STUDIES OF SYNTHETIC PARTICLES AND NERVE ENDINGS ON MASS TRANSPORT AND KINETICS AND INHIBITION OF THE DEGLYCOSYLATED DOPAMINE TRANSPORTER By VERONICA MANLING CHIU A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY WASHINGTON STATE UNIVERSITY Department of Chemistry May 2012 To the Faculty of Washington State University: The members of the Committee appointed to examine the dissertation of VERONICA MANLING CHIU find it satisfactory and recommend that it be accepted. __________________________________ James O. Schenk, Ph.D., Chair ___________________________________ Herbert H. Hill, Jr., Ph.D. ___________________________________ Chulhee Kang, Ph.D. ___________________________________ Barbara A. Sorg, Ph.D. ii ACKNKOWLEDGEMENTS I would like to start by thanking my committee, Drs. Jim Schenk, Herb Hill, Chulhee Kang, and Barb Sorg for their support, encouragement, and guidance. I am especially grateful to my mentor as well as my friend, Dr. Jim Schenk, for the infinite support, patience, and encouragement. Jim, you allowed me to learn, think and find answers on my own, but at the same time you provided help whenever I needed it. You also encouraged me to believe who I am. You taught me how to write a scientific paper and allowed me to write in my own words. You also provided me much help with giving presentations, which I am still learning about. In addition to science knowledge, I learned a lot from you on cooking, food, American culture, and arts. I really enjoyed the time when we gathered and shared food, and of course, your food is always so tasty. I know I am going to miss it! I also enjoyed our talks, and I never met a person who has as much knowledge as you do. I also would like to thank Dr. Barb Sorg for her guidance on brain dissections. I really appreciated your help on my presentation for my interview. In addition, I am grateful to Dr. Nicole Bjorklund, who taught me how to use the RDEV, Paul Schumacher, who helped me on animal handling and electrochemistry, and Peter Lukus, who helped me in various tasks whenever I needed it. I would like to thank the people in the Chemistry department who had helped me throughout the years. I feel so grateful that I have my friends, Lisa, Christina, Elsa, Nelmi, Adam, Kristyn, Jamie, and Nancy, who share my happiness and who are always there when I need a hug. I am especially thankful for my best friend, Rebecca, who will always be there to give me support. iii And last but not least, I am so thankful to have endless support and encouragement from my family: mom, dad, my two lovely sisters (Anita and Bonnie), and my brothers-in-law (Jason and Martin). I love you all! iv STUDIES OF SYNTHETIC PARTICLES AND NERVE ENDINGS ON MASS TRANSPORT AND KINETICS AND INHIBITION OF THE DEGLYCOSYLATED DOPAMINE TRANSPORTER Abstract by Veronica Manling Chiu, Ph.D. Washington State University May 2012 Chair: James O. Schenk Unstirred layer (USL) is present at the interface of solids with solutions, and particles pass through these layers by diffusion. Rotating disk electrode voltammetry (RDEV) has been used in different biological applications. However, it is unknown whether brain tissue preparation in solution affects the mass transport at the electrode, and whether the USL in real biological samples affects the kinetic measurements. In Chapter Two, liposomes, silica, and Sephadex™ were used to model the tissue preparation particles and examined the effect on mass transport at the RDE. Both diffusion and hydrodynamic boundary layers formed between the solution and the electrode surface. In the presence of inert particles, the sensitivity, limiting current, apparent diffusion coefficient, boundary layers thicknesses, and the mixing time decreased with no change on the detection limit (6 ± 2 nM). In addition, the thicknesses of USL’s within the neuronal homogenates do not appear to affect the apparent kinetics of biological mass transport. Results also show that RDEV kinetically resolves transmembrane transport with a timing of approximately 30 ms. v It is known that the glycosylation on the dopamine transporter (DAT) is important for cocaine binding and the function of the DAT. However, the connection between carbohydrate moieties and the function of the DAT is unknown. In Chapter Three, effects of carbohydrate component modification on DA transport velocity were examined by using different glycosidases. DA transport activities decreased after different glycosidase treatments in rats’ striata. Then, α-mannosidase was used for the rest of the studies due to the rapid access to its substrate. After α-mannosidase treatment, Vmax of the DA transport decreased while the Km was unchanged. Moreover, removal of α-mannose abolished the effect of cocaine on the kinetic state of the DAT. Finally, cocaine, bupropion, mazindol, and β-phenylethylamine but not methamphetamine and tyramine block the effects of α-mannosidase on the DAT. Results lead to a conclusion that a derivative of mazindol may be a possible antagonist for cocaine. Methamphetamine is a very addictive drug that will damage the brain. Chapter Four provides a summary on how methamphetamine affects different neurotransmitter contents and its transporter kinetics in different brain regions. vi TABLE OF CONTENTS Page ACKNOWLEDGMENTS ............................................................................................................. iii ABSTRACT .....................................................................................................................................v LIST OF TABLES ...........................................................................................................................x LIST OF FIGURES ..................................................................................................................... xiii LIST OF SCHEMES......................................................................................................................xv ATTRIBUTIONS ........................................................................................................................ xvi CHAPTER 1. GENERAL INTRODUCTION INTRODUCTION .........................................................................................................1 REFERENCES ............................................................................................................11 2. MASS TRANSPORT AT ROTATING DISK ELECTRODES: EFFECTS OF SYNTHETIC PARTICLES AND NERVE ENDINGS ABSTRACT .................................................................................................................22 INTRODUCTION .......................................................................................................22 MATERIALS AND METHODS .................................................................................25 RESULTS ....................................................................................................................30 DISCUSSION ..............................................................................................................33 ACKNOWLEDGEMENTS .........................................................................................37 vii REFERENCES ............................................................................................................38 3. KINETICS AND INHIBITION OF THE DEGLYCOSYLATED DOPAMINE TRANSPORTER ABSTRACT .................................................................................................................55 INTRODUCTION .......................................................................................................56 MATERIALS AND METHODS .................................................................................60 RESULTS ....................................................................................................................65 DISCUSSION ..............................................................................................................66 ACKNOWLEDGEMENTS .........................................................................................70 REFERENCES ............................................................................................................71 4. MECHANISM OF ACTION OF METHAMPHETAMINE WITHIN THE DOPAMINERGIC AREAS OF THE CENTRAL NERVOUS SYSTEM ABSTRACT .................................................................................................................84 INTRODUCTION .......................................................................................................85 HISTORY OF AMPHETMAINE AND METHAMPHETAMINE ............................85 STATISTICS AND EFFECTS OF AMPHETAMINE AND METHAMPHETAMINE USAGE ..............................................................................86 WHY IS IT SO EASY TO MAKE METHAMPHETAMINE ....................................87 DRUG REWARD PATHWAYS IN THE BRAIN .....................................................88 METHODS OF DETECTION .....................................................................................92 METHAMPHETAMINE INHIBITION OF CATECHOLAMINE AND viii SEROTONIN TRANSPORTERS ...............................................................................94 CONCLUSION ..........................................................................................................120 ACKNOWLEDGEMENTS .......................................................................................121 REFERENCES ..........................................................................................................122