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

Ellappan Babu, Sudha Ananth, Rajalakshmi Veeranan-Karmegam, Veena Coothankandaswamy, Sylvia B Smith, Thomas Boettger, Vadivel Ganapathy, Pamela Moore Martin

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)5749-5757
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume52
Issue number8
DOIs
StatePublished - Jul 1 2011

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Retinal Pigments
Glutathione
Epithelial Cells
Retina
Ibuprofen
Xenopus laevis
Oocytes
Oxidative Stress
Cell Death
2-oxothiazolidine-4-carboxylic acid
4-oxothiazolidine

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Transport via SLC5A8 (SMCT1) is obligatory for 2-oxothiazolidine-4-carboxylate to enhance glutathione production in retinal pigment epithelial cells. / Babu, Ellappan; Ananth, Sudha; Veeranan-Karmegam, Rajalakshmi; Coothankandaswamy, Veena; Smith, Sylvia B; Boettger, Thomas; Ganapathy, Vadivel; Martin, Pamela Moore.

In: Investigative Ophthalmology and Visual Science, Vol. 52, No. 8, 01.07.2011, p. 5749-5757.

Research output: Contribution to journalArticle

Babu, Ellappan ; Ananth, Sudha ; Veeranan-Karmegam, Rajalakshmi ; Coothankandaswamy, Veena ; Smith, Sylvia B ; Boettger, Thomas ; Ganapathy, Vadivel ; Martin, Pamela Moore. / Transport via SLC5A8 (SMCT1) is obligatory for 2-oxothiazolidine-4-carboxylate to enhance glutathione production in retinal pigment epithelial cells. In: Investigative Ophthalmology and Visual Science. 2011 ; Vol. 52, No. 8. pp. 5749-5757.
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abstract = "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.",
author = "Ellappan Babu and Sudha Ananth and Rajalakshmi Veeranan-Karmegam and Veena Coothankandaswamy and Smith, {Sylvia B} and Thomas Boettger and Vadivel Ganapathy and Martin, {Pamela Moore}",
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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 Moore

PY - 2011/7/1

Y1 - 2011/7/1

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.

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