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INFORMATION to USERS the Most Advanced Technology Has Been Characterization of a [³H]-5- hydroxytryptamine binding site in rabbit brain Item Type text; Thesis-Reproduction (electronic) Authors Xiong, Wen-cheng, 1962- Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 06/10/2021 06:47:00 Link to Item http://hdl.handle.net/10150/291537 INFORMATION TO USERS The most advanced technology has been used to photo­ graph and reproduce this manuscript from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are re­ produced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. These are also available as one exposure on a standard 35mm slide or as a 17" x 23" black and white photographic print for an additional charge. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. University Microfilms International A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 1338085 Characterization of a [3H]-5-hydroxytryptamine binding site in rabbit brain Xiong, Wen-cheng, M.S. The University of Arizona, 1989 UMI 300 N.Zecb Rd. Ann Arbor, MI 48106 CHARACTERIZATION OF A [3H]-5-HYDR0XYTRYPTAMINE BINDING SITE IN RABBIT BRAIN by Wen-cheng Xiong A Thesis Submitted to the Faculty of the COMMITTEE ON PHARMACOLOGY AND TOXICOLOGY (GRADUATE) In Partial Fulfillment of the Requirements For the Degree of MASTER OF SCIENCE WITH A MAJOR IN TOXICOLOGY In the Graduate College THE UNIVERSITY OF ARIZONA 19 8 9 2 STATEMENT OF AUTHOR This thesis has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under the rules of the Library. Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part made be granted by the head of the major department or the Dean of the Graduate College when in his or her judgement the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED: APPROVED BY THESIS DIRECTOR This thesis has been approved on the date shown below: £21 dwne S David L. Nelson Date Associate Professor of Pharmacology and Toxicology To my husband, my parents, and brothers, with love. 4 ACKNOWLEDGMENTS I wish to express my most sincere thanks to my advisor Dr. David L. Nelson for his supervision, guidance and encouragement. Thanks goes to my graduate committee members Drs. Hugh E. Lai/d II and Henry I. Yamamura for their advice and suggestions. Thanks are also given to Linda Cornfield, Anne Killam, and Georgina Lambert for their assistance. Sincere appreciation goes to Anne Killam for her reading manuscripts and her time. 5 TABLE OF CONTENTS Page LIST OF ILLUSTRATIONS 7 LISTS OF TABLES 10 ABSTRACT 12 INTRODUCTION 13 History of 5-HT receptors 13 Pharmacological classification of 5-HT receptors 16 Second messengers of 5-HT receptors 23 Molecular biology of 5-HT receptors 27 Distribution of 5-HT receptors 31 Rationales of this study 31 METHODS 33 Preparation of membranes for ligand-binding assays 33 [3H]5-HT binding assay—saturation studies 33 [3H]5-HT binding assay—inhibition studies 34 [3H]5-HT binding assay—GTP and cation regulation 35 [3H]5-HT binding assay—NEM inactivation 35 [3HJ5-HT binding assay—Distribution of the 5-HT binding sites in rabbit brain 36 Data analysis 36 Materials 37 RESULTS 39 Blockade of 5-HT^ ant* 5-HTjq binding sites in rabbit CN 39 Saturation analysis of [3H]5-HT binding in rabbit CN 41 Comparison of the pharmacological profiles of [3H]5-HT binding sites in rabbit and bovine CN after blockade of 5-HTj^ and 5-HTjq sites 44 6 TABLE OF CONTENTS—Continued Page Effects of GTP on [3H]5-HT binding to rabbit and bovine CN 64 Interaction of GTP and cations on [3H]5-HT binding 70 Inactivation of the binding sites by NEM 80 Distribution of the [3H]5-HT binding sites in rabbit brain 81 DISCUSSION 86 CONCLUSIONS 94 REFERENCES 95 7 LIST OF ILLUSTRATIONS Figure Page 1. 8-OH-DPAT and mesulergine competition studies with [3H]5-HT binding in the absence and presence of 100 nM 8-OH-DPAT and 100 nM mesulergine in rabbit CN 40 2. [3H]5-HT saturation studies in rabbit CN in the absence and presence of 100 nM 8-OH-DPAT and 100 nM mesulergine 42 3. Competition studies of 5-HT1A~selective drugs with the non-5-HTiA/non-5-HTiQ binding sites in rabbit CN 46 4. Competition studies of 5-HTig-selective drugs with the non-5-HTjA/non-5-HTjQ binding sites in rabbit CN 47 5. Competition studies of 5-HTjQ-selective drugs with the non-5-HTjA/non-5-HTjQ binding sites in rabbit CN 48 6. Competition studies of S-HTg-selective drugs with the non-5-HTiA/non-5-HT;L0 binding sites in rabbit CN 49 7. Correlations between the pK| values of for 3 the non-5-HTjA/non-5-HTic [ H]5-HT binding sites in rabbit and the 5-HT^^, 5-HTjg, 5-HTjq, and 5-HT2 sites 50 8. Correlations between the pKj values of compounds for the non-5-HT1A/non-5-HT2c binding sites in rabbit and bovine CN 54 9. Inhibition curves for spiperone and spirilene against non-5-HT1A/non-5-HTlc [3H]5-HT binding in rabbit and bovine CN 56 10. Chemical structures of spiperone and its analogs 57 LIST OF ILLUSTRATIONS—Continued Figure Page 11. Inhibition curves for ergonovine against 3 non-5-HT1A/non-5-HTlc [ H]5-HT binding in rabbit and bovine CN 60 12. Inhibtion curves for ergotamine and ergocryptine against the non-5-HTi^/non-5- HTjc [3H]5-HT binding in rabbit and bovine CN 61 13. Inhibtion curves for dihydroergotamine, dihydroergDcristine. and dihydroergocryptine against the non-5~HTiA/non-5-HTic [ 5- HT binding in rabbit and bovine CN 62 14. Effects of GTP on the non-5-HT^A/non-5-HTjQ [3H]5-HT binding in rabbit and bovine CN without calcium 65 15. Effect of GTP on agonist inhibition of [3H]5-HT binding to non-5-HTiA/non-5~HTic sites in rabbit and bovine CN without calcium 66 16. [3H]5-HT saturation studies in the presence and absence of 100 uM GTP in rabbit CN without calcium 67 17. [3H]5-HT sturation studies in the presence and absence of 100 uM GTP in bovine CN without calcium 68 18. Effects of GTP on the binding of [3H]5-HT in the precence of 100 nM 8-OH-DPAT and 100 nM mesulergine with or without calcium 72 19. Effects of GTP on agonist inhibition of [3H]5-HT binding to non-5-HTiA/non-5~HTic [3H]5-HT binding in rabbit and bovine CN in the presence of 3 mM calcium 74 9 LIST OF ILLUSTRATIONS—Continued Figure Page 20. [3H]5-HT saturation studies in the presence and absence of 100 uM GTP in the rabbit with 3 mM calcium 75 21. [3H]5-HT saturation studies in the presence and absence of 100 uM GTP in the bovine CN with 3 mM calcium 76 22. Cation effects on the non-5-HT1A/non-5-HTjQ [3H]5-HT binding sites in rabbit and bovine CM membranes 78 23. NEM sensitivity of the non-5-HTj^/non-5-HTjQ [3H]5-HT binding site in bovine and rabbit CN membranes 83 24. Regional distribution of specific [3H]5-HT binding in the absence or presence of 100 nM 8-OH-DPAT and 100 nM mesulergine in rabbit brain membranes 84 10 LIST OF TABLES Table Page 1. Drug affinities for 5-HT1A, 5-HT1B, 5-HTlc, 5-HT^q, and 5-HT2 receptors 17 2. Results of saturation experiments performed in rabbit and bovine CN with [3H]5-HT in the absence and presence of 100 nM 8-OH-DPAT and 100 nM mesulergine 43 3. Correlation between the non-5-HTjA/non-5- HTic [3H]5-HT binding in the rabbit CN and 5-HT1A, 5-HTjg, 5-HT^q, and 5-HT2 binding 51 4.
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