Hydrogen peroxide triggers an increase in cell surface expression of system x_c ⁻ in cultured human glioma cells

System x_c ⁻ exchanges extracellular cystine for intracellular glutamate across the plasma membrane of many cell types. One of the physiological roles of System x_c ⁻ is to provide cystine for synthesis of the antioxidant glutathione. Here we report that hydrogen peroxide (H₂O₂) triggers the translocation of System x_c ⁻ to the plasma membrane within 10 min of the initial exposure. Specifically, we observed a three-fold increase in ³⁵S-L-cystine uptake following a 10 min exposure to 0.3 mM H₂O₂. This effect was dose-dependent with an EC₅₀ for H₂O₂ of 65 μM. We then used cell surface biotinylation analysis to test the hypothesis that the increase in activity is due to an increased number of transporters on the plasma membrane. We demonstrated that the amount of transporter protein, xCT, localized to the plasma membrane doubles within 10 min of H₂O₂ exposure as a result of an increase in its delivery rate and a reduction in its internalization rate. In addition, we demonstrated that H₂O₂ triggered a rapid decrease in total cellular glutathione which recovered within 2 h of the oxidative insult. The kinetics of glutathione recovery matched the time course for the recovery of xCT cell surface expression and System x_c ⁻ activity following removal of the oxidative insult. Collectively, these results suggest that oxidants acutely modulate the activity of System x_c ⁻ by increasing its cell surface expression, and that this process may serve as an important mechanism to increase de novo glutathione synthesis during periods of oxidative stress.