The long-range goal of our research is to elucidate the mechanisms of hyperglycemic and senile cataractogenesis. Though oxidative and osmotic stress have been suggested as key changes associated with opacification, their cellular consequences are clearly understood. Our central hypothesis is that during cortical cataract, oxidative and/or osmotic stress increases free calcium levels in lens fibers, activating proteases. These cause disintegrative globulization of the fiber cells, thus altering the light-transmitting properties of the lens. To test this hypothesis it is essential to examine the mechanisms regulating calcium in cortical fiber cells, and to understand how calcium- mediated proteolysis causes disintegration of these cells. To facilitate such cellular studies, we have recently developed procedures for isolating single fiber cells from the lens cortex and maintaining their viability over the duration of our planned experiments. Our research during the funded period has shown that isolated fiber cells maintain low intracellular calcium, and that exposure to 1 to 2mM calcium causes disintegrative globulization. The globules formed in vitro bear a striking resemble to those observed at the light-scattering centers of cortical cataracts. We will now extend our observations and investigate the processes by which cortical fibers maintain their calcium homeostasis, and the mechanism(s) that elevate free calcium during globulization of isolated fibers. The pathways regulating calcium-dependent and -independent proteolytic activity in cortical fibers will be investigated. Furthermore, to understand the mechanism(s) by which proteolysis causes disintegration of cortical fibers, proteolytic changes to cytoskeletal and noncytoskeletal proteins will be examined, and their contribution to the disintegration of fiber cells will be assessed. Besides providing insight into the fundamental physiology of the lens cortex, our results will elucidate the cellular mechanisms by which cataractogenic conditions perturb the light-transmitting properties of the lens, and may lead to the development of more effective anticataractogenic interventions.
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