze the dioxygenation of arachidonic acid to 5-H(P)ETE but also secondary reactions. 5-H(P)ETE is converted via LT& leukotriene synthase activity to the 5.6epoxy derivative LTA4 (Fig. 5-7). During the reducing agent-dependent hydroperoxidase reaction the ferrous 5-LOX catalyzes the homolytic cleavage of the peroxy bond and thereby undergoes oxidation to the ferric form (Fig. 5-3). For the next substrate turnover the ferric enzyme requires reduction to the ferrous lipoxygenase which may be accomplished by the reducing agent. The reducing agent may constitute a polycnoic fatty [94] acid or a redox-based lipoxygenase inhibitor [78,95,96]. 5-LOX undergoes suicidal inactivation during arachidonic acid oxygenation. This inactivation is irreversible since addition of substrate does not restore catalytic activity 1971.

5.6.2 5-lipoxygenase activating protein (FLAP) When human leukocytes are stimulated with calcium ionophore the 5-LOX translocates from the cytosol to cellular membranes, in particular to the nuclear envelope [98]. In granulocytes and rat basophilic leukemia cells 5-LOX activation was found to correlate with membrane translocation of the enzyme [99]. In these cells, a membrane-bound protein FLAP was detected which is required for leukotriene biosynthesis. Co-transfection studies with FLAP and 5-LOX indicated that the expression of both proteins is necessary for leukotriene biosynthesis [loo]. Inhibition of FLAP function by certain drugs, e.g. MK 886, suppressed membrane translocation of thc enzyme and inhibited cellular leukotriene formation. For a long time it was belicved

5.6 5-Lipoxygenases

123

that FLAP may serve as an anchoring protein for membrane binding of the 5-LOX. However, more recent experiments on resting alveolar macrophages indicated that MK886 inhibited leukotriene formation without influencing the membrane binding properties of the enzyme [ 1011. In addition, a FLAP-independent membrane translocation of the 5-LOX was described in osteosarcoma cells which were transfected with the 5-LOX and this membrane binding was not affected by MK886 [ 1021. These data and additional experiments with inhibitors of the cellular leukotriene biosynthesis 1781 suggested that the MK 886 binding site on FLAP does not directly interact with the 5-LOX. The current view of the biological function of FLAP is that this protein presents the fatty acid substrate to the 5-LOX allowing the 5-LOX reaction to proceed in a more efficient way. This hypothesis is supported by the finding that FLAP appears to be a fatty acid binding protein [ 1031. However, the detailed mechanism of stimulation of the 5-LOX reaction by FLAP, in particular the mode of 5-LOXFLAP-arachidonic acid interaction remains to be investigated.

5.6.3 Molecular biology of 5lipoxygenases The human 5-LOX was cloned from various sources including placenta, lung and DMSO-differentiated HL-60 promielocytic leukemia cells [81,1041 and the rat enzyme was cloned from a rat basophilic leukemia (RBL) cell cDNA library [105]. Amino acids 16-28 and 488-512 display a weak homology to the calcium binding site of lipocortin and the residues 368-383 show a significant homology to the interface binding domain of mammalian lipases [78]. The human 5-LOX gene was mapped to chromosome 10 and spans more than 82 kb. Like the genes of other lipoxygenases it is divided into 14 exons and 13 introns [106]. The transcription initiation site is localized 65 bp upstream of the start codon and there are several GCrich boxes which may constitute putative Sp-1 binding sites. Additional putative Sp-1 binding sites were found in the first intron. The promoter region contains consensus sequences for the binding of the transcription factors NFKB, AP-2 and C-myb. Recently, response elements for the retinoid Z receptor and for the retinoid orphan receptor a l were identified in the 5-LOX promoter [ 1071. Homozygous mice in which the 5-LOX gene was disrupted by homologous recombination in embryonic stem cells were created [l08]. These mice are healthy and develop normally. However, when challenged with stimuli of inflammation they showed selective opposition to certain inflammatory insult.

5.6.4 Tissue distribution and regulation of 5-LOX expression The 5-LOX is mainly expressed in myeloic cells such as granulocytes, monocyte/macrophages, mast cells and B-lymphocytes, but not in T-cells, platelets, endothelial cells and erythrocytes [78-801. However, the recent finding of 5-LOX expression in differentiating human skin keratinocytes indicated the potential of extramyeloic cells to expres the enzyme [ 1091. The eicosanoid-synthesizingcapability of neutrophils can be increased in response

I24

5 Lipoxygenases

to inflammatory stimuli such as granulocyte/monocyte colony-stimulating factor (GM-CSF), tumor necrosis factor-a (TNF,), lipopolysaccharide (LPS) and plateletactivating, factor (PAF). Some of these stimuli do not influence the 5-LOX activity but augment the response of neutrophils to second stimuli [SO]. Since this priming effect by GM-CSF could be mimicked by phorbol esters [ 1 101 protein kinase C appears to be involved. Additional studies revealed that GM-CSF priming also involves activation of a pertussis toxin-sensitive G-protein that apparently is not associated with the phosphatidylinositol turnover [I 111 and that GM-CSF primes phospholipase D to (FMLP) 1 1 12I. subsequent stimulation by N-formyl-methionyl-leucyl-phenylalanine During granulopoiesis, expression of the 5-LOX appears to be upregulated. When differentiation of immature HL 60 cells is induced by DMSO 5-LOX mRNA, 5-LOX protein and the enzyme activity is upregulated [I 131. Interestingly, the increase in steady-state concentration of the 5-LOX mRNA was neither due to transcriptional activation nor to augmented stability of the mRNA. It was concluded that the 5-LOX mRNA level might be controlled by a post-transcriptional mechanism involving destabilization of the nascent mRNA in the nucleus of low level expressing cells or by mRNA transport out of the nucleus [ 1131. A similar upregulation of 5-LOX expression was found when HL 60 cell differentiation was induced with 1,25dihydroxycholecalciferol [1141. Transforming growth factor-8 (TGF-P) stimulated 5LOX activity in differentiated HL 60 cells and this effect was augmented by GM-CSF and TNF-a [115]. The most prominent effect was observed at the level of enzyme activity but there was no appreciable increase in 5-LOX mRNA expression. Undifferentiated MonoMac-6 cells express 5-LOX at very low levels. However, when the cells were stimulated with TGF-8 and vitamin D3, expression of the 5-LOX inRNA and of the enzyme protein was strongly induced [ 1 161.

5.6.5 Biological functions of 5-lipoxygenases As indicated above 5-LOXs are involved in the biosynthesis of leukotrienes which are potent mediators of allergic and inflammatory disorders [ 1 17,1181. The peptidoleukotrienes LTC4, LTD4 and LTE4 have strong spasmogenic activity, particularly in airway smooth muscle cells and in the vasculature. When given as aerosol to men they are much more powerful bronchoconstrictors than histamine or methacholine [I 191. In addition, asthmatic patients show enhanced sensitivity to the bronchoconstrictory effects of peptidoleukotrienes[ 1201. Human pulmonary tissue has the capacity to synthesize leukotrienes and an increased biosynthesis of these mediators was found during asthmatic attack or following antigen provocation of allergic subjects [ 121-1231. In contrast to the peptidoleukotrienes which are of particular importance for atopic disorders, LTB4 plays a major role in inflammation. It exhibits chemotactic activity for leukocytes and induces leukocyte migration and vascular dilatation causing plasma exudation [ 1241. In rheumatiod arthritis LTB4 is present at higher concentrations in the synovial fluid [I251 and elevated concentrations of LTB4 are found in biopsies of patients with ulcerative colitis [ 1261. In psoriasis the chemotactic activity of LTB4 is responsible for leukocyte infiltration [ I 271. The involvement of 5-LOX products in the regulation of the immune system has

been a matter of discussion for several years [128]. Originally, it was believed that neither 5-LOX nor LTA4 hydrolase were expressed in lymphocytes. However, more recently the formation of 5-HETE and LTB4 was detected in human B-cells suggesting the expression of both enzymes. The function of immunocompetent cells is regulated by leukotrienes. Specific binding sites for LTB4 exist on lymphocytes of both helper and suppressor subsets and LTB4 augmented the proliferation of mixed T-cell populations. It also stimulates the release of IL-1 and TNF from human monocytes and was identified as a mediator of natural killer (NK) cell function. In differentiated B-cells LTB4 stimulated the production of immunoglobulins, released soluble CD-23 antigen and amplified the expression of MHC class TI antigen in subnanomlar concentrations.

5.7 12-Lipoxygenases Several review articles on mammalian 12-LOX have been published during the last few years [129-1311. The most recent review by Yamamoto et al. [132] summarized the molecular structure,the catalytic activities and the molecular biology of these enzymes.

5.7.1 Subclassification and enzymatic properties With respect to their enzymatic and molecular biological properties mammalian 12LOXs may be subclassified into two groups (Fig. 5-4): a platelet-type 12-LOX and a leukocyte-type 12-LOX [2, 1321. Leukocyte-type 12-LOXs oxygenate all naturally occurring C18 and C20 and C22 fatty acids. In contrast, the platelet type 12-LOXs strongly prefer C20 fatty acids such as arachidonic acid and eicosapentaenoic acid. Neither lipoxygenase isoforms cross-react with polyclonal antibodies raised against the other subtype. The degree of amino acid identity with 15-LOXs is higher for leukocyte-type 12-LOXs (75-85%) than for platelet-type enzymes (60-65%). Moreover, the structure of leukocyte-type 12-LOX genes is more similar to 15-LOX genes than to the genes of the platelet-type enzymes [ 1321. As with all lipoxygenases both 12-LOX subtypes contain nearly equimolar amounts of non-heme iron [ 1331. Site-directed mutagenesis studies on potential iron ligands of the porcine leukocyte 12-LOX suggested His36 1, His366, His54 1 and the C-terminal Leu663 as direct iron ligands [132]. Mutation of the corresponding amino acid of the human platelet 12-LOX also led to an inactive enzyme species [ 134I. 12-LOX catalyzes the stereo-selective removal of the p r o 3 hydrogen from C- I0 of arachidonic acid derivatives [ 1351 and stereospecific oxygen insertion at C-12. In contrast to the platelet-type 12-LOX which exhibits a singular positional specificity (12-HPETE/l5-HPETE ratio of 99 : I), the porcine leukocyte 12-LOX exhibits a dual positional specificity forming a 9 : 1 mixture of 12- and 15-HPETE [ 1361. The murine leukocyte-type 12-LOX produces 12-HPETEIlS-HPETE in a ratio of about 3 : 1 [137] but the platelet-type enzyme of the same species produces 12-HPETE exclu-

I26

5 Lipoxygenases

sively [54]. 12-LOXs accept 15-HPETE as substrate since it contains a doubly allylic methylene at C-10. As major oxygenation products 14(R), 156)-DiHPETE.X(S), 13s)DiHPETE and 14, I5-LTA4 are formed [ 1321. The leukocyte-type 12-LOX is capable of oxygenating complex substrates such as phospolipids, biomembranes and lipoproteins [ 138-1401. In contrast the platelet-type 12-LOX is inactive with these substrates. As with other lipoxygenases both subtypes of 12-LOXs undergo suicidal inactivation during fatty acid oxygenation. Interestingly, the rate of suicidal inactivation of the platelet-type 12-LOX is much lower than that of the leukocyte-type enzyme [ 132I. The mechanism of suicidal inactivation was studied in more detail for the porcine leukocyte 12-LOX and it was found that the enzyme was rapidly inactivated when incubated with I5-HPETE, the minor product of the oxygenase reaction with arachidonic acid. In contrast, 12-HPETE and 13-HPODE inactivated the enzyme at a much lower rate. It was suggested that 15-HPETE is converted to a reactive 14,15-epoxide which in turn reacts with an amino acid residue to form a covalent adduct [ 14 I]. It remains to be investigated which amino acid is modified and whether site-directed mutagenesis of this residue will lead to a mutant enzyme with higher resistance to suicidal inactivation.

5.7.2 Molecular biology of 12-lipoxygenases The first 12-LOX cDNA was cloned from a porcine leukocyte cDNA library 11421 and later on a leukocyte-type 12-LOX was cloned from bovine trachea epithelial cells [ 1431. An overview on molecular cloning and species distribution of leukocyte-type and platelet-type 12-LOXs is given in Yoshimoto and Yamamoto I 1301. Recombinant 12-LOXs have been expressed in several bacterial, non-bacterial expression systems [ 144-1471. The genes for leukocyte-type 12-LOX were cloned from porcine [ 1481 and murine [54,137] genomic libraries and the genes for the platelet-type enzymes were cloned from human [ 149,I501 and murine 154) libraries. As other mammalian lipoxygenase genes they consist of 14 exons and 13 introns but the size of the introns are different for the leukocyte-type and platelet-type enzymes. The genes for the porcine and murine leukocyte-type 12-LOX are 8 kb in size whereas the human and murine platelet-type 12-LOX genes are about twice as big. In addition, a novel lipoxygenaserelated gene was cloned from a murine genomic library. This gene, which was referred to as Aloxe, encoded a 662 amino acid protein and its amino acid composition was approximately 60% identical to both the platelet-type and the leukocyte-type 12LOX [ 15I]. The genes for porcine, murine and human 12-LOXs contain various consensus sequences for transcriptional regulatory factors. TATA-like boxes were found in several 12-LOX but no CCAAT boxes could be detected [ 1301. GC-rich boxes representing potential SP-I binding sites were present in all 12-LOX genes and AP-2 binding sites were found in the promoter region of the porcine leukocyte, the human platelet and the murine platelet 12-LOX. AP-1 binding sites were detected in the gene of the murine leukocyte-type 12-LOX. The human platelet 12-LOX contains a core sequence for a glucocorticoid responsive element, the functional significance of which is unclear. Southern blot analysis suggested the presence of one copy of human

and porcine 12-LOX gene per haploid genome. The chromosomal localization of the human platelet 12-LOX was assigned to the subband p13. I on chromosome 17 11491. Interestingly, the human 15-LOX was mapped to chromosome 17 too I 149). In contrast, the human 5-LOX gene was localized on chromosome 1 0 and its murine homolog was found on chromosome 6 [ 1521. The murine leukocyte-type and platelettype 12-LOXs were mapped to the central region of mouse chromosome 1 1 [54) closely associated with the Aloxe gene [ 1531. This region has homology to the human chromosome 17. Leukocyte-type 12-LOX-deficient mice have been generated I 1981. For this purpose a 6.9 kb fragment of the leukocyte-type 12-LOX gene containing exons 1-8 was isolated and two reverse-oriented neomycin resistance cassettes were introduced into a Stul restriction site within exon 3. Homozygous leukocyte-type I 2-LOX-deficient mice were bred and peritoneal macrophages of these animals did not express 12-LOX mRNA and protein. 12-HETE formation from exogenous arachidonic acid by macrophages was not detectable. The knock-out mice grew normally and were fertile. There were no obvious defects in erythropoiesis and macrophage function, but the lowdensity lipoprotein oxidizing capability of the macrophages was impaired [ 1981.

5.7.3 Tissue distribution and regulation of 12-LOX expression 12-LOXs are expressed in a variety of mammalian cells and tissues (for reviews see Yamamoto [2] and Yamamoto et a1 [132]. It should be stressed that the platelet-type 12-LOX is not only found in thrombocytes of various species or in the precursor megakaryocytes but also in human and murine skin 11301. On the other hand, the leukocyte-type 12-LOX is found in leukocytes of various species, in human adrenal cells, in porcine pituitary, in canine brain, in bovine trachea, in rat pineal gland, aorta, lung, pancreas and spleen and in murine pituitary, pineal gland, spleen and kidney [130, 1321. The 12-LOX activity of various cells and tissues is regulated by hormones, cytokines and growth factors. mRNA levels of the platelet-type 12-LOX in human erythroleukemia cells were increased three- to four-fold when the cells were differentiated with phorbol esters [ 1541. When human epidermoid carcinoma cells A43 I were cultured in the presence of epidermal growth factor (EGF) for I0 h the 12-LOX activity was augmented and an induction of the platelet type 12-LOX was indicated by Northem and Western blot analysis [ 1551.

5.7.4 Biological functions of 12-lipoxygenases Although 12-LOXs have been known for more than 30 years the biological role of these enzymes is still unclear. In recent years a number of papers from various laboratories have reported interesting biological activities of 12-LOX rnctabolites such a\ 12-H(P)ETE, lipoxins and hepoxilins [ 1321. Hepoxilins are hydroxy-epoxy eicosanoids (Fig. 5-8) which are formed from 12HPETE via enzymatic and/or non-enzymatic reactions (see also Section 1.36. They ex-

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5 Lipoxygenases

Figure 5-8. Chemical structure and biosynthesis of hepoxilins. The two hepoxilin isomers A and B are formed from 12.3-HPETEvia enzymatic and/or non-enzymatic reactions (for details see text)

lipoxin A4

lipoxin B4

*"'

no

I ,

*on

Figure 5-9. Chemical structure of lipoxins. Both lipoxin isomers contain a conjugated tetraene chromophore with local absorbance maxima at 286,301 and 314 nm.

hibit interesting biological activities which are mainly related to modulation of intracellular calcium concentration and opening of potassium channels. In addition, hepoxilin A (HXA) was shown to block norepinephrine release from rat hippocampal slices. A review on the biosynthesis and the biological activities of hepoxilins is given in reference [ 1561. Lipoxins (Fig. 5-9) are bioactive lipid mediators in the biosynthesis of which 12LOXs may be involved (see Section 1.3.4). These trihydroxy eicosanoids, which contain a conjugated tetraene system, are synthesized unicellularly in leukocytes via concerted action of 5- and 15-LOXs or transcellularly by the leukocyte-type 5-LOX and the platelet-type 12-LOX. They appear to be involved in the regulation of immunological and hemodynamic processes. Reviews on the biosynthesis of lipoxins and their potential physiological and pathophysiological role are also available [ 157. 1581.

5.8 Mamrnuliun 15 -1ipuxygenase.r

129

12-HETE exhibits chemotactic activity for human eosinophils, neutrophils and tumor cells [ 1591. It inhibits collagen-induced platelet aggregation but stimulated thrombin-induced aggregation [ 1601. At very low concentrations (3 fh4) I2-HETE stimulated the migration of rat aortic smooth muscle cells [161] and a prolonged constriction of basilar artery was induced by 12-HPETE 11621. 12- and 15-HETE increase the release of mucus proteins from human airways in vitro in nM concentrations [163]. Large amounts of 12-HETE were detected in psoriatic skin. Steric analysis of this compound indicated that the R-isomer was mainly present [ 1641 and, thus, the 12-LOX isoforms described here may not be involved in biosynthesis. On the other hand, a platelet-type 12-LOX producing 12(S)-HETEis expressed in the germinal layer of normal and psoriatic skin [ 1651. In addition, a reduced nicotinamide adenine dinucleotide phosphate dependent monooxygenase was detected in human epidermis and this enzyme produces 12(S)-HETE when incubated with arachidonic acid [166]. Extensive studies have been carried out on the role of 12-HETE in carcinogenesis and metastasis [ 1671. 12-HETE stimulated tumor cell adhesion to endothelial cells, the subendothelial matrix and fibronectin [168]. The ability of tumor cells to synthesize 12-HETE appears to correlate with their metastatic potential [ 1691. Since the leukocyte-type 12-LOXs are capable of oxidizing more complex substrates such as biomembranes and lipoproteins they may exhibit biological activities outside the arachidonic acid cascade (see Section 5.8.4.1).

5.8 Mammalian 15 -1ipoxygenases For mammalian 15-LOXs there is no current review summarizing the protein chemistry, the enzymology and the cell physiology of these enzymes. However, an overview on the molecular biology of 15-LOXs has recently been published [170]. Less current reviews appeared in 1986 [171] and 1991 [172], but in the meantime major advances in structural biology [40] and cell physiology [173] have been made and new hypotheses on their pathophysiological importance have been suggested [ 1741. A recent review on the biological role of 15-LOX products, in particular 15-H(P)ETE and 13-HPODE, was published in 1996 [175] but this article does not cover protein chemical and enzymatic aspects.

5.8.1 Subclassification and enzyme properties As indicated above (Fig. 5-4) mammalian 15-LOX may be subclassified into reticulocyte-type and epidermis-type enzymes. Both types of enzyme have an approximate molecular weight of 75 kDa but only share a small degree of amino acid identity l.591. The best characterized 15-LOX is that from rabbit reticulocytes. It constitutes a single polypeptide chain with a molecular weight of 75 kDa [ 151 and its recently solved 3-D structure indicates two domains (see Section 5.1). For the catalytic activity o f 1 SLOXs the two complete domains are necessary. Truncation from either the N- or the

130

5 Lipoxygenuses

C-terminus led to an inactive enzyme species. The rabbit and the human reticulocytetype 15-LOXs contain 1mole non-heme iron per mole of enzyme. Four histidines and the C-terminal isoleucine were identified as direct iron ligands (see Section 5 . I ). The reticulocyte-type 15-LOX is characterized by a rather broad substrate specificity. All major naturally occurring polyenoic fatty acids such as (5Z.SZ. 1 1Z. 142)eicosa-5,8,11,14-tetraenoicacid (arachidonic acid), (5Z,8Z,11Z, 132,17Z)-eicosa5,8,11, 14,17-pentaenoic acid, (9Z,12Z)-octadeca-9,12-dienoicacid (linoleic acid) and (9Z, 12Z,15Z)-octadeca-9,12,15-trienoic acid (a-linolenic acid) are accepted as substrates. In addition, ester lipids (phospholipids, cholesterol esters and mono-. diand triacyilglycerides)containing polyenoic fatty acid as well as more complex lipidprotein assemblies (biomembranes and lipoproteins) are oxygenated by these enzymes. Reticulocyte-type 15-LOXs exhibit a dual positional specificity; they oxygenate arachidonic acid to a 9 : 1 mixture of 15- and IZHPETE. As other lipoxygenases, 15-LOXs undergo suicidal inactivation. Some years ago it was reported that the fatty acid hydroperoxide formed during the oxygenase reaction oxidizes a methionine residue at the active site of the enzyme to methionine sulfoxide [ 1761. The oxidizable methionine was recently identified in the primary structure and mutated to leucine 1 177I. Surprisingly, the mutant enzyme also underwent suicidal inactivation. These data suggested that methionine oxidation may not be the major cause of suicidal inactivation. The epidermis-type 15-LOX which has been cloned recently from human hair roots is also expressed in the prostate, the lung and in cornea 1591. A PCR screening of 16 different tissues including peripheral blood leukocytes gave negative results. The cDNA encodes for 676 amino acids and the predicted molecular weight is 76 kDa. When expressed in HEK 293 cells the enzyme converts ardchidonic acid exclusively to 15s-HPETE. In contrast to the reticulocyte-type 15-LOX, linoleic acid is less well metabolized. It might be possible that the epidermis-type 15-LOX is the human homolog of the phorbol ester inducible S-LOX of murine skin 11781. More work is needed in the future to further characterize this interesting 15-LOX isoenzyme with respect to its protein chemical and enzymatic properties and to obtain information on its biological role.

5.8.2 Molecular biology of the reticulocyte-type 15lipoxygenases Complete cDNAs have been cloned from rabbit and human reticulocyte cDNA libraries [179,180] and both enzymes share an 81% amino acid identity. From the amino acid sequence no hydrophobic membrane-spanning domains can be predicted. This is in line with the cytosolic localization of the enzyme in rabbit reticulocytes 1151 but does not exclude an additional association with cellular membrane?. The 5' prime untranslated region (UTR) of rabbit 15-LOX is relatively short and there are no indications for a binding motif of cytosolic proteins in the 5' prime 4TR.. In contrast, the 3' prime UTR of the rabbit 15-LOX mRNA is larger and contains a tandemly arranged tenfold repetitive element with the consensus sequence C4AAGC4A/GC1TCTTapproximately 50 nucleotides downstream of the stop codon [ 1801.This sequence, which also occurs in modified forms in the mRNA of other lipoxygenases 11701, has been suggested a\ the translational control element [ 1SO].

5.8 Maminalian 1.5 -1ipoxygenases

131

Structures of 15-LOX genes including the ?prime flanking promoter regions are available for both the rabbit [ 1811 and the human [ 182) reticulocyte-type 15-LOXs. The human gene was mapped to chromosome 17 [149]. As indicated above the intron-exon organization of all mammalian lipoxygenase genes is highly conserved ( 14 exons and 13 introns), but the length of the introns is variable. The rabbit 15-LOX gene [181] is 8 kb in length. The promoter contains a TATA-like motif (TATTTA), two CACCC regions and a CCAAT motif rabbit. Another putative regulatory element in the 15-LOX promoter has been identified as the NF-1 binding site. Intron 7 contains an eight-fold multiple repetitive sequence with homology to the CMV/SV40 immunoglobulin enhancer which is also present in one copy in intron 4. Furthermore, one GC-box (Sp-1 binding site) is present in the promoter region and in intron 2. Two putative STAT6 binding sites have been identified in the rabbit promoter which might be of functional relevance because of the inducibility of the enzyme by IL-4. The promoter region of the human reticulocyte-type 15-LOX contains no typical CCAAT box but a TAITT motif is present 57 nucleotides upstream of the initiating ATG. Two AP-2 binding sites are found at positions -580 and -75214. GC-rich boxes, which are potential SP-1 binding sites, are also present. In addition, two GATA-1 binding sites and a transcriptional silencer sequence were identified. It may be of particular regulatory importance for the expression of the human 15-LOX that three STAT 6 binding sites with the consensus sequence l T C N”N GAA (-723 to -732, 849 to -858 and -944 to -953) and a shear stress responsive element (-1 15 to -120) were identified.

5.8.3 Tissue distribution and regulation of 15-LOX expression The reticulocyte-type 15-LOX is expressed in a variety of mammalian cells and tissues such as rabbit and human reticulocytes, human eosinophils, human airway epithelial cells, polymorphonuclear leukocytes, alveolar macrophages-inflamed tissue, vascular cells, uterus, placenta and various cells of the male reproductive system [172,175]. Rabbit reticulocytes express approximately 4 mg of 15-LOX per milliliter of packed cells [ 151 and, thus, these cells constitute the major source of preparation of the native enzyme. The expression of 15-LOX appears to be highly regulated at pre-translational, translational and post-translational levels. Alveolar macrophages and foamy macrophages of atherosclerotic lesions express 15-LOX [ 183,1841 but the precursor monocytes lack the enzyme [185]. Thus, 15-LOX must be induced during monocytemacrophage transition. When peripheral monocytes are cultured in v i m in the presence of the IL-4 or IL-13 expression of the enzyme is strongly upregulated [185,186]. A similar induction was shown for the human lung carcinoma cell line A549 [187]. Since this induction can be detected at the levels of mRNA, enzyme protein and enzymatic activity, a pre-translational regulation was concluded. As typical example for translational regulation of 15-LOX expression, rabbit reticulocytes, may be discussed. In young reticulocytes the 15-LOX mRNA is present as translationally inactive mRNP (mRNA-protein complex). At this maturation stage no 15-LOX protein can be detected [ 1881. A regulatory protein which is capable of

132

5 Lipoxygenuses

binding to a repetitive motif in the 3' prime UTR of the IS-LOX mRNA and thereby preventing its translation has recently been identified I 1891. This lipoxygenase mRNA binding protein may be cleaved proteolytically at later stages of erythropoiesis and the 15-LOX may be synthesized. In mature erythrocytes the enzyme is not detectable any more because it undergoes suicidal inactivation and proteolytic breakdown. These data indicate that 15-LOX expression only proceeds in a narrow time window during the lifespan of red cells. After being expressed the specific activity of 15-LOX can also be regulated. Increase in the cytosolic calcium concentration induces translocation of the cnzyme to various intracellular membranes [ 1731. This membrane translocation is a precondition for oxygenation of the membrane lipids but also causes a drastic increase in the fatty acid oxygenase activity. A second element of regulation of the enzymatic activity is the cellular hydroperoxide concentration. Lipoxygenases require small amounts of hydroperoxides for activation. In fact, in vitro experiments indicated that 1 5-LOXcatalyzed oxidation of membrane lipids is completely inhibited if the membranes are depleted of hydroperoxides [190]. Thus, the cellular hydroperoxide tone may be regarded as intracellular modulator of 15-LOX activity. In addition to hydroperoxides, there may be other oxidizing molecules capable of converting the inactive ferrous lipoxygenase to an activated ferric form. EPR measurements and X-ray absorption studies indicated that nitric oxide may be capable of oxidizing the ferrous rabbit 15LOX [191]. However, it remains to be investigated whether nitric oxide may function as regulator in vivo. Since rabbit reticulocytes constitute the only source for large-scale preparation of a native mammalian 15-LOX, recombinant expression systems have been developed. Since the recombinant enzyme is only expressed at a low level in bacteria [ 192,1931 the baculovirushnsect cell expression system was employed [ 194,1951. In this system the overall yield of 15-LOX expression varied between 10 and 50 mg of purified enzyme per liter of culture fluid and the specific activity of the final enzyme preparation was comparable to that of the native enzyme. All original enzymatic characteristics of the native 15-LOX were retained in the recombinant enzyme [ 194,1951.

5.8.4 Biological functions of 15-lipoxygenases Although no general concept for the biological role of 15-LOXs is available, there are several hypotheses for the physiological importance of these enzymes: (1) structural modification of complex lipid-protein assemblies,

(2) modulation of intracellular lipid signal transducers and (3) formation of bioactive oxygenated fatty acid derivatives.

5.8.4.1 Structural modification of lipid-protein assemblies. Implication in cell maturation and atherogenesis Mammalian erythrocytes develop from pluripotent stem cells via a cascade of precursor cells, the most mature of which is the reticulocyte. The reticulocyte-erythrocyte

5.8 Mummaliun 15 -lipoxygenuses

133

transition is characterized inter alia by a breakdown of mitochondria and the 15-LOX has been implicated in this process [196]. The biological dynamics of 15-LOX expression during reticulocyte maturation coincides in time with the breakdown of mitochondria and oxygenated fatty acids can be detected in the mitochondria1 membranes [ 1971. Moreover, lipoxygenase inhibitors delay the maturational decline of cytochrome c oxidase which is a marker enzyme of cellular respiration [197]. In order to test this concept transgenic knock-out mice were created. Since no arachidonate 15-LOX has been identified in mice, the gene for the leukocyte-type 12-LOX, which is supposed to be the murine equivalent of the human and rabbit reticulocyte-type 15LOX, was disrupted [ 1981. The transgenic animals did not have any obvious problems with erythropoiesis; all major red cell parameters were normal. These data suggest that, at least in mice, erythropoiesis may proceed in the absence of the leukocytetype 12-LOX. However, it remains to be investigated whether defects may appear if erythropoiesis is challenged in certain ways and whether there are species differences. The reticulocyte-type 15-LOX is expressed in foamy macrophages of atherosclerotic lesions [183] but is not detectable in normal vessel walls. Since the enzyme is capable of oxidizing LDL in v i m to an atherogenic form [199], it was hypothesized that the enzyme may play a pro-atherogenic role in early atherogenesis. Recent studies with a specific 15-LOX inhibitor appear to support this hypothesis [200]. To obtain independent evidence for the pro-atherogenic role of the enzyme, transgenic rabbits were created which overexpress the human 15-LOX in monocytes-macrophages [201]. When these rabbits were fed a cholesterol-rich diet they were protected from lesion development [202]. Similar results were obtained when these animals were cross-bred with low-density lipoprotein (LDL) receptor-deficient rabbits [202]. These data leave us with the paradox that the 15-LOX may act pro- and/or anti-atherogenic. It may be possible that the overall pathogenetic role of the enzyme may change during the time course of atherosclerosis development [ 1741. During early stages, the enzyme may exhibit an anti-atherogenic effect facilitating the metabolism of internalized LDL. At later stages, the 15-LOX may be released from the cells and, thus, may contribute to oxidizing LDL to an atherogenic form (pro-atherogenic action).

5.8.4.2 Modulation of intracellular lipid signal transducers In several cellular systems (renal tubular epithelial cells, human tracheal epithelial cells, bovine pulmonary arterial endothelial cells and human leukocytes), 15-HETE is esterified specifically into the cellular phosphatidylinositol pool [203-2061. The 15HETE-phosphatidylinositolmay be cleaved by a phospholipase C . The resulting oxidized diacylglycerol was suggested as a modulator of protein kinase C (PKC) activity. In epidermal slices, 13-HODE, another 15-LOX metabolite, is incorporated into the cellular phosphatidylcholine (PC) and phosphatidylinositol (PI) pools whereas only minor incorporation into ceramide was observed [207]. Here again, 13-HODEcontaining diacylglycerols were detected and a modulation of PKC activity by these oxidized lipid mediators was suggested. In rat neonatal cardiomyocytes the preferential incorporation of 1SS-HETE into the PI induced supersensitivity towards pool 0agonists [208]. This effect was very specific, because only 15s- and 1 1 S-HETE were capable of inducing supersensitivity whereas other positional and optical isomcrs

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were ineffective. Since calphostine, an inhibitor of PKC, blocked 15-HETE-induccd supersensitivityit was concluded that PKC may be involved.

5.8.4.3 Formation of bioactive oxygenated fatty acid derivatives The major 15-LOX products, 15-H(P)ETE and 13-H(P)ODE, exhibit interesting biological activities which have been reviewed recently [ 1751. 13-HPODE and 15HPETE are regulators of various enzymes of the arachidonic acid cascade (5-LOX and cyclooxygenase-1); activation and inactivation of these enzymes have been described. At the cellular level, 15-HETE and 13-HODE have been implicated in cell proliferation, cell adhesion and metastasis. Exogenous 15-HETE induces contraction of human bronchial smooth muscle cells at submicromolar concentrations, but this effect appears to be restricted to in vitro conditions since inhaled 15-HETE did not exhibit any effect on airway caliber in either normal or asthmatic individuals. In contrast, pre-inhalation of 15-HETE did significantly increase the early allergic response [ 1751. There are several reports suggesting a role of 15-LOX metabolites in hormone synthesis, in diabetes mellitus and in the male and female reproductive system [ 1751. The formation 15-H(P)ETE or 13-H(P)ODE is often elevated in inflamed tissue and it was believed for a long time that these compounds may exhibit proinflammatory activities. However, new experimental data suggested that 1 5-LOX products may act as anti-inflammatory agents. According to a newly developed concept, the in vivo activity of the 15-LOX may be regarded as a protective response to limit or reverse inflammatory symptoms and to maintain basic cell functions [209].

5.9 References C.D. Funk. Biochim. Biophys. Acta 1996; 1304: 65-84. S. Yamamoto. Biochim. Biophys. Acta 1992; 1128: 117-131. J.N. Siedow. Annu. Rev. Physiol. Plant Mol. Biol. 1991; 42: 145-188. G.A. Veldink, M.P. Hilbers and J.F.G. Vliegenthart. In Eicosunoich und Related Compounds in Plants and Animals (A.F. Rowley, H. Kuhn and T. Schewe. eds). Portland Press, London, 1998, pp. 69-96. D.J. Hawkins, A.R. Brash. J. Biol. Chem. 1987; 262: 7629-7634. A.R. Brash, M.A. Hughes, D.J. Hawkins, W.E. Boeglin, W.C. Song, L. Meijer. J. Biol. Chem. 1987; 266: 22926-2293 1. T. Hada, L.L. Swift, A.R. Brash. Biochim. Biophys. Actu 1997; 1346: 109-1 19. A.R. Brash, W.E. Boeglin, M.S. Chang, B.H. Shieh. J. Biol. Chern. 1996; 271: 20949-20957.

I121 1131

E. Andre, K Hou. Comptes Rendus 1932; 194: 645-647. A.L. Tappel. In The Enzymes,Vol. 8 ( P.D. Boyer, H. Lardy and K. Myrback, eds). pp. 275-283. Academic Press, New York, 1963. J.F.G. Vliegenthart, G.A. Veldink. In Free Radicals in Biology (Ed.: W.A. Pryor). Academic Press, New York, 1982, Vol. 5, pp. 29-64. D. Shibata and B. Axelrod. J . LipidMed. Cell Signal. 1995; 12: 213-228. M. Hamberg, B. Samuelsson. Proc. Nut1 Acad. Sci. USA 1974; 71: 3400-3404.

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D.L. Sloane, C.S. Craik, E. Sigal. Biomed. Biochim. Acru 1990; 49: 11-16. H. Kuhn, J. Barnett, D. Crunberger, P. Baecker, J. Chow, B. Nguyen, H. BursztynPettegrew, H. Chan, E. Sigal. Biochim. Biophys. Acta 1993; 1169: 80-89. S. Borngraber, E. Grabenhorst, M. Anton, H. Conradt, H. Kuhn. Protein Expr. Puri~ T c . 1998; 14: 237-246. T. Schewe, and H. Kuhn. Trends Biochem. Sci. I991 ; 16: 369-373. S.M.Rapoport, T. Schewe, B.J. Thiele. Blood Cell Biochemistry Vol. I , Plenum, New York, 1990. D. Sun, C.D. Funk. J. Biol. Chem. 1996; 271: 24055-24062. J. Belkner, R. Wiesner, J. Rathman, J. Barnett, E. Sigal, H. Kuhn. Eur. J. Biochem. 1993,213: 251-261. S.M. Sendobry, J.A. Cornicelli, K. Welch, T. Bocan, B. Tait, B.K. Trivedi, N. Colbry, R.D. Dyer, S.J. Feinmark, A. Daugherty. A. Br. J. Phurmucol. 1997; 720: 11991206 J. Shen, H. Kuhn, A. Petho-Schramm, L. Chan. FASEB .I. 1995; 9: 1623-1631. J. Shen, E. Herderick, J.F. Cornhill, E. Zsigmond, H.S. Kim, H. Kuhn, N. Valentinova, L. Chan. J. Clin. Invest 1996; 98: 2201-2208. A.B. Legrand, J.A. Lawson, B.O. Meyrick, LA. Blair, J.A. Oates, J.A.. J. Biol. Chem. 1991; 266: 7570-757. M.E. Brezinski, C.N. Serhan. Proc. Nutl. Acad. Sci. USA 1990; 87: 6248-6252 R.A. Girton, A.A. Spector, J.A. Gordon. Kidney Int. 1994; 45: 972-980. S.E. Alpert, R.W. Walenga. Am. J. Respirut. Cell. Mol. Biol. 1993; 8: 273-281. Y. Cho, V.A. Ziboh. J. Lipid Res. 1994; 35: 255-262. G. Wallukat, R. Morwinski, H. Kuhn. J. Biol. Chem. 1994; 269: 29055-29060. S. Takata, M. Matsubara, P.O. Alien, P.A. Janmey, C.N. Serhan, H.R. Brady. J. Clin. Invest 1994; 93: 499-508.

Prostaglandins, Leukotrienes and Other Eicosanoids: From Biogenesis to Clinical Application Edited by F. Marks, G. Fürstenberger Copyright © WILEY-VCH Verlag GmbH, D-69469 Weinheim (Federal Republic of Germany). 1999

6 Oxygenation of arachidonic Acid by cytochromes P-450 Emst H. Oliw

6.1 Introduction Cytochrome P-450 designates a group of heme-containing enzymes, which absorb light at 450 nm after reduction and treatment with carbon monoxide. This characteristic property is due to the heme group with its iron in ferric state bound to a cysteine residue. These enzymes are therefore also called heme-thiolate proteins. They can catalyze oxygenation of both foreign and endogenous compounds, e.g. fatty acids, steroids and bile acids, retinoids, prostaglandins and leukotrienes [ 1,2]. The mammalian cytochromes P-450 belong to a gene superfamily, which consists of 14 gene families and 26 subfamilies [2]. Partial or complete sequences of approximately 500 cytochromes P-450 have been published and approximately half of them are mammalian. The available protein sequence information suggests that the gene superfamily has a long history of evolution. The ancestral gene may have existed 3.5 billion years ago [2]. Reviews on the P-450 gene superfamily with updates on new sequences, gene mapping and recommended nomenclature are published regularly (see Nelson et al. [2]). Recent information can also be conveniently obtained from the Internet (for example, http://www.icgeb.trieste.it/P45O/J Oxygenation of fatty acids and other substrates occurs by cytochromes P-450 according to the formula:

RH + 0

2

+ NADPH + H+ A ROH + H20 + NADP'

Actually, the oxidation process is more complicated than this formula suggests. Electrons are transferred from NADPH (or NADH) to cytochromes P-450 via a second enzyme, cytochrome P-450 reductase. During oxidation, one atom of oxygen will be incorporated in the substrate and the other reduced to water. These enzymes are therefore also called monooxygenases. Mammalian cytochromes P-450 are bound to membranes and it has been difficult to obtain suitable crystals for X-ray crystallography. The three-dimensional structure has therefore not yet been determined. Cytochrome P-450 is also present in plants, fungi and bacteria and some of them are soluble and have been crystallized. The 3-D structures of four soluble enzymes have been reported: cytochrome P-450cam (CYP101) of Pseudomonas putidu, cytochrome P-450-BM3 (CYP102) of Bacillus megaterium, cytochrome P-450terp (CUP 108) of a Pseudumonas species and cyto-

144

6 Oxygenation ofarachidonic acid by cytochromes P-450

chrome P-450eryF of Succharopolysporu erythren [ 3-61. One of them, cytochrome P450-BM3, has a remarkably high capacity for oxygenating arachidonic acid with both position specificity and stereo selectivity [7, 81.

6.2 Early work on oxidation of fatty acids by cytochromes P-450 Cytochromes P-450 have traditionally often been studied with fatty acids as substrates. These studies began before cytochrome P-450 was even discovered. oOxidation of saturated fatty acids was described in vivo by Verkade et ul. [91 in the 1930s. About 20 years later liver microsomes were found to catalyse wl- and 02hydroxylation of fatty acids in the presence of NADPH and molecular oxygen 110131. Cytochrome P-450 was discovered and named soon thereafter [ 14,151. Early work on fatty acid metabolism by cytochromes P-450 concentrated on the 01- and o2-hydroxylation of saturated fatty acids by liver microsomes. The rate limiting step in hydroxylation of deuterium-labeled laurate (12 : 0) was found to be hydrogen abstraction [16,17]. The position of the hydroxyl group was the same as that of the removed hydrogen. These observations indicated that a short-lived carboncentered radical was formed during catalysis. o-Oxidation of fatty acids in vivo was found to be small (10-1 5%) in comparison with 0-oxidation under normal conditions [ 18-20], but o-oxidation of fatty acids was increased by starvation and by experimental diabetes [19, 21, 221. In addition to the liver, hydroxylation of fatty acids was soon found in microsomes of kidney cortex from several species [23]. wl- and o2-hydroxylation of saturated fatty acids also occur in the lung, the gut and some other organs. Oxygenation of arachidonic acid by cytochrome P-450 was first reported about 30 years ago. Arachidonic acid was metabolized by a fungal root pathogen of wheat, the take-all fungus, to 19-hydroxyeicosatetranoic acid ( I 9-HETE) and to 18-hydroxyeicosatetranoic acid (18-HETE), apparently by w2- and 03- hydroxylation 124,251. The first structural analysis of mammalian cytochrome P-450 metabolites of arachidonic acid was performed in the rabbit kidney [26]. Low-speed supernatant or microsomes of rabbit renal cortex and NADPH metabolized ['4C]arachidonic acid to many metabolites. These were 14,15-dihydroxy-5,8,Il-eicosatrienoicacid ( 14,I5DHET), 1 1,12-dihydroxy-5,8,14-eicosatrienoic acid (1 1,12-DHET), 19-HETE and 20-HETE. The biosynthesis of these metabolites required NADPH and was inhibited by CO. These findings suggested that cytochromes P-450 might be involved in the epoxidation and o-hydroxylation of arachidonic acid. Early studies also demonstrated that arachidonic acid was converted enzymically and not by lipid peroxidation to six cis-trans conjugated hydroxyeicosatetraenoic acids (HETEs) by rat liver microsomes and NADPH [27]. This oxygenation has been referred to as allylic hydroxylation or hydroxylation with double bond migration. Only a few years ago, a fourth hydroxylation of arachidonic acid was found. The three bisallylic carbons of arachidonic acid (C7, Cloand CI3)can also be hydroxylated

6.2 Early work on oxidation offatty ucids by cytochromes P-4-50

145

by cytochrome P-450 (28-3 1). These metabolites are acid-labile and decompose easily to cis-trans conjugated HETEs. Arachidonic acid can thus be oxygenated by cytochromes P-450 in four ways [Fig. 6.1 J: ( 1 ) Hydroxylation of carbons of the w-side chain to 20-HETE, 19-HETE, Ig-HETE, 17-HETE and 16-HETE. (2) Epoxidation of double bonds to epoxy eicosatrienoic acids (EET), i.e, 5,6-EET, 8,9-EET, 1 1,12-EET and 14,15-EET. (3) Hydroxylation of carbons located between double bonds (i.e. the bisallylic carbons) to 7-HETE, 10-HETE and 13-HETE. (4) Hydroxylation with double bond migration to 5-HETE, 8-HETE, 9-HETE, 11HETE, 12R-HETE and 1SR-HETE. Six cis-trans Conjugated HETEs

A Four EETs

-

t ~

'

"

Arachidonic Acid

"

I)

" Three Bisallylic HETEs

Omega Side Chain Hydroxylation

Figure 6-1. Major routes for oxygenation of arachidonic acid by cytochromes P-450. EETs, epoxyeicosatrienoic acids; HETEs, hydroxyeicosatetraenoicacids.

The first three oxygenation mechanisms are quantitatively dominant, at least in liver microsomes. Arachidonic acid has been intensively investigated due to its biological importance as a precursor of many autacoids in mammals. Linoleic acid seems to have a corresponding function in algae, fungi and plants. Linoleic acid is also a major unsaturated fatty acid in the human diet. This chapter will focus on the oxygenation of arachidonic. Linoleic acid and many other polyunsaturated fatty acids (PUFAs) are metabolized in a similar way. For linoleic acid the corresponding products will be: (1) hydroxylation of carbons of the w-side chain to 18-hydroxyoctadecadienoic acid (18-HODE), 17-HODE, etc., (2) epoxidation to 9,10-epoxy- and 12,13-epoxyoctadecenoicacids, (3) bisallylic hydroxylation to 11-HODE and (4)hydroxylation with double bond migration to 9R-HODE and 13R-HODE.

Epoxyeicosatrienoic acids and their corresponding dihydroxyeicosatrienoic acids

146

6 Oxygenation of arachidonic acid by c.vtochrornes P-4-50

are often abbreviated as EET and DHET. More descriptive abbreviations of epoxyeicosatrienoic acid and dihydroxyeicosatrienoic acid are EpETrE and DiHETrE. respectively, which are also in accordance with the proposed standard nomenclature [32]. In this chapter there is little risk of confusion with epoxides and diols of other unsaturated eicosanoic fatty acids. The abbreviations EET and DHET will be used tbr convenience and HODE for hydroxyoctadecadienoic acids. CYP will be used for cytochrome P-450 in connection with names of enzyme families and subfamilies.

6.3 Oxygenation of arachidonic acid by cytochromes P-450 Mammalian cells can oxygenate arachidonic acid by three different en7.ymes: cyclooxygenases, lipoxygenases and cytochromes P-450. The biological importance of the first two pathways is well established. A series of observations suggest that the cytochrome P-450 pathways can also be of physiological importance.

6.3.1 Hydroxylation of o side chain Early reports showed that purified hepatic cytochrome P-450 from P-naphthoflavonetreated rabbits mainly formed 19-HETE and smaller amounts of 20-HETE [ 3 3 ] ,while ciprofibrate- and clofibrate-induced rat liver microsomes formed 20-HETE as the main metabolite [34,35]. It is well-known that enzymes of the CYP4A gene family can o-hydroxylate a large number of fatty acids, prostaglandins and other eicosanoids [36,37]. CYP4AI 1 from human kidney was cloned and found to metabolize arachidonic acid and saturated fatty acids by o-hydroxylation (38). The mechanism of o-side chain hydroxylation by cytochrome P-450 has been studied in detail. Cytochrome P-450 catalyzes o2-hydroxylation of fatty acids by hydrogen abstraction and insertion of the hydroxyl group with retention of configuration 116,391. The o-hydroxylation of (1R)- and (1s)-[ I-”, lH, 1H;1-14C]octaneto I octanol by microsomes of rat liver and NADPH has also been investigated 1401. The incoming hydroxyl assumed the configuration of the displaced hydrogen. However, there are also many exceptions to this general rule and the incoming hydroxyl may assume the opposite configuration [41,42]. Carbon atoms 16, 17 and 18 of arachidonic acid can also be hydroxylated by heor 3-methylpatic microsomes of P-naphthoflavone-, 3,4,5,3’,4’-pentachlorobiphenylcholanthrene-treated rats 143,441. These results demonstrate that the entire o side chain of arachidonic acid can be oxidized. Whether all these products are also formed in vivo is unknown, but 20-HETE and a glucuronide conjugate of 20-HETE have been identified in human urine [45]. Laethem et al. (46) reported that CYP2E1, which is induced in rat liver by ethanol. metabolized arachidonic acid to 19R-HETE, 19s-HETE and 18R-HETE. In addition. small amounts of the four epoxides of arachidonic acid were also produced. It i\

6.3 Oxygenation of arachidonic acid b.y cytochromes P-450

147

known that CYP2E1 mRNA is increased by diabetes and fasting and these metabolites might be increased in these disorders. However, analysis of a large series of human recombinant cytochromes P-450 showed that almost all enzymes could form 01 and/or w2 hydroxy metabolites of arachidonic acid [47,48].

6.3.2 Epoxidation Arachidonic acid has four double bonds and all of them can be oxygenated to epoxides, which can be rapidly hydrated by epoxide hydrolases to vicinal diols. 1 1,12DHET and 14,15-DHET were first identified in the kidney and liver [26,49,50]. The four epoxides of arachidonic acid were then isolated from microsomal preparations with epoxide hydrolase inhibitors and from purified phenobarbital-induced rat and rabbit liver cytochrome P-450 (CYP2B 1 and CYP2B4, respectively) without epoxide hydrolase activity [33]. Epoxides of arachidonic acid were converted by purified microsomal epoxide hydrolase to diols [33]. Falck et al. [5l] determined the absolute configuration of epoxides formed by phenobarbital-induced, purified rat liver cytochrome P-450. Except for 5,6-epoxy-8,1lI14-eicosatrienoic acid (5,6-EET), the epoxides were formed with significant stereoselectivity. For example, 14,15-EET consisted of 80% R,S and 20% S,R, while 8,9-epoxy-5,11,14-eicosatrienoic acid (8,9EET) and 1 I,12-EET were almost pure (97% of the R,S and S,R enantiomers, respectively). These results were important, since racemic epoxides of fatty acids can also be formed non-enzymatically [52]. Epoxides of arachidonic acid were soon detected in tissues and body fluids, e.g. in rabbit kidney, rat liver, human and rat urine and human and rat blood plasma [53-581. DHETs were also excreted in human urine [58]. Strong evidence for in wivo formation of epoxides was provided by chiral analysis. The absolute configuration of endogenous epoxides in phospholipids of the liver of untreated or phenobarbital-treated rats was determined by chiral high peformance liquid chromatography (HPLC) [59]. 8,9EET and 11,12-EET consisted mainly of the S,R enantiomers (79 and 63% of untreated and 99 and 83% in phenobarbital treated livers, respectively) and 14,15-EET of the R,S enantiomer (77 and 94%, respectively). Phenobarbital treatment thus had a large effect on the stereochemistry. Liver microsomes of phenobarbital treated rats also formed different enantiomers of 11,12-EET than liver microsomes of untreated rats. Karara et al. [60] demonstrated an enantioselective preference for incorporation of 8S, 9R-EET, 1 lS,12R-EET and 14R,15S-EET at sn-2 of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol of rat liver. These enantiomers were also identified after hydrolysis of phospholipids. In the human kidney cortex [61], 8,9-EET was racemic and the R,S enantiomers of 1 1,12-EET and 14,15-EET dominated (80 and 66%, respectively). In quantitative terms, 14,15-EET was the major epoxide (44%), followed by 1 1,lZEET (33%) and 8,9-EET (23%). An arachidonic acid epoxygenase was purified from rabbit renal cortex and designated P-450 2CAA [62]. This enzyme formed I1,12-EET (45%) and 14,15-EET (30%) as the two major products. Laethem and Koop [63] showed that rabbit CYP2Cl transiently expressed in COS-I cells, catalyzed the biosynthesis of 1 I , 12EET, 14,15-EET and 19-HETE (about 48, 16 and 13% of metabolites, respectively).

148

6 Oxygenation of aruclzidonic acid hp cytochromes P-450

while rabbit CYP2C2 in the same system yielded only the two epoxidcs 1 l,I?-EET and 14,15-EET in a ratio of 2 : 1. P-450 2CAA and CYP2C2 formed these two epoxides with only modest stereoselectivity [641. The epoxygenases of the rat and human kidney cortex havc also been investigated [57,65,66]. Rat kidney microsomal epoxygenase formed 8R,9S-, I 1R. 12s- and 14S,lSR-EET with optical purities of 97, 88 and 70%, respectively. The renal epoxygenase was cloned and found to be virtually identical with rat liver CYP2C23 [ 6 5 ] . When expressed in COS-I cells, the recombinant rat kidney epoxygenase formed thc three epoxides with virtually identical optical purity as the enzyme of rat renal cortical microsomes (95, 85 and 75%, respectively). 11,I2-EET was the main product (54%), followed by 8,9-EET (27%) and 14,15-EET (14%). In the human kidney cortex, CYP2C8 was identified as one of the cytochrome P-450 epoxygenase isoforms [66]. The metabolism of arachidonic acid by recombinant CYP2C8 was both regioand enantioselective for the biosynthesis of 1 1 R, 12S-EET and 14R,15s-EET. The identification of the CYP2C gene family as constitutive renal cpoxygenases. which oxygenate the 11,12- and 14,15-double bonds with enantioselectivity, might prove to be of physiological interest. Rat renal cortical epoxygenase activity is induced by salt loading [57]. Whether salt loading will induce epoxygenases in human kidneys is unknown and additional studies are therefore necessary (see also Section 7.4). The substrate specificity of a cytochrome P-450 can be altered by an amino acid mutation at a few key positions in the substrate-heme pocket [67]. Site-directed mutants of CYP2BI have been investigated with arachidonic acid as a substrate [68]. CYP2B 1 predominantly produced 14,15-EET, but mutation of two amino acids. Ile' I' or Gly478,had profound effects on the biosynthesis of epoxides. For example, double mutation of CYP2Bl with Ala at these two positions yielded an active enzyme, which formed 8,9-and 5,6-EET as the two main products. These results suggest that lie"' and G l ~ ~ ~ be ~m important ay for the orientation of arachidonic acid at the active site. An epoxygenase for arachidonic acid has also been purified from chick embryo livers after pre-treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) or 0naphthoflavone, both of which potently induce the epoxygenase activity [69-72]. It should be noted that P-naphthoflavone induces w2-hydroxylation of fatty acids in other species. The chicken liver epoxygenase is apparently induced by aromatic hydrocarbon receptor-dependent inducers, such as TCDD and p-naphthoflavone.Two of the enzymes induced by TCDD in chicken liver were identified as belonging to the CYPlA subfamily and one of them metabolized arachidonic acid to epoxides [72]. Most of the mammalian epoxygenases have low turnover numbers in comparison with other arachidonic acid oxygenating enzymes, e.g. cyclooxygenases and lipoxygenases. However, Bacillus megaterium contains cytochrome P-450-BM3 (CUPI02), which metabolizes fatty acids very rapidly. Arachidonic acid is mainly converted to I8R-HETE (80%) and to 14,15-EET (20%) [7, 81. This is an example of a fairly position-specific epoxygenase. With eicosapentaenoic acid (20 : 511-3) however, cytochrome P-450-BM3 will form both the w3 and the 0 6 epoxides. A cytochrome P-450 with a related position specificity appears to be present in monkey seminal vesicles. Microsomes of this tissue and NADPH converted arachidonic acid to 18R-HETE as the only product and eicosapentaenoic acid to 17S,18R-epoxy-5,8,11,14-eicosatetraenoic acid as the only product [73,74]. The enzyme of seminal vesicles has a rc-

markable position selectivity at least with these two fatty acids as substrates 1741. Zeldin et al. 1751 and Daikh el al. [76] demonstrated that CYP2C8 is one of the primary, constitutive hepatic arachidonate epoxygenases of human liver. This conclusion was based on a comparison of the stereochemistry of endogenous liver epoxides and those formed by recombinant CYP2C8 as well as inhibitory eCfects of polyclonal antibodies against recombinant CYP2C8. It therefore seems likely that CYP2C8 is the main epoxygenase of both human liver and kidney cortex. However, the different cytochrome P-450 isoforms of human livers show a large interindividual variation 1771 and many other enzymes might also contribute. CYPlA2, CYP2C9, CYP3A4 and many other abundant isozymes of human liver can form epoxides [47,48,76].

6.3.3 Bisallylic hydroxylation and hydroxylation with double bond migration It was obvious that biosynthesis of cis-trans conjugated HETEs by cytochromes P450 likely involved abstraction of a bisallylic hydrogen in analogy with the oxygenation mechanism of lipoxygenases (see Sections 1.3 and 5.2). Abstraction of hydrogen from the bisallylic methylene carbon requires at least 10 kcaVmol less energy than abstraction of allylic or methyl hydrogens [78]. Hydrogen abstraction is often the rate-limiting step in cytochrome P-450 catalysis [42]. Linoleic acid was chosen as a model substrate to investigate the mechanism of biosynthesis of cis-trans conjugated hydroxy fatty acids and the results were then extended to arachidonic acid. Linoleic acid was metabolized by liver microsomes from untreated or phenobarbital-treated rats to metabolites formed by epoxidation and w-side chain hydroxylations [79] and to a series of novel metabolites [28]. These were two cis-trans conjugated hydroxy fatty acids, 9-HODE and 13-HODE, 8-hydroxy-9,12-octadecadienoicacid (8-HODE), 1I-HODE and 14-HODE. I 1-HODE (59% R) was found to be a major metabolite, provided that the metabolites were extracted at pH 5-6 [80]. Steric analysis revealed that 13-HODE and 9-HODE consisted mainly of the R enantiomers (>80%), while 11-HODE was almost racemic. 11-HODE was first described in a red algae, where it is formed by other mechanisms [80]. Experiments under 180 2 gas showed that 11-HODE was formed by liver microsomes through incorporation of oxygen from air [28]. 13R-HODE and 9R-HODE also contained oxygen from air. The mechanism of biosynthesis of 9-, 11- and 13HODE was studied with stereospecifically deuterated linoleic acids. When [ 11 R-2H] linoleic and [ l lS?H] linoleic acids were used as substrates, the analysis showed a suprafacial relationship between oxygen insertion and hydrogen abstraction at C1I of the three enzymatic products, 1 lR,S-HODE, 9R-HODE and 13R-HODE. 11-HODE was formed by hydroxylation of CII , a process thus named bisallylic hydroxylation. The two minor products, 8-HODE and 14-HODE, were formed by allylic hydroxylation, i.e. by hydroxylation of the allylic carbons C8and C14, respectively. The experiments with stereospecifically deuterated linoleic acid indicated that 9R-HODE and 13R-HODE were formed by hydrogen abstraction at C I I ,migration of the double bond and suprafacial oxygen insertion at Cg or C13. This sequence was designated

150

6 Oxygenation of urachidonic acid by cytochromes P-450

hydroxylation with double bond migration. In analogy arachidonic acid was found to be hydroxylated at C13, the 0 8 carbon, to 13-HETE by monkey and human hepatic cytochromes P-450 [29]. Many other hydroxy metabolites were formed including 12R-HETE and 15R-HETE. Brash el al. [30] investigated arachidonic acid metabolism by bisallylic hydroxylases of liver microsomes of phenobarbital-treated rats. Two novel metabolites were identified, 7-HETE and 1 O-HETE. The results showed that bisallylic hydroxylases of these microsomes may oxygenate C7, C I Oand C13of arachidonic acid without position selectivity. As described above, the major form of phenobarbital-induced cytochrome P-450 (CYP2B1) will epoxidize all four double bonds of arachidonic acid, while other epoxygenases may show position selectivity. Brash et af. 1301 also made the unexpected observation that the bisallylic hydroxyl oxygen may, under certain conditions, be retained in the cis-trans conjugated products formed by acidic-catal yzed decomposition of 13-HETE, IO-HETE and 7-HETE. As expected, I I-HODE was a prominent metabolite in hepatic microsomes of phenobarbital-treated rats. 1 I -HODE and 18-HODE were formed in approximately equal amounts [28 1. The amounts of 13HETE, 10-HETE, 7-HETE, 19-HETE and 20-HETE were also of the same ordcr of magnitude [30]. The next step was to identify the isozymes of cytochrome P-450 with a large capacity for forming bisallylic hydroxy metabolites of arachidonic and linoleic acids. These cytochromes P-450 were first studied using inducers of hepatic enzymes and then by recombinant isozymes. Drugs from five different classes of inducers of cytochrome P-450 were investigated [311, namely P-naphthoflavone as an aromatic hydrocarbon receptor inducer (e.g., CYPl A), clofibrate as a peroxisome proliferator inducer (CYP4A), the synthetic glucocorticoid dexamethasone a s an inducer of CYP3A, phenobarbital as an inducer of CYP2B and acetone, starvation and isopropanol for CYP2E1. These agents may also induce many other cytochromes P-450 [l]. CYP3AI is also induced by phenobarbital, ethanol and isopentanol [ 8 1,821. With linoleic acid as a substrate, all liver microsomes formed I8-HODE as a major metabolite, while 1 1-HODE and 17-HODE were present in variable amounts. Liver microsomes of j3-naphthoflavone-treated rats hydroxylated [14C]linoleic acid at carbons 8, 11,14,16,17 and 18. In addition, 9-HODE and 13-HODE were formed. Microsmes of phenobarbital-treated rats formed essentially the same metabolites. l l HODE was apparently formed without selectivity by liver rnicrosomes of 11naphthoflavone-and phenobarbital-treated rats. Liver microsomes of rats treated with acetone plus starvation or isopropanol also metabolized linoleic acid to 1 I -HODE along with large quantities of other hydroxy metabolites. These agents induce CYP2E1, which does not show bisallylic hydroxylase activity, as well as other cytochromes P-450, e.g. CYP3Al [81]. Clofibrate did not augment the biosynthesis of 1 1HODE, but induced formation of 18-HODE as expected. Liver microsomes of dexamethasone-treated Sprague-Dawley and Fischer rats showed an augmented bisallylic hydroxylase activity [48]. The two main metabolites of linoleic acid were 11-HODE and 18-HODE. Only small amounts of other hydroxy fatty acids were formed. RP-HPLC analysis showed that dexamethasone treatment consistently increased the formation of I 1-HODE approximately tenfold in compari-

6.3 Oxygenation of arachidonic acid by cytochromes P-4.50

15 1

son with control rats. Troleandomycin, a specific inhibitor of CYP3A, reduced the biosynthesis of 1 1 -HODE. Bisallylic hydroxylations of arachidonic acid were then investigated [28]. Microsomes of dexamethasone-treated rats formed mainly 13-HETE, 7-HETE and 10HETE and only small amounts of 20-HETE and 19-HETE, while control microsomes formed 20-HETE, 13-HETE and 19-HETE as the three major products. These results suggest that bisallylic hydroxylases for linoleic and arachidonic acids could be induced by dexamethasone treatment of Sprague-Dawley and Fischer rats. Dexamethasone induces CYP3A I in the rat. Five human recombinant enzymes, CYPlA2, CYP2C8, CYP2C9, CYP2C 19 and CYP3A4, metabolized linoleic acid to 1 1-HODE,while arachidonic acid was converted to one or several bisallylic hydroxy metabolites. All five enzymes formed 13-HETE. The metabolism by CYP2C9 was particularly interesting. This enzyme formed 13HETE, 1 I-HETE, 15-HETE and 12R-HETE as well as epoxides [48]. The mechanism

A

HETEs 12

Time (min)

Figure 6-2. LC-MS analysis of metabolites formed by CYP2C9 and arachidonic acid. A. Partial reverse-phase chromatogram of metabolites formed under normal atmosphere. B. Partial reverse phase chromatogram of metabolites formed under I8O2 gas. The metabolites were identified by MS-MS with atmospheric pressure chemical ionization and analysis of negative ions. HETEs and EETs have molecular mass of 320 kDa but will give signals at m/z 319 due to the charged carboxyl group, and m/z 319 was followed in. Compounds with oxygen- 18 will give signals at m/z 321, which was followed in. Analysis of m/z 319 of this sample showed almost identical pattern of products (not shown), indicating that oxygen in the products originates mainly from air. EET, epoxyeicosatrienoic acid; HETE, hydroxyeicosatetraenoic acid.

152

6 Oxygenution of uruchidonic acid hy cFtochron1e.y P-450

of biosynthesis of 12R-HETE is still enigmatic. It seems possible that I2R-HETE might be formed by hydrogen abstraction at C ~ Odouble , bond migration and oxygen insertion at C12 in analogy with the biosynthesis of 13R-HODE from linoleic acid. This hypothesis should be tested with stereo specifically labeled arachidonic acid in position Clo. Experiments under oxygen 18 gas supported this mechanism since 17RHETE contained as much oxygen-18 as 13-HETE and the other metabolites. A reversed-phase chromatogram of products formed by CYP2C9 from arachidonic acid and analyzed by liquid chromatography mass spectrometry with MS-MS is shown in Fig. 6-2.

6.4 Metabolism of epoxides The EETs are chemically stable and can be extracted at acidic pH with one exception. i.e. 5,6-EET which will easily form the 56-lactone. In tissues EETs are rapidly hydrolyzed to vicinal diols or incorporated into phospholipids.

6.4.1 Epoxide hydrolases Epoxides of arachidonic acid are converted to vicinal diols by epoxide hydrolases. Purified rat liver microsomal epoxide hydrolases were found to hydrolyLe the four epoxides of arachidonic acid with a V,,, of between 0.09 and 0.25 pmol/niin/mg protein [50], but K,,, values were rather high, e.g. 0.16 mM for 14,15-EET.The corresponding figures for hydrolysis of these epoxides by purified mouse liver cytosolic epoxide hydrolase were between 0.07 and 1.26 pmol/min/mg protein, but the K,,, value for 14,15-EET was only 8 pM [83]. Both microsomal and cytosolic epoxide hydrolases can thus hydrate the epoxides, but the cytosolic enzyme appeared to be much more efficient. The hydration of EETs by cytosolic epoxide hydrolases was investigated by Zeldin ef al. [84], using chiral HPLC to separate the S,S and R,R enantiomers of the vicinal DHETs. Interestingly, acid-catalyzed hydration of the cis'-epoxides did not always lead to equal formation of the SS and RR isomers; 14S,lSR-EET yielded a 3: 1 ratio of these antipodes. Soluble epoxide hydrolase of rabbit liver hydrolyzed racemic 14,15-EET to a 4 : 1 mixture of RR and S S enantiomers of 14,I5-DHET. Incubation with 14S,lSR-EET, the major enantiomer formed in the rat and rabbit kidney, yielded the SS and RR antipodes in a 3 : 1 ratio. 14R,I 5s-EET, the major enantiomer of the liver and human kidney, was hydrated mainly to the RR antipode of 14,lS-DHET (ratio 6 : 1). These results show that the oxirane ring is opened by cytosolic rabbit liver epoxide hydrolase mainly by addition of oxygen at CIS.Similarly, epoxide hydrolase selectively catalyzed addition of oxygen at C9 of 8,9-EET, while II,IZEET was hydrolyzed without regioselectivity. The results have been extended to soluble epoxide hydrolase and a series of fatty acid epoxides and their methyl esters [SSl. The catalytic mechanism of soluble epoxide hydrolases has been investigated in

6.5. Analysis of arochidonic acid metciholitrs

153

detail by Borhan ut al. [86]. cis-9,10-Epoxystearicacid was used as one of two model substrates. [’80]Epoxystearicacid was enzymically hydrolyzed to the diol and “0 was present at either CS (69%) or at C ~ (31%). O The other oxygen was found to be derived from the enzyme itself. When the unlabeled epoxide was incubated with excess enzyme for only one cycle in H2180, the diol did not contain ’*O. Further analysi\ showed that the carboxylate anion of Asp”3 of the enzyme attacked the epoxide substrates and formed an a-hydroxyacyl-enzyme intermediate, which was hydrolyzed by (activated) water (postulated via a histidine) to the active enzyme and the diol. Recently, Greene and Hammock (87) reported that leukotoxin and isoleukotoxin, which designate the 9,lO-epoxide and the 12,13-epoxide of linoleic acid, respectively, were hydrolyzed by soluble epoxide hydrolase to diols with much greater cytotoxic properties than the epoxides. This finding is interesting since leukotoxin/isoleukotoxin has been found in adult respiratory distress syndrome [88]. It will be of interest to monitor the biosynthesis of these toxic diols in human lung disease.

6.4.2 Incorporation into phospholipids Epoxides of arachidonic acid can be incorporated into phospholipids. 8S,9R-, 1lS,12R- and 14R,15S-EET were, for example, demonstrated at the glycerol-sn-2 position of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol in the liver [60]. Exogenous 14,15-EET is rapidly incorporated into phospholipids of mastocytoma cells. The distribution of the epoxide in phospholipids was phosphatidylethanolamine > phosphatidylcholine > phosphatidylinositol [89]. The rate of release of the epoxide from phosphatidylcholine and phosphatidylinositol was higher than the release of arachidonic acid from these phospholipids by phospholipase A2 in agreement with previous reports [52,90]. EETs and DHETs were recently found to be incorporated into phospholipids of vascular endothelial cells [91,92], smooth muscle cells [93], endothelial cells [94] and astrocytes [95]. The incorporation in endothelial cells should be considered in the context of these metabolites as putative endothelium-derived hyperpolanzing factors (EDHFs), as discussed below. Finally, Carroll et al. [96] recently demonstrated that 19-HETE and 20-HETE are stored in phospholipids, at least in the rabbit kidney and released by angiotensin 11. Thus, both HETEs and EETs can be incorporated into the phopholipid pool, but it is unknown whether bisallylic hydroxy metabolites of arachidonic acid also can be stored in this way.

6.5. Analysis of arachidonic acid metabolites 6.5.1 Radioimmunoassay Radioimmunoassay has been used sparingly for analysis of cytochrome P-450derived metabolites of arachidonic acid. However, a fluoroimmunoassay for 14.15-

I54

6 Oxygenation of aruchidonic ucid by cytochromrs P-450

EET and 14,15-DHET was reported recently (971. The method was used to assay these products released from bovine coronary artery endothelial cells and bovine mna glomerulosa cells.

6.5.2 GC-MS and LC-MS analyses As in all other fields of eicosanoid research, mass spectrometry has been the most important tool for structural analysis of metabolites. Gas chromatography-mass spectrometry (GC-MS) has been the primary tool for structural identification of epoxides and other cytochrome P-450-derived metabolites of PUFAs [98]. The mass spectra of fatty acid epoxides provide relatively little information on the position of the epoxide. Analysis of epoxides via 3-pyridinylmethyl esters are more informative [99]. Conversion of the epoxides to diols or chlorohydrin adducts usually gives mass spectra with distinct fragmentation [loo]. A large and useful collection of electron impact mass spectra of eicosanoids has been compiled by Pace-Asciak (1 0 1 1. Instruments for GC-MS analysis and interpretation of mass spectra of fatty acid derivatives and eicosanoids have been described by Murphy in an excellent monograph 11021. Methods for analysis based on deuterated or oxygen 18-labeled internal standards have been used for quantification of epoxides or the corresponding diols by mass fragmentography [ 1031. Recently, EETs and their corresponding diols were identified in nanogram or picogram quantities using capillary GC-MS in the negative ionchemical ionization mode [91,104]. Yamane et al. [ 1051 systematically studied epoxides of polyunsaturated eicosanoic fatty acids and docosahexenoic acid by liquid chromatography-mass spectrometry (LC-MS) (thermospray). In addition, mass spectra of many of the corresponding diols were reported. LC-MS has recently been used in analysis of bisallylic hydroxy fatty acids [106]. LC-MS analysis of 1 1-HODE, 17-HODE and other metabolites from an incubation of linoleic acid with CYP2C9 is shown in Fig. 6-3.

6.5.3 Steric analysis of hydroxy fatty acids Enantiomers of cis-trans conjugated HETEs and other cis-trans conjugated hydroxy fatty acids can be separated by chiral HPLC on R-(-)-N-(3,5-dinitrobenzyol)-aphenylglycine columns with or without derivatization [ 1071. This used to be the method of choice, but much better separations can often be obtained on Chiralcel OB columns [30]. These Chiralcel columns can also be used for bisallylic metabolites. The enantiomers of 7-HETE, 10-HETE and 13-HETE were at least partly resolved by HPLC on Chiralcel OD [30]. Steric analysis of bisallylic hydroxy fatty acids can also be performed after partial hydrogenation of the double bonds, synthesis of (-)-menthoxycarbonyl derivatives and ozonolysis [go]. Steric analysis of 04-, 03- and o2-hydroxy fatty acids can be performed by separation of diastereoisomeric S-phenylpropionic acid derivatives on GC [ 1081. It is also possible to partly resolve the enantiomers of 18-HETE and 19-HETE on Chiralcel OC [ 1091.

Time (min) 2

3

1 1-HODE

"i"

B

I: i 4

mh

Figure 6-3. LC-MS analysis of metabolites formed from linoleic acid by CYP2C9. A. Reversed phase chromatogram from an incubation of linoleic acid with CYP2C9. The major peak contained 13- and 9HODE, while 1 1-HODE and 17-HODE were formed in smaller amounts. B. MS-MS spectrum of 11HODE. C. MS-MS spectrum of 17-HODE. D. MS-MS spectrum of a mixture of 13-HODE ( d z 195 and 179) and 9-HODE (m/z 171). The HODEs will give rise to signals at m/z 295 due to the charged carboxyl group, and MS-MS of this fragment showed signals at m/z 277 (presumably due to loss of water) and m/z 251 (loss of carbon dioxide) in all three mass spectra. Chromatographic conditions as in Fig. 6-2. HODE, octadecadienoic acid.

6.5.4 Steric analysis of epoxy fatty acids and vicinal diols HPLC with separation of the enantiomers of all four epoxides of arachidonic acid was achieved using Chiralcel OB and Chiralcel OD columns [ 1101. The epoxides were analyzed as methyl esters or as pentafluorobenzyl esters. The resolution was improved by hydrogenation. The vicinal rhreo-DHETs, formed by hydrolysis of EETs, were resolved as methyl ester derivatives on Chiralcel OB and Chiralcel OD columns [ 1 1 11. In this case, resolution was also improved by hydrogenation. The pentafluorobenzyl ester derivatives of the hydrogenated vicinal diols yielded better separation on

I56

6 Oxygenation of arachidonic acid hq' cytochromes P-450

the Chiralcel OD column and the elution times were \hortencd. There is also a general method for steric analysis of cis and mm.5 epoxides 01 fatty acids [I 121, which is based on hydrogenation and conversion of the epoxidc to tuo regioisomeric allylic alcohols, followed by ozonoly,i+ of (-)-nienthoxycarbonyl clcrivatives and GC-MS analysis.

6.6 Biological effects EETs can affect cellular function in a wide variety of different tissues and cells. at least in pharmacological concentrations. This area has been subject to inany rcviews [113-1181. In addition, 20-HETE and 19-HETE have also been found to be biologically active, while the newly discovered bisallylic hydroxy metabolite\ have not ye( been fully investigated. Biological effects on only a few organ systems will be discussed. The mechanism behind these biological effects appears to be largely unknown. There is little evidence for the existence of specific cell surface receptor+for EETs and other cytochrome P-450-derived metabolites of arachidonic acid. In s ~ m e cases, the effects of EETs can be explained by inhibition of cyclooxygenasc, by metabolism by cyclooxygenase, by effects on other enzymes of the arachidonic acid cascade or by effects on ion channels. Some investigators report effects at nanomolar or even picomolar concentrations, which might be of physiological relevance and deserve further attention. In recent years the hyperpolarizing action of EETs of certain cells has been discovered and led to speculation of EETs as candidates of the endothelium-derived hyperpolarizing factor (EDHF) as discussed below.

6.6.1 Kidney Cytochrome P-450-derived metabolites of arachidonic acid are formed in critical nephron segments and may participate in receptor-mediated signal transduction as second messengers [ 114, 115, 117, 119, 120; see also Section 7.41. In many spccies, renal cortical epoxygenase activity is high and has been localized to specific cells of the nephron [121,122]. The major products are 11,12- and 14,15-EETs in the rabbit [63] and 8,9-, 11,12-, and 14,15- EETs in the rat kidney 1571. Suggested roles of these metabolites include modulation of the effects of renal hormones and regulation of blood flow and tubular function, but a precise role in kidney physiology has not yet been defined. Possible pathophysiological changes in these metabolites havc nevertheless been reported. For example, in women with pregnancy-induced hypertension, urinary 11,12-, and 14,15- DHET were increased [58l. In several models of experimental hypertension in the rat, there is an increase in renal 20-HETE and mRNA of CYP4A3 and CYP4A8 in the kidney [ 123-1 251. Both racemic EETs and their dihydroxy metabolites inhibited the vasopressinstimulated osmotic water flow in toad bladder via inhibition of adenylate cyclase. 5,6EET and 11,lZEET were most potent. However, 10 pM concentrations were required for activity [126]. Interestingly, 14,15-DHET (0.1 pM) was a s potent as pros-

6.6 Biological effects

157

taglandin (PGE2) in inhibiting the action of vasopressin on collecting ducts of the rabbit [ 1271. 5,6-EET (10 nM-l pM) caused a transient increase in intracellular calcium of proximal tubular epithelium, mimicking the actions of angiotensin 11, suggesting that cytochrome P-450 metabolites are involved in angiotensin 11-induced calcium mobilization 11281. In addition, racemic 14,15-EET ( 1 pM) has been found to inhibit renin release from cortical slices of rat kidney (129). As 14S,l5R-EET is the major enantiomer of 14,15-EET formed by the renal epoxygenases of rabbits and rats, it will be of interest to determine whether the effects on vasopressin and the renin-angiotensin system are stereospecific. Infusion of arachidonic acid into the rat renal artery causes vasoconstriction, which is blocked by inhibitors of cyclooxygenases, revealing a vasodilator response [ 1301. This vasodilatation appeared to be dependent on endothelial cytochrome P-450 as judged from the effects of inhibitors of cytochrome P-450 [131,132]. In the isolated rabbit kidney, 5,6-EET, 8,9-EET and 1 1,IZEET induced vasodilatation, while 14,15EET induced vasoconstriction [ 1321. 5,6-EET was most potent and partly dependent on metabolism by cyclooxygenases for vasoactivity [ 133,1341. Interestingly, the activity of 5,6-EET differed on the renal vasculature in the buffer and the blood perfused rat kidney model [ 1351. In the latter, 5,6-EET exerted vasoconstrictor effects, presumably due to metabolism by cyclooxygenase- 1 and thromboxane synthase of platelets to a vasoconstrictor substance. This action was antagonized by indomethacin. In the isolated rat kidney perfused with physiological buffer the epoxide was a vasodilator and indomethacin was without effect. These observations thus suggested that EETs may modulate renal function. The renal cytochrome P-450 system should be influenced by variations in salt and water balance or by other physiological means. Epidermal growth factor (EGF) (30nM) increased endogenous EET levels fivefold within 10 min in primary cultures of rabbit proximal tubule cells and 5,6-EET, augmented the effect of EGF on thymidine incorporation [ 1361. The only physiological stimuli, which has been shown to increase renal epoxygenases, is salt loading, which results in a 10-12 fold increase in the urinary excretion of the corresponding arachidonic acid metabolites (EETs and DHETs) in rat urine [57]. CYP2C23 appears to be the predominant, constitutive epoxygenase in the rat kidney while salt loading induces a yet unidentified CYP2C isoform that seems to be different from CYP2C23 [57,63,65,137]. Much work has recently focused on 20-HETE and o-oxidation in the kidney. The human kidney contains CYP4A (138; cf. Simpson [37] for a review of the CYP4 family). In the rat kidney, CYP4A2 functions as a renal microvessel arachidonate ohydroxylase, although this enzyme also forms some 11,12-DHET [ 1391. In spontaneously hypertensive rats, mRNAs of CYP4A3 and CYP4A8 increase in the kidneys during the first 4 weeks, which might suggest that expression of these enzymes could be important in the development of hypertension in young rats [ 1251.

6.6.2 Heart The vasodilation of bradykinin on coronary arteries in the rat heart seemed to be dependent on cytochromeP-450 metabolism of arachidonic acid I1401. Effects of EETs on

158

6 Oxygenation of arachidonic m i d by cytochromes P-450

isolated hearts and ventricular myocytes have been reported I 14 I 1. I I , 12-EETdeprc5sed recovery of the contractility after ischemia and elevated Ca, of ventricular myocytes. Only a few cytochromes P-450 have been identified in heart tissue. CYP 1A I ha\ been found in fish heart [142]. A novel arachidonic acid epoxygenase was recently detected in the human heart [ 1431. This enzyme, designated cytochmme P-450-252, forms the 5,6-, 8,9-, 11,12- and 14,15-EETs with little regioselectivity in the following amounts: 21, 24, 18 and 37%, respectively. The 8,9-, I 1,12- and 14.15-EET\ could also be demonstrated in heart tissue. More recently, Wu et (11. [ 1441 cloned a cytochrome P-450 from rat heart myocytes. This enzyme, CYP2J.3, formed 8,9-, 11,12- and 14,15-EET. These investigators also made the intriguing obwvation that infusion of 11.12-EET at a concentration of 5 pM improved cardiac contractility after ischemia. The mechanism of action of 1 I,12-EET in the ischemic heart appears to merit further investigations.

6.6.3 Vascular tree The effects of arachidonic acid and its cytochrome P-450-derived metabolites on the vasculature should be considered in the context of factors which mediate endothelium-dependent relaxation. In addition to prostacyclin and nitric oxide, which are well characterized endothelium-derived relaxing factors, there may be at least one additional and unidentified vasodilator [ 145-1481, This putative substance is designated EDHF and it may be important for vasoregulation, particularly of small resistance arteries (see Campbell et al. [149], Harder et ul. [I501 and Adeagbo I1511 for reviews). EDHF appears to be less reactive than nitric oxide and distinct from prostanoids. An increase in free cytosolic calcium may release EDHF, which may increase conductance to potassium in smooth muscle cells. Some investigators suggest that EETs or 20-HETE might be candidates for EDHF or regulators of vascular tone I 1491511, while others remain sceptical [ 1521. The conclusions drawn from studies with inhibitors of cytochromes P-450 might not be valid, as many of these drugs have other actions, e.g., inhibition of ion channels of smooth muscle [153-1551. Exogenous EETs are rapidly taken up by arterial smooth muscle cells and by endothelial cells in v i m [91,93]. The epoxides are partly hydrolyzed to vicinal diols and released to the medium. Arterial smooth muscle cells metabolize I I , 12-EET to its vicinal diol, 1 1,lZDHET and to a novel metabolite, 7,8-dihydroxyhexadecadienoic acid [94]. The latter was likely formed from 11,IZDHET by two steps of 0oxidation. Both metabolites (1-5 pM) caused vasorelaxation of pre-contracted porcine coronary artery rings. Several investigators have found that arachidonic acid can induce endotheliumdependent relaxation of the rabbit aorta [1561, pulmonary rings [I571 and coronary arteries [ 1581. Proadifen, a cytochrome P-450 inhibitor, blocked this effect [ 156-1 581. suggesting that arachidonic acid was metabolically activated by cytochrornes P-450. However, proadifen may also have a direct effect on potassium conductance [145]. 5,6-EET reduced vascular resistance of the isolated perfused rat caudal artery. the other epoxides being inactive [ 1591. The biological activity of 5,b-EET was dependent on metabolism by cyclooxygenasesin rat caudal artery and cerebral arterioles I I 59>1601.

6.6 Biological effects

159

which suggests biosynthesis of epoxyprostaglandins.Carol1 et al. [ 1331 found that 5,6EET was converted to 5,6-epoxy-PGEl after circulating through the rabbit kidney. Authentic 5,6-epoxy-PGEl was as equipotent as PGE2 as a vasodilator in the isolated, perfused rabbit kidney. Metabolism by cyclooxygenase has also been implicated in the vasodilatory effects of 8,9-EET and 1 1,12-EET on intestinal serosa blood flow [ 1611. 5,6-EET may also be an important mediator of calcium influx in human and bovine endothelial cells [ 1621. The vascular effects of 5,6-EET and the other cytochrome P-450derived metabolites of arachidonic acid have been reviewed [ 1631. Systemic administration of EETs has virtually no effect on systemic or cerebral blood flow in the rat 1921. Epoxides of arachidonic acid may also affect vascular tone by cyclooxygenaseindependent mechanisms. 8,9-EET (50 pM) and 1 1,12-EET (50 pM) caused transient dilatation of pre-contracted cerebral arteries and also increased the frequency of opening of K+ channels at 5 pM [ 1641. All four EETs (0.1-3 pM) increased the openstate probability of the Ca2+-activatedK' channel (Gachannel) in cells from rabbit portal vein, rat caudal artery, guinea-pig aorta and porcine coronary artery [149,165]. 1 1,12-EET hyperpolarized and increased the open-state probability of a kachannel in coronary smooth muscle cells [149]. In renal vascular muscle cells 1 lR,12S-EET (1-100 nM) increased the activity of the I&, channel and relaxed small renal arteries pre-constricted with phenylephrine [ 1661. The vasodilator effect of some EETs may channel. This was studied in detail by Li and therefore involve activation of a ha Campbell [ 1671 who proposed that 1 1,12-EET activates these channels via the asubunit of the G, protein i.e. stimulation of adenylate cyclase. Vessels with injured endothelium due to artery stenosis can synthesize 5,6-, 8,9-, 11,12- and 14,15-EETs and the data suggested that these metabolites may counteract the vasoconstrictory action of thromboxane A2 [ 1681. 14,15-EET (0.25-0.5 pM)promotes adhesion of U937 cells to endothelium and it could therefore be involved in atherogenesis [ 1691. Rabbit aorta, isolated from animals with experimental hypercholesterolemia, generated all four EETs, but normal rabbit aorta failed to do so. All four regioisomers at concentrations from 0.01 to 10 pM relaxed norepinephrine precontracted aortae from normal and cholesterol-fed animals [ 1701. Additional studies suggested that an endothelium-derived metabolite of arachidonic acid is enhanced by hypercholesterolemiaand that it might affect vascular tone [ 1711. Cultured bovine coronary artery endothelial cells form cyclooxygenase-, lipoxygenase- and cytochrome P-450-derived metabolites of arachidonic acid [ 1721. All four EETs were identified and they may contribute to the endothelium-dependentrelaxation caused by bradykinin and arachidonic acid. However, a recent study found that arachidonic acid metabolites were not involved in acetylcholine-induced endothelium-dependent hyperpolarization in guinea-pig carotid artery, rat hepatic and mesenteric arteries [ 152-1551. Further studies are needed to clarify the physiological role of cytochrome P-450 and EETs in the vascular wall.

6.6.4 Central nervous system and the pituitary Cytochrome P-450 is present in many parts of the central nervous system. Biosynthesis of arachidonic acid epoxides was reported in 1992 [ 1731. Slices of mou+e brain liwmed

160

6 Oxygenation of urachidonic acid by cytochromes P-450

two epoxides, 14,15-EET and 5,6-EET. Brain-synthesized 5,6-EET caused transient cerebral arteriolar dilation similar to that caused by authentic 5,6-EET (15.6 pM). The authors suggest 5,6-EET may have a regulatory role in cerebral microcirculation. Microsomes of cat brain formed large amounts of 5,6-, 8,9- and 1 I,12-EETs and only small amounts of 14,15-EET. Two epoxides, 8,9-EET and 1 1,12-EET (50 pM), dilated cerebral arteries which were pre-constricted with serotonin, presumably through activation of KCchannels [164]. Rabbit cerebral arteries were dilated by $6EET (15.6 pM) while 8,9-EET, 11,12-EETand 14,WEET were less active [160]. A series of papers have demonstrated that rat brain astrocytes in primary culture produce EETs [95,173,174].1 I,12-EET and 14,15-EETwere identifiedin one study as well as mRNA of P-450-2C11 in astrocytes [95]. Shivachar et al. [175] also reported the synthesis of 5,6-EET and 14,15-EET and the corresponding vicinal diols. These studies further emphasize that arachidonate epoxygenases are present in the brain and it is conceivable that EETs could modulate &, channel activity of the cerebral microvessels. Microsomes of rat hypothalamus oxygenated arachidonic acid to epoxides in the presence of NADPH [176]. In addition, 5,6-EET (2.5 pM) stimulated the release of somatostatin from the median eminence in vitro. These results were extended by demonstration and quantification of endogenous 8,9-, 11,12- and 14,lS-EETs in the rat hypothalamus [ 1773. Cytochrome P-450 metabolism of arachidonic acid also occurs in the pituitary. Microsomes of rat anterior pituitary formed 20-HETE and at least three epoxides of arachidonic acid (5,6-, 1 1,12- and 14,15-EETs)[ 1781. These cytochrome P-450-derived metabolites affect anterior pituitary cells at relatively high concentrations. Inhibitors of cytochromes P-450 (proadifen and piperonyl butoxide) blocked the release of corticotropins in response to a variety of secretagogs in mouse pituitary tumor cells [179]. 5,6-EET (1 pM) caused efflux of calcium and release of luteinizing hormone [ 1801. At 50 pM concentration, it stimulated the release of prolactin from a rat anterior pituitary cell line GH3 [ 1811 and stimulated the release of growth hormone from pituitary cells in culture at 20 pM [182]. In addition, 5,6-, 8,9-, 11,12- and 14,15-EETs (3 pM) all stimulated release of growth hormone from a population of enriched somatotrophs, with 5,6-EET and 14,15-EET more potent than the other two regioisomers [ 1831. Microsomes of the neural lobe of rat pituitary metabolized arachidonic acid to the major metabolites 8,9-EET, 11,12-EET and 14,15-EET, which were produced in equal quantities and stimulated release of oxytocin and vasopressin over a concentrapM [184]. These concentrations were 50% larger than that of tion range of to PGE2for similar effects on oxytocin and vasopressin release. These studies thus demonstrated that epoxides of arachidonic acid were formed by mouse and cat brain and by rat hypothalamus, pituitary and astrocytes. Little is known about the stereochemistry of the products and the cytochrome P-450 isozymes catalyzing their biosynthesis.

6.6.5 Genital glands and endocrine organs Campbell et al. (185) identified EETs, HETEs and prostaglandins as metabolites of arachidonic acid in rat adrenal glomerulosa cells. Human granulosa cells of the ovary

6.8 References

16I

metabolize arachidonic acid to epoxides and 14,15-EET stimulated estradiol production at low concentrations of 1-5 pM [186]. In addition, 14,I5-EET has been tentatively identified as a metabolite of human reproductive tissue [ 1871. Recently, human placenta, fetal membranes and decidua was found to produce 5,6-EET and 12-HETE as judged from HPLC [ 1881. In addition to prostaglandins, other metabolites of arachidonic acid can apparently be formed in reproductive tissues.

6.7 Summary Arachidonic acid can be metabolized by cytochromes P-450 in the liver, kidney, heart, pituitary, gut and many other organs. Several hepatic or renal isozymes, which catalyze the biosynthesis of epoxides, 19-HETE, 20-HETE and bisallylic HETEs, have been identified. Important human liver and kidney epoxygenases belong to the CYP2C subfamily, human bisallylic hydroxylases belong to the CYP1A2, CYP2C, and CYP3A4 subfamilies and human o-hydroxylases to the CYP4A subfamilies. Arachidonic acid metabolism by these enzymes has also been implicated as physiological regulators in the vascular system and some organs. Yet the overall biological significance of this metabolic route is still largely unknown. The classical experiments with animals made deficient in essential fatty acids [ 1891 indicate that the kidneys, skin and reproductive organs were most vulnerable. These pathological effects may be mainly due to a reduced biosynthesis of prostaglandins but reduced biosynthesis of cytochrome P-450-derived metabolites might also contribute.

Acknowledgements Supported by grants from the Swedish Medical Research Council (6523).

6.8 References [I]

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C. von Wachenfeldt and E.F. Johnson. In Cytochrome P450: Structure, Mechanism and Biochemistry (P.R. Ortiz de Montellano, ed.). Plenum Press, New York, 1995 p. 183-223. D.R. Nelson, L. Koymans, T. Karnataki, J.J. Stegeman, R. Feyereisen, D.J. Waxman, M.R. Waterman, 0. Gotoh, M.J. Coon, R.W. Estabrook, I.C. Gunsalus and D.W. Nebert. Pharmacogenetics 1996; 6: 1-42. T.L. Poulos, B.C. Finzel, 1.C. Gun\alu\, G.C. Wagner and J. Kraut. J . Biol Chcm 1985; 260: 16122-16130. K.G. Ravichandran, S.S. Boddupalli, C.A. Hasemann, J.A Peterson and J. Deiwnhofer. Science 1993: 261: 73 1-736.

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C.A. Hasemann, K.G Ravichandran. J.A Peterson and J. Deisenhofer. J. Mol. Uiol. 1994; 236: 1169-1 185. J.R. Cupp-Vickery and T.L. Poulos. Nut. Struct. B i d . 199.5; 2: 144- 1 5 I . J.H. Capdevila, S. Wei, C. Helvig, J.R. Falck, Y. Belosludtsev. G. Trunn and S.E. Graham-Lorence. J. B i d . Chem. 1996; 271: 22663-2267 I . S. Graham-Lorence, G. Truan, J.A. Peterson, J.R. Falck. S. Wei. C. Helvig and J.H. Capdevila. J. Bio!. Chem. 1997: 272: 1 127-1 13.5. P.E. Verkade, M. Elzas, J. van der Lee, H.H. de Wolff, A. Vcrkade-Sandhergcn and D. van der Sande. Proc. R. Acud. Amsterdum 1932; 35: 25 1-266. K.C. Robbins. Fed. Proc. 1961; 20: 272. K. Wakabayashi and N. Shimazono. Biochim. Biophys. Acrci 1961; 48: 6 1 5-6 I 1 , B. Preiss and K.J. Bloch. 1.B i d . Chem. 1964; 229: 85-88. M. Kusunose, E. Kusunose and M.J. Coon. J . Biol. Chem. 1964; 239: 1374-1 380. T. Omura and R. Sato. J. B i d . Chem. 1964; 239: 2370-2378. T. Omura and R. Sato. J . B i d . Chern. 1964; 239: 2379-2385. M. Hamberg and I. Bjorkhem. J. B i d . Chem. 197 I ; 246: 74 1 1-74 16. I. Bjorkhem and M. Hamberg. Biochem. Biophys. Res. Comrnirn. 1972; 47: 333-340. G.J. Antony and B.R. Landau. J. Lipid Res. 1968; 9: 267-269. F. Wada, M. Usami, M. Goto and T. Hayashi. J. Biochern. 197 I ; 70: 1065- 1070. W. Kam, K. Kumaran and B.R. Landau. J. Lipid Res. 1978; 19: 591-600. 1. Bjorkhem. J. Biol. Chem. 1976; 251: 5259-5266. E. Hemmelgam, K. Kumaran and B.R. Landau. J. Biol. Chem. 1977: 252: 4379-

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Prostaglandins, Leukotrienes and Other Eicosanoids: From Biogenesis to Clinical Application Edited by F. Marks, G. Fürstenberger Copyright © WILEY-VCH Verlag GmbH, D-69469 Weinheim (Federal Republic of Germany). 1999

7 Renal eicosanoids Margarere Goppelt-Striibeand Joachim Fauler

The kidney is a rich source of various types of eicosanoids. These are mainly derived from arachidonic acid which is enzymatically metabolized by cyclooxygenases (also termed prostaglandin GIH synthases) (Fig. 7-l), lipoxygenases (Fig. 7-4) and monooxygenases (Fig. 7-6). Furthermore, arachidonic acid may be transformed nonenzymatically to isoprostanes (Fig. 7-8). The distribution of the metabolizing enzymes within the kidney is distinct, the expression of some enzymes being strictly localized to certain cell types (e.g. COX-2 in distal tubular cells of the normal rat kidney). The local synthesis of the different eicosanoids and their short half-life result in specific and well-defined actions in the kidney. Their effects are dependent on the distribution of specific receptors and the physiological function of the target cells, among them vascular smooth muscle, mesangial, endothelial, various epithelial and resident cells of the monocytic lineage. It is the aim of this chapter to summarize the present knowledge of eicosanoid biosynthesis and function within the kidney with an emphasis on those aspects where physiological and pathophysiological function meets cell biology.

7.1 Renal prostanoids Prostanoids are synthesized in the organism along the cyclooxygenase pathways (Fig. 7-1). They are extremely rapidly metabolized to inactive compounds which are then excreted in the urine. Only minor amounts of the systemically produced prostaglandins are excreted as unchanged prostaglandins. Therefore, the nanomolar concentrations of urinary prostaglandin E2 (PGE2) indicate that the kidney is a particularly rich source of prostaglandins. Under physiological conditions prostaglandins and thromboxane (TXA2) have little or no effect on renal function because their effects on renal hemodynamics or epithelial water and solute transport can be compensated for by other mediators. However, under conditions characterized by hypovolemia, synthesis of prostanglandins becomes vital for renal function. Furthermore, prostaglandins are ubiquitous mediators of inflamation and therefore, they are also involved in renal inflammatory reactions. The functional aspects of renal prostanoids have been thoroughly studied since the 1970s and extensively reviewed [14]. Over the last 10 years, advances in molecular

7 Renal eicosanoids

170

biology and immunohistochemical techniques have allowed research to complemenl these studies by the characterization of the structure, function and localization of individual enzymes and receptors within the kidney.

Membrane-Bound PhosDholipids

1

Phospholipases

Arachidonic acid

1 PGG,

1

Cyclooxygenases (Cox-7 and Cox-2)

PGH,

Thromboxane ( T W ) Prostaglandins (PG)

OH

PGEz

O

OH

G

. OH

-

Prostacyclin (PGI,)

CWH

Y

Figure 7-1. Biosynthesis of prostanoids. Arachidonic acid is liberated from membrane phospholipids by phospholipases, primarily phospholipases Az. COX- 1 and COX-2 catalyze a two-step reaction. cyclooxygenation of arachidonic acid to PGGz and reduction to PGH?. The further metabolism of PGH? depends o n the activity of the distal enzymes PGEl isomerase, PGF?, reductase and PGI2 or TXA2 synthases.

7.1.1 Localization of prostanoid biosynthesis in the kidney The pattern of prostaglandin synthesis along the nephron of the rabbit was determincd by incubating isolated nephron segments with arachidonic acid [ S ] : PGE? was the predominant prostaglandin produced with PGFza synthesis being 50-1 00 times lower except for the glomerulus, where the concentrations were approximately equal. The localization of prostaglandin synthesis obtained by activity measurements correlates with the more recent immunocytochemical localization of COX- 1 and COX-2. the key enzymes of prostanoid biosynthesis (Fig. 7-2).

7.I Renal prostanoirls

I7 I

cox-I Monooxyg. --,

cox-1 Monooxyg

cox-2

Figure 7-2. Distribution of eicosanoid biosynthetic enzymes in the kidney. As outlined in the text, there may be differences between species regarding the localization of certain enzymes. Because most of the functional data have been obtained in the rat, the distribution in this species is depicted. The long-looped nephron and the collecting duct are not drawn to scale. 1, Glomerulus with Bowman’s capsule; 2,3, proximal tubule; 4, descending thin limb; 5 , ascending thin limb; 6, thick ascending limb; 7, macula densa; 8, distal tubule; 9, cortical collecting duct; 10, medullary collecting duct.

The expression of COX-I is most prominent in medullary collecting duct epithelial cells, medullary interstitial cells and cells of the thin ascending limb [6-81. Some staining was also observed in the glomeruli in the endothelial cells lining the terminal portion of the afferent arteriole and possibly in glomerular mesangial cells. Furthermore, COX-1 immunoreactivity was observed in endothelial cells and smooth muscle cells of pre- and post-glomerular vessels. A similar immunofluorescent staining pattern was seen for PGI2 synthase in man, which was intense in endothelial cells of the cortical and medullary peritubular capillary endothelium, in interstitial cells and in glomerular mesangial regions [6]. In most tissues of the organism, COX-2 mRNA and protein are only detectable after stimulation (see Chapter 3). However, COX-2 is present in the normal kidney of untreated animals or healthy men. COX-2 immunoreactivity was localized to endothelial and smooth muscle cells of the arteries and veins [7] and to outer medullary interstitial cells in man and rabbit [7,9] and to the inner medulla in rats [8,lo]. Similar

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7 Renal eicosanoids

species differences were also observed regarding cortical COX-2 localization: i n thc rat, COX-2 protein was localized to the macula densa cells of the distal tubule and to adjacent epithelial cells of the cortical thick ascending loop of Henle [8,I I . 121. Macula densa expression of COX-2 was also observed in rabbits [9], whercas i n man COX-2 immunostaining was detected only in glomerular podocytes (visceral epithclial cells) where it co-localized with thromboxane synthase 171. In other studies. thromboxane synthase was also localized to infiltrating mononuclear cells [ 13). The distinct distribution of COX isozymes indicates involvement of these enzymes in different functional roles of prostaglandins, as will be outlined below. In addition l o being expressed in the normal kidney, both enzymes arc regulated in pathophysiological conditions. A role for COX-2 in kidney development was suggested by examination of kidneys from newborn COX-2 knock-out mice which were severely underdeveloped with few functional nephrons and abundant undeveloped mcsenchymal tissue [ 14,151.

7.1.2 Regulation of prostaglandin synthesis in mesangial cells Most in vitro studies regarding renal regulation of COX mRNA and protein exprcssion have been performed with isolated glomerular mesangial cells. Pure rat renal mesangial cells can be obtained in rather large quantities and cultured over prolongcd times in vitro. These cells retain most of their biological properties over many passages (up to 20) and have therefore been studied in detail by many groups. Human mesangial cells are more difficult to culture and show functional characteristics i n part distinct from rat mesangial cells [16,17]. The most abundant prostanoid of rat mesangial cells is PGE2 (PGE2 > PGF2, > TXA2 > PGI2), whereas human mesangial cells synthesize predominantly PGIz (PG12 > TXA2 > PGF2, > PGEd [2]. Most studies on the regulation of prostaglandin synthesis have been performed with rat mesangial cells. In these cells, COX-2 is induced by a great variety of stimuli that differ in function and in signaling pathways employed. Vasoconstrictors, endothelin, serotonin, angiotensin 11, vasopressin or lysophosphatidic acid, which bind to G-protein-coupled receptors, induce COX-2 mRNA expression within 1 h in ii rapid and transient manner [ 18-21]. The induction is independent of new protein biosynthesis consistent with COX-2 belonging to the group of early response genes. Similar to other cell types, the intracellular signaling pathways that are essential for the induction of COX-2 mRNA in mesangial cells have only partially been characterized as yet [ 19,21,22]. Functionally, induction of vasodilator prostaglandins (mainly PGE, or PGI2) by vasoconstrictors can be envisaged as a counter-regulatory mechanism balancing vascular homeostasis [23]. In contrast, the cytokine interleukin-1 (TL-1) leads to a prolonged induction of COX-2 mRNA and protein which lasts for more than I2 h [24,25 1. These different kinetics might suggest different functional roles for the prostanoids formed under corresponding conditions in vivo. Interestingly enough, induction of COX-2 was not sufficient to increase prostaglandin synthesis in vitro [26], indicating the importance of phospholipase activation in providing free arachidonic acid as a substrate for the enzyme (Fig. 7- 1). Different phospholipase A? (PLA2) isoforms have been isolated from

7. I Renal prosttrrioitls

I73

the kidney: the high molecular weight cytosolic PLAz as well as intra- and cxtrocellular low molecular weight PLAz isoforrns [27,28]. These isoforrns are also pmenr i n mesangial cells 129,301. Platelet-derived growth factor (PDGF), which has been shown to be critically involved in the development of experimental and human glornerulonephritis,transiently induced COX-2 mRNA and protein, but also enhanced COX-I expression, although to a lesser extent and only at later time points [26,311. The later observations clearly shows that, depending on the experimental design, COX-I cannot be considered as a housekeeping enzyme but is regulated, consistent with in vivo data (see below). As in other cell types, induction of COX-2 mRNA and/or protein in mesangial cells is strongly suppressed by glucocorticoids [32]. Partial inhibition of COX-2 is also obtained by the immunosuppressive drug cyclosporine A, which is widely used to prevent transplant rejection (1241 and unpublished results). It may be linked to cyclosporine A-related hypertension, a common adverse effect of this drug.

7.1.3 Cyclooxygenase expression in renal inflammation Large amounts of prostanoids have been described to be synthesized in experimental models of glomerulonephritis.The data obtained from in v i m studies with mesangial cells in culture suggested COX-2 to be the major COX isoform involved in glomerular inflammation. Data on COX isoform expression in animal models are still limited but suggest a role for both isoforrns. In a rat model of glomerulonephritis induced by anti-thymocyte antiserum, COX-2 expression was very transiently upregulated during the first hours of the inflammation, whereas there was a prolonged upregulation of COX-I over several days [33]. A similar increase in COX-] was also observed in other rat models of glomerulonephritis and in biopsies obtained from patients with imunoglobulin A (IgA) nephritis (A. Hartner and M. Goppelt-Struebe, unpublished results). In a very recent study, COX-2 was found to be upregulated in kidney biopsies obtained from patients with active lupus nephritis [34]. Enhanced COX-2 immunoreactivity was found primarily in infiltrating monocytes thus affecting TXA2 synthesis of these cells, with only occasional glomerular COX-2-stained mesangial cells. The functional role of COX upregulation in different disease states remains to be determined. Upregulation of COX isoforms does not necessarily result in enhanced prostanoid synthesis because arachidonic acid, the substrate for both isoforms, may be limiting. Furthermore, due to the distinct localization of the enzymes it is not yet known whether upregulation of COX-2 and COX-I, respectively, leads to the same spectrum of prostanoids, which is dependent on the cell-specific expression of the downstream metabolizing enzymes. Finally it is an open question whether the enhanced levels of prostanoids have to be considered as pro- or anti-inflammatory.

7.1.4 Prostanoid receptors The diverse and in part apparently opposing effects of prostaglandins have become more reasonable upon characterization of the different prostanoid receptors (reviewed

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7 Renal eicosannids

in Breyer et al. [35] see also Section 1.6.2). Pharmacological studies with various agonistic and antagonistic compounds had indicated functionally different receptors. not only for the different prostanoids but also for the major renal prostaglandin, PGE-. Receptors for all prostanoids have now been cloned, their amino acid sequence is known and their coupling intracellular second messenger systems have been investigated [ 36,371. Prostaglandin receptors belong to the family of seven transmembrane receptors which couple to trirneric guanine nucleotide regulatory proteins (G-proteins). Depending on the type of G-protein the receptors couple to different second messenger systems (Table 7-1). So far, only one receptor has been characterized for each of the prostanoids except for PGE?, where four distinct receptors are known which couple to different intracellular signaling pathways and show a very distinct local distribution within the kidney. Table 7-1. Distribution and functions of prostanoid receptors in the kidney Receptor

Localization

Signaling

TP

Mesangial - cells and smooth muscle cells

PI turnover Cd”

IP

Glomerular microvasculature

CAMP?

FP

Mesangium

EP I

Collecting ducts

PI turnover Ca2+f Ca’+?

EP2

Mesdngial cells (rat) Mesangial cells and glomerulus

EP4

CAMP? CAMP?

Collecting ducts Medullary thick ascending limb Collecting ducts

Functional imnlicalion Glomerul ar contraction and vasoconstriction Vasodilation and stimulation of rcnin release Glomerulnr contraction Inhibition of Na’ redbsorption

CAMP? (PI turnover) Ca’+?

Glomerulnr relaxntion. vasorelaxation. immunoniodulation and stimulation of renin release Stimulation of basal HlO absorption Inhibition of NaCl reabborption Inhibition of H20 reabsorption

7.1.4.1 TXAz receptor The TXA2 receptor TP is characterized by its coupling to phospholipase C, generating inosito-l,4,5- trisphosphate (IP3) and diacylglycerol 1381. In glomerular mesangial cells, these and other less well-defined mediators lead to activation of protein kinase C (PKC) and gene expression [39].

7.1 R e n d prostclrioids

175

Because TXA2 is a highly unstable compound, stable analogs of TXA2 such as U46619 are used to study the effects of TXAz in cell culture or in vivo experiments. TXAz is a potent constrictor of vascular smooth muscle and preferentially constricts the afferent arteriole of the kidney [40]. This effect is dependent on Ca2+and can be completely abolished with Ca2+channel blockers [41]. In the glomerulus, TXA2 contracts mesangial cells which leads to a decrease in the area available for filtration, thus reducing glomerular ultrafiltration [42]. Furthermore, in mesangial cells it induces expression of COX-2 [24] and increases the release of vasodilatory PGE2 [43] thereby extending the counter-regulatory role of these two prostanoids. In accordance with these functional studies, TP receptor mRNA was detected in smooth muscle cells of the renal arterioles and in the glomerulus [44]. Immuncytochemical studies confirmed TP receptors in the mesangium, but also on parietal epithelial cells of Bowman's capsule and glomerular podocytes [44]. In rat kidneys, TP receptors were also prominent on cortical tubules, the functional significance of which remains to be determined. Intrarenal cells synthesize only low amounts of TXA2, but under pathophysiological conditions enhanced renal concentrations of TXA2 may originate from infiltrating monocytes and activated thrombocytes. Furthermore, the common precursor of all prostanoids, PGH2, also activates the TP receptor. Increased renal levels of TXA2 have been related to renal dysfunction in various types of experimental and human glomerulonephritis, diabetes mellitus and sepsis [42,45-49]. Inhibitors of TXA2 synthesis ameliorated proteinuria in a variety of models of glomerulonephritis [50]

7.1.4.2 PGF2, receptor The cloned PGF2, receptor FP couples to activation of phospholipase C via a G,protein giving rise to an increase in cytosolic Ca2+levels. The intrarenal distribution of the FP receptor has not yet been reported. PGF2, is one of the major prostaglandins in the glomerulus and increases cytosolic Ca2' levels in cultured mesangial cells [43], suggesting involvement of FP receptor-mediated signaling in glomerular contraction. PGF2, also increased Ca2+levels in the cytoplasm of cortical collecting duct, but possibly by binding to EP receptors [51].

7.1.4.3 Prostacyclin (PGI2) receptor PGI2 receptors (IP) have been cloned from mouse and man. Depending on species and cell type they couple to different types of G-proteins and stimulate both adenylate cyclase and phospholipase C. The potential of one type of receptor to couple to different intracellular signaling pathways may explain the multiplicity and heterogeneity of functional PG12 responses observed in different animals and cell types. The vasodilatory effects of PGI2 in the glomerular microvasculature were related to an increase in intracellular cAMP (cyclic adenosine monophosphate) 1521. Vasodilation mediated via an increase in cAMP levels is also observed with PGEz, but could be shown to be mediated by distinct receptors. Similarly as in other vascular beds, PGIz may thus play a role in counteracting the vasoconstrictor actions of TXA? in regulating renal blood flow. In line with this argument IP receptors were detectable in smooth muscle

176

7 Renal eicosanoids

cells of the walls of interlobular arteries and the afferent arterioles OF the glomeruli [53]. PGI2 is known to stimulate renin release, but no significant staining For IP receptor mRNA was observed in the juxtaglomerular cells of mouse kidneys that were labeled with a probe for renin mRNA [53].These data do not exclude low levels of IP receptors to be also expressed on juxtaglomerular cells, supported by in vifro studies where PG12 stimulated renin secretion from isolated juxtaglomerular cells [ 541. In addition, an indirect regulation of renin release by decreased blood pressure may play a role in PG12-mediatedeffects. An interaction between vasodilating prostaglandins and vasoconstrictors also seems to play a role in the regulation of medullary circulation, most likely involving vascular smooth muscle cells of the respective vessels 15.51. Furthermore, PGll has been reported to act on renal epithelial cells affecting water permeability. Signaling was shown to be mediated by receptors coupled to an increase in cytosolic Ca-+[ 56). These studies were done with stable analogs of PG12, which in addition show agonist activity at the PGE2 receptor EP,. Therefore, a role for IP receptors in renal epithelial function remains to be established.

7.1.4.4 PGE2 receptors PGE2 interacts with four distinct PGE2 receptors (EP) which have been characterired and localized in the kidney [57,58]. As summarized in Table 7-1, these receptors couple to different intracellular signaling pathways immediately denoting the multiple functions ascribed to this prostaglandin. EP receptors show an amazingly distinct local distribution within the ludney that may be related to function (summarized in Breyer et al. [35]). In the future, targeting of the different receptors with specific agonists or antagonists may provide a novel approach to modifying renal function in di\ease states [51]. 7.1.4.4.1 EP1 receptors

The EP, receptor is strongly expressed in collecting duct epithelial cells including cortical and outer and inner medullary collecting ducts, whereas no EP, receptor mRNA was detected in the glomeruli or proximal tubules of the thick ascending limbs [59,60]. Binding of PGE2 to EPI receptors leads to phospholipase C activation and -via IP3- to increased cytosolic Ca2+ levels. In accordance with these receptor characteristics, PGE2 has been shown to increase cytosolic Ca2+ levels in microperfused rat and rabbit collecting ducts [36]. Increased Ca2+levels have been related to the PGE2-dependent inhibition of sodium absorption mediated by EP I receptors, as shown by pharmacological and molecular methods [61]. Inhibition of Na+-K+-ATPaseand reduction of the open probability of the apical sodium channel of collecting duct cells by PGE2 have been described in v i m , most likely by indirect mechanisms, including elevation of cytosolic calcium and activation of PKC. Increased Ca2+levels and activation of PKC are also considered to be involved in PGE2-mediated interference with vasopressin-stimulated osmotic water absorption in collecting ducts [62], in addition to the regulation of CAMP levels by PGE2 via EP3 receptors (see below).

Binding of PGE? to EP, receptors would thus be related to its natriuretic effects by Ca2+-coupledinhibiton of Na+ transport in the collecting ducts. 7.1.4.4.2 EP2/EP4receptors

EP2 and EP4 receptors are physiologically defined by their ability to relax smooth muscle. They are pharmacologically distinguishable, the EP: receptor being activated by the agonist butaprost whereas the EP4 receptor is butaprost insensitive. Both receptors have been cloned and are different in size and amino acid composition [63]. The authentic EP2 receptor has been cloned from human placenta, but was not detectable in human kidney by in situ hybridization under conditions where the expression of the other EP receptors was clearly evident [64]. Expression of EP4, in contrast, is predominantly found in the glomeruli [60,65]. Incubation of cultured mesangial cells with PGE2 leads to an increase in CAMP consistent with the characteristics of the cloned EP4 receptor which activates adenylate cyclase by coupling to a G,-protein. Increased levels of cAMP are related to the dilating effects of PGE2 counteracting the contraction induced by vasoconstrictors such as TXA2, angiotensin I1 or endothelin 1661. Apart from their contractile properties, which are related to the maintenance of glomerular structure and glomerular microcirculation and ultrafiltration, mesangial cells may become actively involved in inflammatory reactions. PGE? is known to regulate expression of certain cytokines negatively by elevation of cAMP levels, e.g. expression of IL- 1. EP4 receptor mRNA staining was also prominent in the renal medulla particularly in transitional cells lining the renal calyx [65]. In situ hybridization has not yet allowed definitive localization of the EP4 receptor to distinct cells of the nephron, although, functionally, expression of this receptor was expected along the nephron because of data showing that PGE2 stimulates cAMP accumulation in various tubular segments (summarized in Breyer et al. [65]). In the collecting duct this leads to enhanced water resorption under basal conditions. In contrast to other EP receptors. which are located at the basolaterlal membrane, EP4 receptors are also present at the luminal membrane allowing interaction with urinary PGE2 [67,68]. 7.1.4.4.3 EP3 receptors

EP3 receptors are prominently expressed in the kidney. In human and mouse kidney, high levels of EP3 receptor expression are detectable in the medullary thick ascending limb cells and in the cortical and outer medullary collecting duct, whereas no expression is found in the inner medullary collecting duct, where EP, is expressed prominently [57,60,69,70]. In rabbit and rat, some species variability with respect to expression in cortical segments of the thick ascending limbs and inner medullary collecting ducts has been reported in agreement with functional effects of PGE, in the respective segments. In the kidney the EP3 receptor is negatively coupled to adenylate cyclase. CAMP levels are critically involved in vasopressin-mediated regulation of water absorption, in addition to the Ca2+-and PKC-dependent effects discussed above. By interference

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7 Renal eicosanoids

with the vasopressin-induced elevation of CAMP levels, PGEz is able lo counleract vasopressin-stimulated water permeability via EP3 receptors. Interestingly, vasopressin has been shown to be an inducer of PGEz synthesis, suggesting a negative feedback loop autoregulating vasopressin action. Although inhibition of activated adenylate cyclase is considered to be the major effect of EP3 receptors, other signaling pathways have to be taken into account because, depending on the species, up to six different isoforms of EP1 receptors have been cloned [63]. These isoforms arise from different mRNA splicing in the cytoplasmic C-terminal domain of the molecule [7 11. Consequently, the binding characteristics of the ligand PGE2 are comparable among the receptor isoforms but they may couple differently to G-proteins. Some isoforms thus stimulate adenylate cyclase or activate phospholipase C. Only very limited data are available regarding the distribution and function of different EP3 isoforms in the kidney [72]. Further studies are necessary to assess the potential role of these isotypes for PGEz-mediated physiologic and pathophysiologic effects in the kidney.

7.1.5 Vasoactive effects of prostanoids: relation to hypertension There is little evidence that prostanoids play a role in those autoregulatory processes. which maintain renal blood flow and glomerular filtration rates at near-constant levels despite wide fluctuations in renal perfusion pressure. Consistently, inhibition of prostaglandin synthesis has little or no effect on renal blood flow in normal human subjects [73]. Under pathophysiological conditions, however, prostanoids may become critically involved in the regulation of renal blood flow, the glomerular filtration rate and the balance of electrolytes. Prostanoids play a dual role in renal regulation of blood pressure. On the one hand, they show anti-hypertensive characteristics by their direct vasodilating effects (PGE, and PGI2), which have also been described in man [74], and by their interference with vasopressin-dependent water and sodium chloride reabsorption [75]. On the other hand, they are pro-hypertensive as vasoconstrictors (PGH2, TXA2 and PGFzJ or by activating the renin angiotensin system (PGI2 and PGE2). Functional studies in the 1970s and 1980s established vasodilating prostaglandins as local mediators counteracting vasoconstrictor effects of norepinephrine or aadrenergic neural stimuli and most prominently the vasoconstrictor effects of angiotensin I1 (Fig. 7-3; reviewed in Walker and Frolich [3] and Levenson et af. [41). In experimental or pathophysiological states associated with enhanced vasoconstrictor influences, renal hemodynamics become dependent on prostaglandin synthesis. Examples are experiments where infusion of angiotensin I1 decreased renal blood flow and the glomerular filtration rate and this effect was enhanced when prostaglandin synthesis was inhibited by COX inhibitors such as indomethacin [3]. Furthermore, counter-regulatory actions of prostaglandins were also suggested by clinical studies showing that indomethacin impaired renal blood flow and the glomerular filtration rate in disease states such as liver cirrhosis with ascites, congestive heart failure or nephrotic syndrome, disorders associated with reduced effective arterial blood volume

7.I Renal prostanoids Basal State Renin

-

Vasoconstriction

f Ang II

\

Vasodilation

179

Normal renal Resistance

f

-L

Vasodilator PG

Increased Levels of Ana II

I

Perfusion pressure + Syrnp. nerve activity 4 Sodium chloride fVasoconstriction

Reninf

+

-

Ang

I

Elevated

114

,

Vasodilation

/

f

Resistance

f

'?4

Vasodilator PG

Inhibition of Cvclooxygenases bv NSAlDs

I

+

Perfusion pressure Symp. nerve activity 4 Sodium chloride

Reninf

-

+

~

Vasoconstriction

Ang II f Vasodilation

NSAIDS~X

Renal Resistance

fl

f

Vasodilator PG

Figure 7-3. Regulation of renal resistance by the renin-angiotensin system and prostaglandins: effect of non-steroidalanti-inflammatory drugs (NSAIDs). Ang 11, angidensin 11.

and elevated levels of angiotensin I1 in the circulation [3,4]. Prostaglandins not only counteract the effects of angiotensin 11, but are also mediators involved in renin release and the subsequent increase in angiotensin II concentrations. In different animal systems, infusion of arachidonic acid resulted in renin release which was blocked by indomethacin. These functional studies were performed in the 1970s. Since then, a large number of studies, even at the molecular level, has been performed, but our understanding of the precise functional role of these mediators in the kidney is still fragmentary and deserves further investigation. The juxtaglomerular cells of the afferent arteriole are the major source of renin. Its synthesis and release is increased by reduced perfusion pressure, enhanced sympathetic nerve activity or a decrease in distal tubular sodium chloride (Fig. 7-3; reviewed in Hilgers and Mann [76]). Prostanoids have been shown to be involved in all three mechanisms though certainly not as exclusive mediators. Other vasoactive mediators, e.g. nitric oxide (NO) or adenosine, interact with prostaglandins to modulate volume homeostasis [77].

180

7 R e n d eicosunoids

The involvement of prostanoids in the pressure control of renin secretion is controversial; experimental evidence has been obtained both in favor and against this concept (discussed in Schricker et al. [78]). In the rat two-kidney one-clip model, renin synthesis induced by low renal arteriolar pressure was inhibited by COX inhibitors [78], but it is not yet known whether prostaglandins are direct mediators of the pressure response or act in a more indirect manner. Studies on the regulation of COX-2 protein expression showed a coordinate increase of COX-2 in macula densa cells and of renin in juxtaglomerular cells in the same glomeruli [ 111. In a detailed temporal analysis, COX-2 mRNA upregulation preceded renin induction, further supporting a role for COX-2 in the regulation of renin. A similar coordinate upregulation of COX-2 [79] and renin [80] was observed in rats fed a low salt diet. Inhibition of COX activity impaired stimulation of renin synthesis in this model [80]. In a more recent study using a specific inhibitor for COX-2, this isozyme was suggested to be related to the increase in the renal renin content induced by low sodium diet [8 11. Intrarenal inhibition of COX-2 activity, however, could not be proven in this study. Therefore, a causal relationship between COX-2 and renin upregulation in vivo remains to be established. Angiotensin II-mediated hypertension in the rat is accompanied by increased levels of TXA2. Whereas TP receptor blockade has little or no effect on renal hemodynamics in normotensive rats, there was attenuation of the pressor and renal vasoconstrictor actions of angiotensin 11 [82,83]. These and other experiments (summarized in Nasjletti 1841) indicate a pro-hypertensive involvement of TXA2 and most likely PGH2 in angiotensin II-mediated hypertension, whereas no such relations were found in models of angiotensin-independenthypertension [84]. Up to now, the discussion of mechanisms of prostanoid effects related to hypertension has been based almost exclusively on data obtained in animals, mostly rats. Thus, caution is recommended in transferring these data to man.

7.1.6 Regulation of water and electrolyte transport by prostanoids: clinical implications PGEz synthesized in the collecting tubules and within the interstitium has considerable impact on medullary hemodynamics which affects sodium and water excretion (for molecular mechanisms see above). Both COX isoforms are detected in the inner medulla. In the rat, dietary salt intake enhanced COX-2 synthesis in the medulla (in contrast to effects observed in the cortex), while no effect on COX-1 expression was observed, suggesting that COX-2 might be involved in medullary PGE2 production, playing a role in promoting the excretion of salt and water in volume overload [lo]. PGE2 affects natriuresis by two different mechanisms: first, PGE2 augments blood flow thereby reducing proximal reabsorption of sodium and, second, PGE2 inhibits sodium reabsorption directly at the ascending limb of the loop of Henle and collecting ducts. This may explain the clinical finding that NSAIDs can partially blunt the effect of loop diuretics. In patients with chronic renal or heart failure, sodium retention can

7.2 R e n d leukotrienes

181

result in weight gain and edema formation and can interfere with antihypertensive therapy (summarized in Murray and Brater [85]) Prostaglandins influence potassium hemostasis via the stimulation of the reninangiotensin system. In patients with pre-existing renal failure, treatment with NSAIDs leads to an inhibition of renin synthesis and a subsequent reduction in aldosterone release which results in a hyporeniemic hypoaldosteronism. The hallmark of this syndrome is hyperkalemia [86].

7.1.7 Cyclooxygenase inhibitors and the kidney NSAIDs, which inhibit COX-1 and COX-2 with preference for the former enzyme, bear little potential of renal toxicity in, healthy persons at therapeutic dosages. Administration of NSAIDs to elderly persons with a high prevalence of arthritis and its associated disability and renal impairment may cause nephrotoxic effects, namely acute ischemic renal insufficiency, acute interstitial nephritis (rare form of renal toxicity) or analgesic-associated nephropathy [85,87,88]. The acute effects are directly related to the inhibition of prostanoid synthesis and are usually fully reversible with discontinuation of the NSAIDs. In contrast, analgesic-associated nephropathy, which is characterized by papillary necrosis and chronic interstitial nephritis, is often irreversible and requires dialysis or renal transplantation. It is caused by long-term treatment with mixtures of NSAIDs and the mechanism is far from clear [89]. Furthermore, chronic inhibition of COX in the elderly or in patients with mild renal insufficiency may lead to hypertension due to sodium retention [90,9I]. The synthesis of prostanoids involved in the regulation of vascular homeostasis and the regulation of water and solute transport in the normal kidney has been largely attributed to COX-1 [92]. The design of selective COX-2 inhibitors thus promised the development of anti-inflammatorydrugs which would spare the kidney and, therefore, should be devoid of the nephrotoxic effects associated with classical non-selective NSAIDs. Detection of COX-2 in the normal kidney, its involvement in blood pressure regulation and its expression in medullary interstitial cells [9], which seem to be an early target of analgesic nephropathy, suggests some caution regarding the safety of specific COX-2 inhibitors until clinical data are available.

7.2 Renal leukotrienes 7.2.1 Biosynthesis Cellular localization of the major enzymes of leukotriene (LT) biosynthesis (Fig. 7-4), 5-lipoxygenase, 5-lipoxygenase-activating protein (FLAP), LTA4 hydrolase and LTC4 synthase, is poorly characterized within the kidney. The major portion of renal leukotrienes is derived from infiltrating polymorphonuclear leukocytes (PMNLs) or resident cells of monocyte-macrophage origin.

I82

7 Renal eicosanoids

Attempts to detect 5-lipoxygenase mRNA or protein in resident glomerular cells have failed. Stimulation of isolated kidneys from rat, rabbit or man with Ca2' ionophore A23 187, the most potent stimulator of leukotriene synthesis, did not induce the release of detectable amounts of leukotrienes. There is, however, evidence that intrinsic renal cells also add to the biosynthesis of leukotrienes, either directly [93] or by transcellular metabolism of intermediates. It is assumed that LTA4, which is derived from neutrophils by their 5-lipoxygenase activity, is released and utilized as substrate by resident glomerular cells to form leukotrienes by the transcellular pathway [94]. LTA4 hydrolase, the rate-limiting enzyme of LTB4 synthesis, is present in glomerular mesangial, endothelial and epithelial cells. Thus, glomerular LTB4 synthesis is dependent on infiltrating PMNLs providing LTA4, and LTA4 hydrolase gene expression and activity in resident cells. Stimulated LTB4 production from isolated rat kidney correlated with renal myeloperoxidase activity, a marker of PMNLs, suggesting that 5-lipoxygenase synthesizing capacity is rate limiting for renal LTB4 production [95]. Conjugation of LTA4 with glutathione is catalyzed by LTC4 synthase, an enzyme distinct from cytosolic and microsomal glutathione S-transferases (GSTs). This enzyme has been partially purified from rat renal microsomes [96]. Transcellular synthesis of cysteinyl leukotrienes was demonstrated in v i m when human PMNLs were incubated with glomerular endothelial cells [94,97]. In the rat, LTC4 is rapidly metabolized by isolated glomeruli to the biologically inactive N-acetyl-LTE4 [98]. In man, inactivation of LTC4 by formation of LTE4 occurs primarily in the blood, but has also been demonstrated in rat renal tubular cells in culture [99]. In naive rats, urinary excretion of cysteinyl leukotrienes is usually below the detection limit of radioimmuno assays (3 pg/ml) [ 1001. Enhanced levels of LTC4, LTD4, LTE4 and N-acetyl-LTE4 were detected under conditions of glomerular inflammation [loo].

7.2.3 Biological activity of leukotrienes in the kidney Cysteinyl leukotrienes are potent constrictors of vascular smooth muscle. Systemic administration of cysteinyl leukotrienes in the rat leads to a reduction in renal blood flow and the glomerular filtration rate [loll. Infusion of LTD4 into the afferent glomerular artery augmented post-glomerular efferent arteriolar resistance, similar to the action of angiotensin 11, thus reducing renal blood flow [ 1021. The reduced glomerular capillary ultrafiltration coefficient was attributed to the effects of cysteinyl leukotrienes on mesanigal cells: in v i m LTC4 and LTD4 contract glomerular mesangial cells [103]. LTD4 receptors have been characterized on human and rat mesangial cells. The Kd for LTD4 binding was determined to be in the nanomolar range [104]. In vitro incubation of mesangial cells with LTD4 led to the formation of inositol-1,4,5trisphosphate. Functionally important is the fact that LTD4 increased mesangial cell adhesiveness for phagocytes that might promote PMNL adhesion in glomerular inflammation [ 1051 Enhanced levels of renal cysteinyl leukotrienes were observed in animal models of glomerulonephritis, e.g. murine lupus nephritis [ 1061, nephrotoxic serum nephritis

7.3 R e n d lipoxins

I83

[loo] or passive Heymann nephritis 11071. A functional role of the increased synthesis of cysteinyl leukotrienes was indicated by the use of a receptor antagonist that ameliorated the reduction in the glomerular filtration rate and renal blood flow in murine anti-GBM (glomelular basement membrane) nephritis [ 1081, nephrotoxic serum nephritis [lo91 and murine lupus [llO]. In addition, there is an additive effect of 12HETE (hydroxyeicosatetraenoic acid) released from activated platelets and cysteinyl leukotrienes on the glomerular filtration rate and renal blood flow in the model of nephrotoxic serum nephritis [ I 1 I]. Under physiological conditions, intrarenal infusion of LTB4 had no acute detrimental effects on glomerular function or the structure of normal rat kidneys [95]. The function of the increased levels of intrarenal LTB4, which were detectable in rat glomerulonephritic kidneys [ 112,1131,is probably only in part due to the chemotactic activity of LTB4 on PMNS, because expression of LTB4 receptors was not always detectable on rat PMNLs [ 1141.

7.3 Renal lipoxins 7.3.1 Biosynthesis of lipoxins Dual lipoxygenation of arachidonic acid by 5-lipoxygenase and 15- or 12lipoxygenase yields a class of ‘lipoxygenase interaction products’ (lipoxins, LXs) (Fig. 7-4;see also Section 1.3.4). 5-Lipoxygenase being absent from glomerular cells, lipoxin synthesis may occur in the kidney by the transcellular pathway via PMNLgenerated LTA4 followed by lipoxygenation by 15- or 12-lipoxygenase located in resident renal cells. Arachidonic Acid

I

5-LOX

4 I

LTC,-Synthase

/

LTA,

I

LTC,

LTA,-Hydm/ase

LTD,

1

LTE,

LTB,

1 -1

PMNLs

Monocytes

12-/15LOX

w\,,

U(B,

Mesangial Cells

Glomerular Endothelial Cells

J

Figure 7-4. Metabolism of ardchidonic acid by lipoxygenases in the glomerulus.

An enzyme with dual 12- and 15-lipoxygenaseactivity has been cloned from rat glomeruli [ 1151, its cellular distribution not being known at present. Metabolism of LTAl

184

7 Renal eicosanoids

to LXA4 by rat mesangial cells was demonstrated in vitro [ 1 161, indicating transformation by 12-lipoxygenase activity in these cells. During glomerular inflammatory reactions, infiltrating macrophages, which are a rich source of 5- and 15-lipoxygenase, may be a source of lipoxins or provide LTA4for transcellular lipoxin synthesis. Furthermore, transcellular biosynthesis of LXA4 by neutrophils and platelets was observed when these cells adhered within the capillary lumen in inflamed rat kidneys [105,117].

7.3.2 Biological activity of lipoxins in the kidney Lipoxins exert distinct effects on renal hemodynamics. In the rat, LXA4 caused vasorelaxation in a COX-dependent manner, possibly by stimulation of endothelial synthesis of prostacyclin. Resistance was reduced selectively in pre-glomerular arteriolar vessels [118]. In addition, LXA4, as well as LXB4 and 7-cis-1l-truns-LX&, displayed vasoconstrictive activity independently of COX-derived metabolites, most likely by acting on mesangial cells or vascular smooth muscle cells [118]. In v i m studies showed partial agonistic activity for LX& at the mesangial LTD4 receptor [ 1191. Because the intrinsic activity of LXA4 is much lower than that of LTD4, interaction between these eicosanoids results in functional antagonism (Fig. 7-5): LTD4-mediated mesangial cell contraction and adhesiveness is attenuated by LX& [119,120]. Cysteinyl leukotrienes are involved in promoting adhesion of PMNLs to endothelial cells by inducing P-selectin expression on endothelial cells [1211. Induction of this adhesion molecule is inhibited by LXA2 and LXB2, again demonstrating the antagonistic modulation of inflammatory reactions by these two types of lipoxygenase products.

Lm, PMNs Chemotaxis?

LTC4 Mesangial Cells Contraction+ GFR ?

4

/

Adhesion t Activation t

/

Endothelial Cells

Endothelial Cells

Adhesiveness ? for Phagocytes

Adhesiveness for Phagocytes f'

P-selectin Expression

+

Figure 7-5. Opposing effects of leukotrienes and lipoxins in glomerular inflammation.

Antagonism of lipoxins is also observed with respect to LTB4. Biosynthesis of LTB4 by neutrophils or in isolated, stimulated glomeruli was inhibited by 15(S)-HETE [1191. Functionally, LXA4 and 15(S)-HETE interfered with LTB4-mediated adhesion of PMNLs [ 1221. These data led to the concept that lipoxins, as antagonists of the pro-inflammatory 5-lipoxygenase products cysteinyl leukotrienes and LTB4, are involved in the resolution phase of glomerulonephritis. In line with this argument, an initial burst of glo-

7.4 Cytochrome P-450enzyme-generuted arachidonic acid metabolites

I 85

merular generation of 5-lipoxygenase products is observed within the first hours of immune complex-mediated experimental glomerulonephritis, leading to glomerular damage and reduced glomerular function amplified by LTB4-mediated PMNL chemotaxis, adhesion and activation. Thereafter, infiltration of macrophages exceeds PMNLs, synthesis of cysteinyl leukotrienes is reduced and synthesis of 12/15lipoxygenase products increases over a longer time course of several days [ 1151. Thus far, specific lipoxin antagonists are not yet available. Therefore, the physiological relevance of lipoxin generation awaits further characterization.

7.4 Cytochrome P-450 enzyme-generated arachidonic acid metabolites In the mammalian kidney, two types of microsomal NADPH (reduced nicotinamide adenide dinucleotide phosphate)-dependent cytochrome P-450 enzyme system metabolize Arachidonic Acid

Epoxygenases

Monooxygenases 7 o/o-l Hydroxylation

Allylic Oxidation

3.

3.

5*,8,9,11,12*,15* HETEs

19-HETE 20-HETE I Oxidation J,

I

Hydrolysis

.L Diols (DHETs)

5,6-EET

20-COOH-AA

e- Arachidonic Acid

+

20-HETE

Figure 7-6. Biosynthesis of monooxygenase products in the kidney. Some HETEs (*) can also be produced by lipoxygenases (see Fig. 7-4). AA, arachidonic acid

186

7 R e n d eicosanoids

arachidonic acid, epoxygenases and monooxygenases (Fig. 7-6; see also Section 6.6. I ) . Most of the metabolites generated by these enzyme systems are highly active compounds that primarily act on kidney vasculature and ion transport (for recent reviews see Harder et al. [ 1231, Makita et al. [ 1241 and Rahman er al. [ 1251).

7.4.1 P-450 arachidonic acid o-hydroxylases In the kidney, P-450-dependent monooxygenases that catalyze fatty acid o or 0-1 hydroxylation belong to the P-450 4A gene family. Most of these enzymes preferentially catalyze o-hydroxylation, some of them exclusively (P-450 4A 1 and 4A4). The highest oxygenase activity is found in the proximal tubules and epithelial cells of the medullary thick ascending limb, as also confirmed by in situ hybridization and immunohistochemistry of several P-450 4A isoforms [ 1261 (Fig. 72). In renal arterioles the enzyme appears to be localized in vascular smooth muscle, although definitive immunolocalization studies have not yet been published. Expression of P-450 4A monooxygenases is regulated by steroid hormones. Treatment of rats with dexamethasone increased P-450 4A mRNA expression and enhanced tubular 20-HETE synthesis approximately twofold [ 1271. Other inducers of P-450 A4 mRNA are the immunosuppressive drug cyclosporine A [128] and clofibrate [127,129]. 20-HETE is the most abundant product formed by isolated microsomal fractions of whole rat kidney [124] and comprises over 60% of all monooygenase products detected. Differences between cortical and medullary microsomal biosynthesis of monooxygenase products have been observed, with 19- and 20-HETE comprising more than 90% of medullary product formation, whereas epoxides (EETs) are the principal cortical products accounting for approximately 60% of total production. No such preferences were observed in rabbit kidney.

7.4.2 Renal epoxygenases Investigation of rat kidney microsomal olefin epoxygenation showed that expoxygenases of the P-450 2C gene family are most prominently involved in the generation of renal arachidonic acid-derived epoxides [ 1301. Principally, four different epoxides may be formed: $6-, 8,9-, 11,12- and 14,15-EETs. Due to distinct isozyme expression, the abundance of these possible regioisomers is variable, 11,12EET being the most abundant product formed by rat microsomal preparations [124]. In human kidney cortex preparations, biosynthesis of 11,12- and 14,15-EETs was predominant [ 1311. Epoxides may be hydrolyzed to their respective diols. Furthermore, allylic oxidation and distraction of H 2 0 forms HETEs. Of the different P-450-produced regioisomers which do not show stereoselectivity, 1 2(R)-HETE has been called to attention because of its biological activity. In contrast to the lipoxygenasederived 12(S)-HETE, 12(R)-HETE inhibits Na+-K+-ATPase[ 1321 and renin release [ 1331.

7.4 Cytochrome P-450enzyme-generated arachidonic acid metabolites

I 87

7.4.3 Functional properties of 19- and 20-HETE Whereas 19-HETE proved to be a stereo-specific vasodilator, the effects of 20-HETE are far more variable. In most instances, 20-HETE showed vasoconstrictor activity [ 127, 1341, although vasodilator effects were observed in the rabbit isolated perfused kidney [135]. The vasodilator effects of 19- and 20-HETE in the perfused kidney were dependent on COX, i.e. 20- and 19-hydroxyarachidonic acids can replace arachidonic acid as substrate of prostanoid biosynthesis [ 1361. The molecular mechanisms of 20-HETE-mediated vasoconstriction are only partially understood. In canine renal vascular smooth muscle cells the vasoconstrictor response to 20-HETE was associated with an increase in cytosolic Ca2+and membrane depolarization [ 1341. Specifically, 20-HETE inhibited the opening of a 117-pS K' channel. Modulation of ion channels is also involved in the actions of 20-HETE in the medullary thick ascending loop of Henle, where it is involved in the regulation of sodium reabsorption. In the rat thick ascending limb K' channel activity was specifically inhibited by 20-HETE. Inhibition of the P-450 metabolic pathway increased the activity of the 70-pS K' channel, which is the predominant of the two apical K' channels operating under physiological conditions in the thick ascending limb, whereas 20-HETE, the major P-450 metabolite in the medullary thick ascending limb, inhibited the activity even at low concentrations (10 nM) [137,138]. 20-HETE also interfered with C1- transport at the same concentration [139,140]. Both, inhibition of the K" channel and C1- transport were dependent on an increase in cytosolic Ca2' (by bradykinin [ 1391 or elevation of extracellular Ca2' [ 138]), suggesting that activation of Ca2+-dependentphospholipases, release of arachidonic acid and metabolism by P450 enzymes might form the chain of events important for the observed physiological response in the rat thick ascending limb. Species differences were observed comparing rats and rabbits: Incubation of rabbit medullary thick ascending limb cells with 20-HETE decreased the K' and Na' content by interference with the Na'-K+-2Cl- cotransporter, but not with K' channels or Na+-K+-ATPase [141]. In contrast, in rat proximal tubules 20-HETE was shown to inhibit Na+-K+-ATPase.As parathyroid hormone- and dopamine-mediated inhibition of the ion exchanger are also dependent on P-450-derived arachidonic acid metabolites, it is likely that 20-HETE is involved in mediating the inhibition of parathyroid hormone and dopamine [ 142,1431.

7.4.4 Functional properties of epoxides Opposing activities, vasoconstrictor as well as vasodilator properties, have been attributed to the epoxides, although, in most non-renal systems investigated, the vasodilator effect seems to dominate, e.g. in rat tail artery, bovine coronary artery or cerebral arterioles [ 1 241. In the kidney, vasoconstriction or vasodilation depend on epoxide isomers and species investigated. In the rabbit, 5,6-, 8,9- and 1 1,12-EETs were vasodilators, whereas infusion with 14,15-EET resulted in a dose-dependent increase in perfusion pressure [ 1361. 5,6-EET was the most potent epoxide and the only one dependent on

188

7 Renal eicosanoids

COX activity. This was partly attributable to conversion of 5,6-EET to a prostaglandin analog, 5,6-epoxy-PGEl, by COX and partly to the release of the vasodilator prostaglandins PGE2 and PGI2 [ 1441. Increased prostaglandin synthesis may relate to an increased Ca2' influx: Epoxides, particularly 5,6-EET have been shown to increase cytosolic Ca2+concentrations in various cell types, among them endothelial, mesangial and proximal tubule cells [ 1241. 5,6-EET action was endothelial dependent, suggesting the involvement of phospholipases and endothelial cell cyclooxygenases. In contrast to the vasodilator properties of the epoxides in rabbit, infusion of 8,9-EET or 5,6-EET elicited vasoconstriction in the rat kidney [145]. Dissimilar renovascular responses of rat and rabbit are also observed after infusion of arachidonic acid. In the rat, vasoconstrictor responses due to the action of PGH2 dominate, whereas in the rabbit, PGH2 is further metabolized to vasodilator prostaglandins [145]. There seems to be a species difference in the ability to metabolize further the primary vasoconstrictory compounds (epoxide and PGH2) to vasodilatory prostaglandins.

7.4.5 Monooxygenase products in animal models of renal hypertension In most cases, investigation of the biological effects of arachidonic acid monooxygenase products still remains somewhat fragmentary and descriptive. The relevant contribution of P-450 metabolites to renal physiology became clearer upon investigation of animal models of renal hypertension. A role for the kidney microsomal arachidonic acid monooxygenases in the development of hypertension in spontaneously hypertensive rats (SHRs) was first put forward by McGiff and coworkers in 1988 and has been confirmed in many studies since then (summarized in McGiff [145]). SHRs develop hypertension between the ages of 5 and 13 weeks; the vascular tone in the pre-glomerular vasculature of these animals is elevated. During this phase, an increase in o-hydroxylase activity and protein was observed resulting in elevated production of 20-HETE [ 1391. Treatment of young rats with SnC12 or hemarginate, which induce heme oxygenase and cause depletion of renal cytochrome P-450, reduced renal cortical microsomal arachidonic acid metabolism and dose dependently prevented the rise in blood pressure [124]. Inhibitors of cytochrome P-450 produced vasodilation of the afferent arteriole and attenuated pressure-dependent vasoconstriction [ 1461. These data, together with the known vasoconstrictor activity of 20-HETE, suggest that 20-HETE serves an endogenous prohypertensive role in the SHRs. In addition to the vascular effects, increased production of 20-HETE in the proximal tubules of SHRs may also modulate sodium transport. In contrast to other strains of rats, the activity of o-hydroxylases but not epoxygenases is regulated by salt in SHRs [147]. Hypertension related to salt handling was investigated in Sprague Dawley rats and Dahl rats which develop genetic salt-sensitive hypertension. In normal rats high salt intake results in an adaptive response, i.e. increased renal salt excretion to prevent progressive salt retention, volume expansion and, as a consequence, hypertension. Investigation of arachidonic acid-metabolizing P-450 enzymes of the rat kidney

7.4 Cytochrome P-450 enzyme-generated arachidonic acid metabolites

1 89

revealed that P-450 2C epoxygenase isoform(s) were upregulated by a high salt diet whereas o-hydroxylases were not changed, suggesting that epoxides, as inhibitors of tubular Na' reabsorption, may play a role in the increased salt excretion. Epoxides thus seem to be part of the compensatory mechanisms of the kidney for handling an excessive salt load. This correlation is substantiated by the finding that inhibition of epoxygenases by clotrimoxazole, together with high salt, led to the development of hypertension [ 1481. In Dahl salt-sensitive hypertensive rats, synthesis of epoxy metabolites was reduced in cortex preparations, whereas in the outer medulla synthesis of the major metabolite 20-HETE was diminished [ 1491. Functionally this may be associated with the elevated C1--reabsorption in the thick ascending limb observed in Dahl saltsensitive rats. In normotensive rats, which were fed a high salt diet, reduction of 20HETE formation in the renal outer medulla by infusion of 17-octadecenoic acid, a monooxygenase inhibitor, also induced the development of hypertension [ 1501. Pharmacological induction of P-450 4A a-monooxygenase by clofibrate prevented the development of hypertension [140]. In contrast to normotensive rats, a high salt diet failed to increase epoxygenase activity in Dahl salt-sensitive rats [ 1241. These animals thus have reduced levels of monooxygenase products to prevent reabsorption of sodium chloride. In consequence, a higher renal perfusion pressure than in normotensive rats is necessary to secrete excess sodium and water. As outlined above there is considerable variation with respect to the contribution of individual P-450 enzymes and monooxygenase metabolites in different strains of rats. However, there is now ample evidence from many studies that, in the rat, monooxygenase products are critically involved in the regulation of vascular tone and the regulation of salt and water balance (Table 7-2). Much less is known about the role of monooxygenase products in man. Table 7-2. Main biological actions of P-450-derived arachidonic acid metabolites in the kidney Epoxides

20-HETE

Regulation of vascular tone, e.g. 5,6-EET (vasodilation in rabbit) and 8,9-EET (vasoconstriction in rat) Vasoconstriction Regulation of sodium balance by inhibition of Na+-K+2C1- co-transporter + decrease in Na+ reabsorption Inhibition of Na'-K+-ATPase

7.4.6 P450-catalyzed arachidonic acid metabolism in man Investigation of P450-catalyzed arachidonic acid metabolism of the human kidney cortex in postmortem subjects showed synthesis of both expoxygenases (14,15-, 11,12- and 8,9-EETs) and o-h ydroxylase products (19- and 20-HETEs) [ 131,15I]. Although qualitatively similar, there was more than 100-fold quantitative interindividual variability. Preliminary studies have suggested that P-450-derived arachidonic acid metabolites may also be related to hypertension in man; however, up to now no conclusive evidence has been provided.

190

7 Renal eicosanoids

7.5 Isoprostanes 7.5.1 Generation of isoprostanes in the kidney In contrast to the arachidonic acid metabolites discussed so far, isoprostanes (IsoPs) are primarily formed non-enzymatically by free radical-catalyzed peroxidation (reviewed in Roberts and Morrow [ 1521 and Morrow and Roberts [ 1531; see also Section 1.3.8). Arachidonic Acid

Y

b: OW

OM

FZ-ISOP

E,-IsoP

h:

@ 0,-lsoP

Irothrornboxanr

cOH

OH

G-

m,

OH

8iso Prostaglandin F2=

Ho'

OH

Prostaglandin F h

Figure 7-7. Non-enzymatic formation of isoprostanes. For comparison, the stereoisomer PGF2, is depicted.

The mechanism of formation is depicted in Figs. 1-10. and 7-7. Free radical-induced abstraction of bis-allylic hydrogens can occur at three different locations thus leading

7.5 Isoprostunes

19 1

to the four regioisomers depicted. Each regioisomer is theroretically comprised of a mixture of eight racemic diastereomers. The bicycloendoperoxide intermediates cannot only be reduced to F2 isoprostanes (Fz-IsoP or 8-Epi-PGF2,) but also rearrange to E2-IsoP, D2-IsoP or isothromboxane. The relative amount of F2, D2, E2 isoprostanes or isothromboxane formed differs between tissues. Thus far, only Fz-IsoP has been investigated in more detail with respect to formation and function, mainly because it was the only synthetic isoprostane available. The essential difference between isoprostanes and COX-derived prostanoids is the orientation of the side chains of the prostane ring: side chains of compounds generated by autoxidation are oriented predominantly in cis, whereas side chains of the enzymatically derived compounds are in trans (Fig. 7-7). There is evidence that small amounts of F2-isoP can be formed in platelets by cyclooxygenases [ 1541, which are, however, an order of magnitude lower than the amount of TXA2 under comparable conditions. Mesangial cells in culture are also able to form Fl-isoP by a COX-dependent mechanism [ 1551, but urinary excretion of F2-isoP in healthy man remained essentially unchanged after treatment with NSAIDs [ 1541 Formation of isoprostanes can occur in vitro, e.g. upon storage of plasma even at temperatures below 0°C. In vivo, F2 isoprostanes are initially formed nonenzymatically in situ on phospholipids, i.e. formation of isoprostanes is not dependent on the availability of free arachidonic acid [156]. Due to the kinked structure of these compounds, formation of isoprostane-containing phospholipids leads to a marked distortion in the membrane symmetry and may thus affect lipid fluidity and lipid protein interactions. Fz-isoP phospholipids are substrates of the low molecular weight PLA2 of bee venom, but it is not yet clear which phospholipases are responsible for the release of isoprostanes from phospholipids in vivo.

7.5.2 Functional properties of isoprostanes There is biochemical and pharmacological evidence that isoprostanes bind to a unique receptor that has, however, not been cloned yet. The receptor seems to be similar to the TXA2 receptor, because the renal vasoconstricting activity of F2-isoP is abrogated by SQ29548, a thromboxane receptor antagonist [ 1571. These results imply that the effects of SQ29548 must not necessarily be related to inhibition of thromboxanemediated biological effects. Renal function of F2-isoP was investigated in rats. Intrarenal infusion of F2-isoP was associated with a marked pre-glomerular vasoconstriction resulting in an increase in afferent resistance and subsequent reduction in the glomerular filtration rate and plasma flow [ 1571. Infusion of the stereoisomer PGS,, in contrast, had little effect on renal hemodynamics under these conditions. Renal microvessels seem to be particularly sensitive to the action of isoprostanes as systemic or intrarenal infusion of isoprostanes did not significantly change systemic blood pressure [ 1571. These findings in rats are consistent with increased urinary levels of F2-isoP in patients with hepatorenal syndrome, a disease that is characterized by renal failure with intense renal vasoconstriction in patients with severe liver disease [ 1581

192

7 Renal eicosanoids

7.6 Perspective The organization of this chapter followed the classical pattern of dealing with eicosanoids, i.e. to sort them by their biosynthetic pathways. This approach also reflects the majority of investigations dealing with one type of eicosanoid at a time. It is certainly justified by improving our understanding of the regulation of biosynthesis, of molecular signaling pathways and of the physiological and pathophysiological significance of individual eicosanoids. It will, however, be the challenge of future investigations to obtain a better understanding of the concerted action of eicosanoids, while also implicating interactions with other bioactive mediators.

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156-161.

1601

[641 [651 1661 [671 1681 [691 1701

1731 [741

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F643-650. K.L. Pierce, D.W. Gil, D.F. Woodward and J.W. Regan. Trends Pharmcol. Sci. 1995; 19: 253-256. M.D. Breyer, L. Davis, H.R. Jacobson and R.M. Breyer. Am. J. Physiol. 1996;270: F912-€9 18 R.M. Breyer, L.S. Davis, C. Nian, R. Redha, B. Stillman, H.R. Jacobson and M.D. Breyer. Am. J. Physiol. 1996;270:F485-F493. P. Mene and M.J. Dunn. Circulat. Res. 1988;62: 916-925. Y.Sakairi, H.R. Jacobson, T.D. Noland and M.D. Breyer. Am. J. Physiol. 1995;269: F257-F265. Y. Ando and Y. Asano. Am. J. Physiol. 1995;269: F1093-FllOI. M.D. Breyer, H.R. Jacobson, L.S. Davis and R.M. Breyer. Kidney Int. 1993; 44: 1372-1378. S. Taniguchi, T. Watanabe, A. Nakao, G. Seki, S. Uwatoko and K. Kurokawa. Am. J. Physiol. 1994;266:C1453-Cl458. A. Schmid, K.-H. Thierauch, W.-D. Schleuning and H. Dinter. Eur. J. Biochem. 1995;228: 23-30. K. Takeuchi, N. Takahashi, T. Abe and K. Abe. Biochem. Biophys. Res. Commun. 1994;199: 834-840. A.J. Donker, L. Arisz, J.R. Brentjens, G.K. Van der Hem and H.J. Hollemans. Nephron 1976;17: 288-296. A. Milot, R. Lambert, M. Lebel, J.R. Cusson and P. Larochelle. J. Hypertens. 1996; 14:765-771. M. Mistry and A. Nasjletti. Am. J. Med. Sci. 1988;295: 263-267. K.F. Hilgers and J.F.Mann. Kidney Blood Press .Res. 1996;19: 254-262. R.C. Harris. J. Hypertens. 1996;14:815-822. K.Schricker, M. Hamann and A. Kurtz. P’iigers Arch. 1995;430: 188-194.

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7 Renal eicosanoids K.F. Badr, G.F. Schreiner, M. Wasserman and 1. Ichikawa. J. Clin. Invest. 1988; 81: 1 702- 1709. S.H. Wu, B.A. Bresnahan and E.A. Lianos. Kidney Int. 1993; 43: 1280-1285. J. Fader, A. Wiemeyer, K.H. Marx, K. Kuhn, K.M. Koch and J.C. Frolich. Kidney Int. 1989; 36: 46-50. C.R. Albrightson, B. Short, G. Dytko, B. Zabko Potapovich, B. Brickson, J.L. Adarns and D.E. Griswold. Kidney Int. 1994; 45: 1301-1310. R.A. Kreisle, C.W. Parker, G.L. Griffin, R.M. Senior and W.F. Stenson. J. Immunol. 1985; 134: 3356-3363. T. Katoh, F.G. Lakkis, N. Makita and K.F. Badr. Kidney Int. 1994; 46: 341-349. R. Garrick, S.Y. Shen, S. Ogunc and P.Y. Wong. Biochem. Biophys. Res. Commun. 1989; 162: 626-633. A. Papayianni, C.N. Serhan, M.L. Phillips, H.G. Rennke and H.R. Brady. Kidney Int. I995 ; 47: 1295- 1 302. T. Katoh, K. Takahashi, D.K. DeBoer, C.N. Serhan and K.F. Badr. Am. J. fhysiol. 1992; 263: F436-F442. K.F. Badr. Kidney Znt. 1992; 38 (Suppl.): S101-SI08. Y.M. O’Meara and H. Brady. Kidney Int. 1997; 58: S56S61. A. Papayianni, C.N. Serhan and H.R. Brady. J. Immunol. 1996; 156: 2264-2272. D.B. Fischer, J.W. Christman and K.F. Badr. Kidney Znt. 1992; 41: 1155-1 160. D.R. Harder, W.B. Campbell and R.J. Roman. J. Vasc. Res. 1995; 32: 79-92. K. Makita, J.R. Falck and J.H. Capdevila. FASEB J. 1996; 10: 1456-1463. M. Rahman, J.T. Wright Jr and J.G. Douglas. Am. J. Hypertens. 1997; 10: 356-365. M.L. Schwartzman, J.L. Da Silva, F. Lin, M. Nishimura and N.G. Abraham. Nephron 1996; 73: 652-663. F. Lin, N.G. Abraham and M.L. Schwartzman. Ann. NYAcad. Sci. 1994; 744: 11-24. M. Nakamura, S. Imaoka, K. Miura, E. Tanaka, S. Misawa and Y. Funae. Biochem. Pharmacol. 1994; 48: 1743-1746. A.E. Sirnpson. Gen. Pharmacol. 1997; 28: 351-359. A. Karara, K. Makita, H.R. Jacobson, J.R. Falck, F.P. Guengerich, R.N. Dubois and J.H. Capdevila. J. Biol. Chern. 1993; 268: 13565-13570. A. Karara, E. Dishman, H. Jacobson, J.R. Falck and J.H. Capdevila. FEBS Lett. 1990; 268: 227-230. T. Satoh, H.T. Cohen and A.I. Katz. J. Clin. Invest. 1993; 91: 409415. W.L. Henrich, J.R. Falck and W.B. Campbell. Am.J.Physio1. 1992: 263: F665-F670. Y.H. Ma, D. Gebremedhin, M.L. Schwartzman, J.R. Falck, J.E. Clark, B.S. Masters, D.R. Harder and R.J. Roman. Circulat. Res. 1993; 72: 126136. M.A. Carroll, M. Balazy, P. Margiotta, D.D. Huang, J.R. Falck and J.C. McGiff. Am. J. Physiol. 1996; 271: R8634869. M.A. Carroll, M.P. Garcia, J.R. Falck and J.C. McGiff. J. Pharmacol. Exp. Ther. 1992; 260: 104- 109. W. Wang and M. Lu. J. Gen. Physiol. 1995; 106: 727-743. W.H. Wang, M. Lu and S.C. Hebert. Am.J. Physiol. 1996; 271: C 103-1 1 1. J.S. Grider, J.C. Falcone, E.L. Kilpatrick, C.E. Ott and B.A. Jackson. Can.J. Physiol. Pharmacol. 1997; 75: 91-96. R.J. Roman, M. Alonso Galicia and T.W. Wilson. Am. J. Hypertens. 1997; 10: 63S67s. B. Escalante, D. Erlij, J.R. Falck and J.C. McGiff. Am. J. Physiol. 1994; 266: (21775C 1782.

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M. Ominato, T. Satoh and A.1. Katz. J. Membr. Biol. 1996; 152: 235-243. C.M. Ribeiro, G.R. Dubay, J.R. Falck and L.J. Mandel. Am. J. Physiol. 1994; 266: F497-F505. M.A. Carroll, M. Balazy, P. Margiotta, J.R. Falck and J.C. McGiff. J. Biol. Chem. 1993; 268: 1226612266. J.C. McGiff. Annu. Rev. Pharmacol. Toxicol. 1991; 31: 339-369. J.D. Imig, J.R. Falck, D. Gebremedhin, D.R. Harder and R.J. Roman. Hypertension 1993; 22: 357-364. D.E. Stec, M.R. Trolliet, J.E. Krieger, H.J. Jacob and R.J. Roman. Hypertension 1996; 27: 1329-1336. K. Makita, K. Takahashi, A. Karara, H.R. Jacobson, J.R. Falck and J.H. Capdevila. J. Clin. Invest. 1994; 94: 24 14-2420. Y.H. Ma, M.L. Schwartzman and R.J. Roman. Am. J. Physiol. 1994; 267: R579R589. D.E. Stec, D.L. Mattson and R.J. Roman. Hypertension 1997; 29: 3 15-3 19. M.L. Schwartzman, P. Martasek, A.R. Rios, R.D. Levere, K. Solangi, A.I. Goodman and N.G. Abraham. Kidney Int. 1990; 37: 94-99. L.J. Roberts and J.D. Morrow. Biochim. Biophys. Acra 1997; 1345: 121-135. J.D. Morrow and L.J. Roberts. Biochem. Pharmacol. 1996; 51: 1-9. Z. Wang, G. Ciabattoni, C. Creminon, J. Lawson, G.A. Fitzgerald, C. Patron0 and J. Maclouf. J. Pharmacol. Exp. Ther. 1995; 275: 94-100. T. Klein, F. Reutter, H. Schweer, H.W. Seyberth and R.M. Nusing. J. Pharmacol. Exp. Ther. 1997; 282: 1658- I 665. J.D. Morrow, J.A. Awad, H.J. Boss, LA. Blair and L.J. Roberts. Proc. Natl Acad. Sci.USA 1992; 89: 10721-10725. K. Takabashi, T.M. Nammour, M. Fukunaga, J. Ebert, J.D. Morrow, L.J. Roberts, R.L. Hoover and K.F. Badr. J. Clin. Invest. 1992; 90: 136-141. J.D. Morrow, K.P. Moore, J.A. Awad, M.D. Ravenscraft, G . Marini, K.F. Badr, R. Williams and L.J. Roberts. J. Lipid Mediat. 1993; 6: 417-420.

Prostaglandins, Leukotrienes and Other Eicosanoids: From Biogenesis to Clinical Application Edited by F. Marks, G. Fürstenberger Copyright © WILEY-VCH Verlag GmbH, D-69469 Weinheim (Federal Republic of Germany). 1999

8 The role of eicosanoids in reproduction H.P. Zahrudnik, B. Wetzku and W.R. Schayer

8.1 Introduction Reproduction is a basic process of life. Sexual reproduction, such as takes place in humans, is naturally more complex than asexual reproduction and therefore requires more sophisticated regulation. Beginning with the maturation of the gametes, through fertilization, pregnancy and parturition, the endocrine, paracrine and autocrine mechanisms and their undisturbed networks are of tremendous importance. Eicosanoids play an important role in the orchestration of local mediators such as cytokines, growth factors and nitric oxide (NO) directed by endocrine messages, for example sex steroids. Effects of eicosanoids which are very important in reproduction are smooth muscle contraction (menstruation and labor), modulation of immune response (implantation and maintenance of pregnancy), physiological inflammatory reactions such as increase of local blood flow, increase of vascular permeability, activation of proteolytic enzymes (ovulation, cervical ripening and labor) and the regulation of vascular tone (pregnancy-induced hypertension). From knowledge of the physiological importance of eicosanoids, their roles in pathological processes and therapeutic use can be deduced and they are now used extensively in clinical medicine. The importance of eicosanoids for reproduction is underlined by the history of eicosanoid research, which started in the reproductive field with the observation of the effects of human seminal fluid on the uterus [ 11.

8.2 Female reproductive system 8.2.1 Ovarian function During the female reproductive phase, ovarian function is cyclical and can be divided into two parts. The first part, the follicular phase, consists of the ripening of follicles, selection of one follicle, maturation of an egg and, finally, rupture of the Graafian follicle to release the egg into the Fallopian tube. During the second or luteal phase, a

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8 The role of eicosanoids in reproduction

corpus luteum develops which produces progesterone and, in case of fertilization, is essential for the early support of the embryo. In cycles without fertilization, the corpus luteum needs to undergo luteolysis which is followed by menstruation. Most interest has focused on the participation of eicosanoids in ovulation, corpus luteum regression and steroidogenesis (see Fig. 8-1 for an overview). PGE, PGF,, LT's

PGE, PGF,,

PGF,,

PGE, PGF,,

LT's

LT's

PGE,

LT's.15-HETE

cox-2

Menstruation

Ovulation

Follicular Phase

Luteal Phase

Figure 8-1. The possible role of eicosanoids in cyclical ovarian function. Size and typing of arachidonic acid metabolites represents the importance of the particular eicosanoid according to current knowledge.

8.2.1.1 Follicular phase: ovulation Ovulation is the liberation of the mature oocyte from the Graafian follicle. This process is triggered shortly after the mid-cycle luteotropic hormone (LH) surge by the complementary action of LH and follicle-stimulating hormone (FSH). It is accompanied by the 'rupture' of healthy tissue at the surface of the ovary which is underlaid by a variety of biochemical events and resembles an acute inflammatory reaction with hyperemia, edema formation and invasion of leukocytes where eicosanoid production is typically increased [2]. Human follicular fluid contains prostaglandin F2a (PGF2a), PGE2, 6-keto-PGF1a, thromboxane B2 (TXB2), leukotriene B4 (LTB4) and LTC4 [3] PGF2, concentration increases during the pre-ovulatory phase [4]. Animal studies suggest that the preovulatory increase in prostaglandin production by ovarian follicles plays a critical role in ovulation [5]. Gonadotropins can rapidly induce the prostaglandin production in vitro [6]. Therefore, the pre-ovulatory rise in LH may be responsible for the stimulation of ovarian prostaglandin production. In contrast to other systems, this is not caused by an increase of arachidonic acid availability from membrane phospholipid stores, but by increased activity of cyclooxygenase (COX) [6]. This increased activity requires RNA and protein synthesis [7]. It is also known that inhibitors of COX can inhibit or delay ovulation which can be reversed by adding PGF2, [8]. COX-2 is induced shortly before follicular rupture and might therefore be responsible for the increased prostaglandin production accompanying or causing

8.2 Female reproductive system

20 1

follicular rupture [9]. Pre-ovulatory COX-2 induction has even been proposed as a molecular determinant of the species-specific length of ovulation [ 101. This hypothesis is further supported by recent experiments with mice having a deletion of the COX-2 gene which failed to ovulate [ 1 11. PGFza and PGE2 are the prostaglandins for which an important role in ovulation could be observed in many human and animal studies (reviewed in Gelety and Chaudhuri [5],Murdoch er al. [12] and Olofsson and b u n g [13]). PGE2 induces the maturation of the oocyte and increases local blood flow and vascular permeability, causing local edema which precedes ovulation. PGF2,, however, appears to be the important eicosanoid for follicular rupture, since it induces proteolysis and plays a role in collagenolysis. PGF2, also increases ovarian contractility and antagonizes the PGE2-induced hyperemia. Further, in ovaries of most species a high abundance of PGE2-9-keto-reductasewas observed which can convert PGE2 to PGF2, [14]. The interconversion of primary prostaglandins may also be significantly related to ovulation. Concerning lipoxygenase products, there are few studies investigating their role in follicle ripening and rupture. Lipoxygenase (LOX) activity is increased shortly before ovulation [ 151. Lipoxygenase inhibitors either blocked ovulation [ 15,161 or increased prostaglandin production and ovulation rate [ 171. Immunohistochemically, the presence of 5- and 12-lipoxygenase could be demonstrated in granulosa cells [18]. After gonadotropin stimulation, the production rate of leukotrienes, prostaglandins and hydroxyeicosatetraenoic acids (HETEs) increased, but with different time courses implicating different functions for the ovulation process [ 19,201. LTB4 synthesis increases quickly after human chorion gonadotropin (hCG) treatment, reaching its peak well before PGE2 and PGF2, and may cause chemotaxis of leukocytes and then stimulate their release of lysozymes. LTC4 showed a first peak early and a second one several hours after the gonadotropins. Peptidoleukotrienes might be important for increased vascular permeability and for contraction of blood vessels after ovulation has occurred. Synthesis of 15-HETE correlated with steroid synthesis and ovarian function, suggesting that it may play a role in mammalian ovulation [19]. Further, cytochrome P-450-derived metabolites of arachidonic acid, for example epoxyeicosatrienoic acids (EETs), may regulate steroidogenesis of granulosa cells [2 11. In conclusion, eicosanoids are produced by growing pre-ovulatory follicles and ovulation can be blocked pharmacologically by inhibitors of eicosanoid biosynthesis in a diversity of vertebrate species. Over the last 20 years it has become dogma that COX derivatives of arachidonic acid, in particular PGE2 and PGFz,, are obligatory local ovarian mediators of ovulation. However, several recent studies have cast some doubt on the absoluteness of this doctrine, indicating that ovulation can progress to completion without a pre-ovulatory rise in ovarian prostaglandin production. It appears that auxiliary, i.e. ‘fail-safe’, processes can assure ovulation [ 121.

8.2.1.2 Luteal phase: luteolysis The corpus luteum is a transient endocrine organ which is essential for the preparation of a receptive endometrium and the survival of the fertilized gamete during the earliest stages of pregnancy. At the end of the lifespan of this gland, highly

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8 The role of eicosunoids in reproduction

coordinated apoptotic reactions lead to its degradation. In rhesus monkeys, intraluteal infusion of COX inhibitors in the first half of the luteal phase caused a decreased progesterone production and a shortening of the luteal phase, which suggests a luteotrophic role for local prostaglandins [22]. The concentration of PGE2 in the human corpus luteum is increased during the early and decreased during the late luteal phase, whereas PGF2, increases during the late luteal phase in relation to PGE2 1231. PGE2 seems to mimic the luteotrophic effects of LH in inducing luteinization via cyclicAMP-coupled signal transduction resulting in increased progesterone production, whereas PGFz, is luteolytic (reviewed in Gelety and Chaudhuri [5]).In non-primate species, PGFz, deriving from the uterus is a wellestablished luteolytic factor, since hysterectomy leads to the persistence of a corpus luteum. In primates and humans, cyclic ovarian function is not affected by hysterectomy [5], which is probably caused by anatomical differences, as the uterus and ovaries of primates have a separate blood supply. Injection of PGF2, directly into the ovary of rhesus monkeys caused a fall in systemic progesterone concentration and an earlier onset of menstrual bleeding without affecting LH levels [24]. COX-I was shown immunohistochemically in luteal cells [25,26] and its expression increases from early to late luteal phase [26]. COX-2 expression in luteal cells was stimulated by LH and gonadotropin-releasing hormone (GnRH) [27]. The presence of most prostaglandin receptors in the ovary was shown by ligand-binding assay and the expression of PGFz, (FP), PGE2 (EP) and TXAZ (TP) receptors was also analysed by protein and mRNA analysis (reviewed in Olofsson and h u n g [ 131). However, only FP and EP seem to be important for ovarian physiology. FP is upregulated during the luteal phase and could be associated with apoptosis in mouse luteal cells [28]. The identity of the prostaglandin E receptor subtype and its signal transduction pathway requires further elucidation. There is even less known about the role of lipoxygenase-derived eicosanoids in corpus luteum function. Immunohistochemically, 5-lipoxygenase was expressed predominantly in small luteal cells, whereas 12-lipoxygenasecould be found mainly in large luteal cells. Positive staining for both enzymes increased from early to late luteal phase [26]. It appears, therefore, that the regulation and lifespan of the corpus luteum may involve the local production of luteotrophic and luteolytic prostaglandins as well as interactions with other paracrine regulatory factors [5].

8.2.2 The Fallopian tube The physiological importance of the Fallopian tube lies in ovum pick up and subsequent transport of the ovum through the ampulla within a few hours of follicular rupture. The tuba1 ampulla is the place where the ovum meets the sperm and fertilization takes place. Ciliary activity and secretory production of the epithelium (endosalpinx), as well as muscular contractions of the Fallopian tube are necessary for fertilization and ovum transport. Both compartments of the oviduct, the smooth muscle layer and the endosalpinx can synthesize arachidonic acid metabolites [29]. The follicular fluid seems to be

8.2 Female reproductive system

203

important for movement of the fimbriae towards the egg after follicular rupture, possibly because of its content of chemotactic leukotrienes [3]. PGF2, increases fimbrial activity [30]. These mechanisms may support ovum pick up. In rabbits, PGE2 and PGF2, accelerate ovum transport (reviewed in Gelety and Chaudhuri [5]). In human tubd preparations, PGE2 inhibits the activity of the longitudinal and circular muscle layers, whereas PGF2, promotes propulsion activity [31, 321. Additionally, PGE2 and PGF2, stimulated ciliary activity in v i m [33]. The activity of the Fallopian tube is further regulated by steroids. Estrogens increase tubal motility, which is accompanied by increased PGFza production and PGF2, bindmg, whereas progesterone decreases PGF2, production and tubal motility [34, 351. In endometriosis, there is an altered PGE2/PGF2, production pattern within the Fallopian tube which may play a role in the reduced fertility rate in these patients [36]. However, hitherto little is known about the definitive role of eicosanoids in tubal function, which is probably caused by the scarcity of Fallopian tubes for study. A critical role of prostaglandins in tubal function is contradicted by some studies which showed a decrease in tubal muscular activity after addition of COX inhibitors [37], whereas others failed to observe this effect [38]. Prostaglandins may be produced during spontaneous contractility as in myometrium, but they are probably not vital for tubal function. Ectopic pregnancy localized in the Fallopian tube is a complication unique to human gestation [35]. The successful termination of intrauterine pregnancies by local PGE2 or PGF2, administration suggests this principle as a possibility for the termination of ectopic pregnancies. Recent studies showed that local injection of PGF2, into an ectopic pregnancy during laparoscopy was highly successful in terminating the pregnancy in selected patients with early and intact ectopic pregnancies and resulted in good subsequent fertility rates [39,40].

8.2.3 Menstruation Menstruation is an event restricted to humans and subhuman primates, in which uterine bleeding and contractions occur. Menstruation describes a catabolic process. This process is triggered by functional interactions between the central nervous system, the hypothalamus-pituitary gland and the ovaries. The first menstruation (menarche) occurs between the ages of 9 and 17 years and the last menstruation (menopause) between the age of 45 and 55 years. Background information about cyclical dependency of endometrial vasculature was given by Markee [41]. In his classic studies he described cyclic changes in endometrium transplanted into the eyes of rabbits. This correlated well with the ovarian cycle. He also described the marked vasodilatation that followed estrogen stimulation. In the late luted phase of the cycle, Markee [41] described a marked shrinkage in stromal tissue, as a result of fluid resorption. Approximately 50% of the explants demonstrated a vasodilatation at this time, accompanied by movement of leukocytes into the adjacent stromal tissue. Just preceding the actual onset of bleeding Markee [41] observed a period of intense vasoconstriction, followed by extravasation of blood into the surrounding stromal tissue. Finally, bleeding occurred from relaxed

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8 The role oj'eicosanoids in reproduction

arterioles, capillaries and post-capillary venules. Subsequent investigations of the mechanisms underlying menstrual bleeding have sought to identify the substances responsible for vasodilatation and vasoconstriction. A breakthrough in understanding was made possible when Pickles [42] reported that extracts of human menstrual fluid were able to cause contraction of human myometrium in v i m and that these extracts contained high levels of PGF2, and PGE2 [43]. Much evidence has accumulated that, besides PGF2, and PGE2, arachidonic acid itself plays a major role in the normal process of menstruation as well as in the disorders menorrhagia and dysmenorrhea [44]. Cell death or damage are known to release phospholipases which are responsible for an increased concentration of eicosanoids [45]. Enzymes which can be found just before the beginning of menstruation in the extracellular compartment of the stroma and epithelium and in the basal membrane of arterioles are responsible for the breakdown of damaged tissues. These enzymes are acid hydrolases [46] of lysosomal origin. The liberation of these hydrolases depends on the disappearance of the membrane-stabilizing effect of progesterone in the late luteal phase. This process triggers the prostaglandin synthesis. As mentioned above, the endometrial vasculature shows rhythmic changes due to alternate constriction and relaxation of the blood vessels. Since eicosanoids are responsible for the tonus and the permeability of blood vessels in many tissues, these substances are leading candidates for mediating the endocrine information of sexual steroids at local uterine level. Eicosanoids seem to be the main paracrine-acting uterine mediators for menstruation [47]. Prostaglandin synthesizing capacity increases throughout the menstrual cycle. Local administration of PGF2, to explants of human endometrium induced dose-dependent constrictions of vessels in the immediate vicinity without inducing bleeding [45].Stromal cells of the endometrium produce large amounts of PGE2 and PGF2, and, moreover, supply a significant amount of the substrate arachidonic acid for the synthesis of PGI2 preferentially produced in myometrium. In cell culture experiments with human endometrial cells we were able to demonstrate a tremendous increase in PGF2, synthesis when we added estradiol and progesterone in concentrations comparable to the corpus luteum phase [47]. It is not only the synthesis but also the metabolism of prostaglandins which is under the control of sexual steroids. The prostaglandin dehydrogenase (PGDH) activity in glandular epithelial cells shows approximately the same cyclical variability as progesterone (reviewed in Zahradnik [47]). A problem in understanding the mechanism of menstrual bleeding is the nature of the vascular damage that allows the loss of blood to occur. Topical application of leukotrienes to the microvasculature of the everted hamster cheek pouch produces an intense dose-dependent constriction of arterioles. This phenomenon was followed by a dose-dependent extravasation of macromolecules. Leukotrienes were >1000 times more effective than histamine in promoting plasma leakage [45]. Thus, it seems possible that leukotrienes produced by white blood cells are responsible for extravasation at the onset of menstrual bleeding. Leucocytic infiltration is a characteristic feature of late secretory and menstrual endometrium [48]. Summarizing the known data, we can outline the following events leading to menstruation: Estradiol induces the endometrial production of progesterone receptors.

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Progesterone in physiological concentrations stimulates the endometrial synthesis of prostaglandins. At late luteal phase, PGFza is produced in such quantities that the local vascular constriction promotes the pre-menstrual endometrial changes. At the very early stage of menstruation, the vasoconstriction causes a hypoxic situation in endometrial tissue. This and other local factors are responsible for the increased liberation of prostaglandin precursors. These precursors are also available to the myometrium, leading to an increased production of prostacyclin (PGI2). PGI2 induces, probably together with PGE2, a reactive vasodilatation and inhibition of platelet aggregation. This process leads to the decreased endometrial hemostatic response at the beginning of menstruation by disturbing the balance between proaggregatory and anti-aggregatory components of the endometrial vasculature. Simultaneously, leukotrienes increase the permeability of the endometrial vasculature leading to extravasation, then menstruation takes place (Fig. 8-2).

Figure 8-2. Distributionof arachidonic acid metabolites in human endometrium and myometrium. C-Endoperox., cyclic prostaglandinsendoperoxides (PGG2, PGHz)

In some cases the blood loss per menstruation is higher (>8Oml) than normal (c60ml). Dysfunction of the utero-ovarian axis is the reason for such bleeding disturbances (menorrhagia). Eicosanoids are involved in the pathogenesis of rnenorrhagia. In endometrial tissue from women with menorrhagia, the ratio between PGF2, and PGE2 was changed in favor of PGE2 [49].Furthermore, a significant increase in PGI2 synthesis could be measured in endometrium from menorrhagic women [50].These findings allow speculation about the pathogenesis of menorrhagia (Fig. 8-3). Different and little-known etiological factors stimulate the endometrium to produce more arachidonic acid, leading to an increased synthesis of cyclic endoperoxides (PGG2, PGH2). The increased availability of substrates enables the myometrium to produce more vasodilating and anti-aggregatingPGI2, causing an increased blood loss per menstruation. Moreover, an increased supply of PGG2 and PGH2 in the endometrium is followed by a shifting of synthesis of prostaglandins in favor of PGE2.

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Figure 8-3. Proposed dysregulation of paracrine-acting arachidonic acid metabolites in menorrhagia (for details see text).

The uterus-contracting potency represented by the PGF2, release is reduced. Nonsteroidal anti-inflammatory drugs (NSAIDs), such as mefenamic acid, are able to normalize the amount of cyclic endoperoxides and, consequently, the ratio of PGF2, and PGE2 or the production rate of PGI2. The loss of menstrual blood will be reduced by approximately 30% (reviewed in Zahradnik [47] and Fraser et al. [51]). In up to 70% of all women, menstruation is more or less painful [52]. The pain may be secondary to various forms of pelvic pathology or may be idiopathic in origin (primary dysmenorrhea). Objectively, one can measure an increased uterine pressure during subjectively expressed menstrual pain [53].

Figure 8-4. Proposed dysregulation of paracrine-acting arachidonic acid metabolites in dysmenorrhea (for details see text).

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Prostaglandins are always involved in painful uterine contractions.Thus, the central role of prostaglandins in the pathogenesis of dysmenorrhea is well established [54]. In dysmenorrheic women, the concentration of the vaso- and myometrial contracting PGF2, in menstrual blood is higher than in eumenonheic women. Furthermore, the ratio between PGF2, and PG12 is significantly altered in favor of PGF2, (Fig. 8-4). Consequently, all pharmacological measures normalizing the PGF2,:PGI2 ratio are able to treat dysmenorrheic pain [52,54,55]. Summarizing our current knowledge, it is likely that prostaglandins play a central role in the pathogenesis of dysmenorrhea. The role played by the lipoxygenasederived eicosanoids requires further investigation. Normalization of disturbed absolute or relative amounts of uterine prostaglandins is followed by disappearance of painful menstruation.

8.2.4 Endometriosis Endometriosis is one of the problems that affects women’s reproductive health about which the least is known concerning etiology and pathophysiology. The overall incidence of endometriosis in white women of reproductive age is approximately 3%. The incidence rate increases in successive age groups through to the age of 44 years, but declines in women aged 45-49 years [56]. Per definition, endometriosis means the occurrence of endometrium in ectopic locations most commonly observed in visceral and parietal peritoneal surfaces. Despite intensive research, the pathophysiological mechanisms of endometriosis and endometriosis-associated reproductive failure remain incompletely understood. Painful inflammatory response and immunological reaction may be considered the body’s first line of defense against the development of endometriosis [57,58]. Eicosanoids are involved in both of these defensive reactions of the body. Prostaglandins may play an important role in endometriotic pain. Their levels are increased in menstrual fluid and in endometrial and endometriotic tissue of patients with endometriosis [59]. In accordance with these findings, symptoms of endometriosis can be prevented or markedly alleviated by a prostaglandin synthesis inhibitor. Nevertheless, concerning the pathogenesis of endometriosis, the role of prostaglandins is poorly understood. In in vitro experiments with slices of normal myometrium, adenomyosis, normal ovaries and endometriotic cysts, the PGI2 production in adenomyosis and endometriotic cysts was significantly higher than in normal myometrium and normal ovaries. A direct relationship between the degree of painful menstruation and prostaglandin production in tissue of endometriosis could be observed [60]. Not only endometriotic tissue itself, but also peritoneal endometriotic implants release significantly more PGF2, and PGE2 than normal peritoneum [61]. PGE2, on the other hand, seems to be the most potent known stimulator of aromatase in endometriosis. As PGEz is known to increase intracellular CAMP levels, estrogen biosynthesis in endometriosis via aromatization may be primarily regulated by locally produced PGEz [621. Local estradiol production can be regarded as a promoter of the growth of endometriotic implants.

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Peritoneal macrophages might be an important production site of PGE;! and PGFza in endometriosis. We could demonstrate that women with endometriosis had significantly more peritoneal macrophages than controls. These cells released significantly more PGF2, and PGE2 [63], which might influence the above-mentioned aromatase activity and alter uterine and tubal contractility, thereby affecting fertility. If this statement is correct, the eicosanoid content of peritoneal fluid of women with endometriosis should be different to that of normal women. This is not uniformly the case. Some investigators failed to find a difference in the level of any of the prostaglandins or products of the lipoxygenase pathway of arachidonic acid metabolism [64,65]. They postulated that there is no correlation between symptoms, underlying diagnosis and the concentration of eicosanoids in the cul-de-sac fluid from patients. Others were able to find elevated prostaglandin levels [66-701 and lipoxygenase-derived metabolites [70,71] in peritoneal fluid of endometriotic patients. If minimal and mild endometriosis is treated with laser surgery (Nd-YAG laser), 3 months later the peritoneal fluid volume and its PGE2 concentration, initially significantly higher than in a control group, turned out to be normal. Within 1 year, 30 out of 52 patients achieved pregnancy [72]. However, the increased eicosanoid content in peritoneal fluid from women with endometriosis seems not to be correlated with the amount of PGF2, in follicular fluid [73]. Therefore, it may be supposed that the elevated levels of peritoneal fluid eicosanoids in endometriotic patients do not influence the ovarian function itself. There must be another reason for reduced fertility in women with endometriosis. One explanation could be that, in patients with endometriosis, tubal prostaglandin production and reaction is altered in the proliferative phase of the cycle [36]. Thus, the mechanism of the reception of the ovum through the Fallopian tube might be significantly disturbed (see Section 8.2.2). In summary, the relationship between eicosanoids and endometriosis is as follows. Macrophages and lymphocytes are the dominant cells in the peritoneal fluid of endometriotic patients. These cells and endometriotic tissues produce eicosanoids that are responsible for pain and inflammatory reactions and partly responsible for infertility. The cellular changes and their activation in peritoneal fluid, together with the increased prostaglandin and leukotriene concentrations, may also explain the fact that local immunological abnormalities play a crucial role in the etiology or pathophysiology of endometriosis.

8.3. Pregnancy 8.3.1 Implantation Successful implantation is a complex event requiring the correct development of the early embryo, the preparation of a receptive endometrium, the attachment of the embryo to the epithelial surface, the recognition by the mother of the presence of the embryo, decidualization of the endometrium and the maintenance of the corpus

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luteum [74]. Disorders of implantation have serious consequences for human fertility and may even be the cause of life-threatening complications in later pregnancy, for example pre-eclampsia. There have been studies on the involvement of eicosanoids, mainly prostaglandins, in the implantation process for 30 years. Prostaglandins seem to be involved in at least two important aspects of early pregnancy. Firstly, increased synthesis of prostaglandins appears to be necessary at the site of implantation to establish and facilitate the embryo-maternal dialogue (see Fig. 8-5). Secondly, once attachment and early invasion have occurred, the release of prostaglandins from the endometrial surface as a whole needs to be suppressed to prevent corpus luteal regression (see Section 8.2.1.2) and the onset of menstruation (see Section 8.2.3).

0

Blastocyst

Glandular Epithelium

Stromal Cells

v Increased vascular permeability Decidualization

Figure 8-5. Eicosanoids and platelet activating factor (PAF) during implantation: Possible amplification of embryonic signals through a paracrine interaction between PAF and PGE2 (for a detailed description see text, adapted from Psychoyos et aZ.[75]). EP: PGE2 receptor.

The increase in endometrial prostaglandin production observed in various species around the time of implantation or at the implantation site reflects the crucial role that prostaglandins might play in this process (reviewed in Smith [74], Psychoyos et al. [75] and Van der Weiden et al. [76]). In many species, both epithelial and stromal cells produce prostaglandins under steroid control. In human endometrium kept in organ culture, estrogen stimulated and progesterone decreased its prostaglandin production [77]; the latter effect could be reversed by addition of the antiprogesterone RU 486 [78]. Separation of glands from stromal cells in vitro showed that both compartments produced PGEz and PGF2a, but the amount of both and their production was dependent on the time of the menstrual cycle and varied according to the study protocol (for reviews see Smith [74] and Psychoyos et al. [75]).

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Immunohistochemical analysis showed COX protein expression in epithelial cells [79,80]. Although the production of PGF2, by endometrial cells is higher than of PGE2 1811, the importance of PGF2, for implantation is not clear, since few PGFbinding sites could be found in the endometrium [75,82,83]. PGF2, is probably metabolized locally to PGE2 [84]. On theother hand, stroma cells stimulated by progesterone express PGE receptors [85] which can be inhibited by RU 486 [86]. The importance of prostaglandins in the implantation process is underlined by the observation that COX inhibitors can delay implantation (reviewed in Psychoyos et al. (751). In rodents, there was even an inhibition of implantation after indomethacin administration, which could be overcome by introducing PGE2 into the uterine lumen [84]. However, far too high a dosage of NSAIDs would be needed for therapeutic use as an anti-conception drug in humans. This is underlined by recent studies with mice having a homozygous deletion of the COX-1 gene which had normal pregnancy rates [87]. On the other hand, wild-type blastocysts failed to implant in COX-2 knock out mice 12231. Recent data indicate that there is a paracrine interaction between the proinflammatory mediator plateletactivating factor (PAF, l-alkyl-2-acetyl-glycero-3phosphorylcholine) and PGE2 at the endometrial level 1761. Uterine phospholipase A2 (PLA2) activity shows a peak just prior to implantation [88] which results in increased arachidonic acid mobilization from membrane phospholipids and causes increased eicosanoid and PAF production. PAF antagonists can inhibit implantation 1891 and the instillation of PAF into uterine horns of mice cause a decidual reaction [90]. Further, PAF is produced by blastocysts (reviewed in Psychoyos et al. [75] and Van der Weiden et al. [76]). PAF causes an increase in vascular permeability [75] and can stimulate PGE2 production [91]. PGE2 is produced by epithelial cells and stimulates PAF production by stromal cells which, in turn, stimulates PGE2 production by epithelial cells resulting in an amplification system (Fig. 8-5) [75]. The role of lipoxygenase-derived eicosanoids for implantation has not been investigated as intensively as of COX products. Leukotrienes seem to be important for the decidual cell reaction in concert with PGE2, since a specific leukotriene receptor antagonist (FPL 557 12) could prevent decidualization [92] which could be antagonized by LTC4 [93]. Endometrium produces peptido-leukotrienes and 12HETE in vitro with a maximum in the secretory phase 194,951. LTC4-binding sites were found mainly in luminal epithelium and stromal cells [96]. 5- and 12lipoxygenase could be shown in epithelial cells and stromal cells from human endometrium, but the highest staining intensity was observed in cells lying in the stroma which might be macrophages 1971. There are other locally active factors such as growth factors and cytokines which have been shown to be important for implantation. Leukemia inhibitory factor (LIF) was found to be vital for implantation, since mice with a homozygous deletion of the LIF gene fail to implant, though this can be reversed by additon of LIF 1981. In endometrial cell culture, LIF did not show any effect on PGE2 and PGF2, production [99]. suggesting that both systems act independently. In conclusion, eicosanoids play an important part in the events leading to implantation, a process which, according to recent models, shows many similarities to an inflammatory reaction [loo]. The possible role of endometrial PGE2 and LTC4

8.3. Pregnancy

21 1

together with PAF is an increase of vascular permeability resulting in local fluid accumulation, decidualization of endometrial stromal cells and modulation of the maternal immune response to fetal antigens.

8.3.2 Placenta In all mammals, the placenta constitutes a unique and vital organ for the intrauterine development of the fetus. It supplies the growing fetus with nutrients and oxygen and functions as a kidney- and liver-like organ for the disposal of waste metabolites. Placental hormones and local mediators redirect the mother’s organism and metabolism firstly to recognize the pregnancy and secondly provide the necessary nutrients. A specific immune balance between acceptance of foreign antigens and protection against the invasiveness of the trophoblast is necessary in hemochorial placentation as in humans where fetal tissue (trophoblast) comes into direct contact with maternal tissue (decidua and blood vessels). Early studies revealed that cell membranes of pregnancy-specific tissue contain high concentrations of arachidonic acid [ 1011. The eicosanoid production of placenta was studied intensively with many different in vitro systems (reviewed in Zahradnik et al. [102]). In short-term culture, the eicosanoid production rate of placental explants (expressed as per milligram wet weight per hour) was in the picogram range for prostaglandins and leukotrienes and in the nanogram range for HETEs [ 103-105]. Since a sufficient blood flow within the placental circulation is vital for fetal nutrition and development, short- and long-term tissue culture and placental perfusion experiments mainly studied the production of the vasoactive prostaglandins PGI2 and TXA2. These experiments showed the importance of prostaglandins for vasoregulation within the placental vasculature [ 1061. The relative potency of TXA2 suggests that it is probably the major vasoconstricting eicosanoid in the villous vascular tree and which is also effective in umbilical vessels [106]. PGI2 is produced in higher quantities by umbilical vessels, so it seems to play an important role in maintaining the vasodilated state of the umbilical vessels throughout pregnancy [ 1071. In pregnancies where high impedance of the uteroplacental circulation is observed by Doppler ultrasound velocity measurements, for example in idiopathic intrauterine growth retardation (IUGR), PGI2 production by umbilical arteries in vitro is decreased relative to thromboxane [ 108-1 1 11. Additionally, growth factors such as insulin-like growth factor 1 act in part via suppression of the synthesis of vasoconstrictive prostaglandins 11 121. However, COX inhibitors do not affect the resting pressure in the perfused cotyledon which suggests that prostaglandins are not the major determinants of resting tone [113]. TXA2 receptors are expressed mainly in the placenta and this has been shown functionally [ 1141 and immunohistochemically with the strongest signal observed in trophoblast cells [ 1151. The production of PGE2 by the placenta may play a role in regulating the immunological tolerance in the intervillous space, where maternal and fetal tissue come into direct contact [ 1161. Steroids influence the prostaglandin production of the placenta and umbilical vessels. Estradiol in high concentrations (>200 ng/ml) stimulated PG12 production without affecting TXA2 synthesis. Placental PGE2

2 12

8 The role of eicosanoids in reproduction

secretion was inhibited by estradiol. These effects could be blocked by progesterone, which itself did not show any direct effect on prostaglandin synthesis [ 117,1181. As mentioned above, prostaglandins constitute only a minor part of all arachidonic acid metabolites in placental short-term tissue culture. Experiments with addition of [3H]-labeled arachidonic acid and subsequent analysis of the metabolite pattern by high-performance liquid chromatography (HPLC) (Fig. 8-6) revealed that the majority of eicosanoids produced by the placenta are 12-HETE and cytochrome P-450-derived metabolites [105,119-1211. So far, there have been only a few studies investigating the importance of these arachidonic acid metabolites in the reproductive tract, despite their abundance. 1ZHETE did not show any effect on placental vasculature in v i m [106]. Its production rate varies in pathological states such as pre-eclampsia [lo51 or maternal diabetes mellitus [ 1211, where a decrease or increase was observed. It might possibly play a role in immunological reactions at the fetal-maternal interface (trophoblast-maternal blooddecidua). 5,6-EET is a potent vasodilator in the kidney [122], an effect which, however, could not be observed in the placental cotyledon [ 1061. Epoxides might be involved in regulating steroidogenesis [21].

I

o

I

I

20

1

I

40

l

I

60min

Figure 8-6. Reverse phase HPLC of eicosanoids released by the placenta in short-term tissue culture. After 30 min pre-incubation ['HI-arachidonic acid was incorporated over 4 h and arachidonic acid metabolites were collected after 20 min stimulation with ionophore A23187 (for details see Schafer et al. ~ 9 1 ) .

The physiological importance of placental leukotrienes has never been investigated thoroughly, but it is known that LTB4, LTC4 and LTD4 have a vasoconstrictive effect on the vasculature of the villous tree which is, however, rather weak compared with TXA:! [106]. The synthesis of prostaglandins in the placenta seems to be compartmentalized. Separated villous core (Hofbauer cells, fibroblasts and endothelial cells) and trophoblast produced ten times more TXA2 than intact villi, which may support decreased platelet aggregation in the intervillous space [ 1231. Additionally, TXA2 synthesis by intact villi could be inhibited by addition of aspirin (acetylsalicylic acid), which showed no effect on the separated trophoblast [ 1241. Therefore, the trophoblast seems to modulate TXA:! production by the villous core, indicating that there is a

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paracrine regulation of eicosanoid production between the different cellular compartments of the placental villi. Immunohistochemically, COX- 1 was found to be expressed in virtually all cell types of the placenta [ 1 15,1251, whereas COX-2 was mainly observed in Hofbauer cells and endothelial cells (Fig. 8-7) [ 1251. Immunoreactive prostacyclin synthase was found in endothelial cells, whereas thromboxane synthase was expressed in Hofbauer cells (Fig. 8-7) [126]. These observations suggest that production of the various prostaglandins within the placenta is regulated by the individual cell and may therefore depend on the activation status of this cell. Isolated Hofbauer cells from first trimester placentas produced PGE2 which is stimulated by progesterone [ 1271. Incubation of macrophages from term placentas showed stimulation of PGE2 and TXA2 synthesis by lipopolysaccharide and decreased PGE2 production rate during incubation in a low oxygen atmosphere [128]. With their position close to fetal capillaries and trophoblast, their prostaglandin production might have very important regulatory functions for vascular tone (TXA2) and immunology (PGE2).

Figure 8-7. Irnmunohistochemical staining for COX- 1, COX-2, prostacyclin synthase (PCS) and thromboxane synthase (TXS) in term placenta. lmmunohistochemistry was performed using the biotinstreptavidin-peroxidase detection system and diaminobenzidine as peroxidase substrate; therefore positive cells are stained brown (for details see Wetzka et al. [ 125,1261.

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8 The role of eicosunoids in reproduction

In conclusion, vasoactive prostaglandins are important mediators within the fetoplacental circulation and effect fetal growth, but more research is needed concerning the regulation of their production in the different placental compartments. PGE2 is probably involved in the immunobalance at the fetal-maternal interface. The physiological significance of 12-lipoxygenase- and cytochrome P-450-derived eicosanoids, which are produced in great abundance, is not yet known.

8.3.3 Pregnancy-induced hypertension and pre-eclampsia Pregnancy-induced hypertension (PIH) and pre-eclampsia are potentially severe complications of human gestation, occurring in approximately 5% of pregnancies. The clinical symptoms are hypertension, proteinurea and edema, which may be accompanied by growth retardation of the fetus. In advanced stages of the disease the patients also show cerebral symptoms, ranging from hyper-reflexia and headaches to general fits. Further, there is increased platelet activation leading to thrombopenia. Recent theories suggest that the beginning of the disease lies in a pathologically poor invasion of fetal trophoblast cells into the uterine arteries [ 1291. This is followed by a decreased blood supply to the placenta which then probably causes the general vasoconstriction in order to increase the placental perfusion. During the course of the disease a dysfunction of endothelial cells develops, resulting in a disorder of plateletvessel-wall interaction which can explain many of the typical clinical symptoms [ 1301. Therefore, studies have focused on the most potent vasoactive prostaglandins-the antagonists PGI2 and TXA2-which have been investigated extensively in this disease (for a review see Zahradnik et al. [102]). It is now generally accepted that preeclampsia resembles a state of prostacyclin deficiency [I 1 I], whereby the absolute production rate of PGI2 and TXA2 is not as important as their ratio. Analysis of TXA2 and PG12 metabolites in urine, which is more reliable than analysing serum because of the short half-life of prostaglandins, showed a decreased ratio of 6-keto-PGF1,:TXB2 (renal origin) and also of 2,3-dinor-6-keto-PGF1,: 1 1-dehydro-TXBz (systemic origin) [ 131-1331. Another pathological change is the decreased affinity of platelet PGI2 receptors, despite normal receptor capacity in pre-eclamptic patients [ 1341. In vitro studies investigating the prostaglandin synthesis in placental tissue culture showed that, in the majority of studies, a decreased PG12RXA2 production ratio by placentas deriving from pre-eclamptic compared to normal pregnancies, in spite of very different culture systems (Fig. 8-8) (reviewed in Zahradnik et al. [102]; for recent studies see Arbogast et al. [lo51 andWalsh and Yang [1351). In addition, preeclamptic placentas produce much less 12-HETE than normal placentas (Fig. 8-8) [ 1051. 12-HETE possibly acts as an immunosuppressive factor during pregnancy and its decreased production may contribute to the pathophysiology of pre-eclampsia. HETEs further influence prostglandin production in vitro [ 136,1371. Cytochrome P450-derived metabolites of arachidonic acid were found to be increased in the urine of pre-eclamptic patients [ 1381. They might be involved in the regulation of vascular tone and fluid balance [139]. The observed imbalance of the PG12:TXA2 system in vitro and in vivo led to the therapeutic strategy of using aspirin for the prevention of PIH. The therapeutic principle

8.3. Pregnancy

PGlp

TXAp

TXAp /PGIp

215

12-HETE

Figure 8-8. Release of PGI?, TXAz and 12-HETE after normal pregnancies (open bars) and pregnancies complicated by PIH (widely hatched bars) and pre-eclampsia (narrowly hatched bars) by placenta in short-term tissue culture. Eicosanoid concentrations were analysed in the incubation media by enzyme immunoassays. Results are shown as mean f.SEM in pg/mg tissue wet weightlh (for details see Arbogast et al. [IOS]).

is known from the prevention of thrombocyte aggregation in atherosclerosis: Lowdose aspirin (1 mgkg body weight) inhibits TXA2 production by platelets with low effect on endothelial PGI2 production, which partly corrects the prostaglandin imbalance in pre-eclampsia [ 1401. The clinical studies performed in the last 5 years showed an overall beneficial effect of low-dose aspirin in patients at high risk of developing pre-eclampsia. The babies had a higher birth weight and the pregnancy could be maintained well into the third trimester, since the clinical symptoms of the mother were less severe [141,142]. If a prophylactic treatment with aspirin is required, one should begin early in the second trimester [ 1421 and the compliance of the patients should be controlled [1431. The application of aspirin is, however, not suitable for treatment of the already manifest disease. The observed side-effects of low-dose aspirin were negligible for both mother and fetus [141]. The expression of both isoforms of COX have also been investigated in intrauterine tissues from pre-eclamptic patients. An increase in COX-2 expression might be expected, because the course of the disease shows similarities with an inflammatory reaction, for example elevated plasma levels of inflammatory mediators such as tumor necrosis factor (TNF) and interleukin-6 (IL-6) El441 and activated granulocytes [ 1451. However, two recent studies did not observe different COX-2 mRNA and protein expression in the placenta [ 115,1251and placental bed [ 1251. The

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amount of COX-1 mRNA and protein increased in placental bed biopsies after preeclampsia [ 1251. These results suggest that COX-2 inhibitors such as meloxicam may not be useful in the prevention or treatment of pre-eclampsia. Additionally, COX-2 inhibitors can be expected to be teratogenic, since deletion of the COX-2 gene in mice resulted in a lethal inhibition of renal development [ 111. Another therapeutic option is the inhibition of thromboxane synthase. Studies looking for thromboxane synthase expression in placenta showed either an unchanged [ 1261 or increased expression [ 1 151 in pre-eclampsia, whereas prostacyclin synthase expression was unchanged [ 115,1261. Since the substrate of thromboxane synthase, PGH2, has a similar vasoconstrictive effect to TXA2 and can act via the thromboxane receptor, thromboxane synthase inhibitors were developed which showed thromboxane receptor-blocking activity as well. There have been few and rather small clinical studies with these compounds [ 1461; therefore their clinical usefulness remains to be proven [142]. A recent study (160 patients) with Trapidil showed a beneficial effect on the incidence of PIH, the necessity of preterm delivery and IUGR. Trapidil increased PGI2 production in vivo and in virro [147]. In summary, P M and pre-eclampsia are characterized by an imbalance of the PG12:TXA2 ratio favoring the vasoconstrictive TXA2. Aspirin, as well as thromboxane synthase/thromboxanereceptor blockers, might be useful for prevention of this pregnancy-specific disease.

8.3.4 Parturition In pregnancy, the growing fetus represents a semi-allogeneic graft which is protected against expulsion by suppression of local defense reactions. As the time of parturition approaches, this delicate balance is altered towards the selective use of inflammatory reactions providing effective mechanisms for delivering the baby. At the hormonal level this shift is characterized by the retreat of effectors which maintain uterine quiescence (progesterone, prostacyclin, nitric oxide and relaxin) [ 1481 and the growing influence of labor-promoting agents such as estrogens, glucocorticoids, oxytocin, prostaglandins, corticotropin-releasing hormone (CRH), endothelins, PAF and cytokines (Fig. 8-9) [148-1511. The precise mechanisms by which the orchestra of hormones and local mediators is coordinated to act on the main target sites, the cervix and the myometrium, are not yet known. In a very complex and poorly understood manner, hormones and other signal molecules of fetal and maternal origin interact in order to control the three phases of the birth process: cervical ripening, activation of the myometrium and stimulation of strong, synchronized contractions [148,150]. Different species vary significantly in the mechanisms leading to parturition (for a comprehensive review, see Challis and Lye [148]). In the human, according to a recently postulated model (Fig. 8-10) [149], induction of labor is coupled to growing fetal cortisol production. It was suggested that cortisol attenuates progesterone-mediated inhibition of placental CRH expression, whereas maternal progesterone plasma levels are not altered. CRH then further drives cortisol biosynthesis in the fetal adrenal gland, thus establishing a positive feedback loop. Concomittant stimulation of fetal dehydroepiandrostendione (DHEA) synthesis

8.3. Pregnancy

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Pregnancy

Figure 8-9. Factors involved in the maintenance of pregnancy and the onset of human labor.

Figure 8-10. Hypothetical model of a placental clock driven by placental CRH and fetal cortisol which stimulates the effectors of labor (from Karalis et al. [149]; reproduced with permission of Nature Medicine). DHEA, dehydroepiandrostendione; E2, estradiol; PROG, progesterone; CORT, cortisol; HT, hypothalamus; PG, pituitary gland; OT, oxytocin; GR, glucocorticoid receptor; ER, estrogen receptor; PR, progesterone receptor; PG, prostaglandin.

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8 The role cf eicosanoids in reproduction

supplies the placenta with substrate for increasing formation of estrogens which, in turn, sensitize the uterus for contractions by inducing the expression of contractionassociated proteins (gap junctions and oxytocin receptors) and stimulating the intrauterine formation of prostaglandins and oxytocin. The participation of prostaglandins and other eicosanoids has been most extensively investigated in cervical ripening, stimulation of myometrial contractions and the induction of pre-term labor. Arachidonic acid contributes to 10-30% of the total fatty acid content of intrauterine tissues. The rate-limiting steps in eicosanoid formation are the release of arachidonic acid and, in some tissues, due to high extracellular arachidonic acid levels, COX activity. The birth process consists of dynamic and complex events which are frequently not amenable to scientific investigation. Therefore, most findings have been obtained from in v i m experiments. Further, one has to keep in mind that eicosanoids often exert their actions at discrete loci and that individal features of patients may be of crucial influence. Thus, only a few common principles for eicosanoid action in parturition are known today. Since small changes of local mediator concentrations are unlikely to be reflected in accessible body fluids and measurements of local, intrauterine concentrations are difficult to perform, a final model of parturition has not yet been proposed.

8.3.4.1 Cervical ripening During pregnancy, the cervix remains firm and closed. When parturition approaches the cervix becomes soft and stretchy, contractions begin and the cervix can dilate and allow the baby to pass through. At approximately the thirty-eighth week of pregnancy, the diameter of the endocervical canal is 1-2 cm. During effective labor, the cervix dilates to 10 cm in diameter. Thus, during gestation and parturition the uterine cervix undergoes profound morphological changes. These changes are the result of a complex biochemical process [ 1521. In the human uterine cervix, collagen and elastic fibres maintain the shape of the cervix and help to keep it closed. Elastic fibres also contribute to the gradual and sometimes reversible opening of the cervix. Interstitial collagen fibres in cervical tissue normally do not stretch easily and are stiff. Changes in the cervix during pregnancy cause rearrangement of the collagen fibres so that the tissue assumes the characteristics of a soft, easily distensible tissue [ 1521. During late pregnancy these components are influenced by degradative enzymes, leading to changes in the synthesis of extracellular matrix proteins and glycoproteins [ 1531. For the process of cervical ripening complex endocrine and paracrine changes are necessary. This cascade of changes appears to culminate in the activation of both myornetrial contractility and cervical ripening in a coordinated and synchronized manner [ 1541. Prostaglandins, mainly PGE2, probably play a crucial role in mediating maternal and fetal messages into the fundamental changes at tissue level provoking cervical ripening. PGI2 and PGE2 are produced in the cervical tissue. This production increases markedly at term and with the onset of parturition [155]. To underline the local significance of PGE2 for the ripening of the cervix, it must be emphasized that this prostaglandin strictly applied within the cervical canal can achieve significant maturation of the cervix within 5 h without measurable uterine contraction (reviewed

8.3. Pregnancy

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in Zahradnik [ 1501). Cervical maturation in humans seems to result from increased collagenase activity, causing a breakdown of the collagen. Concomittantly there is an increased synthesis of the ground substance, as reflected in increased amounts of glycosaminoglycans. The principle changes that occur in the cervix during parturition are increased vascularization, mass and water content and decreased organization of collagen and dermatan sulfate. These changes in collagen content correlate with changes in proteolytic enzyme activity [ 1481. There is direct evidence that interstitial collagenase increases markedly during cervical dilatation in human parturition [ 1561. PGE2 is responsible for the extrusion of cytoplasmic vesicles located in cervical myofibrocytes into the extracellular spaces of the human cervix. These vesicles are matrix lysosomes. The enzymes in these lysosomes are separated from the surrounding ground substance by a unit membrane. Having escaped from the cells, the stability of the membranes of lysosomes is no longer under direct cytoplasmic control. The lysosomal enzymes can then penetrate into the connective tissue more effectively [157]. As regards the strong interrelation between PGE:! and collagenase production, it is evident that PGE2 has a central role in the maturation process of the cervix uteri. Although the exact mechanisms controlling cervical dilatation during the birth process are unknown, additional factors seem to be involved. In human endometrial explants, progesterone abolishes the release of matrix collagenase, an effect that can be prevented by RU 486 [158]. This suggests that the inhibition of the influence of progesterone could be a key mechanism for cervical maturation at the beginning of labor. The physiological event can be mimicked pharmacologically by antiprogesterones such as RU 486 or onapristone [ 1591. Clinical observations suggest that estrogens might be involved in pre-partum cervical priming. This priming process is probably not caused directly by estrogens, but the cervix depends on an adequate estrogen supply for preparation of cervical ripening. The idea that relaxin may play a leading part in cervical maturation is a fascinating prospect. However, clinically the role of relaxin in cervical maturation remains obscure, although intravaginal administration of relaxin induced significant cervical maturation before labor induction [ 1601. The effect of relaxin on cervical growth and softening seems not to be mediated through prostaglandins [ 1611. Leucocyte infiltration and degranulation in human cervical tissue are similar to those seen in inflammatory reactions. This supports the notion that cervical maturation is similar to an inflammatory reaction [162]. There is increasing evidence that cytokines also play a central role in cervical ripening. IL-8 was observed to be released from human chorio-decidual cells in vitro and IL-8 concentrations rise during labor [163]. After local application of TL-1 and L-8 in a gel, significant cervical softening and dilatation without induction of labor was seen in pregnant guinea-pigs. These effects were similar to the physiological maturation of the cervix and to the effects seen after the application of onapristone [159]. The role of prostaglandins in this context is not yet known. Prostaglandins produced within the cervix and by intrauterinetissues are believed to have a major impact on the regulation of changes in collagen at term [157]. In contrast to these findings, animal and human studies with RU 486 have shown that the softening

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effect of RU 486 is not correlated to increased cervical prostaglandin production. In addition, naproxen did not inhibit the cervical effect of RU 486 [ 164,1651. Although their physiological role is still under discussion, the therapeutic use of PGE2 or PGEl analogs for cervical priming is now generally accepted [ 166,1671. Pre-induction ripening by hygroscopic dilators and intracervical PGE2 was equivalent as measured by changes in the cervical score. The change in cervical score, however, was not predictive of successful induction. PGE2 was more frequently associated with induction success. Hygroscopic dilators were associated with a higher incidence of post-partum maternal and neonatal infection because of longer duration of labor [ 1681. Even when PGE2 gel was used in patients with prior cesarean delivery, there was no significant difference in the incidence of uterine rupture between the PGE2 group and the control group. Indicators of maternal and perinatal morbidity were not significantly higher in the prostaglandin-treated group [ 1691. PGE2 gel can be given intracervically or intravaginally in different doses. Compared to intracervical therapy, PGE2 given intravaginally was more likely to change the cervix significantly and stimulate regular contractions. Although each was safe, instillation of PGE2 gel was better at a higher intravaginal dose than a lower intracervical dose because of its greater ease of administration and the higher likelihood of cervical change [170]. Not only does the native PGE;! as gel, but also PGEl as tablets seem to be effective for cervical ripening and labor induction [ 171-1 731. These data taken together suggest that cervical ripening, the prerequisite for normal parturition, is not caused by any muscular contribution from the cervix or myometrium, but rather by biochemical changes that begin long before the onset of labor. These biochemical changes are triggered partly by eicosanoids and vice versa.

8.3.4.2 Labor Arachidonic acid metabolites are considered important paracrine regulators in human myometrial contractions [ 1481. PGF2, and PGE2 are potent pharmacological stimulators of uterine contractility, causing labor at any stage of gestation. Other wellknown uterotonins are oxytocin, PAF and endothelins. However, the physiological role of PGFza and PGE2 in myometrial contractions has been questioned, since they caused much weaker contractions than oxytocin and endothelins in vitro [174,175]. Other eicosanoids such as 5-HETE, 12-HETE and leukotrienes have only weak or no contractile effects on human pregnant myometrium [176,177]. On the other hand, changes in prostaglandin levels in blood and urine have been observed during labor [178-1811. It can be concluded that, in human parturition, there is no vaginal delivery without endogenous prostaglandin synthesis [ 1821. For these contradictory results, it is not yet possible to define precisely whether prostaglandins act as initiating factors of parturition in normal vaginal deliveries or whether their formation serves other purposes or is a mere consequence of the traumatic processes occuring during labor (for critical discussions of this issue, see Olson et al. [I511 and Kelly [183]). For example, another potential role for PGE2 is the regulation of the utero-placental blood flow. New data suggest that PGE2 is a vasodilating agent in uterine vessels, increasing uterine blood flow to sustain the contractions. In contrast, it constricts placental vasculature, leading to the closure of placental resistance vessels at parturition. These

8.3. Pregnancy

22 1

opposite effects are probably mediated by different receptor subtypes [ 1841. For many years the amnion has been assumed to be the main source of prostaglandins initiating the onset of labor. However, in recent years findings have been accumulated which demonstrate that the contribution of the amnion to intrauterine prostaglandin production is low [ 1851 and that prostaglandins from the amnion and amniotic fluid are inactivated during their passage through intact fetal membranes. In the chorion laeve, high concentrations of uterotonin-degrading enzymes, including 15-hydroxy prostaglandin dehydrogenase (PGHD), have been found [ 1861. In transfer experiments with intact fetal membranes, very little [3H]PGE2 crossed the membranes without being metabolized [ 1871. These results show that the chorion acts in part as a barrier preventing an overflow of prostaglandins and other uterotonic agents from the amniotic fluid to the decidua or myometrium under physiological conditions. Further, it was demonstrated that no more than 2% of the total output of PGE2 from the fetal membranes originates in the amnion [185]. However, recent findings suggest that decreased chorio-decidual PGDH expression [ 1 881 or an increase of the prostaglandin synthetic capacity of the chorion after COX2 induction [ 1891 may result in a substantial increase in prostaglandin levels reaching the myometrium during labor. As a consequence of these new findings, the decidua at the feto-maternal interface and perhaps the myometrium itself are now regarded as the physiologically relevant sources of prostaglandin output during labor. Whereas decidual eicosanoid biosynthesis has been extensively investigated, the contribution of the myometrium to intrauterine eicosanoid production is relatively unknown. Flow cytometric analysis of human term decidual cell suspensions has shown that 47% of decidual cells are of bone marrow origin, including a large population of macrophages (approximately 19% of total decidual cells). The non-bone marrow-derived decidual cell fraction consists mainly of stromal cells [190]. Due to its cellular composition, its high capacity for arachidonic acid metabolism [119,185] and its proximity to the uterine vasculature and the myometrium, the well-vascularized decidua is likely to represent a physiologically relevant source of eicosanoids. The production of PGF2a, PGE2, TXA2, HETEs and EETs has been demonstrated in human term decidua [ 119,191- 1971. The biosynthetic capacity for eicosanoid formation has been reported to be higher in bone marrow-derived cells, particularly macrophages, than in decidual stromal cells [192,195] and COX activity was also mainly found in a macrophage-rich population of human third trimester decidua [198]. Production of PGF2, exceeded that of PGE2 [194,195]. However, although decidual macrophages displayed the highest prostaglandin production rates, it was estimated that, due to their relatively low abundance, they contribute only between 24% (PGE2) and 38% (PGF2,) of the overall prostaglandin biosynthesis by term decidua. Nevertheless, bone marrow-derived cells of human decidua are likely to play an important role in the physiology of spontaneous labor at term. It has been suggested that the decidua is maintained in a state of functional quiescence throughout pregnancy and that it is activated in the course of parturition [ 193,1971. The biochemical mechanisms involved in decidual activation are not yet sufficiently elaborated. In contrast to many other cells, the production of eicosanoids in decidual macrophages and stromal cells may not be under the control of

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phospholipase Az, but rather be dependent on the availibility of extracellular arachidonic acid. Intracellular arachidonic acid is metabolized mainly to epoxygenase and lipoxygenase-derived metabolites [ 1 921. Effective regulation of prostaglandin production in decidua will thus be under the control of COXs rather than phospholipases. Various cytokines, for example IL-1p, are well-known inducers of COX expression in decidua 1194,1991. The significance of COX-2 expression in the induction and progression of labor is currently still unclear [ 189,199,2001. First results indicate that increased synthesis of COX-2 in intrauterine tissues may be involved in elevated prostaglandin production after labor [ 194,1971 and that COX-2 expression may be upregulated by glucocorticoids and cytokines [201]. Sites of production of cytokines are bone marrow-derived cells in the decidua itself [202]. Further research is needed to clarify whether this regulation principle is of importance in normal labor or is restricted to the pathomechanisms of pre-term labor. Another aspect controlling decidual prostaglandin output is the very rapid metabolization of PGE2 and PGS, in decidual cells [193,203]. It has not yet been investigated whether activation of decidua is associated with an inhibition of these degradation reactions. The biological role of eicosanoids generated by cytochrome P-450-dependent monooxygenases has been only incompletely characterized. It has been reported that arachidonic acid epoxides (EETs) modulate hormone secretion in some organs [21,204-2061. Thus, it is possible that EETs are also involved in decidual hormone production. The main site of action of uterotonins, the human pregnant myometrium, is itself able to produce eicosanoids, for example PGI2, PGE2, PGFz,, 12-HETE and EET, with prostacyclin being the main arachidonic acid metabolite in this tissue [ 119,191,2071. The expression of several enzmynes of the arachidonic acid cascade including PLA2, COX-1, COX-2, 5-lipoxygenase and prostacyclin synthase has been demonstrated in human pregnant myometrium [208,209]. However, further research is needed to clarify to what extent myometrial arachidonic acid metabolites participate in the onset and maintenance of labor and how biosynthesis of eicosanoids is controlled in myometrium. The effects of prostaglandins on the myometrium are mediated by specific receptors which, in turn, activate a variety of intracellular signalling pathways (for a review see Challis and Lye 11481). One of the major effects of PGF2, is the increase in intracellular calcium, which is the main intracellular effector of contractions. The mechanism of stimulation of myometrial contractions by prostaglandins is different from oxytocin action. Furthermore, eicosanoids have been suggested to regulate the appearance of gap junctions (for a review see Zahradnik [ 1501).

8.3.4.3 Pre-term labor Pre-term labor is one of the most urgent obstetrical problems in industrialized countries, since it is a major source of perinatal morbidity and mortality. It has been estimated that intrauterine infections are a leading cause of up to 30% of pre-term deliveries [2101. According to a widely accepted hypothesis, the cytokine network in fetal membranes is activated by bacterial endotoxins and cytokines released from gestational tissues (for example, IL-lp and TNF,), which in turn stimulate the

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223

production of intrauterine prostaglandins causing uterine contractions [21 11. The stimulatory effects of cytokines on prostaglandin production in fetal membranes and the decidua have been demonstrated in numerous in v i m experiments [196,212, 2131 (Fig. 8-11). New findings demonstrate that the increased prostaglandin biosynthesis is based on the induction of COX-2 enzyme activity [194,1991. A key role in this process was attributed to decidual macrophages, since they produce cytokines as well as prostaglandins [195,202].

Figure 8-11. Hypothetical model of a cytokine-prostaglandin network at the chorio-decidual interface that is involved in the pathophysiology of infection-induced pre-term labor (from Mitchell et al. [211]; reproduced with permission of W. B. Saunders Co.).

Other causes of pre-term deliveries are poorly defined. Recently, some pre-term deliveries without infections were explained by a relative deficiency of PGDH in chorionic trophoblasts, allowing prostaglandins synthesized in the fetal membranes to escape metabolism in the chorion [188].

8.4 Male reproductive system The pharmacological actions of lipid extracts from human seminal fluid were first discovered by von Euler in 1935 [214]. The effective substances were called prostaglandins, since the site of their production was presumed to be in the prostate. The concentrations of PGE2, PGE, and their 19-hydroxy-derivatives in seminal fluid are higher than in all other body fluids. For this reason they were isolated for the first time in pure form from this source 12151. 19-Hydroxy-PGE has not been found in

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8 The role of eicosanoids in reproduction

other tissues. Later it was recognized that the biosynthesis of PGE mainly takes place in seminal vesicles, where particular high activities of prostaglandin endoperoxide PGE-isomerase (9-keto-prostaglandin-isomerase) are present [2161 and that the contribution of the prostate to prostaglandin production is low. Prostaglandins are not enzymatically inactivated in seminal fluid. In spite of these early findings the biological functions of eicosanoids in the male reproductive tract have still not been fully evaluated. Possible actions include effects on sperm function, maturation and capacitation, mediation of ejaculation and induction of immune tolerance towards paternal antigen within the female reproductive tract.

8.4.1 Acrosome reaction The participation of eicosanoids in the acrosome reaction, the crucial step of fertilization, has been investigated in various animal species and in humans. The term ‘acrosome reaction’ describes the process of penetration of a spermatozoon into the glycoprotein-containing zona pellucida of the egg preceding the fusion of both gametes. During this process the acrosome at the head of the spermatozoon releases proteolytic enzymes, for example hyaluronidase, thus dissolving the glycoprotein coat and locally producing a channel through which the spermatozoon can enter the egg. The acrosome reaction is initiated by zona pellucida components, which probably trigger calcium entry into the spermatozoa.The subsequent intracellular mechanism by which fusogenic mediators are activated has not yet been unequivocally elaborated. It has been demonstrated by several investigatorsthat arachidonic acid and certain prostaglandins (for example PGE2) are able to induce the acrosome reaction and that phospholipases Az, C and D as well as protein kinase C play important roles [217]. However, the exact role of eicosanoids in this process remains controversial [218,219].

8.4.2 Immunosuppressive actions of PGE An immunotolerancefor spermatozoa within the female reproductive tract is essential for their survival and the success of internal fertilization, since seminal fluid has a high antigenic potential. Human seminal plasma contains several immunosuppressive factors including prostaglandins. Recent findings [220] demonstrated powerful effects of PGE and 19-hydroxy-PGE on the balance of cytokines released by antigenpresenting cells. The induction of immunotolerance may be explained by stimulating IL- 10 and inhibiting IL- 12 production. However, defense against sexually transmitted diseases (gonorrhea, herpes, papilloma and H N ) may also be impaired by these mechanisms.

8.4.3 Erectile dysfunction Approximately 10% of the adult male population in industrialized countries suffers from erectile dysfunction, provoked mainly by psychosocial stress. Erection is a

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complex physiological event characterized by vasodilation, increased blood vessel permeability and edema. PGE, has been identified as an important mediator in this process due to its vasodilative properties. Thus, it represents a target for therapeutic intervention. Recently, these findings have been clinically applied in the diagnosis and treatment of erectile dysfunction by means of intracavenous injection of PGEl (Alprostadil) [221]. Besides PGEl, other potent vasodilators acting via local prostaglandin modulation (papaverin and phentolamine) or combinations of these three drugs are in use [222].

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Prostaglandins, Leukotrienes and Other Eicosanoids: From Biogenesis to Clinical Application Edited by F. Marks, G. Fürstenberger Copyright © WILEY-VCH Verlag GmbH, D-69469 Weinheim (Federal Republic of Germany). 1999

9 The role of eicosanoids in inflammation and allergy Eva Wikstrom Jonsson and Sven-Erik Dahlkn

9.1 Introduction Asthma is a major global public health problem. The prevalence and perhaps also severity of the disease is increasing, particularly in urbanized areas around the world. In many Western countries the prevalence of asthma is around 5% in the adult population and 10% or higher among children below the age of 10 years [ 13. Acute attacks of asthma are characterized by difficulties in breathing due to airway narrowing. The main events leading to obstruction of the airways are smooth muscle contraction and inflammatory changes in the mucosa, leading to mucosal edema and increased mucus secretion. Furthermore, bronchial hyper-responsiveness is a feature of asthmatic disease. The bronchial hyper-responsiveness seen in asthmatics has been attributed to several different mechanisms, for example airway inflammation, airway remodeling and sensory nerve activation. The recognition of asthma as a chronic inflammatory disorder of the airways has greatly focused etiologic and therapeutic research during the last few years. Airway inflammation is one specific example of the general ability of tissues to respond with an inflammatory reaction. The cardinal signs of inflammation are rubor (redness), tumor (swelling), calor (heat) and dolor (pain). These are brought about by a series of microvascular events, leading to the accumulation of, for example, complement factors, immunoglobulins and white blood cells. An acute inflammatory reaction can be elicited by different mechanisms: infection, mechanical injury, chemical injury and allergic reactions for example. The biological purpose is to defend the affected area against pathogen invasion and to restore normal function. The redness and heat are signs of arteriolar dilation and increased blood flow. The increased blood flow is thought to promote plasma and leukocyte extravasation by increasing the delivery of blood components and, furthermore, by increasing the intravenular hydrostatic pressure [2]. The swelling, edema, results from increased plasma extravasation, which may occur directly through gaps between adjacent endothelial cells in post-capillary venules [3-51, presumably as a result of endothelial cell contraction [6,7]. Furthermore, plasma leakage is thought to occur when emigrating leukocytes push through endothelial junctions [3,8]. The pain or itch associated with the inflammatory response is caused by irritation of sensory nerves. This overview will focus on allergic inflammation and intends to summarize some of the results that have contributed to our current understanding of the role of differ-

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ent eicosanoids in allergic inflammation and asthma. The account is introduced by an overview of the formation and biological activities of different eicosanoids relating to allergic inflammation and asthma. In this context, the eicosanoid receptors will be presented. Agonism or antagonism at some of these receptors is a promising strategy for therapeutic interventions in the twenty-first century. Thereafter, the chapter will mainly deal with recent findings that have established a role for leukotrienes in different forms of airway obstruction, the possibility that thromboxane A2 (TXA2) contributes to the development of bronchial hyper-responsiveness and the indications that certain prostaglandins may be endogenous anti-inflammatory modulators.

9.2 Formation of eicosanoids in allergic inflammation Allergic inflammation is initiated by activation of mast cells via immunoglobulin E (IgE) antibodies. The mast cell membrane contains Fc receptors which bind IgE specifically [9]. Classic anaphylactic activation of mast cells is triggered by cross-linking of two or more adjacent IgE molecules by specific antigen (or, experimentally, by anti-IgE antibodies). This aggregates the Fc receptors and initiates intracellular events leading to secretion of mast cell mediators [ 10-121 (Fig. 9-1). Mast cell-cytotropic IgG antibodies may also initiate mast cell mediator release [10,13]. Some of the compounds released from mast cells are pre-formed and associated with mast cell granules, for example chemotactic peptides, proteoglycans, proteases and biogenic amines such as histamine. The eicosanoids, in contrast, are newly synthesized from precursors in the cell membrane upon activation of the mast cell. More than half a century ago, two seemingly unrelated findings were made, namely that human semen contained a smooth muscle stimulant with previously unknown properties [ 141 and that anaphylactic animal lungs liberated another spasmogenic principle [ 151. The biological activities were named prostaglandin and slow-reacting substance (SRS), respectively. After a long period of research on these intriguing factors, it is today evident that they belong to the large family of compounds formed in different reactions from arachidonic acid. The term eicosanoids [ 161 formally encompasses metabolites of all fatty acids with 20 carbon atoms. However, only metabolites of arachidonic acid (cis-A 5,8,11,14 eicosatetraenoic acid) will be considered here. Prostaglandins and thromboxanes are formed via the cyclooxygenase (COX) pathway (Fig. 9-2), whereas leukotrienes and lipoxins are formed via the lipoxygenase pathway (Fig. 9-3). For more information on the biosynthetic pathways, please refer to chapters 1-5 in this volume. It was described early on that antigen challenge was an effective cause of the release of SRS from perfused animal lungs [151. Brocklehurst [ 171 demonstrated allergen-induced liberation of SRS from lung tissue of asthmatics and introduced the name SRS-A (slow-reacting substance of anaphylaxis). In the years before the discovery of the leukotrienes, SRS-A was primarily considered as a prominent mediator of allergy and immediate hypersensitivity [ 181. Following the structural identification of SRS-A as being composed of the cysteinyl leukotrienes LTC4,LTD4and LTE4 [ 191,

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Figure 9-1. Mast cell activation. Upon cross-linking of IgE bound to the mast cell surface by Fc. receptors or other chemical or physical stimuli, the activated mast cell releases inflammatory mediators. These are either preformed, such as histamine or de n o w synthesized, such as thromboxane A? (TXA2), prostaglandin D2 (PGD2) and the cysteinyl leukotrienes LTC4, LTD4 and LTh.

it was possible to show that human lung tissue was an important source of leukotrienes [20-221. Endogenously formed cysteinyl leukotrienes also seem to mediate arteriolar constrictionand plasma leakageinduced by antigen in the hamster cheek pouch [23,24]. In addition, activated mast cells may release several COX products, such as prostacyclin (PGIz), PGE2, and, in particular PGD2 [25-271. In, for example, guinea-pig lung, TXA2 is a major product of the COX pathway [28]. It can be released by antigen challenge and as a consequence of activation of cysteinyl leukotriene receptors [29-331. In man, formation of TXA2 was first identified in platelets [34] and was later described in alveolar macrophages [35], polymorphonuclear leuko-cytes [36], vascular tissue [37] and isolated endothelial cells [38]. Measurements of leukotriene B4 (LTB4) in bronchial lavage fluid [39,40] and in the nasal cavity [41] suggest that LTB4 is also generated in the cascades which are initiated by allergen exposure. The cellular source of the released LTB4, however, has not been identified, since the mast cell and other cells with high-affinity receptors for IgE appear to release insignificant amounts of LTB4. For example, IgE-dependent activation of human lung tissue has not been found to be an effective stimulus for enhanced formation of LTB4 [22,42]. However, it is possible that LTB4 is formed from other cells in the lung by transcellular metabolism of LTA4 [43] liberated from mast cells and basophils activated by the allergen. Alternatively, the mediators and cytokines released during the initial response to the allergen may stimulate the recruitment of additional inflammatory cells (macrophages and polymorphonuclear cells)

9 The role of eicosanoids in inflammation and allergy

236

I

PGF,

PGI,

9; receptor an onists

DP

FP

EP-

Figure 9-2. Cyclooxygenase products, pathways and receptors. Arachidonic acid i s metabolized to either prostaglandins or thromboxanes. These compounds, together called prostanoids, act at P (prostanoid) receptors. The letter preceding the P states the most important prostanoid binding to the receptor. Thus, PGDz acts mainly at the DP receptor, PGFza at the FF' receptor, PGEz at the EP receptor, PGIz at the IP receptor and TXA2 mainly at the TP receptor (see also Sections 1.6.2 and 7.1.4). This represents a simplified scheme, since several different prostanoids can act, for example, at the TP receptor causing airway contraction. Furthermore, several subtypes of EP receptors exist. The action of the prostanoids can thus be inhibited either by inhibition of the COX enzyme by non-steroidal anti-inflammatory drugs (NSAIDs) or by selective receptor antagonists.

F>y inhibitors

LTA,

LTC4+ LTD4+ LTE,

LTB, I

Figure 9-3.5-Lipoxygenase products, pathways and receptors. The enzyme 5-lipoxygenase (5-LO), which is amenable to inhibition, metabolizes arachidonic acid to leukotriene A4 (LTA4). LTA4 is further metabolized to LTB4 and the cysteinyl leukotrienes LTC4, LTD4 and LTE4. LTB4 acts at the BLT receptor, whereas several different cysteinyl leukotriene receptors (CysLT receptors) exist. At present, selective receptors. antagonists are only available for the BLT and CJJ.VLT/

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which then secrete LTB4 in the airways. The lipoxins were originally isolated from human leukocytes [44] and have since then been described to emanate from various cell-cell interactions, as reviewed in Serhan [45; see also Sections 1.3.4 and 7.31. One of the major compounds which is formed is lipoxin A4 (LX&). The stimuli which evoke formation of LXA4 [46] seem to differ from the stimuli promoting biosynthesis of cysteinyl leukotrienes. Allergen challenge has not been shown to elicit LXA4 formation, whereas there is data suggesting that aspirin challenge of aspirin-intolerant asthmatics may cause release of LXA4 [47]. It has been found that granulocytes from eosinophilic donors form lipoxins [48]. Lipoxins have also been detected in bronchoalveolar lavage fluid from patients with sarcoidosis [49] and intraluminally after atherosclerotic plaque rupture by percutaneous transluminal coronary angioplasty [50]. Recently, lipoxins have been implied in glomerulonephritis;see [51 and Section 7.31. It is worth noting that the profile of products formed from arachidonic acid is influenced by the stimulus that is applied to the mast cell. For example, PGD2 but not 15-hydroxyeicosatetraenoic acid ( 1 5-HETE) is released from the lung when mast cells are activated in IgE-dependent reactions [22,26]. In contrast, if lung tissue is exposed to high levels of free arachidonic acid, there is a selective stimulation of 15lipoxygenase activity without increased production of leukouienes or prostaglandins [52]. Furthermore, inflammation may induce dramatic quantitative and qualitative changes in eicosanoid synthesis, both as a consequence of induction of new enzymes, e.g. COX-2 (see Chapter 3) and as a result of new cells infiltrating the tissue. More recently, it has been discovered that hydroperoxides generated, for example during lung injury, may give rise to a series of eicosanoids, the isoprostanes [53]. These compounds are structurally very similar to conventional prostaglandins (see Sections 1.3.8 and 7.5). The only difference is that the o side chain of the molecule connects to the prostaglandin cyclopentane ring in trans rather than cis configuration. So far, isoprostanes appear to exert their biological activities through the same receptors as conventional prostanoids. Lipid peroxidation may also yield biologically active isoleukotrienes [54]. However, this new field has not matured sufficientlyto be reviewed here. As a final example of yet another group of eicosanoids, it should be mentioned that an endogenous ligand for a cannabinoid receptor turned out to be an arachidonyl amide and was given the name anandamide [55; see also Section 1.3.91. The great number of alternative products synthesized from arachidonic acid suggests that, presumably for evolutionary reasons, it has been practical to utilize arachidonic acid as a substrate for the synthesis of many different biologically active messengers. Such highly conserved use of a simple cellular precursor is a quite common general strategy in many areas of metabolism, e.g. the use of cholesterol for synthesis of steroids and other compounds. The eicosanoids have a great number of potent biological effects on airways and blood vessels. Consequently, several members in this family of compounds have been proposed to function as mediators or modulators of inflammation, airway obstruction and bronchial hyper-responsiveness. There is often an inclination to describe every individual eicosanoid as a pro-inflammatory mediator. However, there are drugs and drug candidates that may interfere with the production or action of eicosanoids in different ways (Figs 9-2 and 9-3) and the results of such studies rather support the vicw

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9 The role of eicosunoids in inflummution and allergy

that different eicosanoids have distinct and sometimes opposing functions. For example, certain leukotrienes indeed mediate airway obstruction, whereas PGE2 has properties which indicate that it protects against asthma [56].

9.3 Biological activities and receptors with relevance for asthma and allergic inflammation 9.3.1 COX products E series prostaglandins and prostacylin (PGI2) are potent vasodilators at low concentrations [57-601. Furthermore, they potentiate the pain- and edema-producing effects of histamine and bradykinin [58-631. PGE2 and PGI2 also potentiate the leukocyte extravasation and accompanying plasma leakage evoked by different chemotactic mediators [64,65]. The ability of vasodilator prostaglandins to enhance edema and leukocyte emigration has been attributed to their capacity to increase blood flow in venules at inflamed sites [2]. Vasodilatingprostaglandins (PGE2 and PGI2) have also been found to inhibit effector functions such as mediator release from basophils,neutrophils, macrophages, mast cells, lymphocytes and lung cells, most likely via the second messenger CAMP (adenosine monophosphate) [66-771. As will be discussed, inhibition of mediator release may in fact be the predominant function of PGE2 in allergic inflammation (vide infra). TXA2 is a potent constrictor of airway and vascular smooth muscle [78-811. TXA2 may also, as may PGD2 and PGFza, enhance immunologically induced inflammatory mediator release from some tissues in vitro [82-841. In addition, PGS, may antagonize edema formation [86], most likely as a result of its vasoconstrictiveproperties. The pharmacology and molecular biology of prostanoid receptors has recently been reviewed [86; see also Sections 1.6.2 and 7.1.41. Prostanoid receptors were originally classified according to functional data obtained with the primary prostaglandins and the use of some experimental compounds which exerted antagonistic properties. Although the natural agonists show rather limited selectivity and may activate almost any prostanoid receptor in concentrations that may still be reasonable, each of the naturally occurring primary prostaglandins nevertheless has a receptor where it is more potent in comparison with the other prostaglandins. These receptors are termed P receptors, with a preceding letter indicating the most potent prostanoid which is an agonist at the receptor (Fig. 9-2). This gives DP, EP, FP, IP and TP receptors for PGD2, PGE2, PGFz,, PGI2 and TXA2, respectively. Among the EP receptors, there are at least four subtypes (EPI, EP2, EP3 and EP4), whereas there is no convincing evidence for subtypes of the other receptors. Differences in pharmacology between TP receptors from different species have been discussed, but is not considered a basis for distinction of separate receptors [86]. All postulated prostanoid receptors have been cloned and found to belong to the family of G-protein-coupled receptors with seven transmembrane domains. There are

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several potent and selective TP receptor antagonists available, including for studies in humans. Experimental drugs that display some selectivity as antagonists of DP, EP, and EP4 receptors exist. For the remainder of the prostanoid receptors, there are as yet no useful pharmacological tools to investigate their functions in either man or in experimental models. There are compounds that show some degree of agonist selectivity for the different prostanoid receptors, but, with the exception of some potent thromboxane/endoperoxide mimetics, which are TP receptor agonists (e.g. U-44069 or U46619), there are no compounds sufficiently selective and potent to be useful in human studies. For pharmacological manipulation of prostanoids in the lung, TP, DP and EP receptors are probably of primary concern. Thus, the bronchoconstrictive actions of TXA2, PGD2 and PGF?, are mediated by the TP receptor. The DP receptor is preferentially activated by PGD2 and mediates vasodilatation. The EP receptors mediate the relaxation of airway smooth muscle and inhibition of inflammatory cells in response to PGE2, and EP2 receptors are probably most important in this context. Several of the receptors, namely DP, EP2, EP4 and IP receptors, couple to adenylyl cyclases via G,proteins, resulting in increased intracellular levels of CAMP (see Section 1.6.2). It is known that EPI, EP3 FP and TP receptors use phospholipase C pathways and G,protein transduction to induce inositol trisphosphate generation and increased levels of cytosolic calcium. As an indication of the importance of different EP receptor subtypes and the selective mediation of different responses by one subtype, mice lacking the EP3 receptor by homologous recombination fail to show a febrile response to PGE2, interleukin-1p (IL- 1p) or lipopolysaccharide [87].

9.3.2 LTB4 Leukocytes are the primary targets for the biological activity of LTB4. LTB4 is a potent stimulus for activation of leukocytes, eliciting chemokinetic and chemotactic responses in vitro [88]. In vivo, LTB4 increases leukocyte rolling and adhesion to the venular endothelium [89]. This initial chemotactic response is followed by their emigration into the extravascular space [89]. During a short-lasting exposure to LTB4, polymorphonuclear leukocytes are mainly recruited. With prolonged exposure to LTB4, as presumably occurs when LTB4 is formed in vivo, other granulocytes, including eosinophils, are found in tissues or exudates after challenge with LTB4[90]. It has been shown that LTB4 is a chemoattractant for IL-5-primed eosinophils [91] and LTB4 may stimulate production of IL-5 in T lymphocytes [92]. In addition to effects on leukocyte adhesion and migration, LTB4 stimulates the secretion of superoxide anion and release of different constituents from leukocyte granules [93,94]. It has been observed that chemotaxis is mediated at lower agonist concentrations of LTB4 than those required for degranulation and superoxide generation [93-951. It has also been found that LTB4 may affect expression of low-affinity receptors for IgE on B lymphocyte cell lines [96] and IgE synthesis induced by It-4 [97]. It may also affect the secretion of IgM, IgG and IgE [97-991. Possibly relating to these effects of LTB4 on B cells, it was recently observed that sensitized 5-lipoxygenasedeficient mice produced lower levels of IgG and IgE upon ovalbumin challenge than

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9 The role of eicosanoids in inflammation and allergy

wild-type mice [ 1001. Furthermore, the observation that LTB4 is an agonist for the nuclear transcription factor PPARa (peroxisome proliferator-activated receptor a, see Section 1.6.3) has created considerable interest [loll. The finding may imply a role for LTB4 in the control of central events in lipid metabolism and inflammation, but also the possible existence of a feedback loop which may respond to increased leukotriene production by enhanced leukotriene catabolism. The structure-activity relations for this effect of LTB4 and the influence of antagonists of LTB4 on the response remain to be determined, but the observations nevertheless point at the possibility that LTB4 also has intracellular and nuclear targets, which may participate in long-term control of gene expression. With respect to the effects of LTB4 on the lung, it has also been established that LTB4 has contractile activity in animal models such as the guinea-pig lung parenchyma [31,96,102-1O4]. The response is indirect, involving the release of TXAz [31,102,103] and most likely histamine as well [103]. This indicates that certain tissues may contain elements with the ability to release spasmogenic mediators when exposed to LTB4. However, LTB4 has not been found to contract isolated human bronchi [ 1051 or to induce bronchoconstrictionfollowing inhalation [ 106-1081. In a dog model, LTB4 was found to increase airway reactivity to acetylcholine [109], whereas among non-asthmatic humans there was no change in bronchial hyperresponsiveness to histamine following the inhalation of LTB4, alone or in combination with PGD2 [106]. Studies in the upper airways [e.g. 1101 nevertheless suggested that leukotrienes induced vascular and cellular reactions. In fact, subsequent studies showed that inhalation of LTB4 affected cell traffic and possibly also plasma extravasation in the human lung [ 107,1081. LTB4 acts at a specific receptor, which is designated the BLT receptor [ 1113. The developed antagonists appear to block the effects of LTB4 and its immediate metabolites at a common BLT receptor and there is currently no basis for a definition of subclasses of receptors for LTB4. The 5S, 12R position of the hydroxyls in LTB4 appears critical for biological activity [96,104]. For example, 5s-HETE and 5S, 1 2s-dihydroxyeicosatetraenoic acid (SS,12S-DHETE) are both less potent than LTB4 in the guinea-pig lung parenchyma [96,104] and the responses elicited by these compounds are also different with respect to time course and mode of action [96,104]. In contrast, a SS,12R-DHETE with 6trans and 8 4 s double bonds (LTB4 is 6 4 s and 8-trans; otherwise identical structure) formed by the LTA4 hydrolase in Xenopus laevis was recently found to share the mode of action of LTB4in the guinea-pig lung parenchymal strip [ 1121. Radioligand binding experiments have been useful in exploration of the properties of BLT receptors. Thus, specific (3H)-LTB4 binding has been demonstrated in many tissues including human polymorphonuclear leukocytes (PMNLs) [ 113,1141. Structurally related metabolites displaced LTB4 with a potency which correlated with their activities in chemotaxis assays [115]. The binding sites in PMNLs were selectively inhibited by guanine nucleotides, suggesting that BLT receptors were Gprotein coupled (116,1171. A novel chemoattractant-like receptor called CMKRLI was cloned [ 1181 and later identified as the BLT receptor [119,120]. It was possible to isolate the cDNA for this BLT receptor in retinoic acid-differentiated HL-60 cells

9.3 Biologicul activities und receptors with relevance for asthmu and allergic injlammutionn

24 1

[120]. The cDNA encoded a 352 amino acid cell-surface protein which was G-protein coupled and mediated chemotaxis. Northern blotting experiments of human tissues displayed a preferential expression of mRNA for the BLT receptor in PMNLs [120]. There was also some expression in the spleen and thymus, whereas most other tissues examined, including the lung, showed no or insignificant expression of mRNA for the BLT receptor [120]. In further exploration, it was recently shown that the LTB4 receptor, among other receptors, mediated entry of human immuno-deficiency virus- 1 (AIDS virus, HIV-1) into CDCpositive cells [121]. Some selective and relatively potent antagonists of LTB4 have been developed. A few compounds have entered into early clinical testing in man. The compound LY2931 11 (VML 295) was recently found to inhibit LTB4-induced neutrophil responses in vivo and allergen-induced neutrophil activation, but it had no effect on allergeninduced early or late phase airway obstruction in asthmatics [122]. The results with LY-293 111 in asthmatics argue against an important role for LTB4 as a mediator in asthma, but do not exclude that LTB4may be involved in other pulmonary reactions. In view of the proficient synthesis of LTB4in activated neutrophils, it would seem to be of interest to investigate the effects of 5-lipoxygenase inhibitors and antagonists of LTB4 in pulmonary syndromes characterized by neutrophil accumulation and activation.

9.3.3 Cysteinyl leukotrienes Cysteinyl leukotrienes have a number of effects which have a bearing on allergeninduced bronchoconstriction and allergic inflammation. The exquisite spasmogenic potency of cysteinyl leukotrienes on isolated human bronchi [ 123-1281 has been extensively confirmed in bronchoprovocation studies of normal subjects [ 129-13 11. The cysteinyl leukotrienes are in fact the most potent endogenous bronchoconstrictors so far known. Accordingly, cysteinyl leukotrienes have generally been found to produce bronchoconstriction in concentrations 100-10 000 times lower than those required with histamine or methacholine. Although LTE4 sometimes displays a spasmogenic activity inferior to that of LTC4 and LTD4 in animal models, such as guinea-pig ileum [132-1341, it is clear that each of the three cysteinyl leukotrienes have closely similar potencies as constrictors of human bronchi in vitro [42,127]. Moreover, their bronchoconstrictive effect is apparently mediated by the same receptor, because in vitro studies show that potent receptor antagonists such as zafirlukast (ICI2042 19/AccolateTM)antagonize each of the cysteinyl leukotrienes to the same degree [127]. It has also been observed that prolonged exposure to LTE4 may produce enhancement of the responsiveness of smooth muscle to histamine [135,136], but the importance of these findings remains to be established. When injected intravenously in guinea-pigs, LTC4 and LTD4 caused biphasic changes in blood pressure [123,137]. Local injection of these two cysteinyl leukotrienes also increased accumulation of the plasma protein tracer Evans blue in the skin [ 123,1371, suggesting an increase in microvascular permeability. In the hamster cheek pouch, it could be established that LTC4 and LTD4 indeed caused extravasation of plasma proteins in post-capillary venules [89]. In addition, LTC4 and LTD4 induced arteriolar constriction, but the plasma extravasation was not a consequence of

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9 The role of eicosanoids in injammation and allergy

the vasoconstriction [89]. In the guinea-pig, it was shown that LTC4, LTD4 and LTE4 were each capable of causing accumulation of Evans blue in the airways [ 1381. The plasma extravasation occurred in all airway segments, ranging from the most peripheral small bronchi to trachea and there was evidence of Evans blue accumulation in superficial as well as deep layers of the airway mucosa. Experiments in isolated perfused hearts also disclosed a depressive effect on cardiac contractility [ 139,1401. The effect correlated with coronary vasoconstriction [141], but a direct negative inotropic effect on the myocardium may also be involved [139,142]. Although the first generation of antagonists of cysteinyl leukotrienes are being introduced as a new therapy in asthma, this field is only in its infancy and the classification of cysteinyl leukotriene receptors primarily rests on functional data. None of the receptors for cysteinyl leukotrienes have been isolated or cloned and their structures remain unknown. On the basis of primarily functional studies in smooth muscle assays, two main classes of receptors have been outlined [ 11 11. Following the lead provided by the compound FPL 557 12 [ 1431, a great number of antagonists for cysteinyl leukotrienes have been developed. The currently developed antagonists generally inhibit the action of LTC4, LTD4 or LTE4 on human airway smooth muscle preparations [128], as well as the action of LTD4 or LTE4 on the guinea-pig trachea or ileum [134,144]. The name CysLT, receptor has been introduced for the group of functional receptors for cysteinyl leukotrienes which is susceptible to antagonism by the current class of antagonists [ 1111. It is well documented that the CysLT, receptor antagonists block the bronchoconstrictive effects of the cysteinyl leukotrienes, but it is less certain to what extent they block other effects of these mediators. For instance, the effects of LTC4 and LTD4 on human pulmonary veins [145] or ferret spleen [ 1461 and the contractile effects of LTC4 on guinea-pig trachea or ileum [134,144], have been found to be resistant to the available antagonists of cysteinyl leukotrienes. The receptors mediating these responses have been designated CysLT2. However, at present there is no selective CysLT2 antagonist, which makes it possible that CysLT2receptors comprise a heterogeneous group. The current classification introducing two main classes of receptors for cysteinyl leukotrienes is a first step supported by the available evidence but is nevertheless likely to represent an oversimplification.The observation that the contractile response to LTD4 in the guinea-pig lung parenchyma is poorly inhibited by both potent CysLT1 antagonists such as ICI 198615 as well as by the combined CysLTllCysLT2 antagonist BAY u9773 [147,148], raises the possibility of a third subclass, CysLT3. A particular problem in studies of cysteinyl leukotriene receptors is that the natural ligands bind with high affinity to the ubiquitous enzymes in pathways generating or metabolizing the primary leukotrienes [149]. It has recently been described that the cysteinyl leukotrienes may bind to microsomal glutathione S-transferase-1 (MGST- 1) [150] and that MGST-2 [151] and MGST-3 [152] may also be involved in the synthesis of cysteinyl leukotrienes. In addition, data demonstrating that leukotrienes are generated close to the cell nucleus [153], together with the findings that there exist specific transport mechanisms for leukotrienes [ 1541, all represent factors which may explain why published data on binding of the natural ligands has been confusing.

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9.3.4 Lipoxins A number of different biological activities have been attributed to lipoxin & (LXA4), lending it potential to serve as a regulator of inflammatory responses. For example, LXA4 may regulate blood flow, as shown by dilation of arterioles in vivo [155-1571. LXB4 has also been shown to induce vasodilation in some tissues [156,157], but has vasoconstricting properties in other tissues [ 1581. The mechanisms underlying lipoxininduced vasodilation are at present not clear [159], but in some systems LXA4 has been shown to elicit formation of PGE;! or PGI2 [158,160], which, as previously discussed, can have inhibitory actions on inflammatory responses. In fact, LX& has been shown to inhibit leukocyte-dependentedema in the hamster cheek pouch in vivo [161]. Furthermore, LX& evokes contractions of guinea-pig lung parenchymal strips [46,157,162-1641. It has also been shown to induce formation of the bronchoconstrictor TXA2, although the contraction elicited by LXA4 is mainly a direct effect of LXA4 itself [ 1651. These observations indicate that lipoxins are potent hemodynamic regulators. Furthermore, they can induce formation of other potent eicosanoids with pro- as well as anti-inflammatory actions, again a reminder of the importance of considering mediator interactions in this complex field. Not only do the lipoxins, as well as many other eicosanoids, have direct or indirect effects on blood flow. In addition, they seem to have direct effects on cells belonging to the immune system. Examples are effects on secretion from leukocytes [44] and inhibition of cytotoxic actions of human natural killer cells [166]. LX& is also a chemoattractant for human neutrophils [ 1671. The hypothesis has emerged that lipoxins exert their activity as chalones (inhibitors) of the immune response [168]. The counter-regulatory actions reported for lipoxins in different tissues are in fact increasing in number [45]. In leukocytes, LX& is described as acting at a G-protein-coupled receptor with seven transmembrane-spanning regions. A human cDNA encoding a functional highaffinity receptor for LXA4 has in fact been identified [169]. In contrast, in smooth muscle cells of various types, there are indications that LXA4 and cysteinyl leukotrienes act at common receptors [157,162-164,170-1721, which have not yet been structurally identified.

9.4 Cysteinyl leukotrienes as mediators of allergen-induced airway obstruction and bronchial hyper-responsiveness From the first reports demonstrating that prostaglandins were formed in the lungs and had biological activity on bronchi and blood vessels [173], the hypothesis emerged that these compounds contributed to asthmatic airway obstruction. However, the results that have since accumulated make it likely that COX products are of minor im-

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9 The role of eicosanoids in inflammation and allergy

portance as primary mediators of spontaneous or induced airway obstruction in man. In contrast, it is now established that cysteinyl leukotrienes mediate major components of allergen-inducedairway obstruction. When assessing the contribution of eicosanoids or any other tentative mediator to the symptoms or pathological changes in allergen-induced airway obstruction in man, it is helpful to consider the criteria required to prove that a substance functions as a mediator [ 1741, namely that the substance must have the biological activity investigated and be endogenously formed and that inhibition of its action or formation blocks the symptoms in question.

9.4.1 Biological activity The mediator candidate must possess biological activity in man, evoking the response in question. In the case of multiple co-mediators, the mediator candidate must have biological effects which may explain certain components of the biological response in question. The species issue is very important to consider. There are many examples of how a factor which may be demonstrated to mediate a response in experimental animal models fails to have the same role in humans. Cysteinyl leukotrienes were soon shown to be very potent bronchoconstrictors in bronchoprovocation studies of asthmatics [175,1761. Asthmatics have also been found to be hyper-responsive to inhalation of LTC4, LTD4 and LTE4 [175-1771. In addition, there are indications that LTD4 may cause greater airway narrowing than methacholine [178]. On the other hand, at least for LTC4 and LTD4, there are observations demonstrating that the hyper-responsiveness of asthmatics to these leukotrienes relative to methacholine was less than in non-asthmatics [175]. This is a rather unique property of a bronchoconstrictor in asthmatics and raises the interesting hypothesis that chronically elevated production of leukotrienes in asthmatics induces changes in the airway effector cells or at the receptor level. There are in fact indications that repeated challenge with cysteinyl leukotrienes is associated with tachyphylaxis mediated by local generation of COX products [179]. However, concerning LTE4, the opposite observation has been reported, namely that asthmatics were particularly hyper-responsive to this leukotriene [1801. In addition, there are reports suggesting that cysteinyl leukotrienes, among other lipoxygenase products, can increase mucus secretion [ 181-1 841 and, perhaps of particular significance in the context of asthma, stimulate eosinophil migration into asthmatic airways [lSS]. The cysteinyl leukotrienes also have the ability to increase microvascular permeability and induce arteriolar vasoconstriction [89]. Thus, the cysteinyl leukotrienes have a wide range of biological effects, which all support the potential to mediate important components of asthmatic airway inflammation.

9.4.2 Endogenous formation The mediator must be synthesized and/or released in association with the biological response in question at the site where it is hypothesized to act. This is most often

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245

shown by recovery of the mediator or relevant metabolites originating from the mediator candidate in samples of biological fluids collected during the course of the response. For mediators of asthmatic reactions this usually although not necessarily always involves formation by cells which reside in pulmonary tissues or infiltrate the lung. Alternatively, the putative mediator should be recovered from human airways or other relevant body fluids and should be present in biologically active amounts. In this context, demonstration of the presence of enzymes for the formation of a particular compound does not prove that the substance is formed in the tissue or cell under in vivo conditions, although it certainly supports this possibility. Soon after the discovery of the leukotrienes and the structural elucidation of SRSA, it was possible to obtain in vitro evidence that cysteinyl leukotrienes were generated in asthmatic lung tissue [42]. When methodology had been sufficiently developed, it was possible to establish that allergen-induced airway obstruction, at least during the early phase, was associated with increased urinary excretion of LTE4[ 1861891. The findings that bronchoconstriction induced by histamine [ 1901 or methacholine [ 1911 had no influence on urinary LTE4, supported the idea that the leukotrienes were released as a primary and early consequence of the allergen challenge. For the early reaction, it appears most likely that the leukotrienes originate from the pulmonary mast cells. The recent observation of an apparent correlation between the postchallenge urinary excretion of LTE4 and the PGD2 metabolite 9a, 11P-PGF2 supports this hypothesis [192]. Recent studies also show an increase in urinary LTE4 during the late phase of the asthmatic response [188,189,193]. It is known that the eosinophil, which is often increased in the tissue and activated during the late phase, is one of the most effective producers of LTC4 [ 1941.

9.4.3 Influence of inhibitors of leukotriene synthesis or CysLT, receptor antagonists on allergen-induced airway obstruction The third criterion for the identification of a mediator is the unambiguous demonstration that specific interventions which attack a purported mechanism also result in significant inhibition of the response in question. In fact, this is the most important criterion for a mediator of inflammation. For example, for technical reasons it is often difficult to measure eicosanoids in different media during a reaction, but when a selective receptor antagonist nevertheless blocks an evoked reaction, it is difficult to escape the conclusion that the substance in question is mediating the studied response. Drugs which inhibit the action or formation of cysteinyl leukotrienes have been collectively termed anti-leukotrienes (Fig. 9-3). These can be divided into different categories based on their mode of action, namely cysteinyl leukotriene receptor antagonists (see Section 12.5) and inhibitors of leukotriene synthesis (direct inhibitors of 5-lipoxygenase or inhibitors of 5-lipoxygenase-activating protein, FLAP; see Section 5.6). These different categories can then be further subdivided based on their chemical structures and more detailed mode of action at a molecular level. One selective 5-lipoxygenase inhibitor, zileuton, is now registered in the US for

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treatment of asthma. It was indeed the first compound to enter the final stages of clinical development, which means that most published studies of the effects of anti-leukotrienes in asthmatics have been conducted with this particular compound 11951. Furthermore, the CysLT, receptor antagonists montelukast and zafirlukast have been registered for treatment of asthma in many parts of the world and pranlukast has been registered in Japan (see Section 12.5). The compounds are administered orally and have been found to cause significant (25-1000 fold) shifts in the dose-response relation for inhaled LTD4 in normal subjects or asthmatics [196-1991. After the structural elucidation of SRS-A, it was possible to obtain in vitro evidence that cysteinyl leukotrienes mediated a major part of the Schultz-Dale contraction to allergen in human bronchi [42]. When more specific pharmacological tools became available, it was possible to demonstrate that cysteinyl leukotrienes indeed mediated major components of both the early and the late response to allergen challenge of asthmatics. Thus, between 50 and 75% of the early fall in forced expiratory volume in 1 second (FEV,) has been found to be blocked by pre-treatment with either potent CysLT, receptor antagonists [42,200,201] or effective inhibitors of leukotriene biosynthesis [193,2021. Likewise, in studies using cumulative challenge with allergen as an end-point, a highly significant increase in the PD20 for allergen was and P D z ~ obtained after leukotriene antagonism [203, 2041. During the late asthmatic reaction, the airflow obstruction is considered to be related to global inflammatory changes in the airways following allergen challenge. The cysteinyl leukotrienes promote eosinophil recruitment [ 1851 and may also affect secretion of cytokines [91,92,205,206], which are considered to promote key events in the inflammation during the late asthmatic response. The vascular actions of cysteinyl leukotrienes would add to the inflammatory response. In v i m , LTC4 and LTD4 have also been found to stimulate secretion of mucus in both animal and human airways [183,207,208]. Such an effect, if also present in vivo, would have the potential to contribute to airway obstruction, in particular if the leukotrienes diminish mucociliary clearance at the same time, as suggested by one study [209]. The findings in bronchoprovocation studies were initially variable with respect to the extent of protection against the late reaction provided by anti-leukotrienes. However, in more recent studies, where the drugs have had sufficient bioavailability and have been potent enough to affect the early phase, inhibition of the late phase has invariably been observed [188,193,200,210].The extent of the inhibition has been substantial (Fig. 9-4). A few studies concern the interaction between leukotrienes and other mediators in man. Such interactions may also contribute to the late asthmatic reaction. In a study of normal subjects, it was demonstrated that inhalation of a dose of LTD4 which caused no bronchoconstriction per se produced a sevenfold shift to the left in the dose-response curve determined for PGF2, 10 min after inhalation of LTD4 [211]. A study in asthmatics found evidence of synergistic interactions between single doses of inhaled LTC4 and histamine as well as PGD2 [212]. Since the activation of mast cells results in concomitant release of histamine, PGD2 and LTC4, it would seem clinically relevant to explore this particular interaction in more detail.

9.4 Cysteinyl leukotrienes as mediators of allergen-induced airway obstruction and bronchial hyper-responsiveness

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Hour, post allergen Figure 9-4. Effect of the CysLT! receptor antagonist zafirlukast on the early and late asthmatic response. The fall in forced expiratory volume in one second (FEVI) after allergen challenge is significantly inhibited by zafirlukast during both phases. Modified from Roquet et al. [188].

Interestingly, a recent allergen bronchoprovocation study [ 1881 using combined treatment with a histamine HI receptor antagonist loratadine and the CysLT, receptor antagonist zafirlukast showed not only that the cysteinyl leukotrienes were major mediators of both the early and the late asthmatic response, but also that histamine was involved in both phases of the response. Both the early and the late asthmatic responses were inhibited. In fact, loratadine and zafirlukast caused significantly more effective inhibition of the late asthmatic response than either drug alone [ 1881. Taken together, it is evident that cysteinyl leukotrienes contribute significantly to both the early and late phases of the asthmatic response. The large effect of antileukotrienes on the response to allergen challenge supports this; despite the many mediators with bronchoconstrictive properties that are generated after exposure to allergen, there is a hierarchy among the mediators with respect to their importance in induced airway obstruction. This brings us to a shift in paradigm from the despairing hypothesis that a great number of inflammatory mediators all contribute equally to every expression of airway inflammation to the awareness that a relatively limited number of principal mediators dominate when a given response such as airway obstruction is studied. This new view may have considerable implications for the creation of new therapy in asthma induced by allergen and other trigger factors.

9.4.4 Allergen-induced bronchial hyper-responsiveness Exposure to environmental allergens results in increased bronchial responsiveness to a great number of stimuli [213]. In fact, this may be the predominant effect of natural exposure to low doses of allergen [214,215]. Bronchial hyper-responsiveness has

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been attributed to the resulting allergic inflammation [216] and irritation of sensory nerves [217]. Structural changes in airways of asthmatics [218] may also increase airway responsiveness. The role of cysteinyl leukotrienes in bronchial hyperresponsiveness is less well established. A few studies have addressed whether or not inhalation of cysteinyl leukotrienes may lead to acute or chronic changes in airway reactivity to other agents. The findings and study designs have been variable and it is likely that there is much more to learn in this area. In normal subjects, some studies have demonstrated small but distinct increments in airway responsiveness to methacholine or histamine following inhalation of LTD4 [178,219,220]. However, another group found no changes in histamine responsiveness after healthy volunteers inhaled either LTD4 or LTE4 [221]. In asthmatics, inhalation of LTD4 or LTE4 has been reported to produce increased airway responsiveness to histamine [221,222]. In one of the studies [221], there was a more than threefold increase in histamine reactivity at 7 h following bronchoprovocation with LTE4 and there was still a significant increase in histamine responsiveness 4 days after the inhalation of LTE4. However, one study failed to detect increased histamine responsiveness following inhalation of LTD4[223]. Recently, increased infiltration of eosinophils into the airway mucosa of asthmatics was observed following inhalation of LTE4 [185] and inhalation of LTD4 increased the number of eosinophils in induced sputum samples from asthmatics [224]. The capacity of cysteinyl leukotrienes to promote eosinophil recruitment has recently been confirmed in experimental models [205,225], although the mechanisms involved still have to be defined. The response may involve leukotriene-induced release of IL-5 from epithelial cells [205], but the release of other chemotactic factors as well as direct chemotactic effects of cysteinyl leukotrienes are also possible [225]. There is also experimentaldata in vitro [226-2291 and in vivo [230] supporting the idea that cysteinyl leukotrienesmay be involved in airway smooth muscle proliferation and remodeling. Experimental studies of bronchial hyper-responsiveness in 5-lipoxygenasedeficient mice have generated findings which suggest that leukotrienes contribute to different aspects of the process in this particular model [loo]. Thus, the antigeninduced allergenic sensitization was suppressed in animals which lacked the 5lipoxygenase and the airway reactivity to methacholine was reduced by disruption of 5-lipoxygenase. It was recently observed that a few hours exposure of healthy volunteers to organic dust induced a remarkable bronchial hyper-responsiveness, expressed as a tenfold drop in the PD20for methacholine 24 h post exposure and this change was associated with increased urinary excretion of LTE4 12311, indicating that cysteinyl leukotrienes are indeed formed during the induction of hyper-responsiveness by environmental factors, even without development of acute bronchoconstriction. In asthmatics, a period of treatment with the lipoxygenase inhibitor AA-861 [232] or the selective CysLT] antagonist pranlukast [233] was found to attenuate baseline bronchial hyper-responsiveness. In one of the first allergen-provocationstudies with a potent CysLT, receptor antagonist [200], zafirlukast was found to cause a small but significant attenuation of post-allergen responsiveness to histamine. However, the finding has been criticized [234], since histamine responsiveness was evaluated during the course of the late reaction (at 6 h). The magnitude of protection was indeed

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relatively small and in the same order as in studies which have failed to obtain a significant effect of anti-leukotrienes on post-allergen reactivity to histamine or methacholine [ 188,193,210,2351.Taken together, there are several observations suggesting that cysteinyl leukotrienes may be involved in bronchial hyper-reactivity, but more long-term treatment studies with anti-leukotrienes are required in order to establish the clinical importance of such a mechanism.

9.5 Cysteinyl leukotrienes as mediators of airway obstruction induced by other factors The mechanisms that initiate airway obstruction following exercise, inhalation of dry cold air or bronchoprovocation with adenosine [236], sulfur dioxide [237] or plateletactivating factor (PAF) [238,239] are clearly different from those that trigger the early phase of allergen-induced bronchoconstriction. Nevertheless, for all of these trigger factors, it appears as if the contributions of different eicosanoid mediators to the primary bronchoconstrictor response are closely similar or identical to the mechanisms described for allergen-induced airway obstruction (Fig. 9-5). Accordingly, cysteinyl leukotrienes mediate significant parts of the airway obstruction evoked by these different challenges, whereas COX products do not. This suggests that different trigger factors converge on one and the same effector mechanism, resulting in release of leukotrienes. Such a common feature may be due to activation of one and the same cell type, e.g. the mast cell, but may also be explained if different cell types, alone or by transcellular metabolism, produce the same set of 5-lipoxygenase products. Both of these alternatives may explain why anti-leukotrienes are effective in preventing bronchoconstriction induced by many different factors which are known to trigger episodes of airway obstruction in asthmatics. Allergen Adenosine Exercise Cold Air \

ASA (in ASA-intolerant)

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I I Obstruction of Airways Cysteinyl leukotrienes

Figure 9-5.Cysteinyl leukotrienes as common mediators of airway obstruction. Several different types of stimuli can activate mediator-releasing cells. Release of cysteinyl leukotrienes is a final common event in many of these reactions which, in the end, cause obstruction of airways via bronchoconstriction and inflammatory changes. ASA, acetylsalicylic acid (aspirin); PAF. platelet-activating factor.

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9.5.1 Exercise A couple of studies [240,241], have detected increased levels of urinary LTE4 in association with the airway response following exercise [ 19I], whereas it is well documented that several different anti-leukotrienes provide a major inhibition of bronchoconstriction induced by exercise [242-2461. Anti-leukotrienes also protect against airway obstruction induced by inhalation of dry cold air [247], which is a closely related challenge since the mechanisms triggering the response to exercise involve changes in airway temperature or osmolarity or both. It should also be acknowledged that subjects with strong responses to exercise challenge demonstrate a high degree of bronchial hyper-responsiveness, which means that comparatively small elevations in the levels of local mediators may be sufficient to produce significant bronchoconstriction. Taken together, a significant component of exercise-induced bronchoconstriction is mediated by cysteinyl leukotrienes.

9.5.2 Adenosine Asthmatics demonstrate hyper-responsivenessto adenosine both in vitro [248] and in vivo [249]. Several observations have suggested that this effect of adenosine is due to activation of mast cell mediator release [250,251]. For example, in bronchi from asthmatics, pre-treatment with a potent CysLT, receptor antagonist blocked the effect of adenosine [248]. It was recently possible to obtain evidence that anti-leukotrienes attenuated adenosine-induced airway obstruction in asthmatics in vivo [236]. Thus, adenosine also seems to act via induction of release of cysteinyl leukotrienes.

9.5.3 Sulfur dioxide In subjects 20-44 years of age who are hyper-responsive to methacholine, the prevalence of airway hyper-responsiveness to sulfur dioxide has recently been described to be 20-25% [252]. Bronchoconstriction triggered by inhalation of sulfur dioxide or other air pollutants has been attributed to different mechanisms, such as increased cytokine production and induction of inflammatory enzymes, for example COX [253]. The recent observation that the CysLT, receptor antagonist zafirlukast was able to inhibit the effect of inhaled sulfur dioxide partially [237] suggests that cysteinyl leukotrienes are also generated during the reaction to sulfur dioxide, perhaps in response to release of a neuropeptide in the airways.

9.5.4 PAF Inhalation of PAF is followed by bronchoconstriction and an increase in bronchial responsiveness to direct challenges [254,255]. The magnitudes of these changes have differed between investigations, but nevertheless it was found that challenge of asthmatics with PAF was associated with substantially increased urinary excretion of LTE4 [256]. It was also found that challenge of isolated human cells with PAF in vitro

9.5 Cysteirzyl leukotrienes as mediators of airway obstruction induced by other facturs

25 I

induced leukotriene generation [256]. The increased urinary LTE4 following inhalation of PAF was significantly reduced by the specific PAF receptor antagonist UK74505 [257]. Moreover, two different CysLT, receptor antagonists have been found to inhibit the airway response to PAF [238,239]. These findings are of interest, because it has been a paradox that PAF has no direct contractile effect on human airway smooth muscle [105,258] but may cause airway obstruction in vivo (vide supra). It is therefore now possible to define the mode of action of PAF on human airways more precisely. Namely, PAF acts indirectly and may cause airway obstruction and bronchial hyper-responsiveness secondary to evoked release of leukotrienes. This indirect mode of action of PAF has gained further support from recent experiments in 5lipoxygenase-deficient mice, which are protected from the lethal cardiovascular shock which follows injection of PAF in animals with intact 5-lipoxygenase [259]. Furthermore, a recent study in mildly asthmatic patients [260] indicated that PAF-induced systemic and pulmonary effects were mediated by release of leukotrienes.

9.5.5 Intolerance to aspirin and other NSAIDs Intolerance reactions to aspirin were described only a few years after the introduction of aspirin as an analgesic almost a century ago. Nevertheless, it was not until the discovery that aspirin inhibited the formation of prostaglandins in 1971 [261] that it was realized that aspirin intolerance might be related to eicosanoids. Szczeklik e f al. [262] showed that the propensity to develop adverse reactions to aspirin and other NSAIDs correlated with the ability of the drugs to inhibit the COX enzymes. There is no evidence that immunological mechanisms contribute to the intolerance. However, a few NSAIDs, for example pyrazolone derivatives [263], have sensitizing properties which may cause IgE-mediated type I hypersensitivity. In these cases, there is no crossreactivity with other, structurally unrelated NSAIDs and these cases should be regarded as allergen induced and not be confused with NSAID intolerance. The only hypothesis put forward to explain NSAID intolerance that rests firmly on scientific ground is the COX theory [262]. Subjects with asthma and aspirin intolerance may develop severe bronchoconstriction upon accidental ingestion of an NSAID. The reaction develops largely (although not identical to) as the early airway response following allergen provocation. However, in contrast to the allergen-induced reactions, there is no evidence that the NSAID intolerance is associated with dual responses. For example, bronchoprovocations with inhaled lysine-aspirin produce bronchoconstriction in aspirin-intolerant asthmatics without systemic symptoms and late reactions [26&266]. Challenge with aspirin in aspirin-intolerant asthmatics is associated with increased urinary excretion of LTE4 [190,192,267,268] and the PGD2 metabolite 9 a , l lP-PGF2 [192], suggesting activation of pulmonary mast cells. Likewise, nasal provocation with aspirin in aspirin-intolerant subjects results in release of additional mast cell markers such as histamine and tryptase [269]. Interestingly, some subjects with systemic mastocytosis also display NSAID intolerance [270]. However, it is not yet understood how the COX inhibition results in the activation of mast cells in intolerant subjects. For example, it has not been possible to obtain evidence that inflammatory cells isolated from aspirin-

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intolerant asthmatics respond abnormally to NSAIDs ex vivo [2711. Mechanistically, it has often been assumed that the cyclooxygenase inhibition shunts excess arachidonic acid into the 5-lipoxygenase pathway, but this hypothesis is, for several reasons, very unlikely. For example, the pools of arachidonic acid used for synthesis of prostanoids and leukotrienes are different and located at different membranes in the cell [ 153,2721. In addition, synthesis of leukotrienes is not initiated by arachidonic acid alone, but additional stimulation is required [22,273,274]. One particular caveat with the shunting hypothesis is that there is no experimental proof that it occurs for endogenous arachidonic acid. The hypothesis that appears most likely to explain the mast cell activation and consequent intolerance reaction in NSAID-intolerant subjects postulates that these subjects, for unknown reasons, are particularly dependent upon bronchoprotective PGEz as an endogenous inhibitor of their mast cells. The removal of PGE2 synthesis after COX inhibition should, according to this hypothesis, result in mast cell activation. Leaving the speculations about how the intolerance reaction is initiated, it is well documented that anti-leukotrienes provide a high degree of protection against bronchoconstriction induced by bronchial [266] or oral [275-2771 challenge with aspirin or other NSAIDs [278]. The increased urinary excretion of LTE4 following aspirin challenge [266] provides further support for the role of cysteinyl leukotrienes in aspirin-induced airway obstruction. There are also indications that cysteinyl leukotrienes may be important mediators of baseline bronchomotor tone in aspirin-intolerant asthmatics. Firstly, several independent observations have documented that aspirin-intolerant asthmatics have an overproduction of cysteinyl leukotrienes, expressed as an increased basal (i.e. prior to aspirin challenge) excretion of urinary LTE4 compared with other asthmatics [190,267,279]. It should be noted that the basal levels of urinary LTE4 generally overlap considerably between asthmatics and non-asthmatics, whereas aspirinsensitive asthmatics have basal values of urinary LTE4 that are significantly (two to five times) higher than those of other asthmatics. Furthermore, it was recently observed that there was a marked overexpression of the LTC4 synthase in bronchial biopsies of aspirin-intolerant asthmatics [280]. In fact, there may be a genetic polymorphism contributing to the development of aspirin-induced asthma [28 11. The source of the basal levels of urinary LTE4 may not be the same as for the increased excretion observed after aspirin challenge, because the levels of the mast cell marker 9a, 11p-PGF2 were not elevated in aspirin-intolerant asthmatics that had increased basal levels of urinary LTE4 [192]. On the basis of the well-established association between eosinophils and aspirin-intolerant asthma [282] and the indications that circulating eosinophils have enhanced capacity to generate leukotrienes (vide supra), the findings together would seem to fit with the hypothesis that the eosinophils are one major source of the baseline production of leukotrienes. This adds to the clinical indications that aspirin-induced bronchoconstriction and the persistent airflow obstruction may be different and mechanistically unrelated components in the syndrome. There are also reports that aspirin-intolerant asthmatics are particularly sensitive to inhalation of cysteinyl leukotrienes [283] and in particular LTE4 [284]. Therefore, it may be that aspirin-intolerant asthmatics both display a basal overproduction of leukotrienes and an increased bronchial responsiveness to these compounds.

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9.6 Treatment of asthma with anti-leukotrienes The suggestions from bronchoprovocation studies that cysteinyl leukotrienes mediate essential components of asthma have indeed been corroborated when the effects of anti-leukotrienes have been assessed in the treatment of asthma. The CysLT,antagonists montelukast, pranlukast and zafirlukast are registered for treatment of asthma in Europe, the US and many countries around the world, whereas the 5lipoxygenase inhibitor zileuton is approved for use in the US alone. The effects which have been consistently observed in treatment trials are summarized in Table 9-1. It is beyond the scope of this chapter to dwell on the clinical use of anti-leukotrienes, suffice to say that anti-leukotrienes may be used alone as first-line therapy or as an adjunct to other asthma medications to obtain improved control of asthma. Table 9-1. Effects of anti-leukotrienes ~~~

Acute bronchodilation Prevention of induced bronchoconstriction Chronic improvement in pulmonary function Reduced use of bronchodilators Fewer asthma symptoms (day and night) Fewer asthma exacerbations Improved quality of life

When anti-leukotrienes are administered to asthmatics with baseline asthma symptoms and compromised baseline pulmonary function, an acute bronchodilator response has consistently been observed [285-2871. The bronchodilation produced by a CysLT, antagonist has also been found to be additive to that produced by shortacting B2 agonists [285,286]. With repeated administration of anti-leukotrienesto different groups of asthmatics, chronic improvements in pulmonary function have been demonstrated as well, expressed both as increased morning and evening peak expiratory flow rates (PEFR), and as increased FEV, recorded at the clinic visits [288-2951. In some studies the long-term improvement has been greater than the acute bronchodilator response to the first tablet of the drug, suggesting a time-dependent improvement of certain components of the chronic airway inflammation. The improvement of pulmonary function has generally been associated with favorable changes in other asthma outcome measures. Thus, asthma symptoms have diminished both during the day and at night and the rescue use of bronchodilators has been reduced. Some recent studies have also shown improvements in asthma-specific quality of life scores. It is particularly encouraging that several studies have documented a reduced number of asthma exacerbations during treatment with anti-leukotrienes. Of considerable practical importance with respect to the clinical use of this new class of drugs are the findings that addition of anti-leukotrienesto patients maintained on conventional asthma therapy with moderate to high doses of inhaled glucocorticoids has resulted in significant further improvements of pulmonary function and increased asthma control [296]. Preliminary evidence suggests that this is associated

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with a reduction in the number of circulating eosinophils and decreased levels of surrogate markers of inflammation, such as serum eosinophil cationic protein (ECP) and exhaled nitric oxide (NO) [295]. The findings are in line with the demonstrated inability of glucocorticoids to inhibit leukotriene formation in vivo [297-3001 and further support the idea that cysteinyl leukotrienes mediate components of the airway inflammation which are not blocked by glucocorticoids. In addition to the effects of anti-leukotrienes on the lower airways, there is also evidence that cysteinyl leukotrienes are involved in some if not all manifestations of rhinitis. Accordingly, both when allergen has been applied topically or when subjects have been exposed to naturally occumng allergens, the nasal reactions have been attenuated by anti-leukotrienes [301-3031, Interestingly enough, in one study of aspirin-intolerant asthmatics, evidence was also provided that anti-leukotrienescaused a return of smell in subjects otherwise suffering from chronic loss of smell [296]. In studies of asthmatics there have generally been very similar effects of 5lipoxygenase inhibitors and selective CysLT, receptor antagonists on evoked or persistent airway obstruction, whereas, at least in bronchoprovocation studies, selective antagonists of LTB4 have been ineffective [122]. This adds support to the current concept that cysteinyl leukotrienes rather than LTB4 are the most important leukotrienes in asthma. As anti-leukotrienes have only been introduced as a new treatment for asthma very recently, it is not possible to provide definite statements on where anti-leukotrienes are the treatment of choice. However, since the anti-leukotrienes appear to have a very favorable side-effect profile and are administered orally once or twice daily, it is likely that this new class of drugs will create new opportunities to treat inflammatory and allergic diseases in the lower and upper respiratory tract.

9.7 COX products as modulators of asthma and allergic inflammation 9.7.1 Effects of prostaglandins and thromboxane All primary COX products may produce bronchoconstrictor responses when inhaled. There are however few recent investigationsthat have compared airway responsiveness to different prostanoids in large groups of subjects tested with the same methodology. Inhalation of PGI2 [304] and PGE2 [305] may sometimes also cause bronchoconstriction, but the mechanisms have not been established. For PGEz, the interpretation of such observations is complicated, since inhalation of this compound can trigger transient cough [306]. However, the normal response to PGEz appears to be bronchodilation [307]. Prostaglandins have the potential to both promote and counteract inflammatory processes in the body. This dual mode of action seems particularly expressed in acute allergic inflammation, where NSAIDs have been reported to either suppress [308-

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3101, enhance [310-3 131 or lack [314-3171 effect on immunologically induced edema formation in different models. In a model of immediate type allergic inflammation in the hamster cheek pouch, inhibition of COX by indomethacin reduced the vasodilation, but nevertheless caused a marked potentiation of the antigen response [3 181. Furthermore, indomethacin as well as diclofenac enhanced histamine release evoked by mast cell activation [3 18,3191. Exogenously added PGE2 reversed the enhanced histamine release [318,3191. There were also indications that PGE2 modulates the release of the chemotactic LTB4 [23,318,320]. These in vivo findings further support the hypothesis that there are anti-inflammatory prostaglandins and that inhibition of prostaglandin biosynthesis may enhance the release of endogenous mediators, thereby potentiating allergic inflammation. The anti-inflammatory effects of systemic treatment with vasodilating prostaglandins have been described in several settings [321-3251 and may be related to inhibition of mediator release. It has not been possible to study the effects of TXA2 directly due to its short halflife. Stable thromboxane mimetics such as U-46619 have been valuable in characterizing the effects of TP receptor activation qualitatively. However, the potency and duration of action of such slowly metabolized analogs may not reflect those of the endogenous agonist TXA2. The rank order of potency observed for the constrictive effects of prostanoids is U-46619 >> PGD2 > PGF2,. Since TP receptor antagonists block not only the effects of thromboxane mimetics but also inhibit bronchoconstriction induced by inhalation of PGD2 or PGF2, [326], it appears that prostanoid-induced bronchoconstriction in man is principally although not exclusively [327] mediated by activation of TP receptors. There are also indications that some prostanoids, in addition to direct effects on the airway smooth muscle, also affect airway neurotransmission [328]. It was originally reported that asthmatics were hyper-responsive to inhalation of PGF2, [329,330]. With the exception of aspirin-intolerant asthmatics, who may be hyporesponsive to PGF2, [3311, the responsiveness to inhaled PGF2, appears to correlate with methacholine responsiveness [332]. Furthermore, experimental data from the pig suggest that PGD2 is involved in vascular reactions to allergen in the bronchial circulation [333]. However, the effects of prostanoids on different effectors in human airways are not conclusively established.

9.7.2 Release of COX products Most COX products have been reported to be elevated following allergen challenge, but the data are sometimes difficult to evaluate. One issue concerns measurements of plasma levels, where artifactual formation is known to give misleading results. Furthermore, some prostanoids, such as PGF2, and PGE2, may be produced locally in response to the agent that primarily induced the contraction, e.g. histamine or leukotrienes [334]. Nevertheless, it is established that increased formation of TXA2 occurs in vivo after bronchoprovocation with allergen. This has been indicated by measurements of plasma TXB2 [335] and established in studies of the urinary excretion of 1 1 -dehydro-

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TXB2 [ 190,3361. Since histamine-induced bronchoconstriction has no effect on urinary excretion of 11-dehydro-TXB2 [ 1901, the release of TXA2 presumably occurs as a relatively early event following contact with the allergen. However, it is not known whether cells in the lung or an extrapulmonary source such as the circulating platelet is involved. In man, synthesis of PGD2 is almost exclusively confined to the mast cell [26]. This presumably explains why increased levels of PGD2 have been demonstrated in the airways following allergen challenge [337]. Interestingly, the urinary excretion of the PGD2 metabolite 9a, 11P-PGF2 has recently been found to be elevated after bronchoprovocation with allergen [ 1921. Measurement of this metabolite in urine provides a sensitive, non-invasive marker of mast cell activation in vivo during both the early and late asthmatic responses [ 1891.

9.7.3 Influence of inhibition of COX products on allergen-induced bronchoconstriction There is thus good evidence that TP receptor activation produces bronchoconstriction in asthmatics and that the two most potent endogenous TP agonists, TXAz and PGDz, are formed in increased amounts during allergen-induced bronchoconstriction. The first pharmacological studies indeed seemed to support the idea that COX products were significant mediators of the response to allergen. For example, pre-treatment with indomethacin was reported to inhibit the late response in 10 out of 11 subjects studied [338], without having a major effect on the early response. More recent studies [336,339], however, have not confirmed these observations on either allergeninduced early or late responses. These results rather suggest that COX products are unimportant mediators in causing these asthmatic responses. Even more compelling evidence against a role for TP receptors or thromboxane in allergen-induced airway obstruction has been produced in studies using either a potent TP antagonist [340] or a thromboxane synthesis inhibitor [341]. It is therefore concluded that neither TXA2 nor other known COX products are likely to contribute to more than a very limited and subordinate component of allergen-induced bronchoconstriction in asthmatics. The COX products may however mediate completely different events. The possibility that COX products are involved in airway hyper-responsivenesswill be discussed later in the next section.

9.7.4 COX products in airway hyper-responsiveness Evidence has been obtained in both animal models of airway hyper-responsiveness [339,342,343] and in human subjects with asthma that COX products are involved in the airway hyper-responsiveness that develops after inhalation of stimuli such as allergens. There is, however, little convincing evidence that COX products are important in causing the ongoing, persisting airway hyper-responsiveness that is charactcristic of asthma. It seems as if TP receptor activation has the potential to enhance airway responses

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to other agents both through direct spasmogenic effects and through local modulation of neurotransmission. On the other hand, it is difficult to know which endogenous compound is the most important activator of this receptor in vivo. Most often, TXA2 has been implicated in the pathogenesis of airway hyper-responsiveness in asthmatic subjects [232]. This assumption is supported by TXA2 being the most potent endogenous TP agonist and by experimental findings in models of airway hyperresponsiveness in dogs [ 109,344-3461 and primates [347]. There are also implications for the involvement of TXA2 in the late cutaneous response to intradermal allergen [348] or the late asthmatic response after inhaled allergen in humans [335]. A recent controlled study, however, showed no effect of the potent thromboxane synthetase inhibitor CGS 13080 on airway hyper-responsiveness to inhaled histamine measured at 24 h post-allergen [339].

9.7.5 Anti-inflammatory and anti-asthmatic effects of PGE2 Studies using exercise challenge have provided further insight into the potential role of some COX products in the phenomenon called refractoriness, i.e. the diminished bronchoconstrictor response that occurs when the challenge is repeated within a few hours. Accordingly, pre-treatment with indomethacin [349,350] or another NSAID such as flurbiprofen 1791 abolished the exercise refractoriness. Repeated bronchial challenge with LTD4 is also associated with tachyphylaxis [ 1791. Moreover, flurbiprofen attenuated both the tachyphylaxis to repeated challenge with LTD4 and the crosstachyphylaxis that was observed between exercise and LTD4 [ 1791. Interestingly, histamine, which also mediates a component of the exercise-induced bronchoconstriction, will also show a diminished bronchoconstrictor response if the provocation is repeated within 6 h [351]. It is hypothesized that the findings during exercise challenge are indicative of a general scheme. Namely, that cysteinyl leukotrienes are mediators of bronchoconstriction whereas COX products are modulators of different airway functions. In humans, it seems that the latter function most often has a bronchoprotective role which opposes the effects of leukotrienes and other bronchoconstrictors. It appears very likely that E-prostaglandins are the mediators of the tachyphylaxis to repeated challenge with exercise, LTD4 or histamine, but it remains to be demonstrated that these responses are associated with in vivo release of PGE2 in humans. In further support of the hypothesis that E-prostaglandins mediate the tachyphylaxis, pre-treatment of asthmatics with PGEl, in doses which do not cause bronchodilation, reduced airway responsiveness to both histamine and methacholine [352]. Furthermore, inhaled PGE2 also protects from allergen-induced bronchoconstriction [306] and exercise-induced bronchoconstriction [353]. Inhalation of PGE2 has also been shown to inhibit aspirin-induced bronchoconstriction [354,355]. The indications that endogenous PGE2 mediates the tachyphylaxis to repeated exercise challenge was supported by the effects of treatment with COX inhibitors. The reduction in bronchial hyper-responsiveness to histamine and methacholine that was observed following administration of oral PGEl [352] could theoretically in part be due to functional antagonism at the level of the airway smooth muscle. In some in vitro systems, PGE2 may cause heterologous desensitization of receptors [356], How-

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ever, there was no significant bronchodilation at the time when PGE2 inhibited the effects of the directly acting bronchoconstrictors [352]. In addition, the finding that inhalation of a non-bronchodilating dose of PGE2 also produced almost complete inhibition of exercise-induced bronchoconstriction [353] seems difficult to explain in terms of effects on the airway smooth muscle. For example, a subthreshold dose of a bronchodilating p2 agonist would not produce the same result. Moreover, the bronchoprotective effects of PGE2 are not limited to exercise challenge. Thus, inhalation of a low dose of PGE2 also caused substantial inhibition of both the early and late phases of allergen-induced airway obstruction, as well as an abolition of the postallergen challenge reduction in bronchial hyper-responsiveness [306]. Since it is known that PGE2 is quickly metabolized into inactive compounds in vivo, the findings also suggest that a brief activation of the particular EP receptors involved, produced a long-lived inhibition of some effector cells. The findings with PGE2 in bronchoprovocation studies may be explained by the well-known ability of PGE2 to inhibit the activation of inflammatory cells [66]. The effect is, at least in vitro, due to EP2 receptor-mediated elevations of intracellularCAMP (see Section 1.6.2), which leads to inhibition of release of inflammatory mediators such as histamine and leukotrienes [318], as well as reduced secretion of oxygen radicals [75]. Although further work is required to prove whether this mechanism explains all or some of the effects of inhaled PGE2 in asthmatics, it remains the most attractive hypothesis [56]for unifying the current observations. It is documented from experimental in vivo studies that endogenous PGE2 is indeed a potent inhibitor of allergen-induced mediator secretion and effector responses such as plasma exudation and leukocyte traffic [3 181. It is concluded that one major effect of PGE2 on the airways includes inhibition of stimulation-evoked responses such as mediator secretion from epithelium and inflammatory cells. In addition to its effects on inflammatory cells and its ability to cause relaxation of airway smooth muscle, PGE2 may also inhibit the release of acetylcholine from airway cholinergic nerves [357]. This effect is thought to occur through stimulation of presynaptic receptors and would obviously add another beneficial anti-asthmatic action. Furthermore, the protective effects of PGE2 may not be limited to the airways, because the allergen-induced late cutaneous reaction in atopics was inhibited by the stable PGE2 analog misoprostol [358] and PGE2 also reduced ischemic reperfusion injury in the heart in one model [359]. Therefore, it appears that the potential of PGE2 as a tool for pharmacological intervention with asthmatic and inflammatory responses deserves more attention. At the moment, the initial cough response to PGE2 is the main limiting factor for its use in asthma, but there should be several ways to diminish or eliminate this side-effect, for example by developing subclass-specific EP receptor antagonists.

9.8 Conclusions The introduction of anti-leukotrienes for the treatment of asthma appears to herald a shift in paradigm, where asthma may be treated with medications that specifically

9.9 Referencess

259

inhibit a final common path in cascades of different events contributing to airflow obstruction and bronchial hyper-responsiveness (Fig. 9-5). This strategy for treatment is similar to the specific pharmacological therapy used in many other complex disorders, e.g. mental illness or cardiovascular disease. Similar to development in these areas, it is likely that it will take quite some time before clinical experience has resolved how to use these new drugs, singularly and in combination with existing therapy. There are also wide gaps in our understanding of basic mechanisms relating to how leukotrienes and their receptors are regulated. With respect to prostanoids, their role in asthma and inflammation are less clearly defined. This may in part relate to the preoccupation of research with the hypothesis that COX products mediate asthma. Although it remains a distinct possibility that TXA2 or PGD2 contribute to the development of bronchial hyper-responsiveness, it may prove to be more rewarding to pursue the hypothesis that PGE;! and related compounds in fact represent an endogenous system with the function of protecting against asthmatic reactions and diminishing inflammation. Finally, further research on the less-established members of the eicosanoid family, for example lipoxins, other lipoxygenase products, monooxygenase metabolites, isoprostanes and isoleukotrienes [54],is bound to lead to new understanding that may create novel strategies for treating and explaining different facets of asthma and other inflammatory diseases. As indicated in this chapter, although the contours of the eicosanoid system are getting sharper, newly discovered metabolites and pathways will presumably lead to further precision of the role of different eicosanoids in health and disease.

9.9 References R. Beasley, U. Keil, E. Von Mutius and N. Pearce. Lancet 1998; 351: 1225-1232. T.J. Williams. Br. Med. Bull. 1983; 39(3): 239-242. A. Thureson-Klein, P. Hedqvist, A. OhlCn, J. Raud and L. Lindbom. Pathol. Immunopathol. Res. 1987; 6: 190-206. G. Majno and G.E. Palade. J. Biophys. Biochem. Cytol. 1961; 11: 571406. D. Hultstrom and E. Svensjo. J. Pathol. 1979; 129: 125-133. G. Majno, S.M. Shea and M. Leventhal. J. Cell. Biol. 1969; 42(3): 647-672. I. Joris, G. Majno, E.J. Corey and R.A. Lewis. Am. J. Pathol. 1987; 126(1): 19-24. A. Thureson-Klein, P. Hedqvist and L. Lindbom. Tissue Cell 1986; 18: 1-12. A. Froese. Prog. Allergy 1984; 34: 142-1 87. D.D. Metcalfe, M. Kaliner and M.A. Donlon. CRC Crit. Rev. Immunol. 1981; 3: 2374. S.J. Galli, A.M. Dvorak and H.F. Dvorak. Prog. Allergy 1984; 34: 1-141. T. Ishizaka and K. Ishizaka. Prog. Allergy 1984; 34: 188-235. E.L. Becker and K.F. Austen. In Textbook of Immunopathology, 2nd edn (P.A. Miescher and H.J. Miiller-Eberhard, eds), pp. 117-135. Grune & Stratton, New York, 1976. U. Von Euler. J. Physiol. (Lond.) 1936; 88: 213-234. C.H. Kellaway and E.R. Trethewie. J. Exp. Physiol. 1940; 30: 121-145.

260

1371 1381

[411 1421

9 The role of eicosunoids in inj-lummation and allergy E.J. Corey, H. Niwa, J.R. Falck, C. Mioskowski, Y. Arai and A. Marfat. Adv. Prostaglandin Thromboxane Leukotriene Res. 1980; 6: 19-25. W.E. Brocklehurst. J. Physiol. 1960; 15I : 4 16-435. R.P. Orange and K.F. Austen. Adv. Immunol. 1969; 10: 105-144. H.R. Morris, G.W. Taylor, P.J. Piper and J.R. Tippins. Nature 1980; 285(5760): 104106. R.A. Lewis, K.F. Austen, J.M. Drazen, D.A. Clark, A. Marfat and E.J. Corey. Proc. Natl Acad. Sci USA 1980; 77(6): 37 10-37 14. L. Sautebin, T. Vigano, E. Grassi, M.T. Crivellari, G. Galli, F. Berti, M. Mezzetti and G. Folco. J. Pharmacol. Exp. Ther. 1985; 234(1): 217-221. M. Kumlin and S.E. DahlCn. Biochim. Biophys. Acta 1990; 1044(2): 201-210. P. Hedqvist, J. Raud and S.E. Dahltn. Adv. Prostaglandin Thromboxane Leukotriene Res. 1990; 20: 153-160. P. Hedqvist, J. Raud, U. Palmertz, M. Kumlin and S.E. Dahlen. Adv. Prostaglandin Thromboxane Leukotriene Res. 1991;537-543. K. Strandberg, A.A. Math6 and S.S. Yen. Int. Arch. Allergy Appl. Immunol. 1977; 53(6): 520-529. R.A. Lewis, N.A. Soter, P.T. Diamond, K.F. Austen, J.A. Oates and L.J. Roberts II. J. Immunol. 1982; 129(4): 1627-1631. I.D. Lawrence, J.A. Warner, V.L. Cohan, W.C. Hubbard, A. Kagey-Sobotka and L.M. Lichtenstein. J. Immunol. 1987; 139(9): 3062-3069. M. Hamberg, J. Svensson, P. Hedqvist, K. Strandberg and B. Samuelsson. In Adv Prostaglandin Thromboxane Res (B. Samuelsson and R. Paoletti, eds), pp. 495-501. Raven Press, New York, 1976. G. Folco, G. Hansson and E. Granstrom. Biochem. Pharmacol. 1981; 30(17): 24932495. P. Sirois, S. Roy, J.P. Tetrault, P. Borgeat, S. Picard and E.J. Corey. Prostaglandins Med. 1981; 7(4): 327-340. P.J. Piper and M.N. Samhoun. Br. J. Pharmacol. 1982; 77(2): 267-275. S.E. Dahltn. Acta Physiol. Scand. 1983; 118(4): 415-421. S. Mong, H.L. Wu, M.A. Clark, J.G. Gleason and S.T. Crooke. J. Pharmacol. Exp. Ther. 1986; 239(1): 63-70. M. Hamberg, J. Svensson and B. Samuelsson. Proc. Natl Acad. Sci. USA 1975; 72(8): 2994-2998. J. MacDermot, C.R. Kelsey, K.A. Waddell, R. Richmond, R.K. Knight, P.J. Cole, C.T. Dollery, D.N. Landon and LA. Blair. Prostaglandins 1984; 27(2): 163-179. I.M. Goldstein, C.L. Malmsten, H. Kindahl, H.B. Kaplan, 0. Ridmark, B. Samuelsson and G. Weissmann. J. Exp. Med. 1978; 148(3): 787-792. G.G. Neri Serneri, R. Abbate, G.F. Gemini, A. Panetta, G.C. Casolo and M. Carini. Prostaglandins 1983; 25(6): 753-766. A. Griesmacher, G. Weigel, W. Schreiner and M.M. Muller. Thromb. Res. 1989; 56(5): 61 1-623. S.E. Wenzel, J.Y. Westcott and G.L. Larsen. J. Allergy Clin. Immunol. 1991; 87(2): 540-548. G.C. Kane, M. Pollice, C.J. K i l t ) . J . Cohn, R.T. Dworski, J.J. Murray, J.R. Sheller, J.E. Fish and S.P. Peters. J. All~,i;y Clin. Immunol. 1996; 97(2): 646-654. A. Miadonna, A. Tedeschi, E. I D2> Fza> U-46619 U-51093 E, U-44069

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Table 10-2. Defects in prostanoid metabolism related to vascular disease Factor or process affected PGI2 metabolism

Nature of defect Phenotype observed Accelerated Myocardial infarction, recurrent venous thrombosis and thrombophilia Diminished Bleeding tendency

Platelet receptor for PGI2

Inactive

TX A>-synthetase

Inactive

Platelet and vascular cyclooxygenase

Inactive

Platelet 12-lipoxygenase

Inactive

Early onset of severe atherosclerosis, some patients asymptomatic Increased bleeding tendency; myeloproliferative syndrome and thrombocytosis in one patient Myocardial infarction, peripheral vascular disease; some patients asymptomatic Myocardial infarction; myeloproliferative syndrome in one patient

Heredity Familial or non-familial, acquired and inborn Non-familial, inborn or acquired Familial, inborn

Familial or non-familial, acquired and inborn

Familial or non-familial, inborn Non-familial, acquired

10.2 Polyunsaturated fatty acids, vascular system and atherosclerosis There is a distinctive inverse correlation between the dietary intake of polyunsaturated fatty acids (PUFA) i.e. the precursors of eicosanoids, and the frequency of atherosclerotic diseases. Sinclair [ 131 first discovered the difference in morbidity of atherosclerosis and myocardial infarction between populations not-consuming or consuming coconut oil which is rich in saturated fatty acids. Among the consuming population atherosclerosis was common. Essential polyunsaturated fatty acids to be taken up with a vegetable diet are linoleic and a-linolenic acid. These are the precursors of the n-3 (or 0 - 3 ) [ 141 and n-6 (or 0 - 6 ) polyunsaturated fatty acids (see Chapter 1 , Fig. 1) which are found in phospholipids, cholesterol esters and triglycerides. Only small amounts are present as free fatty acids. As major components of cellular membranes PUFAs modulate the function of receptors [ 151, ion channels and membrane-bound second messenger systems. Once liberated from cell membranes by phospholipases C ~ PUFAs O are processed to eicosanoids. PUFAs, as well as the corresponding diacylglycerols, also serve as interand intracellular signals (see Chapter 1). In the vascular system PUFAs are components of impermeable membranes like the blood-brain barrier. Normal food contains variable amounts of PUFAs (Table 10-3). The rate-limiting step in PUFA biosynthesis is A-6 desaturation of a-linolenic acid and linoleic acid. Inhibition of A-6 desaturation was observed to correlate with high plasma cholesterol level [ 161, high-intake of saturated fatty acids, aging [ 171, male gender [18], diabetes [19], smoking [20], very high alcohol intake [21] and coronary artery disease. On the other hand, intake of PUFAs may reduce total cholesterol level.

10.2 Polyusaturated fatty ucids, vascular system and atherosclerosis

277

Table 10-3. Content of predominant PUFA in food Food Meat, eggs and dairy products

Oats Breast milk Seafood Primrose oil Tofu, soybeans, canola oil and nuts

Predominant PUFA types y-Linolenic acid ( 1 8:3 n-6; GLA), dihomo-ylinolenic acid (20:3 n-6; DGLA) and arachidonic acid GLA Arachidonic acid, GLA and DGLA Docosahexaenoic acid (22:6 n-3) GLA a-Linolenic acid

triglycerides, blood pressure, platelet aggregation, inhibition of smooth muscle cell proliferation, etc. Diagnostic andor therapeutic studies indicate that the levels of alinolenic acid- and linoleic acid-derived PUFA are in fact responsible for the most desirable actions of essential fatty acids [22]. The addition of co-3 PUFA, as found in fish oil, to the diet lowers the triglyceride level, an effect that is particularly pronounced in marked hypertriglyceridemia [23]. If an increased intake of PUFA is accompanied by a reduction in saturated fat intake, low density lipoprotein (LDL) reduction occurs. While fish oil is ineffective in the treatment of hypercholesterolemia, it can be quite useful in the treatment of those severely hypertriglyceridemic (triglyceride >lo00 mg/dl) patients for whom attempts to correct secondary causes sufficiently (through diet, exercise and fibrate) failed [24]. In patients with type V hyperlipidemia, the use of fish oil supplements is an important therapeutic option [25]. Putative mechanisms of dietary 0-3 PUFA on lipoprotein metabolism include inhibition of VLDL triglyceride synthesis, decreased apolipoprotein B synthesis, enhancement of VLDL turnover with an increased fractional catabolic rate of VLDL, depression of LDL synthesis and reduction of postprandial lipemia [26]. The reduced platelet aggregation response and prolonged bleeding times seen in Greenland Eskimos consuming a fish oil-rich diet suggest an important mechanism by which 0 - 3 PUFAs could affect coronary heart disease [27]. Studies with pigs fed high cholesterol diets with and without cod liver oil showed less coronary atherosclerosis in the cod liver oil group [28]. The Atherosclerosis Risk In Communities Study compiled data from four US communities ( 1 5 000 participants, both black and white) on six hemostatic factors: fibrinogen, factor VII, factor VIII, von Willebrand factor, protein C and anti-thrombin I11 [29]. Dietary intake of 0-3 PUFA showed inverse correlations with levels of fibrinogen, factor VIII and von Willebrand factor and a positive correlation with protein C (whites only). A reduced incidence of vein graft occlusion was observed in patients after coronary artery bypass grafting who received fish oil [30]. Two large multicenter controlled trials revealed a significant benefit of PUFA intake in the prevention of atherosclerosis development and its complications. In the Diet and Reinfarction Trial (DART), a significant decline (29%) in all cause mortality was seen when individuals were instructed to eat fish oil after a first myocardial infarction [31]. The second widely known trial on diet composition and atherosclerosis (Lyon Trial) proved a striking cardioprotective effect upon high intake of a-linolenic acid in the so-called Mediterranean diet [ 3 2 ] . Current indications concerning PUFA

278

I0 Prostanoids in the cardiovascular system

intake are reviewed in ref. [33]. Other important information concerning intake of PUFAs and health status in view of cardiovascular disorders may be found in Phillipson et al. [341 Connor [35], Dyerberg et al. [36], Weiner et al. 1371, Shahar et al. 1381, Eritsland et al. [391, Burr et al. [40], De Lorgeril et al. [41] and Stone [42].

10.3 Endothelium, vascular wall, platelet aggregation and eicosanoids The above mentioned effects of PUFA can be attributed at least partially to their function as eicosanoid precursors. Eicosanoids indeed play an important role in the regulation of endothelial function. For a long time endothelium was regarded merely as a passive, watertight barrier between the vascular wall and superfusing blood. Furchgott and Zawadzki first demonstrated the role of endothelial cells in the regulation of vascular tone in 1980 [431. Since then physiological processes such as selective permeation, vasomotor control, vascular growth, thrombogenesis-thromboresistance,metabolic and immunological activity were found to be controlled by the endothelium. Moreover, cardiac endothelium-both in endocardia1 and coronary microvessels-is believed to play an important role in the regulation of cardiac function (see below). Endothelial pathology has been implicated in the development of atherosclerosis, hypertension, vasospastic disorders, diabetes, respiratory distress syndrome, inflammation, autoimmune disorders, thrombosis and tumor growth. Since then, endothelial release and endothelial-directed actions of eicosanoids have also been reconsidered. (Extended reviews of endothelial function are found in references [44] and [45]). Modulation of vascular tone by the release of endothelial autacoids, including prostanoids, was shown first in excised arteries and later in arterioles of various organs. Arteriolar tone determines vascular resistance and, thus, perfusion rates of any specific organ. In pathologial situations, for instance in coronary heart disease, impaired myocardial perfusion may result from a critical reduction of the lumen of conduit coronary arteries due to intimal thickening and slowly progressing hyperplasia of the media or acute artery closure due to plaque rupture. Here prostanoids have been suggested to play a crucial role. Thromboxane A2 (TXA2) for example, was shown to enhance the plaque rupture and the following thrombotic occlusion of the vessel. On the other hand, TXA2 is considered to be a strong promoter of smooth muscle cell proliferation and arterial remodelling. At the border between fluid shear stress and adjacent vascular wall, the endothelium acts as a signal transduction interface to generate appropriate vasomotor signals. Responses of endothelial cells to stimulation range from ion channel activation with concomitant electrophysiological changes and metabolic events to specific gene regulatory mechanisms. Hemodynamic forces are associated with changes in phosphorylation levels at focal adhesion sites, upregulation of COX-I , PGIz-synthase [46] and constitutive (c)-NOS activity. In the vascular system eicosanoids are produced under physiological conditions mainly by COX-I. From PGHz generated by this enzyme, PGI2 is synthesized in endothelial cells. In contrast TXA2 is mainly synthesized in platelets and only to a small

10.3 Endothelium, vascular wall, platelet aggregution und eicosunoids

219

extent in the vasculature. TXA2 interaction with the TP receptor results in increase of cytosolic Ca2+concentration and protein kinase C activity through formation of inositol - 1,4,5- trisphosphate (IP3) and diacylglycerol, respectively. The main effects in blood vessels are the activation and aggregation of platelets-together with a decrease of intracellular CAMPconcentration-and a contraction of smooth muscle cells (SMC). Constrictory effects of aggregating platelets can also be ameliorated by the release of other mediators such as 8-epi-PGF2, or serotonin. TXA2 and other platelet-derived vasoconstrictors are involved in critical flow reductions only in case of severe localized atheromatous stenoses. Cycling flow reductions may depend on mechanical plugging through activated and aggregating platelets and subsequent constriction which is dependent on release products. This may be prevented by COX inhibitors such as aspirin (acetylsalicylic acid). It may contribute to the reduction of the reocclusion rate after revascularizationprocedures. That is also a basis for beneficial effects observed after administration of TXA2 synthase and/or receptor inhibitors in peripheral vascular disease (PVD). Previously it was suggested that the balance between the antagonistic effects of PG12 and TXA2 is decisive for regulation of platelet, peripheral, and coronary vasomotor function [47]. This concept was expanded by incorporation of the whole spectrum of vasodilatory (nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF), nitrosothiols, etc.) and vasoconstrictory (endothelin, PGH2, platelet-activating factor (PAF), etc.) mediators. PGI2 is released mainly to the vascular lumen exerting inhibitory actions on platelet adhesion (at high concentrations), aggregation and release of pro-aggregatory and vasoconstrictorcompounds such as 5-hydroxytryptamine (5-HT), TXA2 and ADP. The abluminal release of PGI2 causing relaxation of the arterial smooth muscle cells (SMC) is relatively small. In addition, SMC itself generates relevant amounts of PG12. During SMC activation PGI2 release is enhanced. This inhibits subsequent SMC proliferation as well as extracellular matrix production, Growth factors and cytokines may influence SMC proliferation not only directly but also via cyclooxygenase-2(COX-2) activation. Enhanced PGI2 inhibits further growth factor release. All these effects of PG12 are thought to be mediated by the IP receptor and an increase in intracellular CAMP. It has been suggested that a number of vasoactive drugs exert at least part of their anti-atherosclerotic action by stimulating PGI2 formation and release. These drugs include ATP, bradykinin, histamine, catecholamines, Ca2+antagonists, angiotensinconverting enzyme (ACE) inhibitors, phosphodiesterase inhibitors and nitrovasodilators [48]. ACE inhibitors were shown to act both, in vitro as well as in vivo, thus generating anti-atherogenic effects but also side-effects such as flushing, etc. For Ca2+ blockers, in particular the dihydropyridines, the PGI2 inducing effect is even more pronounced. Synergism of PG12 with endogenous NO actions has been observed. This concerns also NO donors. Mutual regulation of enzyme activity of COX and NO synthase was found. Peroxynitrite-produced by the reaction of 0 2 - radicals with NO-was shown to stimulate COX-1 and COX-2, serving as a substrate for the peroxidase activities of these enzymes [49]. Common inhibitory actions on platelet aggregation of PGI2 and nitrates were shown. Synergism of therapeutic actions of these compounds was observed in peripheral vascular disease [SO]. However, a stimulating

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effect of NO on PGI2-synthesizing enzyme could not yet be demonstrated unequivocally [511. Although the PGI2iTXA2 balance is important in maintaining vascular tone, its contribution to organ perfusion under physiological conditions seems to be of minor importance only. An increase in peripheral resistance and in venous pressure reflecting a decrease in venous compliance, has been reported in response to cyclooxygenase inhibition [52, 531. Higher doses of COX inhibitors augment ventricular pre-load and afterload. Indomethacin, for example, increases myocardial 0 2 consumption together with an increase in coronary resistance [54]. Moreover, COX inhibitors may cause a metabolic shift towards the production of leukotrienes thereby inducing bronchoconstriction (asthma) as a side-effect. Some experimental studies showed that stimulators of PGI2 production might reduce myocardial infarction size in experimental models [55]. Free oxygen radicals inactivate the enzymatic PG12 production-the phenomenon that may be a consequence of reperfusion injury after ischemia. Vascular rings from atherosclerotic vessels exhibit an impaired on-demand PGI2 release in vitro. Moreover, higher concentrations of PGI2 were needed to prevent platelet adhesion at the site of active atherosclerosis. Blood pressure and hemodynamics regulate PGI2 synthesis. Unspecific stimuli and tissue damage due to hyperlipidemia, irradiation, mechanical irritation etc. cause a temporarily enhanced synthesis, providing a physiological defense mechanism followed by a sometimes prolonged period of diminished production. On the other hand, higher production of PGI2 in patients with severe atherosclerosis was reported [56]. This may be due to intercellular interactions [57]. Microparticles of cell membranes shed during platelet activation contain phospholipids and soluble phospholipase A2 (PLA2) activity. Therefore, they may deliver arachidonic acid to the endothelial cells. This stimulus was shown to induce cyclooxygenase-2 gene expression directly, independently of TXA2, PAF or a-adrenergic receptor activators. The production of PG12, thus induced in the endothelium, counteracts the platelet activation, constituting another compensatory mechanism and explaining the above mentioned observations. PGI2 downregulates thrombogenicity of the vascular wall for hours or even days after a single application. Whether this long-term effect occurs via downregulation of tissue factor expression or induction of vascular endothelial growth factor, as hypothesized, remains to be shown. Expression of cell adhesion molecules (selectin P and E) as well as neutrophiVmonocyte rolling and attachment are key events in initiation and promotion of the atherosclerotic plaque. This phenomenon, as well as the interaction of neutrophils and platelets, is largely influenced by eicosanoids. PGI:! was shown to inhibit those interactions dose-dependentlywithout significant cooperation with NO.

10.3.1 Aspirin interaction with COX-1. Role in cardiovascular prevention. ‘Resistance to aspirin’ Vane et al. [58] demonstrated that acetysalicylic acid (ASA, aspirin) inhibits prostaglandin formation. Subsequent studies revealed a similar mode of action for other

10.3 Endothelium, vascular wall, platelet aggregation and eicosanoids

28 1

non-steroidal anti-inflammatory drugs, such as indomethacin. In 1974 it was demonstrated that aspirin induces an acetylation of the active site of COX, i.e. at Ser529and Ser5I6in human COX-1 and COX-2, respectively [59]. Due to this covalent modification COX-1 becomes inactive while the reactivity of COX-2 is altered that 15RHETE instead of PGH2 is formed (see ref. [60] and Chapters 1 and 3). The clinical use of low dose aspirin as an antiplatelet drug in the secondary prevention of atherosclerotic diseases is based on the assumption that the different cellular sources of PG12 and TXA2 contribute to the preventive effect of aspirin in coronary and cerebrovascular events. Platelets once derived from megakaryocytes lose their ability of adaptation by means of gene inductiodprotein synthesis. Then, if COX- 1 constitutively expressed in platelets is irreversibly blocked with aspirin, it remains inactive for the entire platelet lifespan. In contrast, in nucleated cells, such as vascular endothelium, the synthesis of new enzyme protein renders the inhibition of COX-1 reversible. Thus, a relatively selective inhibition of COX-1 in platelets results from the irreversible inactivation of the enzyme and the the relatively short lifetime of aspirin in human circulation (1 5-20 min). It also explains the cumulative nature of enzyme inhibition in anucleated platelets upon low but daily doses of aspirin (50-75 mg). This treatment does not evoke side-effects and does not interfere with constant production of endothelial PGI2. Thus, the effective balance between the counteracting effects of TXA2 and PG12 is shifted towards the actions of PGI2. On the other hand, balance of individual plasma aspirin levels after drug ingestion and platelets’ sensitivity are difficult to assess and show wide variations depending on the respective preparation. Moreover, if other drugs such as nitrates, ACE inhibitors and Ca” channel inhibitors are used in the treatment of cardiovascular diseases it should be considered that ASA administered at doses above 50 mg/day may interfere with their effects. An unwanted effect of chronic aspirin treatment is ‘resistance’. Experiments of Bunting [61], Marcus [62] and Karim and coworkers [63] have shown that in witro platelet-endothelium interaction may result in the transcellular delivery of substrate for eicosanoid production: quick recovery of endothelial COX activity (possible COX-2 involvement) after aspirin administration, as well as close contact between endothelium and activated platelets allowing a transcellular transport of prostaglandin peroxides to platelets may lead to prompt restoration of TXA2 synthesis. A recent study by Cipollone and colleagues [64]suggests that similar effects may also exist in vivo in humans. This aspirin-insensitiveTXA2 generation may contribute to a number of clinical aspirin failures. Further studies involving selective COX-2 are required to verify the postulated role of induced COX formation in endothelial cells, and to develop more efficient methods of non-steroidal anti-inflammatory drugs (NSAID) based secondary prevention.

10.3.2 Eicosanoids and restenosis The problem of restenosis after endovascular interventions should be mentioned considering the role of eicosanoids in vascular pathology and endothelial function. The introduction of percutaneous transluminal coronary angioplasty (PTCA) by

282

10 Prostanoids in the cardiovascular system

Andreas Gruentzig in 1979 opened new perspectives in the treatment of coronary artery disease. Together with peripheral interventions, implantation of intravascular devices and other endovascular techniques, PTCA is gaining wider and wider clinical applicationhndications while becoming a relatively successful and safe method of treatment. Nevertheless, outcome after PTCA is still limited by several factors. One of them is recurrence of atherosclerotic occlusion, i.e. restenosis. The rate of restenosis is generally estimated now at 30-50%. Pathology of restenosis consists of a cascade of events starting during PTCA. Damage to endothelium, adhesion and aggregation of platelets, release of mediators such as TXA2, growth factors and other vasoactive substances are followed by proliferation and migration of smooth muscle cells, deposition of extracellular matrix and vascular remodelling. The latter is the main morphologic compound of restenotic changes. A variety of anti-restenotic strategies were investigated both in animal models and humans. Some of these approaches include anti-platelet agents, anti-thrombotics, anti-coagulants, thrombolytics, antiproliferatives, anti-inflammatory drugs, vasodilators, antioxidants, lipid-lowering regimens, chimeric toxins, anti-sense nucleotides and gene therapy [65]. Other approaches include implantation of various endovascular devices. Although few methods have met undisputed success in preventing restenosis, the general rate of restenosis did not change significantly. Pharmacological approaches to the prevention of restenosis in relation to eicosanoid research include application of aspirin and other NSAIDs (ebselen and sulfinpyrazone), TXA2 receptor antagonists (vaviprost, sultroban and S- 1452), TXA2 synthase inhibitors (ridogrel), PGI2 and analogs (iloprost, ciprostene and beraprost) and fish oils (w-3 fatty acids such as eicosapentaenoic acids), (see Table 10-4). Unfortunately, none of these studies showed significant inhibition of restenosis in humans in spite of significantly positive effects in a variety of animal models. Table 10-4. Selected large-scale trials on eicosanoid-related drugs in the prevention of restenosis (NS = no significance.)

Compound ASAlDipyridamole

TXA2 receptor antagonist TXAZsynthase inhibitors PGIz and analogs

Study ‘Canadian study’ [66] Taylor er nl. [67] Antiplatelet Trialists’ Collaboration [68] Mufson et nl. (ASA 80-1500 mg/day) [69] CARPORT trial (vapiprost) [70]

Patients ( n ) 376 216 ca. 800 495 697

NS

M-HEART I1 trial (sultroban) [71] ridogrel-no large-scale trials available PGIz [72] Ciprostene [73]

Effect NS NS 4%,p = 0.02 NS NS

286 31 1

NS 39 to 23% reduction

814

NS

503

NS NS

(p =0.004)

Fish oil and EMPAR trial [74] eicosapentaenoic acid FORT trial [75] OMACOR [76]

10.4 Function ofeicosunoids in the heart und kidney

283

10.4 Function of eicosanoids in the heart and kidney 10.4.1 Heart muscle activity, contractility, ejection fraction While cardiac myocytes have only a low capacity to synthesize eicosanoids [77], prostanoids are released both by coronary vascular [78] and endocardia1 [79] endothelium, and TXA2 and leukotrienes are released in clinical situations of ischemia and/or reperfusion from platelets and leukocytes, respectively. These prostanoids markedly influence the inotropic status of the myocardium. Direct inotropic actions of prostanoids and their importance for the control of pump function of the heart were discussed in detail by Schror et al. [80]. Here attention shall be directed only to basic aspects of the problem and to some new information regarding arachidonic acid metabolites and myocardial contractility. In the heart, as in the rest of the circulation, prostanoids play an important role in the regulation of vascular tone and platelet activity. During myocardial ischemia prostanoid production in endocardium increases dramatically compared to vascular endothelium [8 11. Inhibition of TXA2 synthesis and treatment with PGI2 were reported to reduce infarct size [82]. PGI2 and analogs were suggested to improve the contractile status of myocardium during recovery from ischemia (myocardial stunning) via poorly defined ‘cytoprotectiveeffects’ [83]. Direct functional, i.e. contractile actions of prostanoids on myocardium are not consistent and clear. There are large differences arising from the source of myocardium investigated (species) and the experimental model. According to one of the hypotheses prostanoids may modulate inotropic actions of other agonists [84]. In vivo an improvement of left ventricular ejection fraction (determined by radionuclide ventriculography) with PGEl has been seen at doses not inducing afterload reduction. This indicates a beneficial effect on myocardial contractility.

10.4.2 Myocardial infarction TXA2 is thought to play a role in plaque instability, coronary vasoconstriction and, thus, in myocardial infarction. Myocardial ischemia is associated with enhanced release of eicosanoids into the coronary effluent. In contrast to non-ischemic conditions, there is a significant overflow of TXA2 as well as fatty acid peroxides and leukotrienes. Release of these substances may have deleterious effects on the course of ischemia, resulting in the facilitation of arrhythmia, tissue necrosis and exacerbation of ischemia probably because of local vasoconstriction and platelet plugging. Cysteinyl leukotrienes were also shown to be potent coronary vasoconstrictors in isolated heart preparations. Eicosanoids in ischemic myocardium are synthesized not only by myocardial cells but also by invading platelets, neutrophils and macrophages [85]. Platelets from survivors of myocardial infarction are abnormally sensitive to aggregating agents and produce more TXA2 than controls [86]. Elevated TXB2 (metabolite of TXA?) levels

284

10 Prostunoids in the cardiovascuhr system

were demonstrated in patients with Prinzmetal’s angina and unstable angina. Urinary 2,3-dinor metabolites of TXA2 are increased and PGI2 metabolites are decreased in coronary artery disease patients. Decreased responsiveness of platelets to PG4 due to receptor downregulation may favour local pro-thrombotic condition.

10.4.3 Kidney, eicosanoids and cardiovascular regulation The kidney is critically involved in blood pressure regulation and represents a key organ of eicosanoid metabolism and excretion and also of significant biosynthesis (see Chapter 7) PGE2 and PGS, are produced in collecting ducts and PGA2 in interstitial cells of the renal medulla. Formation of prostaglandin in the medulla is three- to fourfold higher than in the cortex. On the other hand, prostaglandin metabolizing enzymes are present mainly in the renal cortex. Hence, urinary eicosanoids and metabolites (e.g. PGE2, TXB2 and 6-keto-PGFI,) may result from renal prostaglandin synthesis. Additionally, 13,14-dihydro-15-keto metabolites may be produced in pulmonary circulation and only excreted by the kidney. Intrarenal prostaglandins play an important role in renal protection [87, 881. Renal PLA2 is stimulated by hypotension and ischemia, as well as by noradrenaline, angiotensin I1 and anti-diuretic hormone (ADH). Renal prostaglandin synthesis counteracts adverse effects of the above mentioned factors. Important renal vasodilators are PGD2, PGE2 and PGI2. They oppose vasoconstrictive actions of noradrenaline and angiotensin II, antagonize ADH and block distal tubule sodium reabsorption. They may be particularly important in decreasing the vasoconstrictor activity of angiotensin II on afferent arteriole and glomerular mesangial cells [89] Production of prostaglandin promotes renal vasodilatation, maintains intrarenal hemodynamics and enhances water and sodium excretion. At the same time as they modulate the actions of the renin-angiotensin system they stimulate renin excretion, maintaining balance in the system [90]. Infusion of PGEl prevents ischemia-induced acute renal failure in animals and may play a role in the preservation of the donor kidney in renal transplantation [91]. The renal vasodilatory response to mannitol during hypoperfusion also seems to be mediated through prostaglandin activation. PGI2 increases renal blood flow,hence temporarily increasing renal function. While renal function returns after cessation of therapy to the previous state, significant (micro)albuminuria improvement persists for weeks and even months. The renal protective effects of prostaglandin may be seen in the ischemic kidney, which in contrast to normal kidney is highly sensitive to NSAIDs. ASA inhibits COX, which is resynthesized in the kidney after 24-48 h. During ischemic stress NSAIDs compromise renal function, resulting in decreased renal blood flow and glomerular filtration and increased renal vascular resistance [92]. Diuretic-induced sodium excretion is attenuated and hyperkalemia may occur [93]. Adverse effects of NSAIDs have been demonstrated in animal models of haemorrhage, endotoxemia, increased venous pressure and low cardiac output and in humans with mild renal dysfunction with concomitant congestive heart failure, ascites and systemic lupus erythematosus

WI.

10.5 Isoprostanes-novel indicators of cardiovascular disease

285

TXA2 induces mesangial cell contraction. It decreases glomerular filtration by lowering the effective glomerular surface area and filtration constant ( K J . Renal levels of thromboxane are increased in experimental acute renal failure and sepsis. In animal experiments the administration of specific TXA2 synthase inhibitors prevents the deterioration in renal function induced by injection of endotoxin. During chronic inflammatory processes the kidney is also a source of free radicals originated from activated leukocytes which support the formation of vasoconstrictory isoprostanes, in particular 8-epi-PGF2, (see below). Leukotrienes are also released from endotoxinactivated leukocytes. Like TXA2, leukotriene C4 (LTG) and LTD4 induce mesangial cell contraction and decrease glomerular filtration. In 1967 Bartter [95] described a renal syndrome associated with hyperplasia of the juxtaglomerular apparatus in the renal cortex, hyperkalemic alkalosis, hyperreninemia, hyperaldosteronism and inappropriate secretion of ADH. The urinary excretion of PGE2 and PG4 was considerably elevated. In spite of increased plasma volume, the patients were not hypertensive. There was no elevation of blood pressure after administration of either angiotensin I1 or noradrenaline. Platelet aggregation in these patients was reduced and platelet CAMPelevated. Symptomatic treatment with indomethacin abolishes hyper-reninism, hyperaldosteronism, elevated plasma kinin levels and hyperkaliemia but does not modify the proposed cause of this renal dysfunction, i.e. inhibition of C1- reabsorbtion in distal tubules [96].

10.5 Isoprostanes-novel indicators of cardiovascular disease In 1967 Nutgeren was the first to report on the non-enzymatic (auto-oxidative) formation of prostaglandins, i.e. the generation of PGEl from 8,11,14-eicosatrienoicacid [97]. Using a gas-chromatographic assay of tetranor-prostaglandin metabolites, the same investigator measured almost ten times more of the product than expected [98]. It was supposed that at least part of this ‘extra’ mass could be due to so-called isoprostanes [99]. Although only few data are as yet available, the pathology and clinical significance of isoprostanes were recently reviewed in [ 100, 1011. Isoprostanes are formed in a non-enzymatic way by auto-oxidation of PUFAs (see Chapter 1). The proposed mechanism of isoprostane synthesis involves formation of lipid free radicals from phospholipid-bound arachidonic acid, non-enzymatic cyclization, addition of 0 2 and hydroperoxide formation at C-15, reduction by tissue peroxidases and cleavage from phospholipid by PLA2. In 1989 Morrow et al. [lo21 first isolated F2-type isoprostanes (isomeric to PGF2,) from normal human plasma. As it was suspected, in analogy to spontaneous rearrangement of PGG2 to PGD2 and PGE2, isoprostanes of the D and E series were also found [103]. The formation of other isoeicosanoids (isoleukotrienes) was reported by Harrison [ 1041. Possible formation of isoprostanes from fatty acids other than arachidonic acid should not be excluded either. The golden standard for isoprostane measurement is gas chromatography-mas/ spectrometry (GC-MS) [1051. Radio immuno assay/enzyme immuno assay ( R I M I A ) techniques were also

286

10 Prostanoids in the cardiovascular system

developed, but they suffer from antibody cross-reactivity, which often makes extensive sample preparation necessary. Normal plasma from healthy volunteers shows presence of 8-epi-PGF2, in a concentration range of 40-100 pg/ml. Normal human urinary levels of 8-epi-PGF2, may range between 50 and 100 ng/mmol creatinine. Isoprostanes was found to be very potent contractile agents. 8-epi-PGF2,, the most extensively studied and predominant isoprostane in mammals, has been shown so far to be a potent SMC constrictor in different vascular beds [lo61 and a stimulator of human lymphatic contractility [ 1071. 8-Epi-PGF2, was shown to induce SMC proliferation. The action of this isoprostane on SMC is exerted either via the TXA2 receptor [ 1081 or a separate isoprostane receptor [ 1091. This problem is reviewed in reference [41. 8-Epi-PGF2, was demonstrated to induce platelet shape change and intracellular Ca” and inositol phosphate release. Different isoprostanes were checked for their influence on platelet aggregability. The pro-aggregatory properties of e.g. 8-epiPGF2, were shown [110]. Other compounds had only negligible effects. They are probably dependent on increased expression of Gp IIbIIIa complex, but further studies should elucidate the way it might work. An involvement of isoprostanes in various pathologic conditions has been established. According to the nature of their formation they could become useful biomarkers of lipid peroxidation in vivo. Elevated levels were found to correlate with oxidative injury in scleroderma [ 1111, plasma of smokers [112], Se and vitamin E deficiency [113], oxidized LDL [I 141 and coronary reperfusion [115]. In human atherosclerotic tissue, in particular from smokers, their presence is negatively correlated with vascular PG12 production. The formation of F3-isoprostanes correlated well with other indices of lipid peroxidation such as hydroperoxides and thiobarbituric acid reactive substances. These effects are reviewed in ref. [ 1161. Although the contribution of COX to the formation of 8-epi-PGF2, in vivo appears to be negligible under physiological circumstances, the current assumption that plasma and urine levels of this product solely reflect non-enzymatic lipid peroxidation still requires validation in clinical settings of platelet and/or monocyte activation [ 11.

10.6 Prostaglandins as drugs in the therapy of cardiovascular disorders 10.6.1 Mechanisms of action PGI2 and PGEl inhibit platelet activatiodaggregation by increasing intracellular concentrations of CAMP. Both compounds have been used in clinical trials to reduce progression and thrombotic complications of PVD. The anti-platelet action of PGEl was the basic concept for introducing this substance into clinical therapy of atherosclerosis. In the meantime a variety of other mechanisms has been discovered, which might be of importance, especially concerning the long-term benefit seen after treat-

0s

0s

Pulmonary hypertension

Peripheral arterial disease

0s

Chronic administration, pilot study and MC

DB

0s

~

~~

Healing of ulceration and clinical improvement [128, 1291

Improved hemodynamics and reduced mortality in pilot study [ I261

Reduction in frequency, duration and severity of disease lasting up to 6 weeks after 72 h lasting infusion, pain relief and increased skin temperature [ 124, 1251

Relief in advanced ischemic disease: rest pain, healing of refractory ulceration and increase in muscle blood flow-prolonged effects [ 1221; significant increase in absolute and relative walking distance, persisting up to 6 months [ 1231

Sustained lowering of advanced pulmonary hypertension until availability of lungheart transplant [121]

Preservation of platelet number and function[ I 181, reduction of blood loss within first 18 hours after operation [ 1 191 and controlling bypass hypertension [ 1201

Outcome

MC - multicenter; DB - double-blind; PC - placebo controlled, 0 s - open study.

Throtnbangitis obliterans

Severe congestive heart failure refractory to diuretics and digitalis

Raynaud’s phenomenon

DB

Cardiopulmonary bypass

DB PC

Design

Indication

Tahle 10-5. Cinical annlications of PO17 in cardiovascular medicine

MC trial was stopped when more deaths were occurring in PGIz group (complications concerning central vein line and incidence of sepsis) [ 1271

As above

Not understood mode of action and difficulties with proper dose regimen

No prolonged effects and necessity of constant infusion

Associated decrease in perfusion pressure and systemic blood pressure

Disadvantages

Reversal of vasospastic component

Decrease in pulmonary artery pressure and pulmonary vascular resistance

Vasculitis in course of SLE

Pulmonary hypertension

i.v. Intracavernous injection in comparison with papaverin

9 129

1411

i.v. taprostene i.v. i.v.

30

Tissue ischemia during aortic No beneficial effects and even exacerbation due to redistribution 19 surgery of microcirculatory flow

Cardiopulmonary bypass

d-Transposition of the great Improvement of systemic arterial saturation and dilation of arteries and intact ventricular ductus arteriosus septum in infants Aortic arch abnormalities Effective dilation of ductus arteriosus

i.v.

i.v.

3

7

Some beneficial effects on platelet preservation; problems due to 13 marked vasodilatory effect

I401

i.v.

213

Increase in walking distance and improvement of quality of life Increase in pain-free walking time and no change in ankle brachial index and platelet half-life

1421

t I391

i.v. pro-drug incorporated [ 1381 in lipid microspheresAS-0:3

80

Increase in walking distance and improvement of quality of life

PVD

[I371

i.v.

20

[135, 1361

[ 1341

[131, 132, 1331

[ I301

Decrease in pulmonary artery pressure, pulmonary capillary wedge pressure and systemic vascular resistance index, increments in cardiac index and stroke volume index and creatinin clearance

15

i.v.

Method of administration References

Number of patients 3

Severe chronic heart failure cardiogenic pulmonary hypertension

Erectile dysfunction (impotence)

Effect

Disorder

Table 10-6. Various clinical trials on FGE. use in clinical disorders

111 and IV

Healing of ulcers and improvement of clinical symptoms (calf cramps) and increase in transcutaneous oxygen tension

42

30 No significant improvement of rest pain or promotion of ulcer healing Significant clinical improvement, limitation of leg ulceration and 364 improved motor conduction velocity in PGEl group

Reduction in number of amputations, rest pain and use of 57 analgesics and improvement or healing of ulceration; no effect on stage improvement Higher number of healed ulceration 120

SLE. \ystemic lupus erythematosis PVD. peripheral vascular disease

Essential arterial hypertension Modest and transient decrease in mean arterial pressure and systemic vascular resistance and no change in renin activity and catecholamines Decrease in number of major amputations Thrombangitis obliterans Improvement of ulcer

[ 1471

[ 1461

[I451

i.v.

8

F G.

2 R a

5 1581

m

E

i?

s.

2

z

A

E

ro 3

!$E

oc

ss

b

o\

P

‘r

[155, 156, 157,

Lumbar sympathetic [ 1591 block, continuous epidural block and PGEl ointment

Oral misoprostol-stable PGEl analog

[ 1541

l1521

i.v. versus placebo

i.V.

[150, 1511

i.V.

i.v. PGEI incorporated in [ 1481 lipid microspheres versus placebo i.v. alprostadil [I491

i.v.

i.v. versus placebo

i.a. versus ATP

5

Side-effects: cutaneous erythema, desquamation, pain, edema and 24 fluid retention in majority of patients with no significant improvement in terms of occlusive disease onset

Ischemic skin ulcers in course Symptomatic improvement, healing of 35/65 refractory ischemic 12 ulcers, with persisting beneficial effect and well-tolerated of Raynaud phenomenon treatment Hepatic venoocclusive reduction in number of events 57 disease after allogeneic bone marrow transplantation

Venous ulcers

Diabetic neuropathy and leg ulcers

Ischemic ulcers in course of PVD

PVD-stage

290

10 Prostanoids in the curdiovasculur system

ment with PGEI, such as the following:

(1) Stabilization of endothelial lining, reflected by a decreased number of circulating endothelial cells and a decreased thrombogenicity with PGEl cooperating with endogenous EDRF/NO (endothelium-derived relaxing factorhitric oxide). (2) An inhibitory action on SMC such as a decrease in proliferative activity associated with a diminished extracellular matrix formation. (3) An increase in fibrinolytic capacity. (4) Effects on white and red blood cells (stabilizing cell membrane and prolonged viability). (5) Some effects on lipid metabolism by decreasing the arterial wall cholesterol ester content and upregulation of LDL receptors. (6) Moderate lowering of serum cholesterol level, seen in hypercholesterolemics only. It is assumed that the vascular effects in particular, together with the hypolipidemic actions of PGEl at the vascular level, might be of major importance underlying the long-term benefit. Moreover Peskar et al. [ 101 have demonstrated that biologically inactive metabolites of PGEl are finally converted to the active compound 13,14dihydroPGEl (PGEo). This compound was shown to possess long-acting PGE1like action on vessels, inhibiting ADP-induced platelet aggregation as well as the vascular influx of LDL and its accumulation (80-100% bioactivity of parent compound). For PG12 a desensitization mechanism after prolonged continuous administration at the receptor level has been described. Initially known from in vitro studies, this effect was discovered in a patient suffering from peripheral vascular disease and experiencing severe thrombocytosis and platelet activation despite ongoing PGI2 therapy. Intraand post-infusion rebounds have been differentiated. This phenomenon can be avoided by intermittent therapy. As it was recently shown, it also occurs at the vascular wall level. A recent report showed that desensitization to PG12 as well as analogs, such as iloprost is completely reversible [I 171. In the case of PGEl desensitization is far less if detectable at all.

10.6.2 Side-effects PGE I administration sometimes produces local irritation at the infusion site, vasodilatation and headache. Vasodilatory effects are dose dependent and show a large substance-specific individual and interindividual variability. They are generally more pronounced for PGI2 and analogs than for PGE,. Recent reports show rare incidence of prostaglandin-associated gastric mucosal hyperplasia in children upon long-term treatment [160]. In patients with liver disease who were treated with either intravenous (i.v.) or oral PGE I gastrointestinal and/or cardiovascular side-effects were observed and a few of them developed arthritis. High-dose oral PGEl or PGE2 sometimes incurred arthritis and/or gastrointestinal adverse effects. From those treated on a long-term base with PGEl some developed clubbing and cortical hyperostosis resem-

10.6 Prostaglandins as drugs in the therapy of cardiovascular disorders

29 1

bling hypertrophic osteoarthropathy [ 1611. However, all the side-effects together do not exceed 10% of treated subjects.

10.6.3 Route of administration and dosage Due to potential hazards for the patient (e.g. athero/thromboembolism after puncture) and no proven advantage, intraarterial route of administration is no longer used. Instead, intravenous administration is the route of choice. It has been claimed that PGE, is degraded to a major extent during first lung passage. Surprisingly, however, clinical results as well as various platelet function tests and coagulation parameters, revealed no apparent difference after intravenous infusion. In patients suffering from PVD stage 11, according to Fontaine, PGEl was able to decrease, to a comparable extent, vascular platelet deposition after intravenous (and intraarterial) therapy. Similarly, a significant prolongation of platelet survival was noted, again revealing no difference. The decrease in platelet uptake is assumed to be predominantly a vascular and not a drug effect (thromboresistance). Extensive clinical and pharmacological studies showed no dose dependency concerning any of the positive clinical effects studied so far. No controlled and reliable clinical studies are yet available. Usually PGE, is administered in PVD at a rate of about 5 ng/kg/min for 2-6 h daily. PGI2 dosage ranges between 3 and 5 ng/kg/min, while iloprost is usually administered at 0.5-2 ng/kg/min. The side-effects are clearly dose dependent, although within a large variation range, and they limit increase in dose. Although no definite clinical studies are yet available, in PVD the benefit seems to be more pronounced the longer the respective prostaglandin is administered (with an optimum of about 3 4 weeks). Continuous infusion of I series prostaglandins (but not E series) has been shown to induce desensitization at the receptor level (described above). This mechanism may start with wide interindividual variations after 72 h and is more pronounced the longer the therapy lasts. Receptor desensitization was observed both in platelets and vesselwall cells.

10.6.4 Gene therapy-experiments with prostaglandins in cardiovascular disorders Xu and colleagues [ 1621 were probably the first to report on the experimental transfer of human the gene into a human endothelial cell line for enhancement of prostacyclin synthesis using a retroviral vector. Subsequent studies in animal models with various vectors provided evidence for beneficial effects of prostaglandin gene therapy, for instance in preservation of graft patency or prevention of neointimal hyperplasia. It was also shown that COX-1 gene transfer using liposomes and adenoviral vectors [ 1631 into injured vascular wall prevents thrombosis.

292

10 Prostanoids in the cardiovascular system

10.7 Methods of clinical prostaglandin research The investigation of eicosanoids in the cardiovascular system is afflicted by serious methodical problems due to the small quantities and the short half-life of these compounds. Measurement of prostaglandins is either direct (bioassay) or indirect via metabolites. Direct measurements of active substances are usually extremely complicated and require non-standard methods. As a consequence, the values reported may vary by orders of magnitude. The determination of prostaglandins in human body fluids is in particular hampered by a number of sampling (venous occlusion and cellular activation) and processing artefacts. As prostaglandins act at the site of synthesis only, they are considered not to be circulating components. To measure the amount released from organs, selective techniques such as microdialysis, catheterization or withdrawal from effluent vessels have been employed. However, these methods produce an artificial increase in the level of prostanoids. Physiologicallevels are extremely low. The endogenous source is difficult to assess and the metabolism may vary. None of the prostaglandin assays gained, therefore, clinical value in diagnosis or treatment monitoring. Instead various stable metabolites are frequently used to evaluate the formation of prostanoids. 6-Keto-PGF1, is the chemically stablc metabolite of prostacyclin (PGIz). Plasma levels in resting, healthy volunteers are 51 pg/ml and the urinary excretion rate is TXB2 is the hy300-500 ng/24 h or 100-300 ng/24 h per gram of creatinine [la]. drolysis product of TXA2. Infusion of TXBz results in the formation of more than 20 metabolites excreted mainly with urine. 2,3-Dinor-TXBz is a major metabolite. Therefore, formation of TXA2may be assessed by the measurement of urinary levels of 2,3dinor-TXB2or TXB2. Plasma or blood levels of TXBz are influenced by blood withdrawal, processing and assay procedure. Measurement requires careful sample hanacid may serve as a dling. Sometimes, the level of 12-keto-hydroxy-heptadecatrienoic reference level of thromboxane synthase activity. A most comprehensive and up-dated overview of analytical methods in eicosanoid research is provided by Lianos [165].

10.7.1 Sample collection and preparation The current analytical methods for various eicosanoids include a separate and extended sample work-up, derivatization and GC-MS or HPLC detection of each compound. The widely employed extraction method of Powell [166] makes use of reversed-phase chromatography with octadecylsilyl-coated silica separating phospholipids, polar substances and proteins from oxygenated metabolites of arachidonic acid. The latter are eluted by acidified mixtures of acetonitrile and methanol [167]. Connecting this column as a pre-column to an analytical HPLC column makes it possible to analyse complex mixtures of eicosanoids [166, 1681. Identification of the metabolites is achieved by comparing the individual retention times with those of reference compounds or by GUMS [ 1691. A single reversed phaseholid-phase extraction step yielded excellent purification at high recovery of 11 -dehydro-TXBz, 2,3dinor-6-keto-PGFI,, PGF2U,PGE2,PGD2 and isoprostanes in urine [ 1701.

10.7 Methods of clinical prostuglundin research

293

10.7.2 Bioassays Almost all active prostaglandins have been discovered by means of bioassays. According to Gaddum [171] a bioassay was the only technique developed by pharmacologists on their own and not borrowed from physiology, biochemistry, pathology or statistics. The general principle of the method is to use isolated biological material and to determine specific effects of certain substances on measurable organ function(s). There is a large number of modifications of different bioassay techniques. Only the few methods that achieved significance for prostanoid research within the cardiovascular system are mentioned here. The so-called impedance platelet aggregation test developed by Born [ 1721 has been used for determination of eicosanoid action in platelets and also as a sensitive indicator of prostanoids in samples. Activity is compared with a synthetic standard. Before receptor binding techniques became available, platelet sensitivity to various anti-aggregatory prostaglandins (PGD2,PGE, and PG12) had been a useful functional indicator. Platelet response to these compounds was decreased with risk factors for atherosclerosis. Aggregatioddisaggregation on collagen strips in extracorporeal circulation [ 1731 was used for determination of prostacyclin action on platelets in vivo. Aggregation as an indicator or quantifiable response to the action of prostanoids was also observed in mesenteric, femoral, pulmonary and gastric vascular beds [174, 175, 1761 as well as in the microcirculation of a hamster cheek pouch [177]. Superfusion, a bioassay technique introduced by Vane [178] is widely used in prostanoid research. It helped to determine (with certain limitations) the release and fate of arachidonic acid metabolites in animals[1791 In the superfusion assay, bathing fluid runs over the external surface of the tissue [180, 1811. Tissue effects such as muscle contraction are thereafter quantified. The blood-bathed organ technique [182] is a dynamic bioassay in which isolated preparations of vascular smooth muscle are superfused with heparinized blood, continuously removed from an anaesthetized animal and returned intravenously. Specificity is usually checked by selective inhibitors (indomethacin and receptor blockers) and different reactivities in different vascular preparations. The superfusion technique is also used to check the ability of different organs to synthesize bioactive compounds. This is the way that PGI2 was discovered, i.e. an extract from an aorta was incubated with PGH2 and dilatory effects of a product were assayed using isolated rabbit coeliac artery. For assessment of prostanoid effects on the heart, the Langendorf preparation of isolated animal heart is used. This technique helped to determine actions on muscular contractility and the composition of effluents from coronary veins.

10.7.3 Physicochemical methods GC-MS and GC tandem mass spectrometry in the negative-ion chemical ionization (NICI) mode, are the most up-to-date and sophisticated analytical methods for the quantification of eicosanoids and isoeicosanoids. An extensive review of these new

294

10 Prostanoids in the cardiovascular system

techniques has been published recently [ 1831. GC-MS assays, utilizing a single deuterated internal standard for the measurement of all prostanoids, are extremely sensitive but may have errors introduced by variable recovery of the myriad analytes through the purification scheme by GC. An assay directed at a single compound may be more reliable due to the ability to use a corresponding isotope-labelled internal standard. Deuterium-labelled standards have different chromatographic retention times and are more stable than '80-labelled standards. Separation of labeled standards and products is performed by GC [169]. HPLC is a widely used and accepted method of eicosanoid analysis [184] An extraction step is usually essential for the quantitative determination of eicosanoids in biological milieu. Details on extraction procedures are given in the sample preparation section. Usually column, particle size and eluting solvents are critical variables to be considered for optimal resolution. The normal (straight) phase support is used for separation or analysis of non-polar metabolites (HETE and arachidonic acid). Reverse-phase C18 silica columns allow separation and analysis of polar peptidoleukotrienes. Radioactively labelled eicosanoids released from cells prelabelled with [3H]- or [''C]-arachidonic acid are reliably identified and quantified by monitoring radioactivity using HPLC coupled to an on-line detector or by thin-layer chromatography and radiodensitometry. Some compounds (HETE) may be directly detected with UV. The use of sensitive diode array detectors allows good confirmation of the identity of compounds by establishing the spectrum specific to the intrinsic UV absorbing moieties. The alternative for those components that do not absorb UV is derivatization (i.e. covalent attachment of chromophores usually at the carboxylic group) with a easily detectable probe such as the para-substituted phenacyl esters. This approach has been used successfully for prostanoids [185, 1861.

10.7.4 Immunochemical methods Attemps have been made to replace the costly and time-consuming biological and physicochernical assay techniques by cheaper standardized methods. Initially, the development of sensitive and reliable immunochemical tests was limited by the availability of specific antibodies with low cross-reactivity [ 1871. Moreover, since the prostaglandins are p e r se not immunogenic, they must be made immunogenic, preferably by a haptedcarrier technique. Presently, immunoassays probably provide the most widely used way to investigate the eicosanoid cascade showing high sensitivity (usually pg/ml range), linearity of standard curve and reproducibility. Using radioimmunoassay (RIA) methods, unlabeled compound or metabolite competes with the same but labeled (iodinated or tritiated) substance for a fixed number of antibody binding sites. As the concentration of unlabeled antigen increases, less of the added radiolabeled antigen will be bound to the antibody. The level of radioactivity bound is, therefore, inversely proportional to the concentration of antigen in the sample or standard [188, 1891. Some RIA kits need special pre-assay steps, like extraction and chromatography depending on the quality of the antibody and the Sam-

10.8 References

295

pling procedure. It has been demonstrated that extraction procedures interfering with the assay can deteriorate the results. Thus, standardization of sampling and processing is a key requirement. For plasma samples ETDA is the anticoagulant of choice; ASA needs to be added to block further metabolism. Storage at -70°C is absolutely mandatory and repeated freezing and thawing of samples causes artificial increases. Other critical points are: separation of free and antibody-bound substrate (magnetic, charcoal and chromatography), and non-specific ex vivo formation of isoprostanes in both samples and substrateslstandards. An non-radioactive method is the enzyme based immunoassay (EIA). The principle is again competition for certain number of binding sites of antibody. But the compound competing with the substrate is not a radioactive tracer but an enzyme conjugate [ 1901 such as prostaglandin covalently attached to alkaline phosphatase. Enzyme activity is determined by a colour reaction.

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Prostaglandins, Leukotrienes and Other Eicosanoids: From Biogenesis to Clinical Application Edited by F. Marks, G. Fürstenberger Copyright © WILEY-VCH Verlag GmbH, D-69469 Weinheim (Federal Republic of Germany). 1999

11 Eicosanoids and cancer Friedrich Marks and Gerhard Fiirstenberger

11.1. Carcinogenesis: a multistage process Cancer is assumed to result from the toxic effects of environmental and endogenous agents. In causing both genetic defects and long-lasting epigenetic alterations such agents create a situation of unscheduled signaling and incorrect signal decoding in the cell, which finally lead to excessive and destructive tissue growth. According to both clinical observations and experimental models, the disease develops in a stepwise fashion starting with an oncogenic mutation, i.e. either activation of a proto-oncogene or deletion of a suppressor gene, in a single cell. This primary mutagenic event is called initiation (Fig. 11-1). Initiation does not immediately result in visible pathological changes. Instead, the initiated cells may remain undetected for a long period of time (even lifelong) unless they become ‘promoted’. The term tumor promotion describes a process by which the clonal expansion of initiated cells into tumors is facilitated (for reviews see DiGiovanni [ 11 and Marks and Furstenberger [2]). Promotion requires a long-lasting disturbance of tissue homeostasis, which gives the initiated cells a selective advantage provided that they respond more easily to mitogenic and/or less easily to anti-mitogenic, apoptosis- and differentiation-inducing or cytotoxic signals than their neighboring cells. The clonal expansion of initiated cells raises the probability of additional genetic alterations, which are required for the progression to malignancy. In tissues, this accumulation of genetic damage correlates with distinct morphological changes progressing from pre-neoplastic over pre-malignant to malignant lesions [3,4]. While the genetic and morphologic analysis of human neoplasia has impressively demonstrated the gradual development of cancer [3], our knowledge of the underlying mechanisms is mainly based on animal experiments which allow a dissection of tumorigenesis into distinct stages. A classical model of multistage carcinogenesis is provided by skin cancer in mice [ 1,231, where each of the stages of carcinogenesis can be induced separately under strictly controlled conditions, e.g. initiation and malignant progression by genotoxic carcinogens and promotion by so-called tumor promoters, which are a subclass of non-genotoxic carcinogens. However, the conditions of tumor promotion may be created by any situation which leads to repeated release of both mitogenic and anti-mitogenic signals in the tissue. In fact, the effects of many non-genotoxic carcinogens belong to this category. In particular, in the mouse skin model, the most active tumor promoters are also at the same time potent irritants

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which induce both an inflammatory and a hyperproliferative response in the tissue. In addition, in man, chronic irritation is suspected to favor cancer development, as was postulated by Rudolf Virchow in the nineteenth century. This can occur either by repeated non-specific tissue damage or by long-lasting deregulation of signaling cascades which are normally involved in the control of tissue regeneration and defense. Staaes of carcinogenesis

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Figure 11-1. Stages of carcinogenesis. The concept of multistage carcinogenesis implies that cancer results from an accumulation of genetic damage starting with a single gene mutation in a single cell (tumor initiation). The probability of additional gene mutations occurring is greatly increased by clonal expansion of initiated cells occumng in the course of a repeated regenerative tissue response resulting in visible pre-malignant lesions (tumor promotion). The latter exhibit an inherent tendency to progress to malignancy both spontaneously (' mutator phenotype'; genetic instability) and under the influence of environmental genotoxic carcinogens.

Tumor promotion by the repeated disturbance of intracellular signaling is best exemplified by the action of the phorbol ester TPA. This extremly active skin tumor promoter specifically mimics the cellular effects of the second messenger diacylglycerol (DAG) which is released together with inositol-1,4,5-trisphosphate from membrane phospholipids by receptor-controlled phospholipase C (PLC) isoenzymes. The major targets of DAG in the cell are protein kinases of the C (PKC) class, which occupy a central position in cellular signal processing in that they convey the signals from a large number of receptors to the cell nucleus [6-81. PKC activation exerts a mitogenic as well as an anti-apoptotic effect in many cell types [9], i.e. it ideally fulfills the requirements of a tumor-promoting stimulus. However, because of

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the multiplicity of the PKC family (including, as yet, 11 isoenzymes), the large number of potential PKC substrate proteins and the extraordinarily high degree of interactions of PKCs with other signal transducing factors, the molecular mechanisms underlying the role of PKCs in tumor promotion is still not fully understood. On the other hand, numerous phorbol ester/DAG-responsive genes thought to be under the control of PKC-regulated signaling pathways have been identified. These genes are controlled by the AP-1 transcription factor family consisting of dimers of Jun, Fos and ATF proteins [lo]. Both the activation as well as the de now synthesis of AP-1 transcription factors are under the control of several signal transducing pathways, in particular of the so-called mitogen-activated protein (MAP) kinase cascades. These cascades represent a series of major signaling pathways leading from the cell’s periphery into the nucleus [ 111. One of the cascades, i.e. the Ras-Raf-Erk cascade, seems to play a key role in tumor promotion, at least in the skin. Upon wounding it becomes transiently activated by growth factors such as transforming growth factoraTGF-a), whereas the tumor promoter TPA makes a short-cut by activating PKC which, in turn, stimulates the protein kinase Raf-1 [12]. As a consequence, a series of genes, including those of the AP-1 proteins are activated, which control cell proliferation and other processes. In addition, programmed cell death is suppressed along this cascade [131.

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Figure 11-2. PGF2, as a tumor promoter in mouse skin. The diagram shows the effect of indomethacin and of combined treatments with indomethacin plus prostaglandins on tumor development in mouse skin induced according to the initiation-promotion protocol. The drugs were applied topically in the doses indicated 30 min prior to each treatment with the phorbol ester tumor promoter TPA. Note the dosedependent anti-neoplastic effect of indomethacin (solid line and open circles) and its specific reversal by PGF?, (solid line and black dots) but not by PGEz. (broken line). For details see Furstenberger et al. [44].

Several components and target proteins of the MAP kinase cascades are subject to oncogenic mutation. In the mouse skin model of carcinogenesis, the oncogenic acti-

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vation of the H-ras gene appears to provide a major initiating event which results in an H-Ras protein that is defective in signal extinction [5].It is easily conceivable then that cells which are initiated and harbor such a deregulated Ras-Raf-Erk kinase cascade respond more sensitively to mitotic and less sensitively to apoptotic signals as compared with normal cells. Moreover, since the biosynthesis of growth factors such as TGF-a is most probably induced along the cascade [ 14,151, a positive autocrine feedback loop [14-171 is expected to develop in tumor cells, which results in a constitutive overexpression of TGF-a and other proteins controlled by the Ras-Raf-Erk cascade (Fig. 11-4). Under the conditions of tumor promotion, the initiated cells will therefore proliferate faster than the surrounding normal cells and finally gain autonomy. TGF-a is a prototype of a ‘wound hormone’ implying a close relationship between tumor promotion and wound healing. In fact, in the mouse skin model the responses to tumor-promoting agents (such as the phorbol ester TPA) have been found to correspond to the symptoms of tissue repair and wound healing even in molecular details. Moreover, repeated wounding or the injection or overexpression of wound hormones such as TGF-a exerts a powerful tumor-promoting effect in mouse skin and other models [2,18-211. T h s shows that tumor promotion can result from an inappropriate overactivation of the endogenous mechanisms of tissue repair and defense and casts new light on a classical phrase, i.e. that ‘cancer is the result of permanently repeated and disturbed tissue regeneration’ [22]. In experimental models, such as mouse skin carcinogenesis, tumor promotion primarily leads to benign tumors. However, a certain percentage of these premalignant lesions inevitably progress to malignancy even without additional genotoxic treatment [23]. Since, on the other hand, malignant progression is due to further gene mutations [4,5], one has to arrive at the conclusion that, in those tumors which are prone to malignant deterioration, endogenous genotoxic mechanisms are activated during tumor promotion, possibly together with impairment of DNA repair ~241. Such endogenous ‘mutator’ mechanisms may include errors in DNA replication and spontaneous hydrolytic alterations of DNA such as depurination and deamination. However, oxidative DNA damage is becoming recognized as a major cause of ‘genetic instability’ [24-261. This mutagenic process is thought to occur through an overactivation or deregulation of metabolic reactions which give rise to reactive oxygen species, i.e. hydroxyl and superoxide anion radicals, singlet oxygen, hydrogen peroxide, organic peroxides and free radicals, resulting in ‘oxidative stress’ [27]. The concept that such reactions play a critical role in cancer development is supported by a steadily increasing body of evidence indicating that antioxidant nutrients can effectively prevent cancer in man and experimental animals [28]. Eicosanoid biosynthesis is a potential source of toxic oxidation products. Thus, environmental carcinogens are activated and endogenous genotoxic compounds such as reactive oxygen species are produced along the corresponding enzymatic pathways. Moreover, eicosanoids have been found or at least suspected to also play a critical role in ‘epigenetic’ stages of neoplastic development such as tumor promotion and the release of metastatic cells, as well as in immune defense against tumors (see below).

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11.2 Immunosuppression by prostaglandins For reviews see Pelus and Stausser [29] and Young [30]. In principle, cancer cells are recognized and destroyed by cytokine-, i.e. interleukin-2 (11-2)-activated cytotoxic T cells and natural killer (NK) cells, as well as by macrophages. On the other hand, tumors are able to develop properties which enable them to subvert immune surveillance, probably by releasing immune suppressive factors and activating mechanisms of host immune suppression. Among the signaling molecules mediating immune suppressive effects, prostaglandins have gained considerable interest since the immune suppressive efficacy of prostaglandin E2 (PGE2) was discovered more than 20 years ago. In the tumor-bearing organism, both the tumor cells themselves as well as immune cells stimulated by tumor cells are sources of PGE2. Thus, a wide variety of tumors in man and experimental animals as well as transformed cell lines have been found to produce PGE2 in excess which may inhibit the cells of the immune system in the microenvironment. PGE2 specifically inhibits the release of cytokines such as 11-2 from those CD4' T-helper subpopulations which promote cell-mediated immunity. In addition, it decreases the expression of IL-2 receptors by T cells, suppresses antigen presentation as well as cytokine and cytotoxic nitric oxide production by macrophages and impairs the antitumor cytotoxicity of NK cells. Moreover, the function of B lymphocytes is modulated by PGE2 in that immunoglobulin M (IgM) production is attenuated while IgE release is increased [313. Among the prostanoids these immunosuppressive effects are restricted to PGE2 and are mediated by cyclicAMP -dependent signal transduction. Tumors also induce host immunosuppressor mechanisms, probably by the release of cytokines such as IL1, tumor necrosis factor-a (TNFa), granulocyte/monocyte colony-stimulating factor (GM-CSF), TGF-I3 and interferon y (IFNy) which stimulate immune suppressive T cells and monocytes /macrophages. The latter cell types in particular respond by increasing their production of PGE2 which suppresses immune functions by the autocrine and paracrine mechanisms mentioned above. Attempts to restore immune defense against tumors by means of inhibitors of prostaglandin synthesis, in particular non-steroidal anti-inflammatory drugs (NSAIDs), were repeatedly made. Encouraging results were obtained using animal models. Even highly malignant tumors in advanced stages could be treated, in particular when the NSAID was combined with immune stimulatory cytokines such as Il2 [32]. However, the results of clinical trials have not yet fully come up to expectations. Depending on the conditions, PGE2 may exhibit opposing effects on immune cells, i.e. either inhibit or stimulate cytotoxic functions of NK cells, T cells and macrophages [33]. Such apparently contradictory observations are conceivable if one considers not only the complexity of the immune system and the eicosanoid network but, in addition, the fact that PGE2 can activate the production of second messengers with opposing cellular functions via different receptor types, i.e. cyclic AMP versus DAG/inositol-trisphosphate or different G-proteins which either stimulate or inhibit CAMPformation (see Section 1.6.2). Therefore, the response of a given cell to PGE2

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is expected to depend on the actual expression pattern of prostaglandin receptor subtypes. Altogether the physiological significance of PGE2-induced immunosuppression is recognized, but the role of this effect in cancer development is still a matter of debate. In particular, attempts to relate PGE2 production to tumor agressiveness and metastasis have not yet revealed a clear-cut picture although a positive correlation of both parameters has been repeatedly observed (see Section 11.6).

11.3 Metabolic activation of carcinogens in the course of prostaglandin synthesis For reviews see Marnett [34] and Smith et al. [35]. Both the initiation of tumorigenesis as well as the progression from the benign to the malignant state are thought to be due to genotoxic events which lead to activation of proto-oncogenes and inactivation of tumor suppressor genes. These mutations are predominantly caused by environmental factors, i.e. chemicals, UV and ionizing radiation and tumor viruses. The great majority of chemical carcinogens have to be metabolically activated to become mutagenic. A major role in this ‘toxification’ is played by cytochrome P-450 monooxygenases which catalyze epoxidation and hydroxylation of xenobiotics [36]. However, the oxygenation of environmental chemicals to genotoxic ‘ultimate carcinogens’ can also occur in the course of other metabolic processes. The most thoroughly studied pathway in this respect is prostaglandin biosynthesis. In particular, the peroxidase activity of prostaglandin H synthase (PGHS) is responsible for carcinogen activation, whereas COX activity is highly specific for polyunsaturated fatty acids (PUFAs) as substrates. A wide variety of chemical carcinogens have been found to become activated by PGHS-catalyzed cooxidation, at least in cell culture and cell-free systems. In the organism, PGHSdependent carcinogen activation might play some role in tissues outside the liver [37]. However, the contribution of this reaction to cancer initiation in man is not known yet. There is some evidence that carcinogen activation can also occur along the lipoxygenase-catalyzed pathways of eicosanoid formation (see Section 1 1.5.5).

11.4. The generation of agressive products in the course of eicosanoid formation Various agressive and genotoxic (by)products can be produced via the enzymatic pathways of eicosanoid biosynthesis, which may cause cell damage and gene mutations if not eliminated by protective mechanisms of the cell. Malondialdehyde, a bona fide mutagen and genotoxic carcinogen, is generated along the COX pathway as a product of PGH2 breakdown occurring either nonenzymatically or catalyzed by thromboxane synthase [38] (see Section 4.2). Throm-

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boxane synthase is highly effective in producing malondialdehyde from PGH2 and, even better, from PGH,. The physiological meaning of this enzymatic reaction is not clear, in particular since, as a direct-acting mutagen, malondialdehyde may be harmful to the cell. Platelets, the major source of thromboxane synthase, are, on the other hand, not endangered by mutations since they have lost their nucleus in the course of differentiation. Malondialdehydealso derives from non-enzymatic peroxidation of PUFAs. In conventional mutagenicity assays malondialdehyde causes both frameshifts and base pair substitutions (of A, C and G to T) by binding covalently to deoxynucleoside residues of DNA. It has been speculated that the C + T transitions found in mutated p53 tumor suppressor genes in human colon cancer could at least partially result from malondialdehyde [34]. The contribution of malondialdehyde generation to carcinogenesis is still an open question, however. The chemistry of lipoxygenase- and COX-catalyzed fatty acid peroxidation implies the generation of organic free radicals and peroxides as well as reactive oxygen species including singlet oxygen and hydroxyl radicals as intermediates and by-products [39]. Such aggressive compounds are strongly suspected to act as endogenous mutagens and carcinogens. Under normal conditions they are expected to be eliminated by protective enzymes such as catalase, superoxide dismutase, glutathione peroxidase, etc., as well as by cellular antioxidants including vitamins C and E and other food components [40]. This protection may be overrun in tumor cells. As shown below, both cyclooxygenases and lipoxygenases may become heavily overexpressed and arachidonic acid release overactivated in the course of neoplastic development. This not only results in an accumulation of biologically highly active eicosanoids in tumor cells, which certainly play important roles in carcinogenesis, but, in addition, the above-mentioned by-products, which may cause genetic defects thought to be critical for cancer development. Thus, deregulation of lipoxygenase- and COX-catalyzed reactions may provide an important determinant of the mutator phenotype, i.e. the genetic instability of tumor cells. The contribution of these metabolic processes to overall lipid peroxidation, which are suspected of playing a key role in carcinogenesis, has not yet been estimated.

11.5 The role of eicosanoids in tumor promotion 11.5.1 COX inhibitors suppress tumor promotion in animal models Neoplastic tissue frequently exhibits pathologically high levels of prostaglandins and other eicosanoids. Moreover, it has been known for more than 20 years now that, in animal experiments, NSAIDs, i.e. inhibitors of prostaglandin formation, suppress the growth of virus- and chemically induced tumors in a variety of organs such as the esophagus, pancreas, urinary bladder, mammary gland, skin and intestine (summarized in Mamett [41] and Levy [42]). Particularly detailed studies have been undertaken for rat colon cancer induced by chemicals such as dimethylhydrazine or az-

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oxymethane. Many different NSAIDs, including indomethacin, piroxicam, ibuprofen, sulindac and aspirin, have been shown to be powerful inhibitors of tumorigenesis in this system when given at an early stage of neoplastic development [42,43]. The inhibitory effect turned out to be reversible, i.e. recurrent tumor growth was observed upon cessation of NSAID treatment. These results indicated that NSAIDs inhibited tumor development from mutated cells rather than tumor initiation. However, the antineoplastic effect of NSAIDs in the colon could not be unequivocally related to a defined stage of tumor development or to an inhibition of prostaglandin synthesis, although a suppression of the prostaglandin level in tumor tissue was observed upon NSAID treatment. On the other hand, this has been achieved, by using a more elaborate animal model of multistage carcinogenesis, i.e. the mouse skin model. Skin carcinogenesis of rodents, in particular of mice, has been one of the most thoroughly investigated systems of multistage tumor development (see above). Studies in this animal model have led to the establishment of the initiation-promotion approach of carcinogenesis. Experimentally, initiation is brought about by a limited treatment of mouse skin with a genotoxic carcinogen (such as UV light, a chemical agent or a tumor virus) that is applied in a small dose said to be ‘subthreshold’, since the animals will develop benign and malignant epidermal tumors only when this initial treatment is followed by repeated chronic application of a tumor promoter. As explained above, tumor promotion is understood as a process that favors the clonal expansion of single initiated cells through repeated stimulation of cellular proliferation and inhibition of programmed cell death. Clonal expansion increases the probability of further genetic damage both by increasing the number of potential target cells and by inducing endogenous genotoxic processes. The most active tumor promoters employed in the mouse skin model are the phorbol esters (such as TPA or PMA) and okadaic acid. Both act by strongly disturbing intracellular signal processing, either by overactivating PKC (such as the phorbol esters) or by inhibiting phosphoprotein phosphatases (such as okadaic acid). The visible consequence of these molecular actions is an inflammatory hyperproliferation of the epidermis which closely resembles regenerative hyperplasia, i.e. a wound response. Repeated wounding exhibits tumor-promoting efficacy of its own. The responses of skin to tumor promoter treatment or wounding depend critically on prostaglandin formation occurring in epidermal cells shortly after treatment. In correspondence with this finding, prostaglandin formation and skin tumor promotion in mice were shown to be inhibited by topical application of the NSAID and COX inhibitor indomethacin [44]. This inhibitory effect could be specifically overcome by PGF2, rather than by other prostaglandins, proving that the effect of indomethacin was in fact due to an inhibition of prostaglandin biosynthesis. Up to now this result provides the first and only case where an anti-neoplastic effect of an NSAID could be unequivocally attributed to the formation of a distinct prostaglandin species. Recently, the renal tumor promoter ferric nitrilotriacetate has been found to induce PGE2 synthesis in the rat kidney and indomethacin to inhibit the hyperproliferative response of the kidney to this tumor promoter [45].In mouse epidermis release of PGF2, is observed 3-4 h after application of phorbol ester [MI. At this point, de novo synthesis of cyclooxygenase-2 (COX-2) becomes visible [46].Only COX-1 is found in untreated skin, whereas the level of COX-2 is below the limit of detection (Figs. 11-3 and 3-2)

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[47]. Thus, COX-2 behaves as a typical pro-inflammatory, early-response or ‘emergency’ enzyme which becomes transiently expressed in epidermal cells upon irritation and wounding (see also Section 3.4).

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Figure 11-3.Correlation between PGF2, formation, expression of COX-2 and the anti-neoplastic effect of the COX inhibitor indomethacin in phorbol ester-treated mouse skin. In this experiment, tumor development in mouse skin was initiated by a single application of dimethylbenz(a)anthracene (100 nmol) and promoted by repeated applications of phorbol ester TPA (10 nmol). The upper broken line shows the average number of benign tumors obtained by this treatment after 18 weeks. The lower broken line represents the negative control (no TPA treatment). The strong solid line with black points shows the tumor yield in animals which were locally treated with indomethacin (550 nmol, acetone solution) at the times indicated, i.e. before or after TPA application at zero time (see abscissa). Note that indornethacin given 3.5 h after TPA completely suppressed tumor development. The other strong line represents the expression of COX-2 and the shadowed double peak the level of PGR, as measured in mouse epidermis upon TPA treatment (arbitrary units). For details see Fiirstenberger et al. [44] and Miiller-Decker efal. [46].

In benign and malignant tumors generated in mouse skin according to the initiation-promotion scheme COX-2 is constitutively overexpressed (see Fig. 3-2, p. 72) and the tissue levels of prostaglandins approach values 50-fold over the controls. Beside the temporal correlation between COX-2 expression and PGF2, synthesis, two other results point to a critical role of COX-2 in mouse s h n carcinogenesis. Thus, skin tumor promotion can be strongly suppressed by a COX-2-specific inhibitor [48] and, in an initiation-promotion experiment, COX-Zdeficient ‘knock-out mice’ showed a diminished rate of skin tumor development. As in the case of indomethacin, this inhibitory effect could be specifically overcome by local application of PGF2, (R. Langenbach, personal communication). It should be mentioned that, besides COX-2deficient mice, COX-1 knock-out mice have also been found to be less sensitive to chemical skin carcinogenesis, indicating a role of both isoenzymes in tumor devclop-

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ment [49]. This notion is supported by observations on chemically induced rat mammary tumors and biopsies of human breast carcinomas, many of which exhibit an overexpression of COX-I in addition to COX-2 [50,511. Moreover, endothelial cells have been shown to become tumorigenic in athymic mice upon transfection with COX-I cDNA [52]. Surprisingly, this COX-I-induced tumor growth could not be inhibited by indomethacin, perhaps indicating a prostaglandin-independent mechanism.

11.5.2 Prevention of colorectal cancer in man by COX inhibitors A preventive effect of NSAIDs on human cancer has been convincingly demonstrated for colorectal carcinoma [41-43,53,54]. This is one of the most frequent and most dangerous neoplastic diseases. The causes are unknown, but dietary factors have been suspected to play a critical role [54].Less than 5% of colon cancer patients suffer from inherited predispositions for colorectal tumorigenesis, such as familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal carcinoma (HNPCC). While FAP is due to a deletion of the APC (adenomatous polyposis coli) tumor suppressor gene (see below), HPNCC is thought to result from mutations of DNA mismatch repair genes [4]. In FAP patients approximately one in lo6 cells proliferates to be a tumor (polyp) when the second APC allele is deleted, probably by environmental influences. In effect, FAP patients develop intestinal tumors in very high numbers. Although they are benign at the start these tumors harbor a high risk of becoming malignant with time if not surgically removed. An animal model of FAP is the so-called Min mouse (‘multiple intestinal neoplasia’) which also carries a heterozygous APC deletion [53]. However, for unknown reasons Min mice develop tumors predominantly in the small intestine rather than in the colon. APC inactivation is not restricted to FAP but is also observed in more than 80%of ‘sporadic’ colorectal polyps and carcinomas, i.e. the most abundant types of large bowel neoplasia. Homozygous APC deletion has already been found in the putatively earliest precursor lesions of adenomas, the so-called dysplastic aberrant crypt foci and is, therefore, considered to represent a specific marker of tumor initiation in the intestine. Germline mutations of other ‘cancer genes’ such as p53 or ras do not give rise to polyposis, despite the fact that such mutations are involved in the progression of colon cancer [4]. Its specific role in intestinal neoplasia has led to the hypothesis that APC acts as a ‘gatekeeper gene’ that controls the balance between cell gain and cell loss, i.e. tissue homeostasis [4]. Such ‘gatekeeper’ functions are thought to be tissue specific. Since approximately 50% of the human population develops colorectal tumors (mostly benign polyps or adenomas) at advanced age, loss of functional APC seems to be by far the most abundant genetic defect related to human neoplasia. Genetic analysis of human colorectal carcinogenesis has provided strong evidence for the multistage nature of tumor development as the clinical progression of the disease could be correlated with an increasing number of specific genetic defects afflicting both tumor suppressor genes (i.e. APC, DCC and p53) and proto-oncogenes (i.e. K-ras) [4,56]. In this respect colon carcinogenesis of man closely resembles multistage skin carcinogenesis of mice with the reservation that, in the intestine, the cells

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carrying a homozygous APC deletion may be understood as initiated, whereas in mouse skin initiation seems to occur predominantly by mutation of proto-oncogenes such as H-ras [5].Dietary factors may act as intestinal tumor promoters in that they display irritating efficacy which evokes chronic inflammation and tissue repair including COX-2-catalyzed prostaglandin formation as do phorbol ester tumor promoters in the skin [48]. Recently, secondary bile acids such as deoxycholate, which are strongly suspected as acting as tumor promoters in the colon, have been found to induce COX-2 expression via a PKC-dependent pathway in esophageal tumor cells [57]. This effect indeed closely resembles the action of phorbol esters on skin. Therefore, the idea suggests itself that COX-Zcatalyzed prostaglandin synthesis may provide a driving factor not only in experimental skin cancer but also in colorectal carcinogenesis, thus explaining the inhibition of tumor development by NSAIDs in both mice and man. In 1983 Waddell and Loughry [58] succeeded in inhibiting tumor development in FAP patients by oral application of the NSAID sulindac, an observation made accidentally but which fitted the animal data perfectly. However, this and subsequent studies were affected by the small number of patients, which did not allow a clear cut statistical evaluation of the anti-tumor effect. In the meantime, sulindac treatment has been repeatedly shown to be very effective, in particular in inhibiting the recurrence of tumor growth in FAP patients which had undergone initial surgery (see, for instance, Giardiello et al. [59] andWinde et al. [60]). These clinical studies coincided with evaluation of epidemiological data showing that persons who have taken NSAIDs regularly over many years, in particular aspirin, exhibited an approximately 50% lower risk of becoming ill with and dying from colorectal cancer [4143,53]. Such observations clearly indicated that, not only FAP but also much more abundant sporadic large bowel cancer, could be effectively prevented by NSAIDs. At least ten studies, both retrospective and prospective, have validated this very impressive chemopreventive effect [43]. Inhibition of tumor growth by NSAIDs has been found to be fully reversible and restricted to early benign stages of neoplastic development in both animal models and man. In other words, upon cessation of NSAID treatment a tumor recurrence is observed and more advanced tumors, in particular carcinomas, seem to be insensitive to NSAIDs. These findings indicate that tumor promotion is a major target of chemoprevention with NSAIDs. While clear-cut evidence exists that the anti-tumor effect of NSAIDs results from an inhibition of prostaglandin synthesis in the mouse skin model [44,48],the situation is less clear for colorectal tumorigenesis. In fact, a vivid debate is going on as to whether or not the anti-tumor effect of NSAIDs can be solely explained by COX inhibition. The pro-arguments may be summarized as follows [41,42,61].

(1) In general, increased prostaglandin levels are found in intestinal tumors of man and experimental animals and prostaglandins exhibit co-mitogenic and antiapoptotic effects in v i m . (2) In the majority of intestinal tumors of azoxymethane-treated rats, Min mice and man constitutive overexpression of COX-2 has been observed. As in mouse skin, COX-2 is not expressed in the normal epithelium. Genetic COX-2 knock-out (or

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treatment with a COX-2 inhibitor) resulted in a dramatic reduction of intestinal tumor incidence in APC-deficient mice indicating a causal relationship between APC deletion on the one side and COX-2 overexpression and tumorigenesis on the other side [62]. (3) Overexpression of COX-2 rendered intestinal epithelial cell lines more resistant to programmed cell death [63]. (4) Specific COX-2 inhibitors not only suppressed tumor formation in azoxymethane-treated rats [64,65] and APC-deficient mice [62,66], but inhibited cell proliferation in vitro depending on the level of COX-2 expression [67]. ( 5 ) NSAID-induced apoptosis in human colon cancer cells is reversed by prostaglandins [68]. (6) Prostaglandins have been shown to activate the peroxisome proliferator-activated receptor y(PPARy) [69] and activation of this receptor increased the multiplicity and size of colon tumors in Min mice [70]. The arguments raised against a critical role of prostaglandin synthesis in the antitumor effect of NSAIDs include the following [71,721. (1) Sulindac sulfone, a major metabolite of the NSAID and COX inhibitor sulindac, has been shown to protect rats from azoxymethane-induced colon cancer and salicylic acid to inhibit colon cell proliferation as well as to induce apoptosis, although both drugs apparently do not inhibit prostaglandin synthesis in virro. (2) Cell lines which apparently do not express COXs have nevertheless been found to be sensitive to NSAID-induced apoptosis and cell growth arrest. (3) The apoptosis-inducing effect of NSAIDs in cell culture could not be overcome by prostaglandin applications. Instead, arachidonic acid, which may be expected to accumulate upon NSAID treatment of cells, has been shown to induce apoptosis of colon cancer cells in vitro via ceramide as a second messenger [73]. (4) A genetic knock-out of COX-2 only partially suppressed tumor development in APC-deficient mice [62].

Against these arguments, it has been noted that, in the cell culture experiments, NSAID concentrations were used which far surpassed the pharmacologically active in vivo doses [61]. The same argument holds true for the apoptotic effect of arachidonic acid and, in addition, may be raised against the proposal that the pharmacological effects of salicylate and aspirin can be explained by the inhibitory effects of these drugs on the activation of the ‘pro-inflammatory’ transcription factor NF-KB [74]. The latter effect is most probably due to inhibition of the protein kinase which phosphorylates and, thus, inactivates the NF-KB inhibitor I-KB,thereby preventing activation of genes under the control of this transcription factor, by millimolar concentrations of aspirin [75]. Moreover, for salicylic acid at least, the assertion that it does not inhibit prostaglandin formation cannot be maintained any longer since it has recently been shown to be a potent COX inhibitor provided that the arachidonic acid concentration was kept at a physiological level in the assay [76]. On the other hand, I-KI~and the cyclooxygenases are certainly not the only cellular targets of NSAIDs, since recently these drugs have been shown to activate p38 protein kinase [77], to suppress

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3 15

cytokine-activated cJun terminal kinase [77] and p42/44MAP kinase [78] and to reduce the activity of the transcription factor complex AP-I [79]. It has to be emphasized, however, that the pharmacologic significance of these effects is not clear, whereas the inhibition of cyclooxygenases, in particular of COX-2, most plausibly explains the anti-neoplastic effects of NSAIDs at least in the intestine and the skin.

11.5.3 Promotion of tumor development by prostaglandins: a widespread phenomenon? In animal experiments overproduction of prostaglandins by tumors and an anti-tumor effect of NSAIDs have been shown for a variety of tissues including the intestine, epidermis, esophagus, lung, urinary bladder, kidney, stomach and mammary gland (for a review, see Marnett [41]). Excessive prostaglandin levels were also found in various tumors of man, for instance in mammary tumors (see, for instance, Robertson et al. [50]). However, as far as the chemopreventive effect of NSAIDs on tumors outside the gastrointestinal tract is concerned, the clinical and epidemiological evidence is still controversial. Indeed, both a prospective and a case-control study revealed a statistically significant reduction in breast cancer in women who had regularly taken aspirin or ibuprofen [80,81], whereas other authors did not observe such an effect [82-851. The evidence for chemoprevention of lung cancer by NSAIDs is even less significant [80]. On the other hand, a rather strong correlation was found for aspirin intake and reduction of esophagus and stomach cancer [86]. This may also explain the reduced incidence of stomach cancer in patients suffering from rheumatoid arthritis [87], i.e. a disease which has been traditionally treated by NSAIDs. Studies on colorectal tumors have revealed a probably causal relationship between tumor development and upregulation of COX-2. These obervations imply that aberrant COX-2 expression may be taken as an indication of NSAID susceptibility of a tumor. Recently, several studies have been published which address this question. Thus, increased levels of COX-2 mRNA and protein were found in biopsies and cells derived from human stomach cancer [SS], squamous cell carcinoma of skin [89,90] and lung cancer [91,92]. In breast cancer tissue both COX-1 and COX-2 were reported to be elevated [51] and preparations from prostate cancer exhibited tenfold higher prostaglandin synthase activity as compared with normal tissue [93]. A highfat diet, which is thought to promote the development of colon, breast and prostate cancer, may lead to overproduction of eicosanoids and an elevation of COX-2 expression which was induced in the colon of rats by the carcinogen azoxymethane [94,95]. In this context, it is intriguing that COX-2 expression has been found to be accompanied by expression of cytoplasmic phospholipase A2 (cPLA2)in the rat liver, which is induced by tumor-promoting peroxisome proliferators [96] and in non-small lung cancer cells harboring a mutated Ki-ras proto-oncogene [97]. Moreover, secretory phospholipase A2 (sPLA2) was found to be overexpressed in polyps of FAP patients [98], in human breast [99], stomach [loo] and liver cancer tissue [I011 and in chemically induced colon tumors of rats [102]. This upregulation of phospholipases which catalyze arachidonic acid release from phospholipids indicates that, in tumor tissue, overexpression of COX may coincide with an increased availability of the corre-

3 I6

I I Eicosanoids and cancer

sponding fatty acid substrate which, in turn, derives from dietary fat. With the exception of the rat liver, aberrant overexpression of COX-2 during carcinogenesis has also been reported for other animal models. Examples are provided by chemically and UV-induced skin cancer of mice [89,90] and chemically generated intestinal [lo31 and mammary [50]tumors of rats. As in human breast cancer tissue [51], COX-1 was also elevated in rat mammary tumors, in addition to COX-2 [50]. These results indicate a more general role of prostaglandin formation in cancer development and open up the possibility of widespread application of NSAIDs for chemoprevention, which will probably depend on the type of drug and the route and schedule of administration, which in turn may be determined by the type of neoplastic disease.

11.5.4 Mechanistic aspects What do we know so far about the mechanisms of COX-2 overexpression in tumor cells and about the role played by prostaglandins in tumor development? As already mentioned (and explained in more detail in Section 3.4) COX-2 is transiently induced in most tissues in response to damage and irritation, with a wide variety of growth factors and cytokines acting as endogenous mediators or ‘wound hormones’. Many of these factors are activators of the Ras-Raf-Erk cascade or related MAP kinase cascades of intracellular signal transduction [ 111. Table 11-1. Tissue levels of eicosanoids in the course of multistage carcinogenesis in NMRI mouse skin. Tumors were generated according to the initiation-promotion protocol with 7,12-dimethylbenz[a]anthracene (DMBA) as initiator and phorbol ester TPA as promoter. The prostaglandin levels were determined by enzyme immunoassay and are given in ng/mg protein. The HETE levels were assayed by GC/MS and are given in ng/g tissue; N24; SD 10 pM [241] discontinued) pa (rabbit): ~ C S=O0.36 pM (U) [242]

Indications In vitro data (status*) a 2 0 Anti-thrombotic N m pa (guinea-pig): a F ICSO= 0.32 pM (U) (preclinical) vc: ICso = 3.1 nM (U) [239]

Structure CAS-number [synthesis]

AH-23848 Glaxo Wellcome, I + ) - ~ UK h . . -

Daltroban, BM-13505, SK&F-96148 Boehringer Mannheim, Germany Sulotroban, BM-I3 177, SK&F-95587 Boehringer Mannheim, Germany

NamdCode originator 2-335 Zeria, Japan

Table 12-3. (Continued)

vein replacement, pa is reduced in the graft but not platelet survival time [256].

In 12 volunteers 1 mg/kg t.i.d for 9 days blocks pb with high efficacy [255]. In patients with dacron

(u,C)

4 days normalizes plasma TXBz-levels and reduces P-thromboglobulin and platelet factor 4 [244]. Sudden death (rat, 30 mg/kg i.v.): 100% inhibition (U) [244]. Infusion of 12.5 m g h i n causes resting myocardial ischemia [249]. In guinea-pig 5 mgkg abolishes bc (ov) 12481. bc (gp): ID5o = 3.0 nMkg [253] In 6-8 weeks old LH rats the compound produces a marked antihypertensive effect [254]. pa (human, er vivo): IClw = 0.125-1 m g k g

tc (mice, p.0.): IDSO= I .45 mg/kg (U) [242] In seven atherosclerotic patients, 800mg b i d . for

pa (guinea-pig, p.0.. e.r vivo): ED 50 = 63 pgkg (U), 26 pgkg (AA) 30mgkg p.0. for 5 days decreases serum lipid (TG) in fructose-induced hypertriglycemic rats. In the rat peripheral arterial occlusion model, 10 mglkg blocks progression of necrosis. In patients with intermittent claudication, 200 mg b i d . for 6 weeks leads to an increased pain-free walking distance of 50 m [243]. Sudden death (rat, 0.03 mg/kg i.v.): 100% inhibition (U) [244].

In vivo data

IC1-192605 Zeneca, UK

ONO-3708 Ono, Japan

GR-32 19 1 GR-32 I9 1B SN-309 Glaxo Wellcome, UK

Name/Code originator Vapiprost

Ha

Antithrombotic, acute respiratory dystress syndrome (phase 11, discontinued)

1 1762 1-64-4 [270,27 I]

102191-05-9 [263]

Antithrombotic, asthma (phase 11, discontinued)

T”’”

&y

87248-13-3 [257] 85505-64-2 (free base)

Ho

-

th-ao (rabbit): pA2 = 8.0 (U) pa (human): pA2 = 8.3 (U) [270] th-ao (rat): pA2 = 8.4 (U) th-ao (pig): pA2 = 9.4 (U) [272] pa (rat): pA2 = 6.8 (U) pa (rabbit): pA2 = 6.3 (U) pa (guinea-pig): pA2 = 8.6 (U) pa (human): pA2 = 8.2 (U) [273]

K, (human) = 37 nM (U) [264] tr (guinea-pig): pA2 = 7.78 (U); (human): pA2 = 7.43 (U) [265]. ao (rabbit): ICSS= 1 nM (U) [266]

Indications In virro data (status*) Anti-thrombotic KD (human) = 0.5 nM [258] pa (human): pA2 = 8.3 (U, SQ) [259] (phase III, discontinued) ba (can): pA2 = 8.8 (U) ca (can): pA2 = 8.67 (U) ma (can): pA2 = 8.86 (U) fa (can): pA2 = 9.01 (U) [260]

---

0

Structure CAS-number [synthesis] / \ / \

Table 12-3. (Continued)

In 17 healthy volunteers, ICI-192605 in the dose range 2-100 mg causes significant inhibition of pa (U) but has no effect on pa (A), the minimum effective dose was 0.03 mgkg p.0. [274]. 100 nM inhibits contractions of guinea-pig-tr and lung parenchyma preparations (U). 100 mg p.0. can protect against exercise-induced asthma. Increases FEVl and specific airways conductance after exercise challenge in ten patients [275].

both groups [269].

In dogs 1 pgkdrnin 30 min before ligations of the coronary artery prevents decreases in respiratory control index and the rate of oxygen consumption of mitochondria from the ischemic area [267]. pa (dog, i.v.): IDSO= 80 pgkg In dogs 10 and 30 pglkg per min inhibits vasospasm [268]. In rat endotoxic shock, 30 mgkg i.v. causes a greater increase in cardiac index and stroke volume index cf controls mean arterial pressure is the same in

Platelet pure plasma taken 12 h post-dosing (human) produces 4&80% inhibition of pa of plateletrich plasma taken from control [261]. In 40 patients 40 mg db (n = 20) is as effective as full body anti-coagulation in maintaining vessel patency post-thrombolytic therapy [262].

In vivo data

5

w

Z

h

3

‘3

-_

rn

s

a

-+

s3

r?

r@ T

m

23

a 6a

2

A

4

I4 1286-14-8 [245,281,282]

8

142945-74-2 [278]

12 1 147-49-7 [276]

a

Structure CAS-number [synthesis]

Antithrombotic (preclinical, ceased)

Antithrombotic (preclinical, ceased)

ceased)

thrombotic

Indications (status*)

In vivo data

pa (rabbit): Icso = 0.3 pM (U) [245] pa (human): ICSO= 0.67 pM (U) [282]

pb: Ki = 2nM (SQ) [279,280]

pa (human): pA2 = 7.6 (U)

P821

tc (mice, p.0.): ID50 = 0.15 mgkg (U) [229] Antigen-induced airways resistance (ov, DNPBSA) is strongly reduced in guinea-pig 12831. Mortality (mice, p.0.): IDSO= 0.53 mgkg (C + E)

tc (guinea-pig, i.v. bolus): ID50 = 2.2 pg/kg (U) [279,280]

pb: K,= 2.3 nM (U) pa (dog, ex vivo):ICioo = 1 mgkg (for 24 h) (2771 ao (rat): pA2 = 8.8 (AP), Icso = 0.3 nM (TX), 0.3 nM (PH), 0.22 nM (PF) [277]

In vitro data

"'Representsthe most advanced stage of development recorded in at least one of the indications listed. Agents: AA, arachidonic acid; AP, 9 , l l -azo-PGHZ; C, collagen; DNP-BSA, dinitrophenylated bovine serum albumin; E, epinephrine; H, histamine; ov, ovalbumin; PF, PGF2=; PH, PGH2; SQ, SQ 29548; TX, TXA2; U,U46619; U*, U44069. Methods and tests: ao, aorta; ba, basilar artery; bc, bronchoconstriction; ca, coronary artery; fa, femoral artery; fsva, fetal stem villous arteries; Ips, lung parenchymal strips; ma, mesenteric artery; ma-(n)p, myometrial arteries (non)pregnant women; MCAO, middle cerebral artery occlusion; pa, platelet aggregation; pb, platelet binding; psc, platelet shape change; pt, platelet; sv, saphenous vein; tc, thrombocytopenia; th, thoracic; tr, trachea; ts, tracheal strips; tiii, umbilical artery; vc, vasoconstriction; wp, washed platelets.

ON-579 Taisho, Japan

ZK 151847 Schering AG, Germany

Name/Code originator FR-10688 1 Fujisawa, Japan

Table 12-3.(Continued)

k 5

0

3.

sg

a,

2. r5

h

a

5

(b

3

g

Re

2

K

0

3

*8 a

2.

i.7-

c

z2

.-

2

t2

w

12.4 Thromboxane receptor antagonists

349

levels of the arachidonic acid cascade, is indicated in such situations. Cyclooxygenase inhibitors, e.g. aspirin, block not only the formation of TXA2 but also the synthesis of PG12, although the activity of endothelial cyclooxygenase, which is primarily responsible for the formation of PGI2, is less strongly influenced when low doses of aspirin are used [199]. TXA2 synthetase inhibitors selectively block the formation of TXA2 but not the formation of the arachidonic acid metabolite PGH2, which also expresses its activity via the TXA2 receptor and, thus, exhibits a pharmacological profile similar to that of TXA2. The development of pure TXA2/PGH2receptor antagonists is therefore an attractive objective as it is to be anticipated that they will block the effects of TXA2 and PGH2 without influencing the formation of PGI2 [200]. In order to be able to study the pathophysiological role of TXA2 and PGH2 it was first necessary to develop chemically stable TXAzPGH2 mimetics. The replacement of the peroxy group in PGH2 with a methyleneoxy group led to the stable TXA2/PGH2analogs U44069 and U46619 [20 1,2021 (cf. Fig. 12-2) whereby the latter developed into the most commonly used standard TXA2 agonist in biological test systems. With these agonists as a basis, further structural modifications to the bicyclic ring system and in the o-chain led to the development of a first generation of TXA2 receptor antagonists. The most prominent representative is SQ-29548 (Bristol-MyersSquibb) [203-2051, which has become the standard TXA2 receptor antagonist. Further optimization of the structure yielded the two developmental candidates S- 1452 from Shionogi and ifetroban (BMS- 180291) from Bristol-Myers-Squibb, both of which are undergoing phase I1 clinical trials for the treatment of cardiovascular diseases (cf. Table 12-3). S-1452 is also being developed for the treatment of asthma. Similar stabilizing and optimization of the structure based on the TXA2 skeleton concluded with the synthesis of the pinane analog Ono-3708 whose development as an anti-thromboticwas terminated in phase II. A second type of TXA2 receptor antagonists lacking the bicyclic ring system was represented by the non-chiral Boehringer compounds sulotroban (BM-13 177) and daltroban (BM-13505). As in the Shionogi compound S-1452 an arylsulfonamide unit imitates the o-chain. Development of the two compounds as anti-thrombotics and for asthma therapy was terminated in phases 11and In, respectively. Various other companies, such as Bayer (ramatroban), Nycomed (linotroban), Kotobuki Seiyaku (KT2-962), Merck KGaA (LCB-2853), Zeria (2-335) and Taisho (ON-579), have employed similar structural approaches. The chiral compounds from Fujisawa (FR-106881) and Schering AG (ZK 151847) still possess prostaglandin-like elements. The latter compound combines the biphenyl-methyloxy group from AH-23848 (Glaxo-Welkome) with a sulfonamide. Interestingly, the distance between the l-carboxylate and the sulfonamide unit is eight to nine atoms in every case except in the Taisho compound (ten atoms). Structure-Activity studies have shown that an extension or a shortening of this distance leads to a loss of activity. From this class of compounds the TXA2 antagonist ramatroban from Bayer has reached the furthest stage of development. It is already in phase I11 and its launch is expected in the near feature. The main indications are asthma, allergic rhinitis and angina. In a clinical trial in asthmatics ramatroban exhibited significant protection against PGD2-inducedbronchoconstriction. With AH-23848 Glaxo had synthesized a TXA2 receptor antagonist which can be

350

12 Synthetic eicosanaids: development and clinical applications

formally derived from the structure of PGD2. Here the o-chain of PGD? was replaced by a morpholine moiety, the 9-hydroxyl function by a biphenylmethylether and the double bond in the a-chain was switched to position 4. Although this compound proved to be highly active its development was discontinued due to teratogenic effects in rats. It was possible to demonstrate that this teratogenic effect was not attributable to the TXA2 antagonistic properties but was linked to the chemical decomposition product 4-biphenylmethano1[206]. To overcome this problem, the successor compound vapiprost [207] has a palcohol in position 11, the metabolic oxidation of which is hindered thus preventing the elimination of 4-biphenylmethanol. Vapiprost showed a long duration of action in humans, which may be explained, at least in part, by a downregulation of TXA2 receptors [208]. In a preliminary assessment in healthy humans vapiprost was well tolerated. Multiple dosing (0.25 mgkg, three times a day or every 12 h) resulted in a cumulative inhibitory effect on U466 19-induced platelet aggregation in the apparent absence of a build-up of plasma concentrations of the drug [209]. In a double-blind, placebo-controlled study with nine subjects suffering from chronic stable asthma and taking inhaled beclomethasone, treatment with vapiprost did not result in any significant improvement in mean forced expiratory volume per second (FEV,). The authors concluded from these data that TXA2 is not important in the maintenance of airways hyper-responsiveness in chronic asthma and that TXA2 receptor antagonists in general are unlikely to provide effective treatment for this group of patients [210]. A completely different and unusual non-prostanoid structure is represented by the TXAzPGH? receptor antagonist seratrodast from Takeda, which has already been launched in Japan for the therapy of bronchial asthma [211j.

12.5 Peptidoleukotriene antagonists In 1979 the peptidoleukotrienes LTC4/D4/E4 were identified by Borgeat and Samuelsson [284] as important metabolites of the 5-lipoxygenase pathway. The physiological activity of a mixture of these peptidoleukotrienes was described as early as 1938 [285] and was attributed to a factor termed ‘slow-reacting substance of anaphylaxis’ (SRS-A). Today it is known that LTC4 and D4 induce intense and persisting contraction of the smooth muscle tissue of the lung. The leukotrienes are 1000 times more potent as bronchoconstrictors than histamine. As elevated levels of leukotrienes had been found in the lungs of asthmatics it seemed obvious that 5-lipoxygenase inhibitors or peptidoleukotriene antagonists should have anti-asthmatic effects. The search for and the development of leukotriene antagonists of this kind has been documented in numerous articles [286-2881. FPL 55712 (Fisons) and LY 171883 (Eli Lilly) belonged to the first generation of antagonists (cf. Table 12-4). The rather disappointing results obtained in various studies with these two compounds are probably attributable to their only moderate efficacy, the unsatisfactory specificity and the minimal bioavailability. Further research efforts had led to BAY x7195 (Bayer) and SKF 104353 (Smith-

SingulairTM, montelukast, L 706,63 1, MK 0476 MK 476 Merck, USA 0

151767-02-1 [317]

\

\

/

107753-78-6 [302]

Asthma (launched)

(launched)

zafirlukast,

Zeneca, USA

Asthma,

Indications (status*) Asthma, allergy (launched)

AccolateTM,

103 177-37-3 [296]

b

NamelCode Structure originator CAS-number [synthesis] OnonT", UItairT", pranlukast, ON0 1078, 8 ONO-RS-4 1 1, SB-205312 ONO, Japan

Table 12-4. Peptidoleukotriene antagonists

bc (LTD4): I 17-fold shift in LTDa dose-response curve 2 h after 40 mg oral dose; five fold shift persists at 24 h [304]. Effective in blocking by allergen following oral [305-3071 or aerosol [308,309] administration; by exercise following oral [310] or aerosol [31 I] administration; cold air [312]. Provides acute improvement in lung function [ 3 131. In a 6 [314] or 13 [315] week study lung function and relevant symptoms of stable asthmatics are improved. Improves symptoms in allergic rhinitis [3 161. bc (LTD4): more than 50-fold shift in LTDl24 h following 40 mg oral dose [3 181. Is effective and well tolerated in exercise-induced bc (10 mglday) [290]. A single oral dose of 100 mg and 250 mg produces significant increases in FEV, (8.6 and 8.5%, respectively), irrespective of the concurrent use of inhaled corticosteroids in asthmatic patients with airflow limitation [319]. 5 mg/day inhibits exercise induced bc in 6-14 year old asthmatics at the end of a once-daily dosing interval [320].

rc (guinea-pig Im): ICso = 0.5 nM (LTD4) tc (guinea-pig): pA2 = 9.3 (LTD4) [3171

bc (LTD4): 26-fold shift in LTD4 dose response curve 3.5 h after 5 days of 450 rng b i d . ; seven-fold shift at 24 h [287]. Antigen-induced bc is blocked 1298.2991. Analgesicinduced bc in aspirin sensitive asthmatics is effectively blocked [300]. In a 4 week chronic study improvement of lung function, symptoms and reduction of P-agonist usage are observed [301].

tc (guinea-pig): PKB= 7.5 (LTDd) [297]

rc (guinea-pig Im): Ki = 0.34 nM (LTD4) tc (guinea-pig): pAz = 9.5 (LTD4) [3031

In vivo data

In vitro data

L

VI

w

B

'4

3.

0

5i

tb

3

3 rn

a

??

z c

5.3

UI

9

4

FPL 557 12 Fisons, USA

Hoffmann La Roche, USA

Cinalukast,

Iralukast sodium, CGP 45715A Novartis, USA

J J J " "

Germany

1283 12-5 1-61 [327]

0

125617-94-9 [324]

@

[*I-

Asthma (phase 11)

Indications (status*) Asthma (phase 11)

bc (LTD4):eight fold shift in LTDJ dose-response curve 2 h after 250 mg oral dose [322]. 250 and 500 mg improves FEV, 5 h after administration to mild to moderate

tc (guinea-pig): pkB = 8.4 (LTD4) [321] asthmatics [3231.

In vivo data

In vitro data

rc (guinea-pig Im): Ic$o = 6.4 nM (LTD4) tc (guinea-pig): pA2 = 9.6 (LTD4) [3281

Asthma rc (guinea-pig Im): (discontinued) ICso = 4000 nM (LTD,) tc (guinea-pig): pA2 = 6.0 (LTD4) [330]

Asthma (phase 11)

Slight inhibition of LTC4 induced bc [33 11 and moderate improvement in lung function in some asthmatic patients [332].

Exercise-induced bc in mild asthmatic patients following oral administration is blocked [289].

rc (guinea-pig Im): Clinical studies are ongoing. K, = 26 nM (LTD3) 13251 tc (guinea-pig): pku = 6.28-7.92 (LTD.4, non-competitive) [326]

6mcF' TW@

143538-27-6 [321]

0

Loo

Structure CAS-number [synthesis]

Name/Code originator BAY x7195 B ayer,

Table 12-4. (Continued)

w

ul

3

f? -. 0

0

2

5

%

3. 0

2

&

a

R'

2

3 B

@

4

tu

VI

107023-41 -6 [345]

,rVCO,”

s m r n 2 ”

s ~ m I o 1 3 ’ z

Asthma (phase 11, discontinued)

Asthma (phase II, discontinued)

Indications (status*) Asthma (phase 111, discontinued)

In vivo data

rc (guinea-pig Im): K, = 5 nM (LTD4) tc (guinea-pig): pA2 = 8.6 (LTD4) [346]

rc (guinea-pig Im): ICso = 3.1 nM (LTD4) tc (guinea-pig): PKB= 8.8 (LTD4) [340]

bc (LTD4): ten fold shift in LTD4 dose-response curve 2 h following 100 pg aerosol dose in normal volunteers [347]. two to three-fold shift following 800 pg aerosol dose in mild asthmatics 13481. Antigen [349] and exercise [350] induced bc is blocked. Lung function in aspirin-sensitive asthmatics is improved [351].

Aspirin-induced bc is blocked (3411. Acute baseline lung function in asthmatics is improved by aerosol [342] or intravenous [343] application. Improves in a 6 week study lung function and some symptoms of asthmatics. In 5% of patients liver abnormalities are detected [344].

rc (guinea-pig Im): bc (LTD4): 4.6-6.I-fold shift in LTD4 bc dose-response Ki = 637 nM (LTD4) [334] curve following a 400 mg oral dose [335]. Small effects in different asthma models (exercise [336], cold air induced [337]). Improves in a 6 week study lung function and some symptoms of mild asthmatics [338].

In vitro data

*Represents the most advanced stage of development recorded in at least one of the indications listed. Methods and tests: tc, trachea contraction; bc, bronchoconstriction; rc, receptor; Im, lung membranes

Pobilukast, SKF 104353 SmithKline Beecham, UK

VenzairTM, % a verlukast, L-688,019, MK 679 Merck, USA 120443-16-5 [339]

I 88107-10-2 [333]

Name/Code Structure originator CAS-number [synthesis] Tomelukast, LY 171883 Eli Lilly, USA HO H

Table 12-4. (Continued)

W

v,

w

6

g.

a

00

3

zi

a

z

m

2.

2

fi

E

3m“

3

3

cll

h,

4

354

12 Synthetic eicosanoids: development and clinical upplicutions

Kline-Beecham) whose structures were based more closely on the natural substance. While the development of SKF 104353 was abandoned-probably due to lack of bioavailability-BAY x7 195 is undergoing phase I1 clinical trials. The clinical data of iralukast, which is in phase 11 and belongs, like FPL 55712 (Fisons) and LY 171883 (Lilly),to the structural class of hydroxyacetophones,are not yet pub1ished.A remarkable feature of iralukast as compared with the natural substance LTD4 is the inverted configuration of the stereocenters to which the thioether and hydroxy groups are attached. Likewise, cinalukast, a member of the structural class of styrylthiazoles, which is novel to the peptidoleukotrieneantagonists, proved to be effective in exercise-induced bronchoconstriction [289]. Montelukast (Merck) has been launched and can be regarded as the successor of verlukast. It is effective and well tolerated in exercise-induced bronchoconstriction (10 mg/day) [290]. So far no intolerance in conjunction with food or other medicinal products has been found [291]. The first peptidoleukotriene antagonists on the market were pranlukast (Ono) and zafirlukast (Zeneca), which are structurally highly different. The Ono compound improved the condition of chronic asthmatics (the relevant parameters were, e.g. baseline FEV and reduced P-agonist usage) in a 4 week multicenter study with 225450 mg p.0. twice a day [292]. Further investigations have shown that pranlukast also protects asthmatic patients against bronchoconstriction induced by Sulpyrin (aspirin analog) or by the allergen of the house-dust mite [293]. The use of pranlukast in seasonal allergic rhinitis is currently under clinical investigation. Zafirlukast is a highly potent competitive LTD4 antagonist. In several dose-finding studies an administration of 20 mg p.0. twice a day or 400 pg by inhalation proved adequate [294,295]. In randomized, double-blind studies the efficacy of zafirlukast was demonstrated in 1380 patients with mild to moderate asthma. It was possible to reduce the number of waking phases during the night, the intensity of asthmatic attacks and the consumption of P-agonists. Various lung function parameters such as FEVI were improved. The good tolerance and safety of zafirlukast was proved in more than 4000 patients, some of whom used the drug for more than 1 year. The adverse reaction rate (headache, nausea and diarrhea) was similar to placebo. Zafirlukast also alleviated the symptoms of seasonal allergic rhinitis.

12.6 Leukotriene B4 antagonists LTB4 was discovered by Borgeat and Samuelsson [352] in 1979. It is one of the most potent chemoattractants and stimulators for polymorphonuclear leukocytes (PMNLs) [353]. Besides the chemotactic motion LTB4 enhances chemokinetic motion and leads to increased adhesion, degranulation, superoxide formation and cytotoxicity of these cells and it is also able to induce neutrophil accumulation in viva Elevated levels of LTB4 can be detected in diverse inflammatory disorders such as psoriasis [354], rheumatoid arthritis [355], asthma [356-3581 and intestinal conditions [359] and it is concluded that LTB4contributes to the initiation or perpetuation of these diseases.

SB 20 1993 SmithKline

CGS 25019C Novartis, USA

Ono, Japan

ON0 LB-457,

VML-295 Eli Lilly, USA

NamdCode originator

IPr,N

150399-22-7 [379,380]

147398-01-4 [375]

134578-96-4 [372]

X",

152608-41-8 Na-salt 161172-5 1-6 free acid [369]

Structure CAS-number [synthesis]

Table 12-5. Leukotriene 8 4 antagonists

Peritonitis (mice) ED5o = 7.1 rngkg, p.0. (LTBI) ee EDXI= 0.58 mg/ear (AA) [3801

collagen arthritis (murine) in 1-10 mg/kg p.0. range [376]. EDl(a = 300 mg for CDI lb up. at 4 h in volunteers [378].

ct: = 2.4 nM (LTB4) CDl Ib up.: ICSO= 0.3 n~ (LTBI) na: ICs0= 0.1 nM (LTB4) [376] Psoriasis rc (human): Ki = 7.1 nM (LTB4) (phase 11, cm: Ic50 = 131 nM (LTB4) discontinued) nd: ICSO= 268 nM (LTB4) [380]

np (rat) ED50 = 3 mgkg at 4 h, 11 mgkg at 18 h ee and MPO release: EDSO= I .4 and 1.2 mgkg p.0. (AA) [377]. Effective versus

np (guinea-pig): EDSO= 25.6 mgkg, p.0. (LTB4) LTB4 induced neutrophil influx in guinea-pigs EDSO= 5.3 mgkg p.0. [373]. Inhibition of LTBd induced neutrophil cm following a 300 mg dose In humans [374].

rc (human): ICso = 0.4 n~ (LTB4) [36 11 cm: IC50 = 2 n~ (LTB4)

rc (human): K, = 3.7 nM (LTB4) cm: lCso = 700 nM (LTB4) ct: ICSO= 900 nM (LTB4) nd (human): ICSO= 1600 nM [373]

60 or 120 mg t.i.d. or 200 mg b.i.d. inhibits CDI Ib up. in volunteers [371].

rc (human): Ki = 25.3 nM (LTB4) cm: ICs" = 20.7 nM (LTB4) CD1 l b up. I C ~ = O3.9 nM [370]

Asthma (phase 11)

Atopic dermatitis, ulcerative colitis, Behget's disease (phase 11) Arthritis (phase 11)

In vivo data

In vitro data

Indications (status*)

s

VI VI

w

z

$

0

00

; b 3

m 3 n

6 E P2. ??

b,

p\,

%

LY 223982

CP 105696 Pfizer, USA

Ticolubant, SB 209247 SmithKline

NamelCode originator

117423-74-2 [385]

158081-99-3 [383]

154413-61-3 [381]

Structure CAS-number [synthesis]

Table 12-5. (Continued) In vitro data

rc (human): ICSO= 8.42 nM (LTB4) ct (human): ICso = 5.0 nM (LTBI) cm: ICSU= 940 nM (LTB4); CD1 Ib up. (human): pA2 = 8.03 [3841

Psoriasis, rc (human): ICSO= 13.2 nM (LTB4) na (human): ICSO= 100 nM (LTB4) asthma ct: ICso = 6 pM (LTB4) [386] (phase 11, discontinued)

Arthntis

rc (human): Ki = 0.88 nM (LTB4) Eczema (preclinical) cm: ICSO= 6.6 nM (LTB4) cm: ICSO= 1.3 nM ( I2R-HETE) nd: ICSU= I17 nM (LTB4) [382]

Indications (status*)

np (rabbit) EDSO= 3 mgkg, iv (LTB4) [386] Minimal systemic absorption, but no effect in psoriasis scores following 0 5 3 % topical application [387].

Neutrophil cutaneous influx (micelgp): EDSO = 4.2 mgkg10.26 mgkg, p.0. (LTB4) 1 m g k g completely inhibits collagen-induced arthritis in mice [383]. Blocks I2R-HETE skin inflammation (75% at 3 mgkg, p.0.) [286]. Effective in humans in the CD1 l b up. ex vivo assay (3611.

ee (mouse): ED511 = 20 pglear, topically and 18.7 mg/kg, p.0. (AA) ee (mouse) EDSO= 114 pglear (PMA) [382]

In vivo data

Ro25-4094 Hoffinann-La Roche, USA

RG 14893 RhBne-Poulenc Rorer, USA

SC-53228 Searle, USA

SC-41930 Searle, USA

NamdCode originator

0

3

0 0

0 0

150596-99-9 [394]

\ ? *O -

141835-49-6 13931

In vitro data

Psoriasis rc (human): Ki = 1 nM (LTB4) (discontinued) ct: ICso = 9 nM (LTB4) cm: ICSO= 2 nM (LTB4) 13941

Asthma rc (human): Ki = 2 nM (LTB4) (discontinued) na (guinea-pig): Ic50 = 0.8 nM (LTB4) [3931

rc (human): ICso = 2.2 nM (LTB4) Psoriasis, inflammatory ct: Icso = 32 nM (LTB4) bowel disease nd: ICso = 19 nM (LTB4) [391]

Psoriasis, rc (human): ICsO= 20 nM (LTB4) arthritis, n d Icso = 1080 nM (LTB4) inflammatory ct ICso = 832 nM (LTB4) [389] bowel disease (phase 11, discontinued)

Indications (status*)

G H

153633-01-3 [391,392]

H,CNH

120072-59-5 [388]

14-

~

Structure CAS-number [synthesis]

Table 12-5. (Continued)

bc (guinea-pig): EDSO = 0.07 mgkg i.v. (1 min pretreatment); EDSO= 0.4 mgkg p.0. (20 h pretreatment) (LTB4) W41

Intradermal LTB4 induced neutrophil ct in guinea-pig EDSO = 0.14 mg/kg, p.0. LTB4 induced neutropeniafneutrophilia in cynomolgus monkeys 3 mg/kg 2 min before challenge inhibits 61% and 73% [393].

ct (guinea-pig): EDSO = 0.07 rng/kg, P.o., duration = 21 h (LTB4) ct (guinea-pig): ED50 = 5.8 mglkg, p.0. (12RHETE) [391] ee (mouse) ED50 < 2.5 rng/kg, p.0. (PMA) [392]

LTB4 induced neutrophil chemotaxis in guinea-pig EDSO = 1.7 mgkg, p.0. duration = 5.5 h [390] ee ED50 = 4.1 FmoYear (PMA) [388]

In vivo data

a

VI 4

W

;rl

4

3.

3

3

Q

4

B

i

2.

0,

??

E

e

o\

9

+

Indications (status*)

162362-36-9 [395]

OH

Psoriasis (phase I)

62

Structure CAS-number [synthesis] rc (human): Ki = 15 nM (LTB,) ct KB = 21 nM [3651

In vitro data

ee + ni (mice) Ic50= 14-35 kg/cm2 (LTBJiloprost) ni (mice) Ic50 = 40 &&m'(AA) [3651

In vivo data

"Represents the most advanced stage of development recorded in at least one of the indications listed. Agents: MPO, rnyeloperoxidase; AA, arachidonic acid; PMA, phorbol myristate. Methods and tests: rc, receptor; cm, calcium mobilization; CDl l b up., CDl l b upregulation; ct, chemotaxis; nd, neutrophil degranulation; np, neutropenia; ee. ear edema; na, neutrophil aggregation; ni, neutrophil infiltration.

ZK 158252 Schering AG, Germany

Name/Code originator

Table 12-5. (Continued)

00

wl

W

12.6 Leukotriene B4 antagonists

359

On the basis of these findings, it might be assumed that LTB4 antagonists exhibit anti-inflammatory effects and, as a consequence, many companies have started rational compound-finding programs (for reviews see Jackson and Fleisch [286], Brooks and Summers [287], Cohen and Yagaloff [360], Sawyer [361] and Fretland and Penning [362]). The structural relationship to the natural substance LTB4 is not readily recognizable in many of the compounds listed in Table 12-5. Among the first active structures were LY 223982 (Lilly) and SC-41930 (Seacle) which were derived from already existing LTC& antagonists such as FPL 55712. Besides other structural modifications the methylation of the phenolic hydroxy group ultimately led to the compound SC-41930, which is selective for the LTB4 receptor (no affinity for the LTD4 receptor at concentrations up to 10 p f ) . The weak LTB4 antagonistic efficacy of this compound and also of LY 223982 (Lilly) in vivo necessitated the development of more potent substances. Structurally similar compounds were also developed by Ono, SmithKline Beecham and Hoffmann-La Roche. Whereas Ro 25-4094 was terminated because of its hepatotoxicity in animals [363], O N 0 4057 is undergoing clinical trials. The development of SB 201993 has been stopped, whereas SB 209247 is in the preclinical phase. The status of development of SC-53228 (Seacle), the successor to SC-41930, in the treatment of inflammatory bowel disease and psoriasis has not been reported. Like CP 105696 (Pfizer), this compound also inhibits 12-R-HETE (hydroxyeicosatetraenoic acid)-induced inflammatory effects, which could be of interest in the treatment of psoriasis. In contrast to all other LTB4 antagonists, CGS 25019C (Novartis) contains a basic amidine instead of the carboxylic acid pharmacophore but the significance of this structural feature for activity is still unclear. A dose-finding study revealed gastrointestinal imtation after administration of >500 mg CGS 25019C, which could limit the clinical application of the substance [361]. CP 105696 (Pfizer) has a novel chemical structure. In a phase I study this compound inhibited LTB4-induced CDl l b upregulation ex vivo. Despite the good tolerance of CP 105696 (40-640 mg) its development was terminated because of its extremely long half-life (ti12 = 420 h) [364]. The knowledge that simple para-benzyloxy-phenylacetamidebinds moderately to the LTB4 receptor led to the development of the naphthalenecarboxylic acid derivative, RG14893. The compound shows good results in vitro as well as in vivo, but its clinical development for the treatment of asthma has been discontinued. Schering AG’s ZK 158252 is derived from the natural substance LTB4. It showed potent anti-inflammatory effects after topical administration in mouse and guinea-pig skin inflammation models [365,366]. Based on its pharmacological profile it was assumed that ZK 158252 might be suitable for topical therapy of psoriasis. The inflammatory skin reaction after topical application of 200 ng LTB4 could be completely antagonized by a single simultaneous application of 1% ZK 158252 and 3% ZK 158252 corresponding to dosages of 455 and 1364 pg/cm2. In a prospective doubleblind, placebo-controlled, intraindividual comparison in 69 patients with guttate psoriasis the topical application of 3% (w/v) ZK 158252 once daily for 10 days had no significant effect on either the increasing size of the psoriatic papules or on erythema, thickness and scaling [367]. A compound already used in a number of human studies is LY 293 1 1 1 (Lilly),

360

12 Synthetic eicasanoids: development and clinical applications

which is structurally derived from LY 223982. In a double-blind, placebo-controlled study in 12 atopic asthmatics [368], bronchoalveolar lavage (BAL) fluid was investigated after oral administration of 112 mg LY 293111 Na salt three times a day for 7 days before application of the antigen. A pronounced reduction in LTB4, LTC4, LTE4, elastase, myeloperoxidase and interleukin-8 were found under these conditions. The resulting anti-inflammatory effect is possibly supported by additional mechanisms. The number of neutrophils in the BAL fluid was also reduced while eosinophils and lymphocytes were practically unaltered. In a second double-blind, placebo-controlled study, LY 2931 1 1 Na salt was given orally to 20 healthy volunteers (48 or 200 mg per day for 7.5 days). Significant suppression of the neutrophil CD 11b expression was seen in immunohistochemical examinations of comparative skin biopsies. There was, moreover, a dose-dependent suppression of LTB4-induced inflammation and hyperproliferation together with significant regression of epidermal neutrophil migration. This study confirmed the epidermal bioavailability and the anti-inflammatory effect of this compound after oral application and, in principle, opened up the possibility of oral treatment of psoriasis.

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Prostaglandins, Leukotrienes and Other Eicosanoids: From Biogenesis to Clinical Application Edited by F. Marks, G. Fürstenberger Copyright © WILEY-VCH Verlag GmbH, D-69469 Weinheim (Federal Republic of Germany). 1999

Index

ACE inhibitors 281, 283 acetylsalicylic acid see aspirin acrosome reaction 223 active lupus nephritis 173 adenosine 179 adenosine-induced bronchoconstriction 250 adenylate cyclase 175, 177 adrenic acid 2 aging 24 airway obstruction - adenosine 250 - allergen-induced 244ff - exercise 250 - platelet activating factor 250 - sulfur dioxide 250 aldehyde reductase 100 allene oxide cyclase 27, 3 1 allene oxide synthase 12,31, 1 11 allergic inflammation 234 allergy 248 allylic hydroxylation 144 Aloxe gene 126 Alprostadil 225 Alzheimer’s dementia 24 amnion 221 analgesic-associated nephropathy 18 I anandamide see arachidonylethanolamide anaphylatic reaction 7 angina 286 angiotensin 38, 179 antarafacial attack 12 anti-leukotrienes 246,254 - aspirin intolerance 252 -asthma 253 antioxidants 22,3 11 - antitumor effect 324 anti-thrombin 111 343 AP- 1 transcription factor 307, 3 17 APC gene 3 14,320,322 aplydilactone 29 Aplysia californica 19 - 12-lipoxygenase 29

- neurotransmitter release 29

apoptosis 3 16 - cyclooxygenase-2 322

- non-steroidal antiinflammatory drugs 322 arachidonic acid - as intracellular mediator 6ff - autoxidation 2 1 - biosynthesis 2 - bisallylic hydroxylation 149ff, 151 -cascade 89 - epoxides see epoxy-eicosatrienoic acids - epoxygenase 147, 186 - - renal hypertension 188 - hydroxylation 20, 146ff, 186 --kidney 186 - w-hydroxylation 146ff, 186 -in plants 30 - monooxygenation 20,143ff, 185 - reincorporation 7, 48 --albumin 48 - - arachidonyl coenzyme A synthase 48 - - lysophosphatide acyltransferase 48 - - phospholipase A2 48 - - proteinkinase C 48 arachidonyl coenzyme A synthase 48 arachidony I-CoA-1y sophospholipid transferase 7 arachidonylethanolamide 4,24ff, 237 - biosynthesis 25 - cannabinoid receptors 24,36 - cyclooxygenases 26, 78 - endothelium-derived hyperpolarizing factor 25 - lipoxygenases 26 - metabolism 26 - signal transduction 24 - tissue levels 25 Armadillo 320 aryl-hydrocarbon receptor 68 aspirin 4, 17,38,212, - antitumor effect 3 15 - cardiovascular diseases 282

376

Index

- intolerance 25 1 - myocardial infarction 275

-

- platelet aggregation.281

- 12-lipoxygenase 325

- pregnancy-induced hypertension 214 - resistance 282 - restenosis 284 -stroke 275 - thromboangiitis obliterans 342 - vasoconstriction 28 1

aspirin-intolerant asthmatics 237 asthma 15, 233, 242, 244, 246, 250 atherosclerosis 24, 38, 133, 275, 278 -aspirin 283 - 15-lipoxygenase 13Iff - risk factors 278 ff Atherosclerosis Risk in Communities Study 279 atopic disorders 124 autocoids 35 autocrine dysregulation 3 19 Bartter syndrome 287 Bcl-2 protein 322 beta-trace protein 97 bisallylic hydroxylase activity - P-naphtoflavone 150 - Clofibrate 150 - glucocorticoid 150 - phenobarbital 150 bisallylic hydroxylation 149ff - arachidonic acid 150 - linoleic acid 149 blood-bathed organ technique 295 bronchial hyper-responsiveness 248 bronchoconstriction 15,22,250 Buerger's disease see thromboangiitis obliterans Ca2+channel inhibitors 283 Ca2' channels 37 Ca2+ionophore A23 187 15 Ca2+mobilization 20, 36 cadherin 320 CaLB (calcium-dependent lipid binding) domain 53 calcium-independent phosholipase A2 see phosholipase A2 cancer 24,38,305ff

- cyclooxygenase-2 7 1,3 12ff

initiation 305

cannabinoid receptor 24,36 carcinogenesis 305 - cyclooxygenases 72,3 12ff - lipoxygenases 323 $-catenin 320,322 P-catenidArmadillo pathway 321 cc4 22 chiral HPLC 154 chirality analysis 154 ff chronic intoxications 24 c-Jun terminal kinase 3 17 Clofibrate 186 Cotrimoxazole 189 colorectal cancer 320 -APCgene 314 - aspirin 315 - chronic inflammation 315 - cyclooxygenase-2 3 15 - dietary factors 315 - genetic defects 314 - non-steroidal antiinflammatory drugs 3 15 - role of prostaglandins 3 15 coronary heart disease - w-3 fatty acids 279 corpus luteum 38,201 corticotropin-relasing hormone 2 16 cPLA2 see phospholipase A:! curcurbic acid 31 cyclic AMP 35, 37,281 cyclic AMP-dependent protein phosphorylation 37,53 cyclin D 326 cyclin-dependent kinase 2 inhibitor p21/Wafl/Cipl 326 cyclin-dependent kinase 4 326 cyclooxygenases 1, 10,65ff -cloning 66 - enzyme reaction 65, 78 - HODE formation 78 - isoforms 66 - mechanism 12 - metabolic activation of carcinogens 3 10 - myocardial infarction 278 - peroxidase activity 65 - X-ray crystallography 82 cyclooxygenase- I 66ff, 96 -aspirin 283

Index

- carcinogenesis 72 -cloning 67 - endothelium 283 - gene knock-out 80,313 - gene structure 67 - gene therapy 293 - glomerulonephritis 173 - glycosylation 74 - induction 69 - - by platelet-derived growth factor 173 - inflammation 80 - inhibitors 81 - kidney 170 - molecular weight 74 -mRNA 72 - NSAID binding site 75 - overexpression in tumors 3I4,3 18 - phospholipase A2 coupling 77 -placenta 213 - platelet aggregation 79 - platelets 283 - skin carcinogenesis 313 - stomach protection 79 - structural elements 73 - subcellular localization 76 - substrates 78 - tumor development 3 13 - vascular system 280 - X-ray crystallography 74 cyclooxygenase-2 96,66ff, 3 12 - acetylation 17,82 - acute inflammation 71 - antiinflammatory function 39 - apoptosis 322 -aspirin 283 -brain 71,SO - cancer 71,80,314ff, 3 I7 - carcinogenesis 72 - colorectal cancer 3 15ff - control of renin secretion 180 - endothelial cells 282 - 15-epi-lipoxinformation 17 - fertility 80 -fever 80 -gene knock-out 80, 172,201,210,313, 316 - gene structure 68 - glomerulonephritis 173 - glycosylation 74 - high- and low-salt diet I80

- high-fat diet 3 17 - immediate early response protein 69 - implantation 2 10

- induction 68ff, 70,72 - - azoxymethane 317

--GnRH 202 - - gonadotropins 200,202 - - interleukin-1 171 - - platelet-derived growth factor 173 - - secondary bile acids 3 15 - - thromboxane A2 175 - - vasoconstrictors 172 - - via MAP kinase cascades 69,3 18 - - forskolin 69

--hormones 68 - - infections 79

- - inhibition by cytokines 72 - - inhibition by glucocorticoids 72 - - lipopolysaccharide 68 --NF-KB 68

- - phorbol ester 68,3 I2,3 18ff --stress 69

- inflammation 79ff - inhibitors 75, 81ff - - anti-tumor effect 3 16 - - nephrotoxic effect 181 - - pre-eclampsia 21 6 - - teratogenic effect 216 -kidney 71,79, 171

- labor 221,222 - 15R-lipoxygenaseactivity 17,82, 283 - luteolysis 202 - metastasis 324, 325

- molecular weight 74 -mRNA 72 - natriuresis 180 - NSAID binding site 75 - osteoarthritis 71 - overexpression in tumors 313, 3 17 - ovulation 80,200 -pain 80 - permanent expression 69,7 1 - phospholipase Az coupling 77, 3 17 - placenta 2 13 - platelets 283 - pre-eclampsia 2 15 - pregnancy 222 - pre-term labor 223 - rheumatoid arthritis 7 1 - skin cancer 3 17

377

378

Index

- skin carcinogenesis 3 12ff - structural elements 73

- subcellular localization 76

cytosolic phospholipase A2 see phospholipase A2

- substrates 78

- suppression by cyclosporine A 171 - transcriptional activation 68 - tumor promotion 3 12 cyclopentenone prostaglandins 9, 39 - as anti-cancer drugs 39, 326 - anti-viral activities 326 - apoptosis 326 cyclosporine A I86 cyclosporine A - related hypertension 173 Cymathere ether 33 cysteinyl leukotrienes - airway constriction 241,244,249 - allergy 124,244 - antagonists (see also anti-leukotrienes) 15, 120,241,245,249,349 - aspirin intolerance 252 - asthma 124,242,244 - blood pressure 242 - bronchial hyper-responsiveness 248 - cardiac contractility 242 - chemotaxis 248 - eosinophil recruitment 246,248 - glomerulonephritis 182 - implantation 210 -kidney 182, 184 - mast cell 245 - metabolism 182 - mucus secretion 246 - placenta 212 - receptors 36, 182,242,245 - rhinitis 154 - sooth muscle proliferation 248 - vasoconstrictor activity 182 cysteinyl lipoxins 16 cytochrome P-450 4,20, 11,91,93, 143ff - allylic hydroxylation 144 - bisallylic hydroxylation 145 - epoxidation 145 - fatty acid o-oxidation 144 - whydroxylation of arachidonic acid 146 ff - kidney 185 - linoleic acid oxygenation 145, 149 - reductase 143 - superfamily 143 cytoprotective effect 337

DAG see diacylglycerol DAG lipase see diacylglycerol lipase dehydro-epiandrostendione 2 16 15-desoxy prostaglandin J2 39,76,326 diabetes 175, 278 diacylglycerol 8,35,56, 306 - lipase 56 didemnilactone 29 Diet and Reinfarction Trial 279 DiHETE 112 13,14-dihydroprostaglandinEl 292 5S, 12R-dihydroxyeicosatrienoicacid 240 DHET see dihydroxy-eicosatrienoicacids 21, 152 dihydroxyhexadecadienoic acid 158 dihomo-y-linolenic acid 2 DiHPETE 112 dihydroxy-eicosatrienoicacids 2 1, 152, 157 - chirality analysis 155 dioxin-responsive element 68 dishevelled 320 dopamine 187 dysmenorrhea 206 dysplastic aberrant crypt foci 3 14 ectopic pregnancy 203 EDHF see endothelium-derived hyperpolarizing factor EET see epoxy-eicosatrienoic acids eicosanoids - families 5 -fish 30 - functions 34 - inactivation 5,34 - invertebrates 27ff - kidney function 38, 169ff - metabolic stability 34 - modulatory function 7 - myometrium 222 - receptors 35ff, 38, 173ff elicitors 31 endocannabinoids 27 endometriosis 207 endothelial function 280 endothelin 22,220,28 1

Index endothelium-derived hyperpolarizing factor 25, 153, 156,281 endothelium-derived relaxing factor 158 enzyme-based immunoassay 297 EPI receptor see prostaglandin E2 receptor EPBP4 receptors see prostaglandin E2 receptor EP3 receptor see prostaglandin E2 receptor epi-lipoxins - antiinflammatory function I8 - biosynthesis 17 - functions 17 - structure 17 epoxidation 147 epoxigenases 20, 142ff epoxy-eicosatrienoic acids 20, 147ff - adrenal glomerulosa cells 161 - atherogenesis 159 - biological effects 156 - central nervous system 160 - cerebral microcirculation 160 - chirality analysis 152, 155 ff. - decidua 221 - epoxide hydrolases 152 - gas chromatography/mass spectrometry 154 -heart 158 - hypothalamus 160 - incorporation into phospholipids 153 - Kca channel 159 f. - kidney 156, 186 - metabolism by PGH synthase 159 -ovary 161 -pituitary 160 -placenta 161, 212 - radioimmunoassay 154 - steroidogenesis 201, 21 2 - vascular system 158 - vasodilator effect 187 epoxy-prostaglandins 159, 188 epoxystearic acid 153 erectile dysfunction 224 erythropoiesis 133 exercise-induced bronchoconstriction 250,258 expoxide hydrolase 21 extraction method of Powell 294 fallopian tube 202

379

familial adenomatous polyposis 3 14 FAP see familial adenomatous polyposis fatty acids 1 ff - autoxidation 1, 21 - -binding proteins 7 - A5-desaturase 2,278 - A6-desaturase 2,278 - - atherosclerosis 278 - - plasma cholesterol level 278 - elongase 2 -essential 1 - 03 fatty acids 2,279 - 06-fatty acids 2 - o-oxidation 144 - polyunsaturated 1, 278 - - atherosclerosis 278 - - blood pressure 278 --infood 278 - - myocardial infarction 278 Fc receptors 234 ferric nitrilotriacetate 3 12 fish oil - hyperlipidemia 279 - prevention of atherosclerosis 279 - restenosis 284 FLAP see 5-lipoxygenase activating protein FMRFamide 29 follicle-stimulating hormone 200 frizzled 320 functional hyperemia 7 gangrene 342 gap junctions 222 GC-MS see gas chromatography/mass spectrometry gas chromatography/mass spectrometry 154,294 gene knock-out - cyclooxygenase-1 80,313 - cyclooxygenase-2 80, 172, 201,2 10, 3 13, 316 - 5-lipoxygenase 123,248 - 12-lipoxygenase 127 - 15-lipoxygenase 133 gene therapy 293 genetic instability 306, 308, 31 1 genotoxic carcinogens 305 glaucoma 336 glomerulonephritis 173, 175

380

Index

- cyclooxygenases I73 - cysteinyl leukotrienes 182

- lipoxins 184 glucocorticoid 254 glutathione peroxidase 110 glutathione S-transferases 101,243 glycogen synthase kinase 3p 320 G proteins 35, 37, 174, 175 GTPase activating proktin 8

- discovery 109

- functions I29 - implantation 210

- in skin tumors 318 - integrin expression 325 - in tumors 323

- immunosuppression 2 14 - metastasis 129, 325 - placenta 21 2 - platelet aggregation 129

- pre-eclampsia 214 halicholactone 29 hamster cheek pouch model 255 heme-thiolate protein 91, 93, 143 heparin-associated thromobcytopenia 342 hepatorenal syndrome 24, 191 hepoxilins 19ff - Aplysia californica 29 -barnacles 30 - bios nthesis 19, 127 2Y - Ca mobilizing effect 20 - functions 20 - potassium channels 20 - presynaptic inhibition 20 - receptors 20 hereditary non-polyposis colorectal carcinoma 3 14 HETE -decidua 221 - placenta 21 1 -receptor 12 - steric analysis 154 5-HETE - apoptosis 324 - in skin tumors 3 18 - uterine contractility 220 7-HETE 150,151,154 8-HETE - in skin tumors 318 8R-HETE -Hydra 30 -starfish 30 8R-HPETE 27 8S-HETE 39 10-HETE 150,151,154 1lR-HETE 30 12-HETE 161,183 - Aplysia californica 29 - apoptosis 324 - chemotactic activity 129

- proteinkinase C 325

- receptor 325 - uterine contractility 220 I2H(P)ETE

- pharmacological effects 13 12R-HETE 150, 151,283 -kidney 186 - psoriatic skin 129 12s-HETE - skin 129 - in tumors 325 13-HETE 150, 151,154 15-HETE - apoptosis 324 - functions 134 - in skin tumors 3 18 -intumors 323 - ovulation 201

15-HETE-phosphatidylinositol 133 15R-HETE 150, 151 15s-HETE -kidney 184 18R-HETE 146,148 19-HETE 146, 150, 151, 153, 156 - kidney 186 - NafK+-ATPase 187 - modulation of ion channels 187 - vasoconstrictor activity 20-HETE 146,150,151,153, 156 - endothelium-derived relaxing factor 158 - vascular system 158 - kidney 157, 186 -pituitary 160 - renal hypertension 188 - vasoconstrictor activity 188 high performance liquid chromatography see HPLC hippocampus 29 HODE 150, 154

Index 9-HODE 13 13-HODE 13 -functions 134 - in diacylglycerol 133 - in tumors 323 HPETE -receptor 12 HPLC 212,296 HPODE 13, 114 Hydra 30 Hydractinia 30 hydroperoxide isomerase 12 hydroperoxide lyase 34, 1I 1 hydroperoxy eicosatetraenoic acid see HPETE hydroperoxy octadecadienoic acid see HPODE hydroxy eicosatetraenoic acid see HETE 12-hydroxy heptadecatrienoic acid 91,94 hydroxy octadecadienoic acid see HODE hydroxy octadecatrienoic acid 3 1 15-hydroxy prostaglandin dehydrogenas see prostaglandin dehydrogenase 19-hydroxy prostaglandin E 223, 224 hydroxylation with double bond migration 144

hypercholesterolemia 159 hyperpolarization 36 hyporeniemic hypoaldosteronism 181 idiopathic intrauterine growth retardation 21 1 IKB 316 immunoglobulin A nephritis 173 immunoglobulin E 234 immunosuppression 224 - by prostaglandin E2 309,322 - non-steroidal antiinflammatory drugs 309 - tumor development 309,322 impedance platelet aggregation test 295 implantation 208 indomethacin 3 12 inflammation 7,233 - cyclooxygenase-2 7 1 initiation-promotion approach of carcinogenesis 3 12,318 inositol- 1,4,5-trisphosphate 35 insects 29 insulin-like growth factor 21 1

invertebrates 27 IP3 see inositol-l,4,5-trisphosphate iPLA2 see phosholipase A2 isoleukotrienes 237,287 isolevuglandins 24 isoprostanes 4, 21ff, 287 -assay 288 - biosynthesis 22, 190 - dioxetane mechanism 23 - functions 22 -kidney 190 - lipid peroxidation 22 - lung injury 237 - nomenclature 22 - platelet aggregation 288 - receptor 191, 288 - release from phospholipids 22 - renal vasoconstriction 191 - scleroderma 288 -smoking 288 - stereochemistry 23 - vasoconstriction 288 jasmonates - biosynthesis 31 - defense reactions 3 1 - stress situations 33 - wound healing 3 1 jasmonic acid 3 1

KETE see ketoeicosatetraenoic acid keto-eicosatetraenoic acid 113 12-keto-eicosatetraenoicacid 29 1Cketo PGF2, 30 15-keto-prostaglandinA13-reductase 6 6-keto-prostaglandin Fia 294 9-keto-prostaglandin isomerase 224 labile aggregation stimulating substance 4 labor 220,222 LASS see labile aggregation stimulating substance leukemia inhibitory factor 210 leukocyte enhancing factor 320 leukocyte degranulation 38 leukocyte motility 37 leukotoxin 153 leukotrienes 13, 121, 236 -A4 14

381

382

Index

- airway tissue 237 - B4 14,39, 120 - - antagonists 241,356 - - chemotaxis 239 - - leukocyte activation 240

--lung 240 - - receptor 240 - biosynthesis 13, 120 -c4 14,120 - - synthase 14, 120 - - in kidney 182 - - y-glutamyl transferase 14 - chemotaxis 15, 239 - D4 14, 120 - - dipeptidase 14 - functions 15 - inflammation 124 - immune system 124 - implantation 2 10 - kidney 181 - mast cell 235 - menstruation 204 - ovulation 201 - placenta 21 1 f. - receptors 15, 36, 240 - - antagonists 2 10 - uterine contactility 220 - ulcerative colitis 124 levuglandins 24 linoleic acid 2, 13, 130 - biosynthesis 2 - bisallylic hydroxylation 149 - cycloxygenases 78 - epoxidation 153 - formation of plant oxylipins 30 -in food 219 - lipoxygenases 13, 114 - octadecanoid formation 13 - oxygenation by cytochrome P-450 145 a-linolenic acid 2, 13, 30,32,33, 77, 130, 279 y-linolenic acid 2 lipid peroxidation 1 1, 22, 190,288, 3 1 1 lipopolysaccharide 96 lipoxidase 4, 109 lipoxins 15ff - airway constriction 243 - airway tissue 237 - antiinflammatory mediators 17, 184 - biosynthesis 15, 128, 183

- Ca2+ionophore A23 1 87 15

- chalone function I7 - chemotaxis 243 - functions I7

- glomerulonephritis 184 - natural killer cells 243 - receptor I7,36,243 - transcellular formation 16 - vasodilation 243

lipoxygenase domain 121 lipoxygenase-like enzyme 1 12 lipoxygenases 4, 109 - antitumor effect 324 - carcinogenesis 323 - domain structure 116 - double or triple oxygenation 1 12 - epoxy-leukotriene formation 113 - implantation 210 - inhibitors 249 - iron binding 116 - hydroperoxidase reaction 1 12 -in plants 30 - linoleate oxygenation 114 - mechanism 12, I10 - octadecanoid formation 13 - overexpression in tumors 323 - ovulation 201 - positional specificity 1 18 - stereoselectivity 11, 115 - substrate binding region 1I8 - suicidal inactivation 112 5-lipoxygenase 14, 114, 120 - activating protein 15, 122, 181 - activation by ATP and Ca2+ 122 - apoptosis 324 - corpus luteum 202 - expression 123 -gene 123 - gene knock-out 123,248 - hydroxyperoxidase reaction 122 - inducers 124 - inhibitor --asthma 246 - in tumors 323 - kidney 182, 183 - leukotriene formation 13, 124, 236 - lipoxin formation 16 - membrane association 122 - suicidal inactivation 122 - tissue distribution 123

Index 8-lipoxygenase 39, I 14, 115, 130 - in tumors 323

8R-lipoxygenase 27 1 2-lipoxygenase 1 1, 1 14, 125ff - Aplysia californica 29 -cancer 325 - corpus luteum 202 - expression 127 -genes 126 - gene knock-out 127 - hepoxilin formation 18, 127 - inducers 127 - in tumors 323, 325 -isofoms 125 - kidney 183 - lipoxin formation 15, 128 - myocardial infarction 278 - properties 125 - substrate specificity I25 - suicidal inactivation 126 - tissue distribution 127 15-lipoxygenases 1 14, 129 - activation by hydroperoxides 132 - activity 125 - antiinflammatory activity 134 - atherosclerotic lesions 131, 133 -calcium 132 - epidermis type 130 - expression 131 -gene 131 - gene knock-out 133 -isofoms 129 -kidney 183 - leukotriene formation 120 - lipoxin formation 15 - 12-lipoxygenase activity 130 - properties 120, 129 - reticulocyte maturation 133 - substrate binding region 120 - substrates 119 - substrate specificity 130 - suicidal inactivation 130 - tissue distribution 131 - two-domain structure 1I7 - X-ray crystallography116 long-term potentiation 8 low-density lipoproteins 39, 133,276, 279 LTA4 hydrolase (see also leukotriene A4) 14,120 - kidney 182

383

LTB4 see leukotriene B4 LTC4 see leukotriene C4 luteolysis 20 1 luteotropic hormone 200 LXA4 hydrolase 16 Lyon Trial 279

malignant progression 305, 308 malondialdehyde 91,94,3 10 MAP kinase 7,55,317,323 - kinase cascades 37,3 18 f. - - tumor promotion 307 mass spectrometry 295 mast cell 234 f. meadacid 2 mediterranean diet 279 menorrhagia 205 menstruation 203 metabolic activation of carcinogens - cytochrome P-450 310 - prostaglandin H synthase 3 10 metastasis 324 1-methyl-adenine 30 Min mouse 3 14,320 monooxygenase see cytochrome P-450 multistage carcinogenesis 305 mutator phenotype 306, 3 1 1 myocardial infarction 278,285 natriuresis 180 neural disease - cyclooxygenase-2 7 1 neuroprostanes 24 neurotransmitter release 38 NF-KB 316 nitric oxide 96, 132, 158, 179,254,281 - synthase 96 nitrovasodilators 28 1 NMDA receptor 8,71 NO see nitric oxide non-genotoxic carcinogens 305 non-steroidal antiinflammatory drugs 4, 38, 66,236,320 - allergic inflammation 255,256 - anti-conception 210 - antitumor effect 3 1 1,3 15, 3 17,322 - apoptosis 316, 322 -asthma 2S2

384

Index

- atherosclerosis 38 - Bartter syndrome 287

-cancer 38 - cardiovascular diseases 282

- classification 8 1 - cyclooxygenase-2 expression 320 - cyclooxygenase inhibition 8 I

- damage of gastric mucosa 336 - fallopian tube 203 - hypertension 18I - implantation 210 - intolerance 25 1

- kidney 179,180, I 81 - kidney damage 286 - luteolysis 202

- menstruation 206 - nephrotoxic effects 181 - pre-eclampsia 2 15 - restenosis 284 - side effects 81 - stomach protection 38 NSAID binding site 75 NSAID see non-steroidal antiinflammatory drugs octadecanoids 4, 13 17-octadecenoicacid 189 okadaic acid 3 12 oleic acid 2 oncogenic mutation 305 osteoarthritis 71 ovulation 200 oxidative stress 22,3 1 1 - cancer development 308 - eicosanoid biosynthesis 308 - genetic instability 308 - mutator mechanisms 308 12-0xo-PDA 3 1 oxylipins 1 oxytocin 220 p38 MAP kinase 316 p53 tumor suppressor gene 31 1 parathyroid hormone 187 parturition 216 PDGF see platelet-derived growth factor percutaneous transluminal coronary angioplasty 283,343

peripheral arterial occlusive disease 342 peripheral vascular disease 293 peroxisome proliferator-activated receptor 39, 76,3 16,323, - antiinflammatory function 39 - 15-desoxy prostaglandin J2 39 - leukotriene D4 39 -8s-HETE 39 peroxygenases 3 1, 1 1 1 pertussis toxin 20 PGEo see 13,14-dihydroprostaglandinEl PGG peroxidase 10 15d-PGJ2 see 15-desoxy prostaglandin J2 PGH synthase see cyclooxygenase PGHS see cyclooxygenase phorbol esters 306,3 12,318 phosphatidic acid phosphohydrolase 56 phospholipase 1, 10,239 phospholipase A1 49 phospholipase A2 37,48 ff, 282 - activation 7,58,3 18 - calcium-independent phospholipase A2 56 - cytosolic phospholipase A2 52 - - activation 54 --calcium 53 - - expression 52 - - Gprotein regulation 55 - - lysophospholipase activity 53 --nucleus 55 - - phosphorylation 53,55 - implantation 21 0 - interaction with cyclooxygenase 76, 173 -kidney 172 - macrophages 57 - menstruation 204 - secretory phospholipase A2 5 1 ff - - expression 52 - - receptor 52 - - stimulation of release 5 1 - upregulation in tumors 3 17 phospholipase C 56, 133, 174 ff phospholipase D 25 f., 56 phospholipid hydroperoxide gluthathione peroxidase 111 PIH see pregnancy-induced hypertension pINF114 17 PKC see proteinkinase C placenta 21 1 plant oxylipins 12. 3 1 plant wound hormones 33

Index plants - arachidonic acid 30 - defense reactions 3 1 - linoleic acid 30 - a-linolenic acid 30 - lipoxygenases 30 - wound healing 31 platelet activating factor 209 f., 25 1, 28 1 - (PAF) acetyl hydrolase 5 1 platelet aggregation 38 platelet-derived growth factor 173 PLC see phospholipase C PLD see phospholipase D Plexaura homomalla prostaglandin A2 27 potassium channels 8,29,36 PPAR see peroxisome proliferator-activated receptor preclavulone A 28 pre-eclampsia 2 I4 pregnancy-induced hypertension 156,2 14ff pre-term labor 222 proadifen 158, 160 programmed cell death 316,322 prostacyclin 4,9, 10,38 - heparin-associated thrombocytopenia 342 - myocardial infarction 342 - pregnancy-induced hypertension 2 14 - Raynaud’s phenomenon 342 - thromboangiitis obliterans 342 - thrombotic microangiopathy 342 - synthase --cloning 93 - - enzymatic reaction 95 - - induction 96 - - inhibition by peroxynitrite 96 - - isolation 93 --kidney 171 - - placenta 213 - - pre-eclampsia 216 - - properties 94 - - tissue distribution 96 - anti-thrombotic activity 343 - bleeding tendency 278 - blood pressure 337 - bronchoconstriction 255 - cervical ripening 2 18 - clinical applications 289 -function 96 - heart muscle activity 285

385

- kidney 175,184

- menorrhagia 205 - menstruation 205

- myocardial contractility 285 - myocardial infarction 278 - nitric oxide 281 - organ transplantation 341 - peripheral arterial occlusive disease 341 - placenta - platelet aggregation 281, 283 - pulmonary hypertension 341

- receptor 36, 175 - receptor desensitization 292

- restenosis 284 - side-effects 292 - structural stabilization 341 - synthetic derivatives 337 - vasoactive drugs 28 1 - vasodilatation 28 1

prostaglandins -amnion 221 - atherosclerosis 282 - biosynthesis 10,65,97ff - estradiol 21 1 -kidney 286 - progesterone 2 11,2 13 - endoperoxides 4, 10,65 - epoxidation 159 - expression of cell adhesion molecules 282 - invertebrates 27 - lactones 27, 29 - metabolism 6 - myocardial infarction 282 - nomenclature 9 - overproduction in tumors 3 17 - ovulation 200 - parturition 218 - placenta 2 1 1 - receptors 175 - renin release 176 - structural analysis 3 - synthases - - tissue distribution 102 - synthetic derivatives 334 -yeast 27 prostaglandin A2 9,28,40,326 -incorals 28 -kidney 286 prostaglandin-associated gastric mucosal hyperplasia 292

386

Index

prostaglandin D 9,238 prostaglandin D synthase - isolation 97 - properties 97 -subtypes 97 - inhibitors 98 - site-directed mutagenesis 98 - sleep-wake rhythm 98 - structure 98 prostaglandin D2 39 - allergic inflammation 256 - bronchoconstriction 256 - mast cell 235 - mast cell activation 256 -receptor 36 prostaglandin D2 1l-ketoreductase 100 prostaglandin dehydrogenase 6 - menstruation 204 - pregnancy 22 1 - pre-term labor 223 prostaglandin E synthase 99 prostaglandin El - acute renal failure 286 - clinical applications 293 - erectile dysfunction 225 - hypolipidemic actions 292 - pediatrics 336 - peripheral arterial occlusive disease 336 - platelet aggregation 288 - seminal fluid 223 - side-effects 292 - vasodilation 225 prostaglandin E2 - acrosome reaction 224 - allergic inflammation 238,255 - antiinflammatory effects 255 -asthma 257 - bronchoconstriction 250 - bronchoconstrictor tachyphylaxis 258 - bronchodilation 255 - bronchoprotection 257 - cervical ripening 218 - collagenase production 219 -cough 259 - cutaneous atopic reaction 259 - cytoprotective effect 336 - decidua 221 - endometriosis 207 - fallopian tube 203 - immunosuppressive effect 224,309, 322

- implantation 209 - in skin tumors 318

- labor induction 220, 336 - menorrhagia 205 - menstruation 204 - receptor 36, 176

-RU486 219 - seminal fluid - uterine contractility 220 prostaglandin endoperoxide - menorrhagia 205 prostaglandin F2a - ariway constriction 238 - endometriosis 207 - decidua 221 - fallopian tube 203 - glaucoma 337 - hyper-responsiveness 255 - in skin tumors 3 I8 -kidney 175 - luteolysis 202 - menorrhagia 205 - menstruation 204 - myometrium 222 - receptor 36, 175 - tumor promotion 3 12,319 - uterine contractility 220 prostaglandin F synthase - enzymatic reaction 101 -isolation 100 - multiplicity 101 prostaglandin G 9,65 prostaglandin H 8, 65 prostaglandin H synthase see cyclooxygenase prostaglandin H-E isomerase see prostaglandin E synthase prostanoids 8 f. - bioassays 295 - biosynthesis 10, 169 - formation by glutathione S-transferases 101 -HPLC 296 - - corpus luteum 202 --kidney 173 - mass spectrometry 295 - radioimmunoassay 296 - receptors 35, 175 proteinkinase A 53 proteinkinase C 7,37,48,53,55, 133, 176, 319 - phorbol esters 306

Index - tumor promotion 306

P-selectin 184 psoriasis 129,356 rabbit aorta contracting substance 4 radioimmunoassay 296 Raf- 1 307,3 19 Ras 8,319 - -Raf-Erk cascade 308,3 18,319 -GAP 8 Raynaud’s phenomenon 342 RCS see rabbit aorta contracting substance reactive oxygen species 22 receptors 35ff, 173ff - cannabinoid 24, 36 - leukotriene 236, 35 - lipoxin 35 -nuclear 38 peroxisome proliferator-activated 39.76, 3 16,323 - prostaglandin 175 - prostanoid 35, 173,236,239,277 regenerative hyperplasia 3 12 relaxin 219 renin 176, 180 - -angiotensin system 179, 286 reproductive system 38 resistance to aspirin 282 respiratory distress syndrome I53 restenosis 283 reticulocyte maturation 133 retinoic acid receptor 39 retinoid transporter 98 rheumatoid arthritis 7 1 RU486 209 salicylic acid 3 16 scleroderma 24 secretion 36 secretory phosholipases A2 see phosholipases A2 selenium 111 self-promotion 322 sepsis 175 serum eosinophil cationic protein 254 skin cancer 305,3 17,3 18 skin carcinogenesis 308ff, 3 I2 - HETE production 3 18

387

prostaglandin production 3 I8 sleep-wake rhythm 102 slow-reacting substance of anaphylaxis (SRS-A) 15,234,349 smoking 24 smooth muscle constriction 36 f. soybean lipoxygenase-1 30, 109 soybean lipoxygenase-3 115 sPLA2 see secretory phosholipases A2 sponges 28 spontaneously hypertensive rats 188 SRS-A see slow-reacting substance of anaphylaxis starfish 30 stearidonic acid 2,33 f. stomach protection 38,79 sulfur dioxide-induced bronchoconstriction 250 sulindac 315 superfusion assay 295 systemic mastocytosis 252 systemin 32 -

TCDD 148 T-cell transcription factor 320 Tethys fimbria 27 2,3,7,8-tetrachlorodibenzo-p-dioxin see TCDD TGF-a see transforming growth factor-a thromboangiitis obliterans 342 thrombotic microangiopathy 342 thromboxane 9, 10 - mimetics 255 - nomenclature 9 - receptor 36, 174 - - blockers - - - pre-eclampsia 2 16 --kidney 174 - synthase 213 - - enzymatic reaction 95 --gene 92 - - in creased bleeding tendency 278 - - induction 92 - - infarct prophylaxis 93 - - inhibitor 93, 257 - - isolation 91 - - kidney 172,287 - - malondialdehyde formation 9 1,94,3 10 - - molecular mass 91 - - restenosis 284

388

Index

- - pre-eclampsia 216 - - primary structure 92 - - tissue distribution 92

thromboxane A2 4,9, 159,343 - airway constriction 238

- angiotensin 11-mediated hypertension 180 -asthma 257 - biosynthesis 91 - bronchial hyper-responsivess 257 - bronchoconstriction 255,256 - cyclooxygenase-2 induction 174 - decidua 221 -heart muscle activity 285 - kidney 174 - mast cell 235 - myocardial contractility 285 - myocardial infarction 285 - placenta 21 1 - platelet aggregation 28 1, 283 - pregnancy-induced hypertension 214 - receptor 36, 174 - receptor antagonists - - allergic rhinitis 348 --angina 348 --asthma 348 - - pre-eclampsia 216 - - restenosis 284 - renal failure 287 - restenosis 284 - smooth muscle cell proliferation 280 - structural stabilization 348 - synthetic derivatives 348 -thrombosis 280 - vasoconstriction 28 1 thromboxane B2 9,294 transcellular eicosanoid formation . - and aspirin resistance 283 - of leukotrienes 15, I82 - of lipoxins 16 transforming growth factor-a 307, 319 Trapidil 216 traumatin 33 TriHETE 112 TriHPETE 112

trioxilins I9 trioxilin B3 30 trophoblast 2 1 I tuberonic acid 3 I tumor invasiveness 324 tumor promotion 305,3 1 1 - cyclooxygenase-2 312 - inhibition by non-steroidal antiinflammatory drugs 312 - okadaic acid 312 - phorbol esters 308,3 12 - prostaglandin F2a 3 12 - secondary bile acids 315 - self-promotion 322 - wounding 308,3 12 ultimate carcinogens 3 10 uterine contractility 37 vasopressin 38, 177 Viennese Initiative for Prostaglandin Screening 276 WinglessfWnt growth factors 320 wound hormone 308,3 19 woundrepair 7 X-ray crystallography - cyclooxygenase-1 7 4 - cyclooxygenases 82 - 15-lipoxygenase 1 16 - lipoxygenases 11 1 - soybean lipoxygenase 115 yeast 27 Zafirlukast 249 Zeste-White kinase 320