ENDOCANNABINOID SYSTEM IN THE PLANARIAN MODEL Katie Lynn Mustonen Thesis Prepared for the Degree of MASTER OF SCIENCE UNIVERSITY OF NORTH TEXAS December 2010 APPROVED: Barney J. Venables, Major Professor Kent D. Chapman, Committee Member Aaron P. Roberts, Committee Member Art J. Goven, Chair of the Department of Biological Sciences James D. Meernik, Acting Dean of the Robert B. Toulouse School of Graduate Studies Mustonen, Katie Lynn. Endocannabinoid System in a Planarian Model. Master of Science (Biology), December 2010, 138 pp., 22 tables, 9 figures, references, 452 titles. In this study, the presence and possible function of endocannabinoid ligands in the planarian is investigated. The endocannabinoids ananadamide (AEA) and 2- arachidonoylglycerol (2-AG) and entourage NAE compounds palmitoylethanolamide (PEA), stearoylethanolamide (SEA) and oleoylethanolamide (OEA) were found in Dugesia dorotocephala. Changes in SEA, PEA, and AEA levels were observed over the initial twelve hours of active regeneration. Exogenously applied AEA, 2-AG and their catabolic inhibition effected biphasic changes in locomotor velocity, analogous to those observed in murines. The genome of a close relative, Schmidtea mediterranea, courtesy of the University of Utah S. med genome database, was explored for cannabinoid receptors, none were found. A putative fatty acid amide hydrolase (FAAH) homolog was found in Schmidtea mediterranea. Copyright 2010 by Katie Lynn Mustonen ii ACKNOWLEDGEMENTS I would like to express my gratitude to my major professor and super-hero Dr. Barney Venables for his patience, understanding and encouragement. I would also like to thank my student colleagues who helped with so many little things and to especially acknowledge Cheryl Waggoner. Our paths were so similar, in reaching this milestone her absence is palpable. I thank my parents Katie H. and Lynn T. Mason for their unwavering faith and support. Finally, I thank my loving husband Ilkka Mustonen, otherwise known as Honey, who accepts all my shortcomings and makes everything possible. iii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ................................................................................................................... iii LIST OF TABLES ............................................................................................................................... vii LIST OF FIGURES .............................................................................................................................. ix Chapters 1. INTRODUCTION ....................................................................................................... 1 The Endocannabinoid System ..................................................................... 1 Cannabinoid Receptors ................................................................... 2 Cannabinoid Ligands ....................................................................... 3 Synthesis and Degradation ............................................................. 6 Entourage Compounds ................................................................... 7 Other Receptor Interactions ........................................................... 8 ECS Functions in Human and Murine Physiology ..................................... 11 Cannabimimetic Behavior ............................................................. 11 Cannabinoid Withdrawal .............................................................. 13 Appetite ........................................................................................ 14 Analgesia ....................................................................................... 15 Opioid Interactions ....................................................................... 15 Inflammation ................................................................................. 16 Excitotoxicity ................................................................................. 19 Cannabinoid Induced Cell Death................................................... 20 Ischemia ........................................................................................ 20 Cancer ........................................................................................... 21 Stem Cells ...................................................................................... 23 Natural History of the ECS ........................................................................ 24 Bioinformatics ........................................................................................... 29 The Planarian Model ................................................................................. 30 Hypothesis Statements ............................................................................. 36 iv 2. MATERIALS AND METHODS .................................................................................. 37 General Information ................................................................................. 37 Sample Procedures for GC/MS Analysis ................................................... 38 Stressed Sample Set ...................................................................... 38 Sectioned Sample Set.................................................................... 38 Enzyme Inhibitors ......................................................................... 39 Extraction and Cleanup ................................................................. 39 GC/MS Procedures ........................................................................ 40 Procedures for Behavior Analysis ............................................................. 41 Cannabinoid and Enzyme Inhibitor Exposures ............................. 42 BLAST Search Methods ............................................................................. 44 3. RESULTS................................................................................................................. 46 Analytical Results ...................................................................................... 46 Stressed Sample Sets .................................................................... 47 Sectioned Animals ......................................................................... 49 Enzyme Inhibitor Exposures .......................................................... 49 Behavioral Results ..................................................................................... 51 Anandamide and OEtA Exposures ................................................ 53 2-AG and oAG Exposures .............................................................. 54 Bioinformatics ........................................................................................... 56 Cannabinoid Receptors ................................................................. 56 Other GPCRs .................................................................................. 57 Metabolizing Enzymes .................................................................. 57 TRP Channels ................................................................................. 59 PPARs ............................................................................................ 60 Results Summary....................................................................................... 60 4. DISCUSSION ........................................................................................................... 62 Analytic Results ......................................................................................... 62 Background Levels ........................................................................ 62 Stressed Animals ........................................................................... 63 v Sectioned Animals ......................................................................... 64 Enzyme Inhibitors ......................................................................... 65 Behavior Results ........................................................................................ 66 Anandamide and OEtA Exposures ................................................ 66 2-AG and oAG Exposures .............................................................. 68 Comparison to Murine Models ..................................................... 68 Bioinformatics ........................................................................................... 69 Cannabinoid Receptors and Associated GPCRs ............................ 70 Metabolizing Enzymes .................................................................. 71 Transient Receptor Potential Channels ........................................ 74 Peroxisome Proliferator-Activated Receptors .............................. 77 Other Observations ................................................................................... 77 Conclusions ............................................................................................... 79 Appendices A. ADDITIONAL INFORMATION REGARDING CANNABINOID PHARMACOLOGY ...... 80 B. COMPLIATION OF LITERATURE VALUES CONCERNING ENDOCANNABINOID- ASSOCIATED LIGANDS AND ENDPOINTS IN INVERTEBRATES ............................... 84 C. BLAST RESULTS ...................................................................................................... 89 D. LEGEND OF ABBREVIATIONS ................................................................................. 96 REFERENCES ................................................................................................................................ 99 vi LIST OF TABLES Page 1 Quantification