Gluthathione (GSH) is known to protect the lens from oxidant stress and maintain lens transparency. GSH levels decrease with advancing age rendering the aged lens susceptible to reactive oxygen metabolites and injury. We have previously shown that GSH is transported intact in an in situ guinea-pig perfused eye model. Subsequent work showed that when bovine lens mRNA is injected into Xenopus laevis oocytes, GSH transport is expressed and that bovine and rat lenses express the transcript and protein for one of the recently cloned hepatic GSH transporters, rat canalicular GSH transporter (RcGshT). More recently, evidence for the presence of an additional, novel Na+-dependent GSH transporter in the rat lenticular epithelium was also obtained using the oocyte expression system. The GSH transport expressed by lens epithelial mRNA was sensitive to BSP-GSH (unlike RcGshT), a finding that was confirmed by inhibitory studies in the in situ eye perfusion model. The goal in the present proposal is to characterize the GSH transporters, and to study their developmental regulation at the molecular level.
The specific aims are to: 1) clone the Na+/GSH co-transporter in the lens. The PI will achieved this by size fractionation of lenticular epithelial mRNA, exclusion or identification of RcGshT, the sinusoidal GSH transporter (RsGshT), gamma glutamyltranspeptidase and cloning the fraction with Na+-dependent GSH uptake followed by sequence analysis. Organ distribution of the newly cloned GSH transporter and kinetics and specificity of transport will also be studied. 2) characterization of RcGshT in the lens. We will study developmental and age-dependent changes in RcGshT transcript and gene product and localization of RcGshT in the rat lens by in situ hybridization and immunohistochemistry. Use of phenobarbital as a tool to increase RcGshT will also be pursued. And finally, 3)he will use vesicles as a model to study GSH transport in rat lens cortical plasma membranes. Specifically, he will determine kinetics of uptake and determine Km and Vmax of multicomponents, study transstimulation of uptake, inhibitor specificity and identify driving force (membrane potential, Na+-dependence, pH dependence). Knowledge gained on the novel GSH transporters will be of value in better understanding of GSH homeostasis in the lens, their developmental regulation, and in designing therapeutic modalities to prevent lens damage associated with old age.
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