SYNAPTIC ACTION OF ANANDAMIDE AND RELATED SUBSTANCES IN MAMMALIAN BRAIN by Chengyong Liao M.Sc., Sichuan University, 1991 B.Sc., Sichuan University, 1984 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Inthe Department of Biological Sciences © Chengyong Liao 2007 SIMON FRASER UNIVERSITY 2007 All rights reserved. This work may not be reproduced in whole or in part, by photocopy or other means, without permission ofthe author. APPROVAL Name: Chengyong Liao Degree: Doctor of Philosophy Title of Thesis: Synaptic Actions of Anandamide and Related Substances in Mammalian Brain Examining Committee: Chair: Dr. Z. Punja Professor ofDepartment ofBiological Sciences, SFU. Dr. R. A. Nicholson Senior Supervisor Associate Professor ofDepartment ofBiological Sciences, SFU. Dr. C. J. Kennedy, Supervisor Associate Professor of Department ofBiological Sciences, SFU Dr. F. C. P. Law, Supervisor Professor ofDepartment ofBiological Sciences, SFU Dr. M. A. 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Simon Fraser University Library Burnaby, BC, Canada ABSTRACT Anandamide and the synthetic cannabimimetic drugs AM 404 and WIN 55,212-2 were found to inhibit the binding of CH]batrachotoxinin A 20-a.-benzoate (BTX) to voltage-gated sodium channels (VGSCs) and also to depress VGSC-dependent release of GABA and L-glutamic acid. These effects occur independently of CB-l receptor activation since they were not attenuated by AM251 at concentrations known to antagonize CB-l receptors, although at higher concentrations AM251 inhibited VGSCs also. These results suggest that anandamide and endocannabimimetics have the ability to depress synaptic transmission by reducing the capacity of VGSCs to support action potentials. In other experiments, I observed that anandamide is synthesized by both resting and depolarized synaptoneurosomes, however, data from filtration and superfusion experiments failed to support the hypothesis that depolarization activates the release of anandamide from these synaptic preparations, even though substantial quantities of anandamide were clearly present in the intracellular compartments. In these investigations, I unexpectedly found that CH]ethanolamine (a potential precursor of anandamide) is accumulated by synaptosomes and synaptoneurosomes during extended incubation and released (as CH]ethanolamine) in a calcium-dependent fashion during a depolarizing challenge with 30 mM KCl. Ethanolamine also stimulates the release of the pH fluoroprobe acridine orange (AO) from synaptosomes and decreases AO release when synaptosomes are depolarized with KCl. However, ethanolamine and other amino alcohols (methylethanolamine and dimethylethanolamine) stimulate resting and KCI- 111 evoked release of eH]-D-aspartate from synaptosomes. All amino alcohols rapidly access synaptic vesicles where they sequester protons, thus accounting for AO efflux. Proton sequestration by amino alcohols increases the AIP-dependent transvesicular membrane potential which in tum enhances D-aspartate uptake into synaptic vesicles. My data suggest a potential role for ethanolamine and related amino alcohols in the regulation of synaptic vesicle filling and release of amino acid neurotransmitters. Depolarization evoked release of ethanolamine from synaptosomes supports the idea that this amino alcohol may also have an important postsynaptic role. Keywords: anandamide; cannabinoids; sodium channels; synaptic vesicles; mammalian brain. IV DEDICATION ~~~~~~~*~.W~~~*~~~~.~5o .OO~~~T~~ffim~~, ~*~~o Inmemory ofmy mom and thanks to my father for bringing me upwith love and support. Thanks also to my wife for always supporting me in hard and happy times. v ACKNOWLEDGEMENTS The thesis is a product of many people's support. First and foremost, I thank my senior supervisor Dr. Nicholson for giving me the opportunity to work in his lab. He gave me direct instruction, contributed great ideas, and provided an enjoyable and encouraging environment in which I could display my creation at my best. I appreciate him giving me support throughout my graduate studies. When needed, I always got his timely and effective help. It was great to have a lot of discussion about the project and related scientific issues with him. I enriched my background in related fields. I was fortunate to be his graduate student. I especially thank my supervisory committee members Dr. Law and Dr. Kennedy for their enormous help and direction at each stage of my research. I learned a lot from Dr. Law for example, chromatography theory relating to HPLC analysis and the way to effectively deliver an oral presentation of my research. I will never forget Dr. Kennedy who provided me with the necessary instruments and apparatus for my research and gave me his encouragement in the hard times of my study. I especially thank my good friend Jian Zheng who introduced me to Dr. Nicholson's lab and helped me to master many important skills for the research ahead. I would like to thank Yanshen Deng for mentoring me and making me familiar with SFU facilities and giving me guidance in my work. I would like to thank my colleague Yin Duan, who assisted me in many aspects such as preparing mitochondria, and other lab work, especially when it was difficult to balance my lab work and taking care of my baby. I thank QiangTu Zhang and Dan Sit VI who assisted me in optimizing my HPLC analysis. I will never forget the help and assistance provided by so many people at SFU such as Xui He, Ying Zhen, Guanghua Gao, Laurance David and Sudip Ghose. I would like especially to thank a very special person who gave me great support in my study, my wife Chaoli Wang. She always stands behind me and always ensured that I had a quiet environment for my study at home. I will never forget the support from my parents. So that I was not distracted in my research, my mom did not tell me about her serious illness before she passed away. I always got strong support and encouragement from my family and Chaoli's family. Without their support, it would have been impossible for me to complete my Ph.D. study. I sincerely thank them. vii TABLE OF CONTENTS Approval ii Abstract iii Dedication v Acknowledgements vi Table of Contents viii List ofFigures xii List of Tables xv Glossary xvi Chapter 1 Introduction 1 1.1 The discovery ofthe endocannabinoid system in mammals 1 1.2 The synthesis