Transport via SLC5A8 (SMCT1) is obligatory for 2-oxothiazolidine-4-carboxylate to enhance glutathione production in retinal pigment epithelial cells

Purpose. To evaluate the role of SLC5A8 in the transport of 2-oxothiazolidine-4-carboxylate (OTC) and to determine whether OTC augments glutathione production in RPE cells, thereby providing protection against oxidative stress. Methods. SLC5A8-mediated transport of OTC was monitored in Xenopus laevis oocytes by electrophysiological means. Saturation kinetics, Na ⁺-activation kinetics, and inhibition by ibuprofen were analyzed by monitoring OTC-induced currents as a measure of transport activity. Oxidative stress was induced in ARPE-19 cells and primary RPE cells isolated from wild type and Slc5a8 ^-/- mouse retinas using H ₂O ₂, and the effects of OTC on cell death and intracellular glutathione concentration were examined. Results. Heterologous expression of human SLC5A8 in X. laevis oocytes induced Na ⁺-dependent inward currents in the presence of OTC under voltage-clamp conditions. The transport of OTC via SLC5A8 was saturable, with a K _t of 104 ± 3 μM. The Na ⁺-activation kinetics was sigmoidal with a Hill coefficient of 1.9 ± 0.1, suggesting involvement of two Na ⁺ in the activation process. Ibuprofen, a blocker of SLC5A8, inhibited SLC5A8-mediated OTC transport; the concentration necessary for half-maximal inhibition was 17 ± 1 μM. OTC increased glutathione levels and protected ARPE-19 and primary RPE cells isolated from wild type mouse retinas from H ₂O _2- induced cell death. These effects were abolished in primary RPE isolated from Slc5a8 ^-/- mouse retinas. Conclusions. OTC is a transportable substrate for SLC5A8. OTC augments glutathione production in RPE cells, thereby protecting them from oxidative damage. Transport via SLC5A8 is obligatory for this process.