title: Transport via SLC5A8 (SMCT1) is obligatory for 2-oxothiazolidine-4-carboxylate to enhance glutathione production in retinal pigment epithelial cells.
abstract: OBJECTIVE
  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(2)O(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(2)O(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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical College of Georgia, Georgia Health Sciences University, Augusta, GA 30912, USA.
authors.forename: Ellappan
authors.initials: E
authors.lastname: Babu
authors.forename: Sudha
authors.initials: S
authors.lastname: Ananth
authors.forename: Rajalakshmi
authors.initials: R
authors.lastname: Veeranan-Karmegam
authors.forename: Veena
authors.initials: V
authors.lastname: Coothankandaswamy
authors.forename: Sylvia B
authors.initials: SB
authors.lastname: Smith
authors.forename: Thomas
authors.initials: T
authors.lastname: Boettger
authors.forename: Vadivel
authors.initials: V
authors.lastname: Ganapathy
authors.forename: Pamela M
authors.initials: PM
authors.lastname: Martin
mesh_terms: D000818:Animals; D017209:Apoptosis; D001692:Biological Transport / Q000502:physiology; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D004347:Drug Interactions; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D007052:Ibuprofen / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D008817:Mice, Mutant Strains; D027501:Monocarboxylic Acid Transporters; D009865:Oocytes / Q000502:physiology; D016877:Oxidants / Q000633:toxicity; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D011761:Pyrrolidonecarboxylic Acid / Q000737:chemistry / Q000493:pharmacokinetics*; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D053778:Thiazolidines / Q000737:chemistry / Q000493:pharmacokinetics*; D014982:Xenopus laevis
chemical_list: D027682:Cation Transport Proteins; D016861:Cyclooxygenase Inhibitors; D027501:Monocarboxylic Acid Transporters; D016877:Oxidants; C476760:SLC5A8 protein, human; C507459:Slc5a8 protein, mouse; D053778:Thiazolidines; D006861:Hydrogen Peroxide; D005978:Glutathione; D011761:Pyrrolidonecarboxylic Acid; D007052:Ibuprofen; C029536:2-oxothiazolidine-4-carboxylic acid