GSH Transport in Immortalized Mouse Brain Endothelial Cells

Publisher Website
Google Scholar
Abstract
We have previously shown GSH transport across the blood‐brain barrier in vivo and expression of transport in Xenopus laevis oocytes injected with bovine brain capillary mRNA. In the present study, we have used MBEC‐4, an immortalized mouse brain endothelial cell line, to establish the presence of Na+‐dependent and Na+‐independent GSH transport and have localized the Na+‐dependent transporter using domain‐enriched plasma membrane vesicles. In cells depleted of GSH with buthionine sulfoximine, a significant increase of intracellular GSH could be demonstrated only in the presence of Na+. Partial but significant Na+ dependency of [35S]GSH uptake was observed for two GSH concentrations in MBEC‐4 cells in which γ‐glutamyltranspeptidase and γ‐glutamylcysteine synthetase were inhibited to ensure absence of breakdown and resynthesis of GSH. Uniqueness of Na+‐dependent uptake in MBEC‐4 cells was confirmed with parallel uptake studies with Cos‐7 cells that did not show this activity. Molecular form of uptake was varified as predominantly GSH, and very little conversion of [35S]cysteine to GSH occurred under the same incubation conditions. Poly(A)+ RNA from MBEC expressed GSH uptake with significant (∼40‐70%) Na+ dependency, whereas uptake expressed by poly(A)+ RNA from HepG2 and Cos‐1 cells was Na+ independent. Plasma 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 apical (luminal) membrane by localization of P‐glycoprotein 170 by western blot analysis. Localization of Na+‐dependent GSH transport to the luminal membrane and its ability to drive up intracellular GSH may find application in the delivery of supplemented GSH to the brain in vivo.