GSH transport in immortalized mouse brain endothelial cells: Evidence for apical localization of a sodium-dependent GSH transporter

We have previously shown GSH transport across the blood-brain barrier in vi vo and expression of transport in Xenopus laevis oocytes injected with bovi ne brain capillary mRNA. In the present study, we have used MBEC-4, an immo rtalized mouse brain endothelial cell line, to establish the presence of Na +-dependent and Na+-independent GSH transport and have localized the Na+-de pendent transporter using domain-enriched plasma membrane vesicles. In cell s depleted of GSH with buthionine sulfoximine, a significant increase of in tracellular GSH could be demonstrated only in the presence of Na+. Partial but significant Na+ dependency of [S-35]GSH uptake was observed for two GSH concentrations in MBEC-4 cells in which gamma-glutamyltranspeptidase and g amma-glutamylcysteine synthetase were inhibited to ensure absence of breakd own and resynthesis of GSH. Uniqueness of Na+-dependent uptake in MBEC-4 ce lls was confirmed with parallel uptake studies with Cos-7 cells that did no t show this activity, Molecular form of uptake was verified as predominantl y GSH, and very little conversion of [S-35]cysteine to GSH occurred under t he same incubation conditions. Poly(A)(+) RNA from MBEC expressed GSH uptak e with significant (similar to 40-70%) Na+ dependency, whereas uptake expre ssed by poly(A)(+) RNA from HepG2 and Cos-1 cells was Na+ independent, Plas ma membrane vesicles from MBEC were separated into three fractions (30, 34, and 38% sucrose, by wt) by density gradient centrifugation. Na+-dependent glucose transport, reported to be localized to the abluminal membrane, was found to be associated with the 38% fraction (abluminal). Na+-dependent GSH transport was present in the 30% fraction, which was identified as the api cal (luminal) membrane by localization of P-glycoprotein 170 by western blo t analysis. Localization of Na+-dependent GSH transport to the luminal memb rane and its ability to drive up intracellular GSH may find application in the delivery of supplemented GSH to the brain in vivo.