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https://theses.gla.ac.uk/ Theses Digitisation: https://www.gla.ac.uk/myglasgow/research/enlighten/theses/digitisation/ This is a digitised version of the original print thesis. Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ [email protected] 5HT RECEPTORS AND a -ADRENOCEPTORS? CLASSIFICATION, 2 LIGAND BINDING, FUNCTIONAL CORRELATES AND ALTERATIONS IN CEREBRAL ISCHAEMIA. A thesis presented for the degree of Doctor of Philosophy By Christine Mary Brown, BSc Institute of Physiology University of Glasgow & Syntex Research Centre Edinburgh May, 1988. ProQuest Number: 10997942 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 com plete 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 10997942 Published by ProQuest LLC(2018). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 FOR MY DAD PAGE TABLE OP CONTENTS 1 List of figures and tables 4 List of Abbreviations 11 Acknowledgements 12 Declaration 13 Summary 14 Chapter Is INTRODUCTION 1.1 General introduction on 5HT 18 1.2 Receptor classification 22 1.3 Classification of 5HT receptors 25 1.4 Clinical significance of recent 43 advances in 5HT research Chapter 2: METHODS 2.1 Binding methodology 45 2.2 5HT Functional assays 63 2.3 Gerbil Stroke Models 65 2.4 Drugs 7 0 AIMS OF THESIS 72 Chapter 3S STUDIES ON 5HT RECEPTOR SUBTYPES 3.1 Introduction 73 3.2 Results 77 3.2.1 Preliminary binding studies 77 3.2.2 In vitro modulation of specific 80 3 [ H]-5HT binding 3.2.3 Specific 5HT binding assays 81 3.2.4 A comparison of the binding of 86 different radioligands 3.2.5 A comparison of the binding in 87 different rat brain regions 3.2.6 5HT binding sites in the 87 1 gerbil brain 3.2.7 5HT binding sites in 88 1 peripheral tissues 3.2.8 Functional 5HT^ binding sites 88 3.3 Discussion 125 1 PAGE Chapter 4: STUDIES ON 5HT RECEPTORS 2 4.1 Introduction 134 4.2 Results 136 3 4.2.1 [ H]-ketanserin binding studies 136 4.2.2 Modifications to the basic technique 138 4.2.3 Specific 5HT binding assays 139 2 4.2.4 A comparison of 5HT radioligands 140 2 4.2.5 A comparison of 5HT binding in 140 2 different rat brain regions 4.2.6 5HT binding sites in the 141 2 gerbil brain 4.2.7 5ht2 binding sites in 142 peripheral tissues 4.2.8 Complex interactions at the 5HT 144 2 binding site 4.2.9 5HT functional assays 146 2 4.3 Discussion 176 Chapter 5: THE EFFECT OF CEREBRAL ISCHAEMIA ON 5HT NEUROPHARMACOLOGY IN THE MONGOLIAN GERBIL 5.1 Introduction 182 5.2 Results 184 5.2.1 Identification of stroke-prone animals 184 5.2.2 Effect of cerebral ischaemia on 5HT 191 levels 5.2.3 Effect of cerebral ischaemia on 5HT 193 binding sites 5.2.4 Functional correlates for changes in 196 5HT binding 5.3 Discussion 214 Chapter 6: 5HT/NA INTERACTIONS IN THE BRAIN AND ALPHA -ADRENOCEPTOR SUBTYPES 2 6.1 Introduction 223 Evidence for a -adrenoceptor subtypes 226 2 6.2 Results 229 6.2.1 Receptor binding assays 229 2 PAGE 6.2.2 A correlation of the affinities of 229 standard compounds for a- adrenoceptor and 5HT binding sites 6.2.3 -adrenoceptor binding assays 230 6.2.4 A comparison of the binding 233 3 characteristics of [ H]-yohimbine in rat cortex , rat hippocampus and human platelet 6.2.5 The effect of chemical modifying 234 agents on a -adrenoceptor binding sites 6.2.6 A^comparison of the binding of 235 [ H]-idazoxan in the rat cortex and hippocampus 6.2.7 A comparison of the binding of 236 3 3 [ H]-yohimbine and [ H]-idazoxan 6.2.8 Effect of assay buffer constituents 238 3 3 on [ H]-yohimbine and [ HJ-idazoxan binding 3 6.2.9 [ H]-Idazoxan binding to human 239 platelet membranes 6.3 Discussion 266 Chapter 7: GENERAL DISCUSSION 274 REFERENCES 279 Appendix Is Theoretical basis for identification 322 of receptors using direct radioligand binding techniques 3 LIST OF FIGURES AND TABLES PAGE Chapter 1 Fig. 1.1 The chemical structure of 5HT 18 Fig. 1.2 5HT formation and metabolism 20 Fig. 1.3 5HT pathways in the rat brain 21 Table 1.1 Proposed classifications of 5HT receptors 26 Table 1.2 Major developments in 5HT receptor 31 binding studies Table 1.3 New tools in 5HT research 33 Table 1.4 Summary of proposals for classification 34 & nomenclature of functional 5HT receptors Table 1.5 Receptors for 5-hydroxytryptamine 44 Chapter 2 Fig. 2.1 Tritium quench curve for the Packard 460C 49 Chapter 3 Fig. 3.1 Breakdown products of [ H]-5HT 90 Fig. 3.2 Effect of washing on recoverable specific 91 [ H]-5HT binding Fig. 3.3 Association and dissociation of [ H]-5HT 92 binding to rat cerebral cortex Fig. 3.4 5HT inhibition of [ H]-5HT binding 93 3 Fig. 3.5 [ H]-5HT binding to rat cerebral cortex 94 Fig. 3.6 Effect of GTP on [ H]-5HT binding 95 Fig. 3.7 Effect of cations on the specific binding 96 3 of [ H]-5HT to rat cerebrocortical membranes Fig. 3.8 Effect of incubation pH on specific binding 97 of [ H]-5HT to rat cerebrocortical membranes Fig. 3.9 Spiperone inhibition of [ H]-5HT 98 binding to rat striatal membranes Fig. 3.10 5CT inhibition of [ H]-5HT binding 99 3 Fig. 3.11 Remaining specific [ H]-5HT binding in 100 the rat cerebrocortical membranes Fia. 3.12 Effect of GTP on buspirone inhibition of 101 3 [ H]8-OH-DPAT binding to rat cerebrocortical membranes 4 PAGE 3 Fig. 3.13 Filter binding of [ H]8-0H-DPAT 102 Fig. 3.14 Correlation between pKi values for 103 3 3 [ H]8-0H-DPAT and [ H]-WB4101 binding in rat cerebral cortex 3 Fig. 3.15 [ H-8-0H-DPAT binding to gerbil hippocampus 104 Fig. 3.16 Inhibition of specific [ H]-5HT binding to 105 gerbil cortex 3 Fig. 3.17 pKi values for [ H]5HT binding 106 Fig. 3.18 [ H]-5HT binding to rat lung membranes 107 + 3 Fig. 3.19 Effect of [K ] on the release of [ H]-5HT 108 from rat frontal cortex slices + Fig. 3.20 Effect of 5HT and 5HT antagonists on [K 1 109 3 evoked release of [ H]-5HT from rat frontal cortex slices Table 3.1 Effect of GTP on affinity values for 110 [ H]-5HT binding to rat cerebral cortex Table 3.2 The effect of incubation medium on the 111 3 specific binding of [ H]-5HT Table 3.3 Effect of equilibrium temperature on 112 [ H]-5HT binding to rat cerebral cortex Table 3.4 Affinity values^determined for the 113 inhibition of [ H]-5HT binding in rat striatal membranes 3 Table 3.5 [ H]-5HT binding to rat cerebrocortical 114 membranes Table 3.6 Affinity values of imipramine, panuramine, 115 desipramine and zimeldine for 5HT , 5HT 3 1A. IB binding sites and [ H]-5HT uptake sites Table 3.7 A comparison of the 5HT binding sites 116 3 1A labelled by [ H]-WB4101 in the rat cortex and rat hippocampus Table 3.8 pKi values for 5HT binding site in the 117 1A 3 rat hippocampus labelled by [ H]-8-OH-DPAT Table 3.9 Affinity values for 5HT binding sites 118 125 IB labelled by [ I]-CYP in the rat striatum 5 PAGE Table 3.10 A^comparison of the binding of 119 [ H]-8-OH-DPAT and [ H]-WB4101 to rat cerebral cortex Table 3.11 Saturation data for 5HT binding sites in 120 different brain areas in the rat Table 3.12 Comparison of the affinities of several 121 drugs for 5HT , 5HT and 5HT binding . 1A IB 1C sites in rat cerebral cortex Table :3.13 Saturation data for 5HT binding sites in 122 different brain areas of the gerbil Table :3.14 Comparison of the affinities of several 123 drugs for 5HT , 5HT and 5HT binding 1A IB 2 sites in gerbil frontal cortex Table :3.15 The effect of chronic dosing of 124 desipramine on 5HT binding sites 1A in the rat hippocampus Chapter 4 Fig. 4, 1 Association and dissociation of 148 3 [ H]-ketanserin binding to rat o frontal cortex at 37 C Fig. 4. 2 Methysergide and ketanserin inhibition of 149 3 [ H]-ketanserin binding 3 Fig. 4. 3 [ H]-ketanserin binding to rat frontal 150 cortex membranes Fig. 4. 4 Effect of GTP on 5HT inhibition of 151 3 [ H]-ketanserin binding Fig.

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