Requirement for GSH in recycling of ascorbic acid in endothelial cells

Ascorbic acid may be involved in the defense against oxidant stress in endothelial cells. Such a role requires that the cells effectively recycle the vitamin from its oxidized forms. In this work, we studied the ability of cultured bovine aortic endothelial cells (BAECs) to take up and reduce dehydroascorbic acid (DHA) to ascorbate, as well as the dependence of ascorbate recycling on intracellular GSH. BAECs took up and reduced DHA to ascorbate much more readily than they took up ascorbate. Although BAECs in culture did not contain ascorbate, ascorbate accumulated to concentrations of 2-3 mM in BAECs following incubation with 400 microM DHA. Extracellular ferricyanide oxidized intracellular ascorbate, which was recycled by the cells. Reduction of DHA, either when added to the cells or when generated in response to ferricyanide, caused significant decreases in intracellular GSH concentrations. Depletion of intracellular GSH with 1-chloro-2,4-dinitrobenzene, diethylmaleate, and diamide almost abolished the ability of the cells to reduce DHA to ascorbate. DHA reduction by thioredoxin reductase was evident in dialyzed cell extracts, but occurred at rates far lower than direct GSH reduction of DHA. These results suggest that maximal rates of DHA reduction, and thus recycling of ascorbate from DHA, are dependent upon GSH in these cells.