EFFECT OF CHLORPYRIFOS OXON ON M2 MUSCARINIC ACETYLCHOLINE RECEPTOR REGULATION BY ELMAR MABUNGA UDARBE Doctor of Veterinary Medicine University of the Philippines Los Baños College, Laguna, Philippines 1999 Submitted to the Faculty of the Graduate College of Oklahoma State University in partial fulfillment of the requirements for the Degree of MASTER OF SCIENCE July, 2004 EFFECT OF CHLORPYRIFOS OXON ON M2 MUSCARINIC ACETYLCHOLINE RECEPTOR REGULATION Thesis Approved: DR. CAREY N. POPE Thesis Advisor DR. CYRIL C. CLARKE DR. CHARLOTTE C. OWNBY DR. DORIS K. PATNEAU DR. AL CARLOZI Dean of Graduate College ii ACKNOWLEDGMENTS My sincerest gratitude goes to my major advisor, Dr. Carey N. Pope for the intelligent supervision, for providing inspiration to do this work. I am also thankful to my committee members, Dr. Cyril Clarke, Dr. Charlotte Ownby and Dr. Doris Patneau for helpful comments on the content and form of this manuscript. I am indebted to the Fulbright-Philippine Agriculture Scholarship Program (FPASP) and the Philippine American Education Foundation (PAEF) whose exchange program deepened my understanding of the U.S. culture and its people and allowed me to promote mutual understanding between the U.S. and the Philippines. I am grateful to the University of the Philippines in Mindanao (UPMINDANAO) for supporting my pursuit for graduate studies, the National Institute of Environmental Health Sciences (NIEHS), Oklahoma State University Board of Regents and Dr. Sidney Ewing, Wendell H. and Nellie G. Krull Endowed professor for the financial assistance. I am also thankful to the following: Ms. Sharon Baker for doing the preliminary work on the project; Dr. Liming Zou for vital inputs on M2 receptor regulation; Dr. Subramanya Karanth, Dr. Jing Liu Pope and Dr. Shaikh for technical and statistical help; Dr. Greg Sawyer, Ms. Tiffany Ricks and Mr. Jon Hart for technical assistance in cell culture techniques; Dr. Tom Bonner and Dr. Jelveh Lameh for providing the CHO cells used in the study, Dr. Laura A. Volpicelli for help with the immunocytochemistry and imaging techniques; Dr. Telugu Narasa Raju and Dr. Reddy for help with the fluorescent microscope; Ms. Betty Handlin for document formatting. My deepest thanks to Ms. Paetra Hauck (IIE), Dr. Adel Tongco, Randy Beckloff and Amy Fair (OSU-ISS) for administrative help during my stay here at OSU; the Filipino community here in Stillwater for being my family away from home; to my roommate Brenda Tubaña for the statistical analysis and camaraderie and all my friends who have helped me survive here in the U.S. just by being there. I want to extend my appreciation also to my greatest friend and husband, Dr. Rex Rhoderick Zamora, for making my adjustment in the US easier and especially for the love, patience and understanding all throughout the conduct of this work. I am also thankful to my family in the Philippines, my mom Elsie, my dad Marvelino and my siblings Leif Marvin and Marielle who continue to inspire me; and to the Zamoras, Dr. Emilio, Ampy and Zandra, for their love and support. Most of all, I am forever grateful to God, my Lord and Saviour, for the gift of life that allowed me to experience all the wonderful things He has created. And most especially for the graces He has bestowed on me. I am nothing without Him. iv TABLE OF CONTENTS Chapter Page Acknowledgments.....................................................................................................................................iii Table of Contents.......................................................................................................................................v List of Figures ...........................................................................................................................................vii Chapter One: Introduction.......................................................................................................................1 Background and Significance........................................................................................................ 1 Insecticides and Pesticides.........................................................................................................1 Organochlorines..........................................................................................................................2 Organophosphates......................................................................................................................3 Chlorpyrifos .................................................................................................................................7 Food Quality Protection Act of 1996 ......................................................................................8 Cumulative risk assessment and common mechanism of toxicity.....................................10 Non-cholinesterase targets.......................................................................................................12 Muscarinic receptors as targets ...............................................................................................14 Effects of Chlorpyrifos oxon on M2 receptors....................................................................17 M2/M4 receptor regulation.....................................................................................................18 Desensitization ..........................................................................................................................20 Internalization............................................................................................................................27 Downregulation as a cumulative effect of OPs....................................................................30 Hypothesis..................................................................................................................................32 Objectives ......................................................................................................................................32 Chapter Two: Materials and Methods..................................................................................................34 Sources of Chemicals ...................................................................................................................34 Cell Culture....................................................................................................................................34 Pre-coating with poly-D-lysine................................................................................................34 HEL 299 cells ............................................................................................................................34 CHO-K1.....................................................................................................................................35 Rat Striatal Neurons..................................................................................................................35 Experimental Design....................................................................................................................36 Drug Treatments...........................................................................................................................37 Radioligand Binding Assays ........................................................................................................38 [3H]Oxotremorine (Oxo-M) binding assay in HEL 299 cell membrane preparations ...38 [3H]N-methyl scopolamine (NMS) binding assay in intact HEL 299 and CHO-M2 cells ......................................................................................................................................................39 Immunocytochemistry .................................................................................................................40 Statistical Analyses........................................................................................................................40 Chapter Three: Results ............................................................................................................................42 v Effect of carbachol and CPO on M2 receptor desensitization..............................................42 Effect of carbachol and CPO on M2 receptor internalization ..............................................42 Radioligand binding ..................................................................................................................42 Immunocytochemistry..............................................................................................................44 Chapter Four: Discussion .......................................................................................................................55 Implications................................................................................................................................65 Chapter Five: Summary, Conclusions and Recommendations........................................................66 Chapter Six: Bibliography .......................................................................................................................68 Chapter Seven: Appendices....................................................................................................................78 Appendix 1. Raw data ..................................................................................................................78 A. The Effect of CCH and CPO on Oxo-M binding (% control) in HEL 299 cells. ....78