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REGULATION of the ENDOGENOUS OPIOID SYSTEM by ACUTE NICOTINE and NICOTINE WITHDRAWAL DISSERTATION Presented in Partial Fulfillme

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REGULATION of the ENDOGENOUS OPIOID SYSTEM by ACUTE NICOTINE and NICOTINE WITHDRAWAL DISSERTATION Presented in Partial Fulfillme REGULATION OF THE ENDOGENOUS OPIOID SYSTEM BY ACUTE NICOTINE AND NICOTINE WITHDRAWAL DISSERTATION Presented in Partial Fulfillment of the Requirements for The Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Michael J. McCarthy, B.S. ***** The Ohio State University 2003 Dissertation Committee: Dr Maria H. Neff, M.D., Ph.D., Adviser Dr Gopi Tejwani, Ph.D. Dr Karl Obrietan, Ph.D. Approved by Adviser Neuroscience Graduate Studies Program ABSTRACT Nicotine addiction emerges as a result of long-term plastic changes in the brain including altered intracellular signaling, gene transcription and receptor function. The endogenous opioids may contribute to aspects of nicotine’s addictive psychopharmacological properties. Presently, the response to both acute nicotine and chronic nicotine withdrawal was evaluated with respect to effects on the regulation of opioid peptides, opioid receptor function and the signaling events that link these processes. Acute nicotine biphasically increased striatal dynorphin content with peaks at 1 hr and 18hr after injection. The mRNA for prodynorphin (PD), as well as the immediate early genes c-fos, c-jun and egr-1 was increased from 30 min to 12 hr following acute injection. Increased p-CREB immunoreactivity and CREB-PD promoter binding was observed in the early stages after acute nicotine. Antagonist pre-treatments revealed that dopamine and muscarinic receptors contribute to the 1 hr and 18 hr dynorphin increases after acute nicotine treatment. For withdrawal studies, mice were treated with nicotine four times daily for 14 days and killed at 0-96 hr after the last injection. During nicotine withdrawal, PD mRNA was increased in the striatum, whereas dynorphin was decreased for 72 hr. Analysis of CREB, adenylyl cyclase and the PD promoter suggest these are coordinated in an atypical manner, resulting in decreased adenylyl cyclase activity concomitant to an increased CREB activity. Preliminary data suggesting a role for the transcriptional repressor protein, DREAM in this interaction is presented. Analysis of the delta (DOR) and kappa (KOR) opioid receptors was assessed for altered function during nicotine withdrawal. While changes in DOR and KOR binding were subtle, evidence for DOR and KOR uncoupling from intracellular signaling ii pathways was observed. Data suggest that regulation of G-proteins and adenylyl cyclase during nicotine withdrawal may contribute to these functional alterations in the DOR and KOR. Taken together, nicotine engages signaling mechanisms that regulate PD transcription. Perturbations of this interaction may disrupt dynorphin release in the striatum during nicotine withdrawal. Alterations in DOR and KOR function were observed during withdrawal. These findings together provide a substrate by which the endogenous opioid system could mediate aspects of the nicotine withdrawal syndrome. iii DEDICATION To my family: Denis, Carol, Kevin, Brian, Anne, Jerry and Sallie. Thanks for keeping me afloat. To all the friends who pulled me through. iv ACKNOWLEDGMENTS Dr Gopi Tejwani, Dr Karl Obrietan, Jacqueline Bresnahan for technical assistance and service on my candidacy and dissertation committees. Adrienne Frostholm, Wolfgang Sadeè, David Saffen, Thomas Boyd, Sari Izenwasser and Charles Murrin for helpful comments and technical assistance. Trina Wemlinger, Amy Burrows, Lili Mo, Hailin Zhang, Brian Wulff, Tina Tran, Erin Newburn and Anne-Marie Duchemin….my helpers, teachers and friends. Most of all, thanks to Maria and Norton Neff whose guidance was critical to the success of this project. I am forever thankful for your generosity, patience and mentorship over the years. v VITA 1974………………..Born in San Fransisco, CA, USA. 1992-1996………….Bachelor of Science, Biochemistry and Psychology (honors), University of New Mexico; Albuquerque, NM, USA. 1996………………..Internship in clinical neuropsychology, National Institutes for Neurological Disorders and Stroke, Bethesda, Maryland, USA. 1997………………..Industrial internship in neuropharmacology at SmithKline Beecham pharmaceuticals, Harlow, England. 1997-present……….Medical Scientist Training Program Fellow, The Ohio State University, College of Medicine and Public Health and Neuroscience Graduate Studies Program, Columbus, Ohio, USA. PUBLICATIONS McCarthy, M.J. and Hadjiconstantinou, M. Altered Kappa Opioid Receptor Coupling During Nicotine Withdrawal. Society for Neuroscience Abstracts 2003. Program No 322.11 Abstract Viewer/Itinerary Planner. Washington DC: Society for Neuroscience, 2003. CD-ROM. McCarthy, M.J. Zhang, H. and Hadjiconstantinou, M. Striatal Transcription Factor Binding During Nicotine Withdrawal. Society for Neuroscience Abstracts 2002. Program No 502.6 Abstract Viewer/Itinerary Planner. Washington DC: Society for Neuroscience, 2002. CD-ROM. McCarthy, M.J. and Hadjiconstantinou, M. Opioid Receptors During Nicotine Withdrawal. Journal of Neurochemistry 81 (supplement 1) Abstract AP04-06 2002. McCarthy, M.J., Duchemin, A.M., Wemlinger, T.A., Neff, N.H. and Hadjiconstantinou, M. Acute Nicotine Enhances the Expression of Opioid Precursors and Immediate Early Genes. Society for Neuroscience Abstracts, Vol. 27 Program No 222.12, 2001. vi FIELDS OF STUDY Major Field: Neuroscience Neuropharmacology Neuroanatomy vii TABLE OF CONTENTS Abstract…………………………………………………...……………………………....ii Dedication…………………………………………………..…………………………….iv Acknowledgments…………………………………………...………...………………….v Vita……………………………………………………………………………………….vi List of Tables…………………………………………………………………………..….x List of Figures…………………………………………………………………………….xi List of Abbreviations…………………………………………………………………….xii Chapters: Introduction..……...……...……...……...……...……...……...……...……...……….…....1 Chapter 1: Review Neurotransmitters Systems and Their Role Drugs of Abuse……........6 1.1 Overview of Nicotine and Nicotinic Receptors…………………………….…......6 Neuroanatomical Substrates of Nicotine Addiction..………………………..6 Molecular Pharmacology of Nicotine….……………………………………14 Nicotinic Receptors in the Midbrain Dopamine Systems….……………..…16 Nicotinic Receptors Involved in Reward……….…………………………...17 Nicotinic Receptors Involved in Withdrawal…………………………….... 18 1.2 The Endogenous Opioid System……………...…………………………..…….. 20 Opioid Peptides….…………………………………………………………21 β- Endorphin….…………………………………………………………....21 The Enkephalins…………………………………………………………....24 The Dynorphins…………..…………………………………………….…..25 The Opioid Receptors………..…………….……………………………….27 Mu Opioid Receptors………………………………………………………32 Delta Opioid Receptors…………………………………………………….35 Kappa Opioid Receptors…………………………………………………...36 Non-Classical Components of the Opioid System…………………………37 Opioid Neurotransmission…………………………………………………38 Opioids and Drugs of Abuse……………………………………………… 39 Morphine and the Opiates………………………………………………….39 The Psychostimulants: Amphetamine and Cocaine………………………..43 Nicotine…………………………………………………………………….45 viii Chapter 2: Materials and Methods…..………………………………………………....51 Animals..…...……………….……………………………………………. 51 Acute drug administrations and treatments………………………………....51 Nicotine withdrawal paradigm……………………………………………...52 Dynorphin radioimmunoassay…………………………………………….. 52 Electrophoretic mobility shift assay…………………………….…………..53 Western Blots…………………………………………………….…………54 In situ hybridization………………………………………………………...55 Northern blots………………………………………………………………57 Receptor binding……………………………………………………………58 Receptor binding autoradiography…………………………………………59 [35S]-GTPgS Binding Autoradiography………………………….…………60 Adenylyl cyclase activity………………………………………….………..60 Chapter 3: Results……………………………………………………..…………………63 3.1 Transcriptional Regulation of Prodynorphin After Acute Nicotine…………………63 Prodynorphin mRNA and dynorphin content………………………………63 Immediate early gene transcription factor mRNA content…………………64 Transcription factor binding...……………………………………………..66 Regulation of CREB and CREB phosphorylation by acute nicotine………70 Acute nicotine and adenylyl cyclase……………………………………….70 Pharmacological analysis…………………………………………………..71 3.2 Transcriptional Regulation of Prodynorphin During Nicotine Withdrawal………...78 Prodynorphin mRNA and dynorphin content……………………………...78 Transcription factor binding………………………………………………..78 Adenylyl Cyclase Activity and cAMP Content……..………………….87 Regulation of CREB and during nicotine withdrawal……………………...87 3.3 Opioid Receptors During Nicotine Withdrawal……………………………………..87 Kappa Receptor Regulation………………………………………………...87 Delta Receptor Regulation………………………………………………….96 Opioid Receptor-Adenylyl Cyclase Coupling……………………………...96 Striatal G-protein content…………………………………………………...97 Chapter 4: Discussion and Conclusions..……………………………………………….105 4.1 Acute Nicotine PD Studies.…………...……………………………………………105 4.2 Chronic Nicotine PD Studies………………...……………………………………..115 Prodynorphin Regulation………………………………………………….115 4.3 Opioid Receptors During Withdrawal……………..……………………………….120 4.4 Conclusions……………………………………………………..…………………..129 Bibliography……………………………………………………………………………131 ix LIST OF TABLES Table 1: Neuroanatomical organizational of the striatum. Table 2: Pre- and post-synaptic distribution of striatal opioid receptors. Table 3: CREB and p-CREB in the striatum after acute nicotine. Table 4: Dopamine receptors and muscarininc receptors contribute to dynorphin increases after acute nicotine. Table 5: Adenylyl cyclase activity and cAMP content in the striatum during nicotine withdrawal. Table 6: CREB and p-CREB in the striatum during nicotine withdrawal. Table 7: Kappa opioid receptor mRNA in the brain during nicotine withdrawal. Table 8: Kappa opioid receptor binding
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