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 not 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 are not electrogenic, uncoupling from the epithelium during isolation results in decrease of resting potential to practically zero (-4 mV). Intracellular calcium in isolated fiber cells is low, and that exposure to 1 to 2 mM calcium causes increase of [Ca2+]j to 1 to 2 uM and protease activity 6 to 8 fold which causes disintegrative globulization. The globules formed in vitro bear a striking resemblance to those observed at the light-scattering centers of cortical cataracts. We have partially purified a novel protease (named CMAC Protease) from lens cortex. This protease is different from Calpain and leucine aminopeptidase. Ca2+ Ringer's solution activates this enzyme 6 to 8 fold in rat lens fiber cells. Bestatin, an inhibitor of CMAC Protease significantly (> 3 hrs) delays fiber cell globulization in Ca2+ Ringer's solution. We will now purify CMAC Protease to homogeneity and investigate its kinetic and structural properties and examine its role in the breakdown of cytoskeletal and non-cytoskeletal proteins. The pathways regulating calcium-dependent and -independent proteolytic activity in cortical fibers will also be investigated. Furthermore, proteolytic changes to cytoskeletal and noncytoskeletal proteins that may contribute to the disintegration of fiber cells will be assessed. We will also investigate the processes by which cortical fibers maintain their calcium homeostasis, and the mechanism(s) that elevate free calcium during globulization of isolated fibers. 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.