PURPOSE: To characterize glutathione (GSH) transport by cultured human retinal pigment epithelial (HRPE) cells. METHODS: Cultured HRPE cells were pretreated with acivicin for GSH efflux and with buthionine sulfoximine for GSH uptake to prevent the breakdown and resynthesis of GSH. Efflux was measured by the linear rate of accumulation of GSH in the supernatant; uptake was measured using [35S] GSH plus varying concentrations of GSH. Molecular forms were verified by high-performance liquid chromatography. HRPE cell mRNA was probed for the presence of the two recently cloned rat sinusoidal and canalicular GSH transporters, (RsGshT and RcGshT), by Northern blot analysis. RESULTS: Glutathione efflux was temperature dependent (undetectable at 4 degrees C), and its averaged 23 +/- 3.3 pmol/10(6) cells/minute or 10% of the total GSH effluxed per hour (total cell GSH = 13.6 +/- 1.5 nmol/10(6) cells). Efflux was not influenced by dithiothreitol or sulfobromophthalein-reduced GSH adduct, agents known to affect liver sinusoidal GSH transport. Glutathione uptake was linear up to 45 minutes and was temperature dependent. The difference between 37 degrees C and 4 degrees C uptake values represented true uptake. Glutathione uptake (2 microCi/ml + 1 mM mass) was Na independent and was inhibited significantly by phenol-3,6-dibromphthalein disulfonate. The kinetics of GSH uptake was assessed by measuring uptake with 35S-GSH and 0.05 to 40 mM extracellular GSH for 30 minutes. Uptake was saturable with Vmax = 18.7 +/- 1.7 nmol/10(6) cells/30 minutes, Km = 12.1 +/- 1.9 mM, n (binding site) = 1. On Northern blot analysis, HRPE cells express mRNA for RcGshT but not for RsGshT. CONCLUSIONS: The similarities in functional characteristics of GSH transport and the presence of RcGshT-like mRNA suggest GSH transport in HRPE cells is mediated by a RcGshT homolog. Although the transporter can operate bidirectionally, it is expected to be a net efflux pump under normal physiologic conditions because the intracellular GSH concentration is much higher.
PURPOSE: To characterize glutathione (GSH) transport by cultured human retinal pigment epithelial (HRPE) cells. METHODS: Cultured HRPE cells were pretreated with acivicin for GSH efflux and with buthionine sulfoximine for GSH uptake to prevent the breakdown and resynthesis of GSH. Efflux was measured by the linear rate of accumulation of GSH in the supernatant; uptake was measured using [35S] GSH plus varying concentrations of GSH. Molecular forms were verified by high-performance liquid chromatography. HRPE cell mRNA was probed for the presence of the two recently cloned rat sinusoidal and canalicular GSH transporters, (RsGshT and RcGshT), by Northern blot analysis. RESULTS:Glutathione efflux was temperature dependent (undetectable at 4 degrees C), and its averaged 23 +/- 3.3 pmol/10(6) cells/minute or 10% of the total GSH effluxed per hour (total cell GSH = 13.6 +/- 1.5 nmol/10(6) cells). Efflux was not influenced by dithiothreitol or sulfobromophthalein-reduced GSH adduct, agents known to affect liver sinusoidal GSH transport. Glutathione uptake was linear up to 45 minutes and was temperature dependent. The difference between 37 degrees C and 4 degrees C uptake values represented true uptake. Glutathione uptake (2 microCi/ml + 1 mM mass) was Na independent and was inhibited significantly by phenol-3,6-dibromphthalein disulfonate. The kinetics of GSH uptake was assessed by measuring uptake with 35S-GSH and 0.05 to 40 mM extracellular GSH for 30 minutes. Uptake was saturable with Vmax = 18.7 +/- 1.7 nmol/10(6) cells/30 minutes, Km = 12.1 +/- 1.9 mM, n (binding site) = 1. On Northern blot analysis, HRPE cells express mRNA for RcGshT but not for RsGshT. CONCLUSIONS: The similarities in functional characteristics of GSH transport and the presence of RcGshT-like mRNA suggest GSH transport in HRPE cells is mediated by a RcGshT homolog. Although the transporter can operate bidirectionally, it is expected to be a net efflux pump under normal physiologic conditions because the intracellular GSH concentration is much higher.
Authors: Bryan W Jones; Rebecca L Pfeiffer; William D Ferrell; Carl B Watt; James Tucker; Robert E Marc Journal: Front Cell Neurosci Date: 2016-04-28 Impact factor: 5.505