A Comparative Study of Purinoceptors in Some Invertebrates and Vertebrates
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A COMPARATIVE STUDY OF PURINOCEPTORS IN SOME INVERTEBRATES AND VERTEBRATES by Gillian Elizabeth Knight G.l.Biol Department Of Anatomy and Centre for Neuroscience, University College London, Gower Street, London WCIE 6BT Thesis submitted for the degree of Doctor of Philosophy University of London ProQuest Number: 10017490 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10017490 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT The main objective of this thesis has been to identify and characterise responses to purine compounds in a variety of invertebrates and lower vertebrates, and to examine whether they act on receptors that resemble the purinoceptor subtypes established in mammals. Tissues were chosen largely from the gastrointestinal or cardiovascular systems. In mammalian systems adenosine and adenosine 5 ’-triphosphate (ATP) activate separate classes of purinoceptors; this also appears to be true for tissues in most invertebrate groups, but there are exceptions, for instance the hearts of the snail Helix aspeTsa, and slug Avion atev, and the snail rectum all possess purinoceptors that do not appear to distinguish the purine nucleosides from nucleotides. However, the discriminating effect of glibenclamide revealed the presence of P^- and P^-like purinoceptors in the gastric ligament of the starfish Astevias vubens. The evolutionary advancement from invertebrates to lower vertebrates was reflected in the greater degree of purinoceptor differentiation. The stomach of the stickleback Gastevosteus aculeatus, exhibited a P2Y” purinoceptor, while there is a P2x”Purinoceptor in the intestine, both mediating contraction; both expressed a P^-purinoceptor, although no subtypes equivalent to those from mammals could be distinguished. Adenosine dilated the aorta of the frog Hana tempovavia, via a receptor resembling the Ag subtype defined in mammals ; a P2x“P^rinoceptor mediating constriction was identified in this vessel comparable to that known in mammalian vessels. A P]^- purinoceptor mediating vasodilatation was also found on the aorta of the garter snake Thamnophis sivtalis pavietalis together with multiple P^-purinoceptors, P2X" P^Y'^^^eptors, both mediating constriction. In addition to pharmacological investigations, some histochemical studies were carried out on invertebrate tissues using the fluorescent dye quinacrine, to visualise structures containing high concentrations of bound ATP. Quinacrine fluorescence was found in the majority of marine and terrestrial invertebrates examined, including the intestines of Lumbvicus terrestres, Golfingia minuta^ Littorina littorea, Helix aspersa. Avion atev and Nactva covalline, and the hearts of Patella vulgavis, Littovina littovea, Nucella lapillus and Mytilus edulis. Some neuronal tissues were also found to exhibit quinacrine fluorescence including the pedal ganglion of Hytilus edulist cerebral ganglion of Helix aspevsa and visceral and pedal ganglia of Cavdium edule. Nerve fibres and nerve cell bodies could be distinguished. Finally, in the general discussion, some speculations about the evolution and development of purinoceptors are considered. ACKNOWLEDGEMENTS I am most grateful to my supervisor, Professor Geoffrey Burnstook, for his enthusiasm, interest and invaluable advice, prior to and during the course of this thesis. My sincere thanks are also extended to the many people who assisted me during this period. In particular, I thank Charles Hoyle, for his patience, continual help, friendship and wisdom. Many friends have provided support, encouragement and laughter, during both the experimental and writing phase of this thesis. My thanks go to Amanda Vials, Robyn Rennick, Anita Sexton and Antonia Brizzolara. I should also like to thank Daphne Christie for excellent editorial assistance and a willing ear; the staff of the animal house, particularly John Cadera; the staff of the photographic department and the many other colleagues who provided practical aid and helped make this thesis both personally rewarding and enjoyable. Finally, I would like to dedicate this thesis to two people whom I love very much, my parents Jean and Dennis Knight. I thank you for your continual support, encouragement and love. CONTENTS Title Page................................................... 1 Abstract..................................................... 2 Acknowledgements..............................................4 Contents..................................................... 5 List Of Tables............................................... 11 List Of Figures.............................................. 11 Publications Arising From This Thesis.......................... 12 Other Publications............................................13 Chapter 1 GENERAL INTRODUCTION................................ l4 1.1 PREFACE............................................. 15 1.2 HISTORICAL BACKGROUND............................... l8 1.3 RECEPTORS FOR ADENOSINE AND ADENINE NUCLEOTIDES...... 20 1 .3.1 Pj^-Purinoceptors.................................... 21 1 .3.2 P2 -Purinoceptors.................................... 26 1 .3.3 Pg-Purinoceptors.................................... 31 1.4 PURINERGIC TRANSMISSION..............................32 1.4.1 Purinoceptor Subtypes Within The Gastrointestinal Tract.............................................. 36 1.4.2 Purinoceptor Subtypes Within The Cardiovascular System............................................. 37 1 .4.3 Other Systemic Purinoceptor Subtypes................. 39 1.5 EFFECT OF PURINE COMPOUNDS ON INVERTEBRATESPECIES 40 1.6 EFFECT OF PURINE COMPOUNDS AND PURINERGIC TRANSMISSION IN LOWER VERTEBRATE SPECIES.............46 Chapter 2 GENERAL METHODOLOGY.................................57 2.1 ORGAN-BATH PHARMACOLOGICAL STUDIES................... 58 2.1.1 Apparatus........................................... 58 2.1.2 Tissue Preparation For Organ-Bath Studies............. 59 2.2 PRESENTATION OF DATA AND STATISTICAL ANALYSIS.......... 6l 2.2.1 Agonists............................................ 6l 2.2.2 Antagonists......................................... 62 2.2.3 Statistical Analysis................................ 62 2.3 PHYSIOLOGICAL SALINES............................... 62 2.4 DRUGS AND SOLVENTS.................................. 63 2.4.1 Preparation Of Solutions.............................65 2.5 LIST OF ABBREVIATIONS............................... 66 SECTION A ....................................................... 68 Chapter 3 GLIBENCLAMIDE ANTAGONISES THE RESPONSES TO ATP, BUT NOT ADENOSINE OR ADRENALINE, IN THE GASTRIC LIGAMENT OF THE STARFISH ASTEEIAS BUBENS..............68 3.1 SUMMARY............................................. 69 3.2 INTRODUCTION........................................ 69 3.3 METHODS............................................. 71 3.3"! Animals............................................. 71 3 .3*2 In VitTo Pharmacology............................... 71 3.4 RESULTS............................................. 71 3.5 DISCUSSION.......................................... 80 Chapter 4 EFFECTS OF ADENINE NUCLEOSIDES AND NUCLEOTIDES ON THE ISOLATED HEART OF THE SNAIL HELIX ASPERSA AND THE SLUG ARION ATER............................. 82 4.1 SUMMARY............................................. 83 4.2 INTRODUCTION........................................ 8] 4.3 METHODS............................................. 85 4.3-1 Animals............................................. 85 4 .3.2 In Vitro Pharmacology................................85 4.4 RESULTS............................................. 86 4.4.1 Exogenously Applied Purine Compounds.................. 86 4.4.2 Electrical Stimulation...............................88 4.5 DISCUSSION.......................................... 101 Chapter 5 RESPONSES OF THE RECTUM AND OESOPHAGUS OF THE SNAIL HELIX ASPEESA TO PURINE NUCLEOTIDES AND NUCLEOSIDES.....................................104 5.1 SUMMARY............................................. 105 5.2 INTRODUCTION........................................ IO6 5.3 METHODS............................................. 107 5 .3.1 Animals............................................. 107 5 .3.2 In Vitro Pharmacology................................ IO8 5.4 RESULTS............................................. 108 5..4.1 Responses To Applied Purine Compounds And ACh.......... IO8 5 .4.2 Electrical Stimulation............................... 110 5.5 DISCUSSION.......................................... 118 Chapter 6 QUINACRINE STAINING OF NEURONES ANDACTIVITY OF PURINE NUCLEOSIDES AND NUCLEOTIDES IN MARINE AND TERRESTRIAL INVERTEBRATES FROMSEVERAL PHYLA....... 122 6.1 SUMMARY............................................. 123 6.2 INTRODUCTION........................................ 123 6.3 METHODS............................................. 125 6 .3.1 Animais............................................. 125 6 .3.2 Histochemistry...................................... 127 6 .3.3 In Vitro Pharmacology................................128