Abstract
Abstract: :
Purpose: Inhibition of rod cGMP phosphodiesterase activity leads to sustained elevation of intracellular calcium (Ca2+), which triggers apoptosis in retinal photoreceptors (Fox et al., 1999), a mechanism, which appears to involve the activation of the cystein–protease calpain (Sharma and Rohrer, ARVO abstract 2003). Likewise, a redox imbalance caused by photo–oxidative stress has been shown to cause apoptosis in photoreceptors (Krishnamoorthy et al., 1999), and oxidative stress has been shown to trigger apoptosis via calpain in other systems. We set out to investigate whether Ca2+–induced apoptosis in photoreceptors is mediated by the interactive actions of calpain and oxidative stress both in vivo (rd mice) and in vitro (murine photoreceptor derived cell line (661W cells); Al–Ubaidi, et al., 1992) to get a more complete picture of photoreceptor cell death in the rd mouse retina. Methods: Oxidative stress markers such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase were analyzed in both the rd mice and the 661W cells. 661W cells were assessed for changes in calpain activity, antioxidant enzymes levels, generation of reactive oxygen species (ROS) and cell survival (MTT assay) in response Ca2+–influx mediated by the Ca2+ ionophore A23187 (5 µM). H2O2 (100 µM) was used as a positive control. Results: A) The activity levels of antioxidant enzymes, SOD and GPx were increased two– to three–fold in the rd mice retinas as compared to the wild type. B) Fluorometric analysis of calpain activity in rd mice showed an age–, and thus Ca2+–dependent increase. Likewise, a multifold increase in calpain activity was observed in 661W cells with A23187 or H2O2 treatment. C) The generation of ROS was evaluated by fluorometric analysis showing a time–dependent increase in the generation of free radicals upon A23187 or H2O2 treatment in 661W cells. D) The assessment of cell viability by MTT assay demonstrated a time– and dose–dependent cell death by A23187 or H2O2 in 661W cells. A23187 (5µM) caused 40 ± 5.8% loss of cell viability and H2O2 (100 µM) treatment resulted in 33 ± 4.2% loss by 24 hrs. Conclusions: Our preliminary results suggest that Ca2+–mediated apoptosis in photoreceptors involves an oxidative stress–dependent calpain activation leading to apoptosis in photoreceptors. Further experiments examining the interaction between these two pathways will be performed. These apoptotic pathways will be further examined in the rd mouse retina, using a retina organ culture system.
Keywords: photoreceptors • calcium • oxidation/oxidative or free radical damage