Characteristics of glutathione biosynthesis by freshly isolated rat kidney cells.

Suspensions of freshly isolated rat kidney cells were used to study glutathione biosynthesis and utilization for amino acid translocation. Incubation of cells isolated from diethylmaleate-perfused kidneys in the presence of glutamate, glycine, and cystine led to replenishment of cellular glutathione and to accumulation of glutathione disulfides in the medium. Cystine could be replaced by cysteine (t0.5 mM) but not by methionine plus serine indicating that the cystathionine pathway was insufficient to support optimal glutathione biosynthesis. Uptake of cystine was associated with enhanced intracellular concentration of acid-soluble thiols independently of whether glutathione biosynthesis was inhibited or not. Incubation of control cells in an amino acid-free medium resulted in a slow decrease in cellular glutathione level which was accentuated in the presence of the inhibitor of glutathione biosynthesis, methionine sulfoximine. Glutathione loss was markedly accelerated by addition of various amino acids or dipeptides to the medium (e.g. glycylglycine, methionine, cysteine, cystine, and glutamate); this effect was abolished by serineborate, a potent inhibitor of y-glutamyltransferase. The uptake of several amino acids by cells isolated from diethylmaleate-perfused kidneys was inhibited by serineoborate; the uptake of methionine, cysteine, and cystine was markedly affected whereas that of glycine was not. The inhibition by serineeborate of cellular uptake of cystine was correlated to a similar inhibitory effect on glutathione biosynthesis. The results indicate major differences in the requirements for glutathione biosynthesis by isolated kidney and liver cells and provide strong support for the functioning of the y-glutamyl cycle in renal cells.