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Pamela M. Martin, Ellappan Babu, Rajalakshmi Veeranan-Karmegan, Veena Coothankandaswamy, Sylvia B. Smith, Thomas Boettger, Vadivel Ganapathy; Transport via Slc5a8 (SMCT1) is Obligatory for 2-Oxothiazolidine-4-carboxylate to Enhance Glutathione Production in Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):928.
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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.
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.
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.
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.
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