A role for γ-glutamyl transpeptidase in the transport of cystine into human endothelial cells: relationship to intracellular glutathione

Abstract

The mechanism of uptake of cystine into human umbilical vein endothelial (HUVE) cells was further investigated. Previous experiments have suggested that the uptake of cystine into HUVE cells is mediated by the X − c anionic amino acid transporter. However, in our experiments, the accumulation of cystine into confluent HUVE cells was not fully inhibited by anionic amino acids, such as glutamate, aminoadipate and homocysteate. On the other hand, inhibitors of γ-glutamyl transpeptidase (γ-GT), such as anthglutin and AT-125, were also effective inhibitors of cystine accumulation in these human cells. Indeed, incubation of HUVE cells with anthglutin effectively depleted cellular glutathione (GSH) in association with inhibition of cystine uptake. A dose-dependent inhibitory effect of anthglutin on the initial rate of accumulation of cystine into control HUVE cells was also confirmed, which was also dependent on the carrier concentration of the disulfide. Additionally, in an effort to relate the uptake of cystine and the levels of GSH in these human cells, the incubation of HUVE cells with diethylmaleate (DEM, 25 μM) was shown to stimulate the uptake of cystine by up to 200% over the first hour of incubation, irrespective of whether DEM was coincubated or preincubated with the cells for 60 min before the addition of cystine. Under these conditions the stimulation was not associated with extensive depletion of cellular GSH. Depletion of HUVE cell GSH by an 18 h incubation in M199 medium lacking sulfur amino acids (M199-medium) also stimulated the uptake of cystine by up to 300%, which was dependent on de novo protein biosynthesis. On the other hand, the depletion of HUVE cell GSH by an 18 h preincubation with buthionine sulfoximine did not stimulate cystine uptake above controls. Again, both the anionic amino acids and the inhibitors of γ-GT were effective in inhibiting the accumulation of cystine into M199-medium-pretreated HUVE cells, in association with impaired resynthesis of GSH. Anthglutin also dose-dependently inhibited the initial cystine uptake rate into these M199-medium-pretreated cells by a proportionally similar extent to its effects in control cells. This also indicates that γ-GT-dependent uptake of cystine accounts for a portion of the induced uptake of this disulfide into these M199-medium-pretreated cells. These results indicate that both the X − c carrier and γ-GT are involved in the uptake of cystine into these human endothelial cells, illustrating another important physiological role for γ-GT in the uptake of cystine from the circulation into the human endothelium. This may be of importance in providing cysteine equivalents for protein and GSH synthesis, both within the endothelial cells themselves, and in other cells associated with endothelial cells which are unable to utilise cystine. The data also show that HUVE cells can up-regulate the uptake of cystine in response to different stress stimuli which are associated with the depletion of cellular GSH, but that the depletion of the GSH per se is not sufficient for this response. Finally, inhibitors of γ-GT may be useful in studies of the functions of GSH in HUVE cells.