Lens transparency is essential for proper visual function. Glutathione (GSH) plays an important role in preventing lens opacification (cataract) by maintaining SH-groups of the lens proteins (membrane components, enzymes and crystallins) in reduced state. The lens is rich in GSH, and the current concept states that low levels of GSH are not consistent with lens clarity, but themselves are cataract inducing. Uptake of GSH by the lens and GSH lens synthesis de novo from sulfur amino acids (SAA) have been demonstrated in vitro, but direct evidence is lacking to conclude that this approximates the situation invivo. A new invivo vascular eye perfusion (VEP) model in the guinea-pig has been developed in our laboratory. The use of this species as an animal model for proposed studies has been justified during the course of our preliminary work. Biochemical characterization of guinea-pig lens suggested that metabolic scheme for GSH homeostasis is similar to that in other mammalian lenses. Using present VEP model, we obtained strong preliminary evidence indicating significant insitu rapid cellular uptake of newly secreted plasma-derived [35S]-GSH by the lens. A rapid in situ GSH lens synthesis from newly secreted blood-borne (35S)-cysteine was shown by radio HPLC analysis. The focus of this proposal is directed at plasma-derived GSH and SAA in the lens. The proposal is designed to test in a comprehensive fashion the hypothesis that blood-to-lens transport of circulating GSH and SAA is essential for the regulation of GSH levels in normal lenses. Towards this end, several experiments are proposed to test the following two hypotheses: I. Plasma-derived GSH is rapidly taken up at the lenticular epithelium by a specific transport system. II. GSH lens synthesis de novo is dependent on blood-to-lens transport of SAA. All transport and metabolic studies in normal guinea-pigs will use the VEP model, and consider for kinetic analysis four eye compartments including plasma, aqueous humor, lens capsule/epithelium and lens/cortex. Molecular forms of uptake during compartmental blood-to-lens transfer will be determined by radio-HPLC. The specificity and kinetic properties of GSH transport system in the lens will be characterized in situ, and GSH de novo synthesis from circulating SAA precursors will be estimated. These studies will help us understand the significance of blood-to-lens transport of GSH and SAA for normal lens function. Defining the role of plasma-derived GSH and SAA in normal lenses may be important in designing therapeutic strategies to decelerate cataract-inducing processes and/or to prevent formation of cataract.
