Transport via Slc5a8 (SMCT1) is Obligatory for 2-Oxothiazolidine-4-carboxylate to Enhance Glutathione Production in Retinal Pigment Epithelial Cells

Abstract

Purpose: : To evaluate the role of SLC5A8 in the transport of 2-oxothiazolidine-4-carboyxlate (OTC), and to determine whether OTC augments glutathione production in RPE cells.

Methods: : Oxidative stress was induced in ARPE-19 cells using H₂O₂, and the effect of OTC on cell death was examined. SLC5A8-mediated transport of OTC was monitored in X. 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. Primary RPE cells from wild type and slc5a8^-/- mouse retinas were cultured in the presence or absence of OTC and used for measurement of glutathione levels.

Results: : OTC protected ARPE-19 cells from H₂O₂-induced cell death. 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 micromolar. The Na⁺-activation kinetics sigmoidal (Hill coefficient = 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 micromolar. Glutathione levels were increased by OTC in primary RPE cells from wild type mice, but this effect was abolished in primary RPE cells from slc5a8^-/- mice.

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.

Keywords: retinal pigment epithelium • antioxidants • age-related macular degeneration