C-13-NMR SPECTROSCOPIC STUDIES OF 2-MERCAPTOETHANOL-STIMULATED GLUTATHIONE SYNTHESIS IN THE INTACT OCULAR LENS

C-13-NMR spectroscopy was employed non-invasively for the real-time me asurement of the rates of glutathione synthesis in intact rabbit lense s supported in organ culture containing L-[3-C-13]cysteine. Supplement ation of the organ culture medium with 2-mercaptoethanol resulted in a dose-dependent enhancement of lenticular glutathione synthesis rates (dose for 50% maximal effect = 125 mu M). At the most effective concen tration (400 mu M) 2-mercaptoethanol increased the rate of glutathione synthesis 163% relative to the rate observed under control conditions . The mechanism of action for this effect was investigated in intact l enses using antagonists of specific amino acid uptake systems. These e xperiments demonstrated that the enhanced rates of glutathione synthes is observed in the presence of 2-mercaptoethanol were due to the affin ity of the mixed-disulfide formed between cysteine and 2-mercaptoethan ol for L system amino acid uptake, thereby providing a mechanism for i ncreasing intracellular cysteine levels by circumventing normal cellul ar cysteine uptake pathways in the lens. Because of the role of cystei ne as the rate limiting substrate in lenticular glutathione biosynthes is, these results suggest a potential strategy to prevent tissue opaci fication associated with depleted glutathione levels.