A cystine-cysteine shuttle mediated by xCT facilitates cellular responses to S-nitrosoalbumin.

We have shown previously that extracellular cysteine is necessary for cellular responses to S-nitrosoalbumin. In this study we have investigated mechanisms involved in accumulation of extracellular cysteine outside vascular smooth muscle cells and characterized the role of cystine-cysteine release in transfer of nitric oxide (NO)-bioactivity. Incubation of cells with cystine led to cystine uptake, reduction, and cysteine release. The process was inhibitable by extracellular glutamate, suggesting a role for system x(c)(-) amino acid transporters. Smooth muscle cells express this transporter constitutively and induction of the light chain component (xCT) by either diethyl maleate or 3-morpholino-sydnonimine (SIN-1) led to glutamate-inhibitable cystine uptake and an increased rate of cysteine release from cells. Likewise, overexpression of xCT in smooth muscle cells or endothelial cells led to glutamate-inhibitable cysteine release. The resulting extracellular cysteine was found to be required for transfer of NO from extracellular S-nitrosothiols into cells via system L transporters leading to formation of cellular S-nitrosothiols. Cysteine release coupled to cystine uptake was also found to be required for cellular responses to S-nitrosoalbumin and facilitated S-nitrosoalbumin-mediated inhibition of epidermal growth factor signaling. These data show that xCT expression can constitute a cystine-cysteine shuttle whereby cystine uptake drives cysteine release. Furthermore, we show that extracellular cysteine provided by this shuttle mechanism is necessary for transfer of NO equivalents and cellular responses to S-nitrosoablumin.