TY - JOUR
T1 - Transport via SLC5A8 (SMCT1) is obligatory for 2-oxothiazolidine-4-carboxylate to enhance glutathione production in retinal pigment epithelial cells
AU - Babu, Ellappan
AU - Ananth, Sudha
AU - Veeranan-Karmegam, Rajalakshmi
AU - Coothankandaswamy, Veena
AU - Smith, Sylvia B.
AU - Boettger, Thomas
AU - Ganapathy, Vadivel
AU - Martin, Pamela M.
PY - 2011/7
Y1 - 2011/7
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=80053309143&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=80053309143&partnerID=8YFLogxK
U2 - 10.1167/iovs.10-6825
DO - 10.1167/iovs.10-6825
M3 - Article
C2 - 21508099
AN - SCOPUS:80053309143
SN - 0146-0404
VL - 52
SP - 5749
EP - 5757
JO - Investigative Ophthalmology and Visual Science
JF - Investigative Ophthalmology and Visual Science
IS - 8
ER -