Redox regulation of ascorbic acid transport: Role of transporter and intracellular sulfhydryls

AbstractAscorbic acid is one of the most sensitive cellular defenses against oxidant damage. However, it requires a sodium‐ and energy‐dependent transporter to enter cells against a concentration gradient. To test the hypothesis that ascorbate transport is sensitive to redox stress, we studied changes in transport of the vitamin in response to sulfhydryl modification of the protein and to GSH depletion in cultured endothelial cells. Transport of ascorbic acid, measured as the uptake of radiolabeled ascorbate, was inhibited by the membrane‐impermeant sulfhydryl reagents thorin, p‐chloromercuribenzene sulfonic acid, and 5,5′‐dithiobis‐(2‐nitrobenzoic acid) in a dose‐dependent manner without significant depletion of intracellular GSH. Sulfhydryl reagents capable of penetrating the plasma membrane, including phenylarsine oxide, p‐chloromercuribenzoic acid, and N‐ethylmaleimide, inhibited transport and lowered cellular GSH. Diamide, which induces disulfide formation, increased ascorbate transport over a narrow concentration range under conditions in which GSH was not depleted. On the other hand, specific depletion of intracellular GSH by several different mechanisms did inhibit transport. Together, these results suggest that the ascorbate transporter is sensitive to redox modulation. This relates in part to sulfhydryl groups exposed on the exofacial ascorbate transporter, and to sulfhydryl groups that are sensitive to changes in the redox state of intracellular GSH.