title: GSH transport in immortalized mouse brain endothelial cells: evidence for apical localization of a sodium-dependent GSH transporter.
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 gamma-glutamyltranspeptidase and gamma-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 verified 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 (approximately 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.
authors.affiliation: Research Center for Liver Diseases and Department of Medicine, University of Southern California School of Medicine, Los Angeles 90033, USA.
authors.forename: R
authors.initials: R
authors.lastname: Kannan
authors.forename: A
authors.initials: A
authors.lastname: Mittur
authors.forename: Y
authors.initials: Y
authors.lastname: Bao
authors.forename: T
authors.initials: T
authors.lastname: Tsuruo
authors.forename: N
authors.initials: N
authors.lastname: Kaplowitz
mesh_terms: D000818:Animals; D001692:Biological Transport; D001921:Brain / Q000098:blood supply*; D002352:Carrier Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D005260:Female; D018014:Gene Transfer Techniques; D005978:Glutathione / Q000378:metabolism*; D007700:Kinetics; D026901:Membrane Transport Proteins; D051379:Mice; D009865:Oocytes / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012964:Sodium / Q000494:pharmacology*; D014982:Xenopus laevis; D005723:gamma-Glutamyltransferase / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; C086860:glutathione transporter; D012964:Sodium; D005723:gamma-Glutamyltransferase; D005978:Glutathione