LOCALIZATION AND EXPRESSION OF ET-1 RECEPTORS IN THE NORMAL AND GLAUCOMATOUS DOG EYE By MARIA E. KÄLLBERG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2003 Copyright 2003 By Maria E. Källberg Dedicated to my family and my friends who made this work come true. ACKNOWLEDGMENTS I would like to thank everyone who helped and supported my scientific work and my graduate education, especially those listed below. Dr. Dennis E. Brooks invited me to Florida and made the dream of my life come true. Thanks to Dr Brooks I will be able to dedicate my professional life to ophthalmology as a researcher and clinician. I thank him for his support through the years. The precious friendship we have developed will always help me whatever challenges life has to offer in the future. Dr. William W. Dawson guided me through the world of vision research and electrophysiology, which for me was unknown territory. He has inspired me not only by his knowledge but also by his way of questioning axioms and looking for new truths. I am thankful to Dr Dawson and his wife, Judyth, for their concern and warm generosity. Dr. Adrian M. Timmers meant a great deal to me and my project. He opened his laboratory for me and patiently taught and helped me through every necessary step of the molecular biology part of my project. His positive and supportive attitude was extraordinary. I would like to thank Dr. Kirk N. Gelatt for sharing with tireless enthusiasm his tremendous knowledge in research and clinical ophthalmology. I am also grateful for the many times he helped me in his own positive way whenever I needed good advice. iv Dr. Don A. Samuelson broadened my view of comparative ophthalmology. He and his laboratory technicians, Mrs. Patricia Lewis and Ms. Mae Chisholm, were very helpful in accomplishing all the challenges in histologic methods that I encountered during my time as a graduate student. I owe Dr. Adolfo G. Garcia many thanks for his contribution to my project. I am grateful to have gotten not only scientific help but also a friendship for life. My dear friend, Dr. András M. Komáromy, has been by my side from the very first page of this new chapter of my life. András made the transition easy by always being there for me whenever I needed help or support. Dr. Franck J. Ollivier, dear friend and colleague, deserves lots of thanks for the wild scientific discussions and many laughs. With all my heart I thank my best friend, Kerstin Bergvall, who helped me through many tough times. Ann Ellis gave me many hours of help teaching me electron microscopy. I would like to thank Dr. Stacy E. Andrew, the ophthalmology residents at the University of Florida, and various ophthalmologists in Florida (especially Dr. Salisbury and Dr. Brogdon) for their contributions. Mr. Harold L. Sapp gave valuable technical assistance. Dr. Carol Detrisac was very helpful in reviewing my histology slides. Dr. George Lambrou, CIBA Vision Ophtha, Novartis, provided very generous financial and technical support. v I would like to thank Ms. Marinela Capanu and the Interdisciplinary Center for Biotechnology Research at the University of Florida for their help with statistic analysis of the data. Dr. Nancy Szabo at the Analytical Toxicology Core Laboratory at University of Florida has been extremely helpful with analyzing my samples and sharing her expertise. Dr. Thomas Yorio, Dr. Ganesh Prasanna, Dr. Ragu Krishnamoorthy, Dr. Martha Stokely, Christina Hulet, and Santos Narayan at the Health Science Center, Fort Worth, Texas deserve a special thanks for their tremendous support. I owe many thanks to the Office of Research and Graduate Studies at the College of Veterinary Medicine, University of Florida. Associate Dean Charles Courtney III and Mrs. Sally O’Connell made it possible for me to become a graduate student and to successfully finish my dissertation. Sally helped me through several storms. Finally I would like to thank my parents for supporting me the whole way through my training and for helping me to realize my dreams. The sometimes overwhelming distance from home was overridden by the closeness of our relationship. vi TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................................................................................. iv LIST OF TABLES...............................................................................................................x LIST OF FIGURES ........................................................................................................... xi ABSTRACT..................................................................................................................... xiii CHAPTER 1 INTRODUCTION ........................................................................................................1 Glaucoma......................................................................................................................1 Role of Endothelin in Glaucoma ...........................................................................1 Canine Glaucoma ..................................................................................................4 Primary narrow-angle glaucoma in American Cocker Spaniels. ...................6 Primary open-angle glaucoma in the Beagle................................................10 Ophthalmic Vascular Morphology and Physiology ...................................................11 Endothelin and Nitric Oxide.......................................................................................16 Synthesis, Secretion and Clearance of Endothelin..............................................16 Nitric Oxide.........................................................................................................19 Endothelin Receptors...........................................................................................20 Endothelin and the Ophthalmic Circulation........................................................24 Endothelin and Aqueous Humor Production and Outflow.........................................26 Purpose of Study.........................................................................................................27 2 MATERIALS AND METHODS ...............................................................................28 Study Design...............................................................................................................28 Clinical Examination of Dogs ....................................................................................29 Endothelin-1 and Nitric Oxide Levels in Aqueous Humor and Vitreous...................29 Endothelin-1 Analysis .........................................................................................29 Nitric Oxide Analysis..........................................................................................30 Localization of Endothelin Receptors in the Retina and Choroid ..............................30 Light Microscopy ................................................................................................31 Transmission Electron Microscopy.....................................................................31 Morphology of Retina and Iridocorneal Angle ...................................................33 Endothelin Receptor Protein Expression in the Retina and Choroid..........................33 Protein Extraction................................................................................................34 vii Endothelin Receptor and Endothelin-1 mRNA Levels in the Retina.........................36 Isolation of Total RNA........................................................................................36 Reverse Transcriptase Polymerase Chain Reaction ............................................37 Real Time Polymerase Chain Reaction...............................................................38 Data Analysis..............................................................................................................40 Morphology .........................................................................................................40 Endothelin-1 and Nitric Oxide Levels in Aqueous Humor and Vitreous ...........40 Endothelin Receptor Protein Expression in the Retina and Choroid ..................40 Endothelin-1 and ET Receptor mRNA Levels in the Retina ..............................41 3 RESULTS...................................................................................................................49 Morphology ................................................................................................................49 Iridocorneal Angle...............................................................................................49 Retina...................................................................................................................49 Endothelin Receptors in the Retina and Choroid .......................................................50 Evaluation of Endothelin Antibodies using Light Microscopy...........................50 Evaluation of Endothelin Antibodies using Transmission Electron Microscopy ......................................................................................................50 Localization of Endothelin Receptors using Transmission Electron Microscopy ......................................................................................................51 ET A receptor................................................................................................51