Table Of Content

CYCLIC NUCLEOTIDES IN THE (rd) RETINAL DEGENERATE CHICKEN RETINA By NANCY RUTH LEE 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 1991 This thesis is dedicated to the memory of Robert J. Ulshafer, Ph.D., my mentor, friend and inspiration for this work. . ACKNOWLEDGEMENTS I thank my advisor Dr. Robert J. Ulshafer for sharing his knowledge of the retina and expertise in electron microscopy, for his patience as a mentor and for his kindness, generosity and sense of humor as a friend. His enthusiasm for his research sparked my interest in the retina and was the inspiration for my disseration project. I am grateful to Dr. William G. Luttge for his support, encouragement and advice as both coadvisor and chairman, which have facilitated completion of this dissertation. I thank committee member Dr. Robert J. Cohen for his technical advice and knowledge concerning various biochemical aspects of the project and for his support and interest throughout the course of the dissertation studies. For her enthusiasm, shared interest in the rd chick model and assistance with dissections, I thank committee member Dr. Susan Semple-Rowland. I am grateful to committee member Dr. William W. Dawson for his technical suggestions and the use of equipment and to previous committee member Dr. Adrian J. Dunn for his technical recommendations I thank Dr. Melvin L. Rubin, Chairman of the Department of Ophthalmology, for the use of the department's facilities iii and equipment throughout the course of my dissertation. I thank Connie Daughtry and Evelyn Clausnitzer for their assistance in the preparation of this manuscript. A special thank you to my husband, Roger Lee, for his continuous patience, love and support, which have made possible the realization of both a career and family life. And a final thanks to Mom and Dad, who made this possible and who have always been there. The present studies were supported by Office of Naval Research Grant No. N00014-88-J-1137 N.I.H. Grant No. EY04590, , and by March of Dimes Basic Research Grant No. 1-1039. IV TABLE OF CONTENTS page ACKNOWLEDGEMENTS iii ABSTRACT vii CHAPTERS 1 LITERATURE REVIEW 1 Transduction in Rods 3 Transduction in Cones 8 Chicken Retina Development and Cyclic Nucleotides 15 The Retinal Degenerate (rd) Chicken 16 Abnormal cGMP Metabolism and Retinal Degeneration 23 LIGHT DECREASES cGMP LEVELS IN THE 2 CONE-DOMINANT CHICKEN RETINA 26 Introduction 26 Methods 28 Effect of Freezing on Retinal cGMP and cAMP.... 29 Effect of Light Adaptation on cGMP and cAMP.... 30 Effect of Brief Light Pulse on cGMP and cAMP... 31 Results 32 Effect of Freezing on Retinal cGMP and cAMP.... 32 Effect of Light Adaptation on cGMP and cAMP 34 Effect of Brief Light Pulse on cGMP and cAMP... 36 Effect of Ischemia on Dark-Adapted Levels of Cyclic Nucleotides 36 Discussion 37 LIGHT MODULATION AND DEVELOPMENTAL TIMECOURSE 3 OF CYCLIC NUCLEOTIDE LEVELS IN THE rd CHICKEN RETINA 41 Introduction 41 Methods 44 Effect of Light Adaptation 44 Development of Cyclic Nucleotide Levels 45 Extraretinal Tissues 46 . Results 47 Effect of Light Adaptation 51 Extraretinal Tissue Levels of cGMP and cAMP. ... 53 Developmental Timecourse 53 Central vs. Peripheral Retinal Cyclic Nucleotide Levels 58 Discussion 61 4 DISTRIBUTION OF cGMP AND cAMP IN THE CONE-DOMINANT RETINAS OF SIGHTED AND BLIND (rd) CHICKENS 68 Introduction 68 Methods 70 Results 72 Distribution of cGMP 72 Distribution of cAMP 77 Discussion 78 cGMP 79 CAMP 83 5 HISTOCHEMICAL LOCALIZATION AND KINETIC PROPERTIES OF cGMP AND cAMP PHOSPHODIESTERASES IN THE rd CHICKEN RETINA 88 Introduction 88 Methods 89 Histochemical Techniques 90 Kinetic Analyses 92 Results 94 Histochemical Localization of cGMP PDE 94 Histochemical Localization of cAMP PDE 101 Kinetic Properties of cGMP PDE and cAMP PDE. 105 . . Discussion 109 Phosphodiesterase in Carrier Chick Retinas 109 Phosphodiesterase in rd Chick Retinas Ill 6 KINETIC ANALYSIS OF GUANYLATE CYCLASE ACTIVITY IN THE rd CHICKEN RETINA 114 Introduction 114 Methods 115 Guanylate Cyclase Assay 115 Results 120 Discussion 126 Speculation 134 7 OVERALL DISCUSSION 138 REFERENCE LIST 145 BIOGRAPHICAL SKETCH 160 vi Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CYCLIC NUCLEOTIDES IN THE (rd) RETINAL DEGENERATE CHICKEN RETINA By Nancy Ruth Lee August 1991 Chairperson: William G. Luttge, Ph.D Major Department: Neuroscience This study investigated the light modulation, retinal distribution and developmental timecourse of cGMP and cAMP levels as well as the kinetic properties and histochemical localizations of cyclic nucleotide metabolizing enzymes in the cone-dominant chicken retina. Comparisons were made between sighted chicks and congenitally-blind rd (retinal degenerate) chickens, in which visual transduction is severely compromised prior to the onset of photoreceptor pathology. In sighted chicks, levels of cGMP and cGMP phosphodiesterase (PDE) activity were concentrated within the photoreceptor cells and were highest in the outer segment (OS) region. Levels of cAMP and cAMP PDE activity were more evenly distributed between outer and inner retina and were lowest in the OS region. Light adaptation significantly reduced levels of both cGMP and cAMP vii in the proximal photoreceptor layers. In the rd retina, defective transduction was found to be accompanied by a severe and photoreceptor-specific deficiency in cGMP levels, rd retinas failed to accumulate normal levels of cGMP during photoreceptor outer segment development. On the day of hatch, photoreceptor levels of cGMP were reduced 75% within the OS region and were unresponsive to light adaptation. Levels of cAMP in rd retinas were not severely affected in association with blindness. Kinetic analysis of cGMP PDE activity in rd retinas revealed a Vmax comparable to sighted chicks and a 28% reduced apparent Km for cGMP, indicating that rd photoreceptors may have an enhanced capacity for cGMP hydrolysis. Kinetic analysis of guanylate cyclase activity in rd retinas revealed a 78% increased Km for GTP and significant inhibition of enzyme activity at GTP levels above approximately 250 uM. The kinetic abnormalities apparent in guanylate cyclase are sufficient to largely explain the 75% deficiency in rd outer segment levels of cGMP. Our results suggest that inadequate synthesis of photoreceptor cGMP levels may underly defective transduction in the rd chick retina and provide in vivo evidence supportive of a cGMP-mediated transduction mechanism in cone photoreceptors. Vlll CHAPTER 1 LITERATURE REVIEW Visual signalling begins with absorption of light by photosensory receptors. Most vertebrate retinas possess two types of photoreceptors, rod cells and cone cells. Rods are extremely sensitive to light and mediate vision in dim light. These cells contain a single visual pigment, rhodopsin, which absorbs light over a broad spectral range, but maximally at 500 nm. Rod vision is therefore achromatic. Cones generally require more light to operate than rods and mediate daylight vision. They possess one of several visual pigments with narrow yet overlapping absorption spectra. Cones are responsible for color vision. Rods and cones are highly specialized to capture light and convert it into a neural signal which may ultimately be transmitted to the brain. Phototransduction takes place in the photoreceptor outer segment, an elongated specialization at the cell's apical end. The outer segment is comprised of a dense stack of 500-2000 flattened membranous disks in which light-absorbing visual pigment molecules are embedded. Surrounding the disks is a plasma membrane which contains light-sensitive ion channels. In rods, the disk membranes are physically separated from the outer plasma membrane with the 1 2 exception of a few basal disks. In cones, the disk membranes are continuous with the plasma membrane throughout the length of the outer segment. At the base of the outer segment is a thin cilium which connects the outer segment to the inner segment. Outer segment membranes are continuously renewed, forming by evagination of the ciliary plasma membrane. The inner segment contains the metabolic machinery of the photoreceptor cell. Numerous mitochondria, densely packed in the apical "ellipsoid" region of the inner segment, generate the energy required for inner segment needs as well as for transduction and membrane renewal processes in the outer segment. A somatic region and connecting fiber joins the inner segment to the cell's synaptic terminal, which possesses specialized ribbon synapses for chemical communication with retinal interneurons. Unlike most other sensory receptors, vertebrate photoreceptors hyperpolarize in response to light stimulation. In the dark, the cation channels in the outer segment plasma membrane are primarily in an open state. A dark current of Na* ions flows into the outer segment, depolarizing the cell. Light stimulation leads to the closure of the outer segment cation channels which reduces the circulating current. The cell hyperpolarizes and neurotransmitter release from the synaptic terminal is subsequently decreased in graded response to light.