Affiliations: Department of Medicine, Vanderbilt University School
of Medicine, Nashville, TN 37232-6303, USA
Note: [] Address for correspondence: Dr. James May, 715 Preston Research
Building, Vanderbilt University School of Medicine, Nashville, TN 37232-6303,
USA. Tel.: +1 615 936 1653; Fax: +1 615 936 1667; E-mail:
james.may@vanderbilt.edu
Abstract: Ascorbic 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
5,5p-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.
