Figure 8.

Functional analysis of Slc5a8 in primary RPE (mRPE) cells isolated from wild type (WT) and Slc5a8^-/- (KO) mouse retinas, and OTC-mediated protection of wild type mRPE against H₂O₂-induced cell death. (A) Wild type and Slc5a8^-/- mRPE cells were used for analysis of [¹⁴C]-nicotinate (30 μM) uptake. Uptake measurements were made in the presence (NaCl) or absence of Na⁺ (NMDG). Data in the right panel represent the values for Na⁺-dependent nicotinate uptake in WT and KO mRPE cells. (B) Dose–response for H₂O₂-induced oxidative stress/cell death in wild type mRPE. (C) Wild type and Slc5a8^-/- mRPE cells were incubated in the presence or absence of two different concentrations of OTC (250 and 500 μM) for 60 minutes, followed by determination of intracellular glutathione concentration (*P < 0.001 compared to WT cells; **P < 0.001 compared to WT cells cultured in the absence of OTC; NS, not significant compared to KO cells cultured in the absence of OTC). (D) Oxidative stress was induced in WT and KO mRPE using H₂O₂ (25 μM) in the presence or absence of 500 μM OTC. Cell viability was analyzed by trypan blue exclusion assay (*P < 0.01 compared to control cells cultured in the absence of H₂O₂ and of OTC; **P < 0.01 compared to WT cells cultured in the presence of H₂O₂; NS, not significant compared to KO cells cultured in the presence of H₂O₂). (E) RT-PCR analysis of mRNA expression of genes involved in glutathione homeostasis.