The Role of Calcium in Light-Induced Photoreceptor Apoptosis

Krueger, Darrel Scott, The Role of Calcium in Light-Induced Photoreceptor Cell Apoptosis. Doctor of Philosophy (Biomedical Sciences), May, 2002; 305 pp., 15 tables, 31 illustrations, bibliography, 421 titles. Retinal degenerative diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD) result in loss of photoreceptors by apoptosis. Photo-oxidative stress accelerates the rate of photoreceptor apoptosis in models of retinal dystrophies. Thus, a better understanding of light-induced apoptosis is important for developing preventative and treatment options for persons with these blinding diseases. The goals of this dissertation were to investigate the effect of photo-oxidative stress on [Ca2+]I in rat photoreceptor cells and determine whether cell death could be prevented by altering cells’ abilities to manage [Ca2+]i. Thirty minutes of light exposure resulted in significant elevation of [Ca2+]i, determined using Fura-2 ratiometric imaging, which increased with additional light exposure. At 120 minutes, the F340/380 ratio was 3-times the beginning baseline ratio. Using multiple techniques indicative of early and late phases of apoptosis, changes consistent with apoptosis were observed, including early labeling (30 Min) with Annexin-V, activation of caspase-3 (2IIr), TUNEL labeling and Propidium Iodidc staining. TUNEL and Propidium labeling were more intense at 3Hrs light exposure, reflecting late phase changes. Apoptosis was confirmed using electron microscopy (TEM). TEM showed mitochondrial swelling, indicative of permeabilization, prior to chromatin condensation. Pharmacological agents were utilized to mediate participation of cellular calcium sources or storage sites in the maintenance of intracellular calcium homeostasis during photo-oxidative stress. Agents were separately added to the media prior to light exposure and their effects on cell viability were assessed using the Formazan assay. Mitochondria were confirmed to be the site of action affected by elevated [Ca2+]I, since prevention of calcium uptake by ruthenium red (1-100-μM) provided significant protection of cell viability in a dose-related manner. The 100- μM concentration resulted in complete maintenance of viability. BAPTA-AM also demonstrated some protection (50%) indicating that reduction of [Ca2+]I independent of source is beneficial is maintaining cell viability. Identification of mitochondrial uptake and cytosolic buffering of calcium as key viability determinants in light-induced apoptosis is a significant discovery for targeting future research for preventing or inhibiting photoreceptor cell apoptosis associated with retinal dystrophies such as RP and AMD.