R. Kannan et al., GSH transport in immortalized mouse brain endothelial cells: Evidence for apical localization of a sodium-dependent GSH transporter, J NEUROCHEM, 73(1), 1999, pp. 390-399
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.