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Α1-Adrenoceptor Subtypes and Signal Transduction Mechanisms in the Rat

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Α1-Adrenoceptor Subtypes and Signal Transduction Mechanisms in the Rat ai-Adrenoceptor subtypes and signal transduction mechanisms in the rat thoracic aorta A thesis submitted by Dean Wenham for the degree of Doctor of Philosophy in the University of London 1994 Department of Pharmacology University College London Gower Street London WC1E6BT ProQuest Number: 10045753 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 10045753 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 a i-adrenoceptors are a heterogeneous family of receptors consisting of the cloned ttia/d-» otib-, ttic-adrenoceptors and the ‘classical’ tissue aiA- and aie-subtypes. This thesis describes a [^H]-prazosin binding study comparing the pharmacological profiles of the cloned mammalian awd-, otib- or aic-adrenoceptors to those of the proposed tissue aiA-,cxib - and ttjc-subtypes described in rat and rabbit tissues. On the basis of agonist and antagonist binding affinities, the cloned aia/d-subtype did not resemble the tissue aiA-subtype. The cloned aib-adrenoceptor closely matched its tissue counterpart and the cloned aic-subtype correlated well to the proposed tissue aic-adrenoceptor and also correlated closely with the rat tissue aiA-subtype indicating that this cloned receptor may represent the ‘classical’ aiA- subtype. The a1 -adrenoceptor agonist-induced contractions of the rat aorta have been suggested to be mediated via either a homogeneous or heterogeneous population of a r adrenoceptors. In this thesis, the subtype(s) of ai-adrenoceptor involved in the contraction of isolated rat thoracic aortic rings has been investigated by using a range of ai-adrenoceptor agonists and selective antagonists. Contractions to noradrenaline were highly sensitive to the ttiA/aic-selective antagonists (WB-4101 and 5-methyl-urapidil) and the aie/aic-alkylating agent CEC. However, contractions to oxymetazoline were insensitive to CEC but highly sensitive to WB-4101. It was concluded that a heterogeneous population of ai-adrenoceptors are responsible for the contractile response in the rat thoracic aorta. The source of Ca^"^ mobilised during ai-adrenoceptor mediated contractions of the rat thoracic aorta was also investigated. Noradrenaline- and oxymetazoline-induced contractions were differentially affected by the L-type Ca^^-channel blocker, nifedipine or by extracellular Ca"^ removal, since the responses to noradrenaline were partially sensitive to these treatments and those to oxymetazoline were almost totally abolished. Further, the initial phasic component of the noradrenaline-induced contraction was least sensitive to extracellular Ca^^ removal compared to the latter tonic component and in addition, IP3 production only occurred during this early component of the contraction. These results suggest that noradrenaline and oxymetazoline may couple differentially to different sources of Ca^^ mobilisation and that intracellular Ca^^ mobilisation might be primarily involved in the rapid phasic component of the contraction to noradrenaline. These studies suggest that the pharmacology of the cloned rat aia/d-adrenoceptor does not correlate to that of the ‘classical’ aiA-subtype but instead, the cloned aic-subtype may represent the recombinant counterpart to this receptor. The a 1-adrenoceptor agonist-induced contractions of the rat thoracic aorta appear to be mediated by a heterogeneous population of a I-adrenoceptors and further, both intracellular and extracellular sources of Ca^"^ seem to be involved with the contractile response. For my parents Contents Page Title 1 Abstract 2 Contents 3 Acknowledgements 8 Publications 9 Abbreviations 10 List of tables 12 List of figures 13 Chapter 1: Introduction 17 1.1 Adrenergic receptors 17 1.2 Pharmacological subdivision of a 1-adrenoceptors 19 1.2.1 Early subdivision of ai-adrenoceptor 19 1.2.2 Subdivision of the ai-adrenoceptor based on ligand binding 19 studies 1.2.3 ai-Adrenoceptor subtype selective antagonists 21 1.2.4 Subdivision of the ai-adrenoceptor based on functional studies 22 1.2.5 ttiH, otiL and an alternative nomenclature for 23 a 1-adrenoceptor subtypes 1.3 Molecular biology of the ai-adrenoceptors 24 1.3.1 Cloning of the a i-adrenoceptor 24 1.3.2 Protein structure of the ai-adrenoceptor 25 1.3.3 Cloning of additional ai-adrenoceptors 25 1.3.4 Correlations between the cloned and the pharmacologically 27 characterised tissue a i-adrenoceptors 1.4 tt|-Adrenoceptor tissue distribution: atypical tissues 29 1.4.1 tti-Adrenoceptor tissue distribution 29 1.4.2 The rat aorta: an atypical ai-adrenoceptor tissue 30 1.5 ai-Adrenoceptor signal transduction mechanisms 31 1.5.1 tt]- Adrenoceptor coupling to G-proteins 31 1.5.2 a 1-Adrenoceptors and the elevation of intracellular Ca^^ levels 33 1.5.3 Other signalling pathways activated by ai-adrenoceptors 36 1.6 Aims of project 38 Chapter 2: Materials and methods 2.1 Materials 39 2.2 Radioligand binding studies 40 2.2.1 Membrane preparation 40 2.2.2 Chlorethylclonidine (CEC)-pretreatments of membrane 41 homogenates 2.2.3 Protein concentration determinations 41 2.2.4 [^H]-Prazosin binding assays 42 2.3 Functional studies 44 2.3.1 Dissection 44 2.3.2 Isometric recording 44 2.3.3 Experimental protocol 44 2.4 Mass measurement of inositol 1,4,5-trisphosphate (IP 3) levels in rat 45 thoracic aortic rings using a radio-receptor binding assay 2.4.1 Preparation of cell extracts from rat thoracic aortic rings 45 2.4.2 Preparation of bovine adrenalcortical binding protein 46 2.4.3 [^H]-IP3 Binding assay 46 2.5 Data analysis 49 Chapter 3: [^H]-Prazosin binding studies of native tti-adrenoceptor subtypes using selective antagonists 3.1 Introduction 50 3.2 Experimental protocol 52 3.2.1 Data analysis 52 3.3 Results 54 3.3.1 Saturation studies and CEC-pretreatments 54 3.3.2 Competition studies 55 3.4 Discussion 71 Chapter 4: Agonist binding affinities: A comparison of the cloned and tissue ai-adrenoceptor subtypes 4.1 Introduction 79 4.2 Experimental protocol 81 4.3 Results 82 4.3.1 Saturation studies 82 4.3.2 Effect of Gpp[NH]p and EDTA on the binding of agonists to 82 the tissue a i-adrenoceptors 4.3.3 Comparison of the affinities of ai-adrenoceptor agonists for 83 the cloned and tissue ai-adrenoceptor subtypes 4.4 Discussion 102 Chapter 5: Functional studies: homogeneity or heterogeneity amongst ai-adrenoceptors mediating contractions of the rat thoracic aorta to ai-adrenoceptor agonists? 5.1 Introduction 108 5.2 Experimental protocol 109 5.2.1 Data analysis 110 5.3 Results 111 5.3.1 Types of responses evoked by the aradrenoceptor agonists 111 5.3.2 Effect of uptake blockade on noradrenaline-induced 112 contractions of the rat thoracic aorta 5.3.3 Effect of antagonists on ai-adrenoceptor agonist-induced 113 contractions of the rat thoracic aorta 5.3.4 Effect of abanoquil and CEC on noradrenaline-induced 114 contractions of rat spleen and epididymal vas deferens 5.3.5 Effect of abanoquil on KCl and 5-hydroxytryptamine-induced 115 contractions of the rat thoracic aorta 5.4 Discussion 135 Chapter 6: The role of extracellular Ca^^ and inositol-1,4,5- trisphosphate in the -adrenoceptor mediated contractions of the rat thoracic aorta 6.1 Introduction 144 6.2 Experimental protocol 146 6.3 Results 147 6.3.1 Effect of nifedipine and Ca^"^-free Krebs treatment on the 147 noradrenaline-induced contractions of rat epididymal vas deferens and spleen 6.3.2 Effect of nifedipine and Ca^'^-free Krebs treatment on 147 noradrenaline- and oxymetazoline-induced contractions of the rat thoracic aorta 6.3.3 Effect of nifedipine and Ca^^-free Krebs treatment on the 148 phasic and tonic components of the noradrenaline-induced contractions of the rat thoracic aorta 6.3.4 Time course for IP 3 production during noradrenaline- 149 and oxymetazoline induced contractions of the rat thoracic aorta 6.4 Discussion 163 Chapter 7:General discussion 172 Appendix 1 178 Appendix 2 181 Appendix 3 183 References 201 Acknowledgements I wish to express my gratitude to my supervisor, Dr. Ian Marshall, for the continued encouragement and advice he has given me during the last three years and especially for the support he has provided by being available for discussions at all the right times. I am also indebted to Dr. Pam Greengrass and all of the staff in the binding laboratories at Pfizer Central Research for giving me the opportunity to work in their lab. and for putting up with me. Thanks also to Dr. Peter Bungay, also at Pfizer, for supplying the [^H]-IP3. I also wish to thank Mark Coller for being a good friend during my numerous visits to Pfizer. My special thanks to Sonia Sooch, Franca Wisskirchen, Sam Masters and Richard Burt for their friendly support and for making day to day life in F28 a memorable experience. I extend my gratitude to Richard for partaking in many interesting debates and for supplying me with some of the latest references and to Sam for photography. I would also like to express my appreciation to Dr. Paul Brickel, Dr. Geoff Swanson, Dr. Ijsbrand Kramer and all members of his lab. for their assistance with some of the molecular studies described in this thesis and also to Dr.
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