Dynamic regulation of GSH synthesis and uptake pathways in the rat lens epithelium

Abstract

Glutathione (GSH) is an essential antioxidant required for the maintenance of lens transparency. In the lens, GSH levels are maintained by a combination of de novo synthesis and or direct uptake of GSH from the aqueous. Previous work in our laboratory has sought to identify and spatially localise the different components involved in GSH synthesis and uptake. Utilizing a high resolution imaging technique, we have mapped the distributions of GSH and its precursor amino acids cyst(e)ine, glutamate and glycine throughout the entire rat lens. An interesting observation from these studies was the marked difference in the localization of GSH and its precursor amino acids in the equatorial epithelium. While GSH was high in the equatorial lens epithelium there was an absence of cystine, glutamate and glycine. These results indicate that precursor amino acids were depleted through GSH synthesis or the source for GSH accumulation in the equatorial epithelium is primarily by uptake from the aqueous. In this paper, we have examined the contributions of GSH synthesis and uptake pathways in the different regions of the rat lens epithelium. We have extended and compared our mapping of GSH and its precursor amino acids to the central lens epithelium and have included labeling for gamma-GCS, the rate limiting enzyme for GSH synthesis. We show that spatial differences in GSH synthesis and uptake pathways exist between the equatorial and central epithelium. Moreover, in a distinct region of the equatorial epithelium, we were able to induce an increase in the labeling of precursor amino acids and gamma-GCS indicating that a dynamic switch from GSH uptake to GSH synthesis in response to depletion of extracellular GSH from the culture media had occurred. Finally, we also describe the identification of a putative GSH transporter which is most likely to mediate GSH uptake in this region.