BIDIRECTIONAL GLUTATHIONE TRANSPORT BY CULTURED HUMAN RETINAL-PIGMENTEPITHELIAL-CELLS

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 sulf oximine 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 [S-35] GSH plus varying concen trations of GSH. Molecular forms were verified by high-performance liq uid 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 ef flux was temperature dependent (undetectable at 4 degrees C), and it a veraged 23 +/- 3.3 pmol/10(6) cells/minute or 10% of the total GSH eff luxed per hour (total cell GSH = 13.6 +/- 1.5 nmol/10(6) cells). Efflu x was not influenced by dithiothreitol or sulfobromophthalein-reduced GSH adduct, agents known to affect liver sinusoidal GSH transport. Glu tathione uptake tvas linear up to 45 minutes and was temperature depen dent. The difference between 37 degrees C and 4 degrees C uptake value s represented true uptake. Glutathione uptake (2 mu Ci/ml + 1 mM mass) was Na independent and was inhibited significantly by phenol-3,6-dibr omphthaleim disulfonate. The kinetics of GSH uptake was assessed by me asuring uptake with S-35-GSH and 0.05 to 40 mM extracellular GSH for 3 0 minutes. Uptake was saturable with V-max = 18.7 +/- 1.7 nmol/10(6) c ells/30 minutes, K-m = 12.1 +/- 1.9 mM, n (binding site) = 1. On North ern blot analysis, HRPE cells express mRNA for RcGshT but not for RsGs hT. 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 transporte r can operate bidirectionally, it is expected to be a net efflux pump under normal physiologic conditions because the intracellular GSH conc entration is much higher.