Mechanisms of Use of Extracellular Glutathione by Lung Epithelial Cells and Pulmonary Artery Endothelial Cells

Cells in most culture media use cystine as the primary source of the cysteine precursor needed for glutathione (GSH) synthesis. As a result, GSH levels in many cultured cells may be limited by the rate of uptake of cystine into cells. We have shown that incubation with extracellular GSH can result in the reaction of GSH with cystine to generate cysteine, and that bovine pulmonary artery endothelial cells and lung type II epithelial cells transported cysteine more efficiently than cysteine. Cysteine transport was not affected by the presence of GSH. In cells incubated with GSH in RPMI-1640 there was a cystine-dependent increase in intracellular GSH levels. The increases in GSH were not prevented by the presence of acivicin, an inhibitor of the gamma-glutamyl transpeptidase reaction. Incubation with oxidized glutathione (GSSG) did not result in significant increases in intracellular GSH levels. We conclude that a primary mechanism by which extracellular GSH may increase intracellular GSH levels in cultured cells is by reducing cystine to cysteine, which is then rapidly transported and used as a substrate for intracellular GSH synthesis.