Glutathione Transport in Human Retinal Pigment Epithelial (HRPE) Cells: Apical Localization of Sodium-dependent GSH Transport

Abstract

The study was undertaken to identify and localize GSH transport in non-transformed cultured human retinal pigmented epithelial cells (HRPE). In confluent monolayers exhibiting high transepithelial resistance (TER 700–1000Ωcm−2), apical and basolateral GSH uptake were determined after introducing35S-GSH (+1m M GSH) to the apical side or basal side in NaCl (Na+-containing) or choline chloride (Na+-free) buffers. Cells in growth medium or in incubation buffers were pretreated with acivicin to inhibit γ-glutamyltranspeptidase (GGT). GSH efflux was measured after labelling the intracellular GSH pool by incubation overnight with35S-cysteine and quantitating the release of labelled GSH into the medium. Uptake of GSH was found at both the apical and basolateral membranes of HRPE cells. Inhibition of γ-glutamyltranspeptidase (GGT) with acivicin did not alter mean GSH uptake (nmol per million cells per 30min) significantly at the apical (1.63±0.32 vs 1.45±0.30; with and without acivicin respectively) or the basolateral (1.17±0.21 vs 1.44±0.38) membranes. Transport was verified to be in the form of intact GSH by HPLC. Uptake was unaffected by the removal of Na+at the basolateral membrane while apical uptake exhibited partial but significant (approximately 40%) Na+-dependency. Net GSH efflux (nmol per million cells per min) to the apical side of HRPE cells was higher than to the basolateral side in the presence of sodium. Transepithelial flux in the basolateral to apical direction was approximately 17-fold higher than the apical to basolateral direction resulting in a net flux of GSH to the apical side. In conclusion, HRPE cells exhibit GSH transport by Na+-dependent and Na+-independent mechanisms. The Na+-dependent GSH transporter is localized to the apical membrane of HRPE cells.