GSH transport in human cerebrovascular endothelial cells and human astrocytes: evidence for luminal localization of Na+-dependent GSH transport in HCEC.
The purpose of the present study was to identify and localize glutathione (GSH) transport in an in vitro tissue culture model of blood-brain barrier (BBB). The localization of Na+-dependent GSH transport in an immortalized cell line of human cerebrovascular endothelial cells (HCEC) and asymmetry of transport in Transwell studies were investigated. Initial studies with cultured HCEC established a significant (45%) Na+-dependency for GSH uptake in cultured HCEC pretreated with acivicin, an inhibitor of gamma-glutamyltranspeptidase (GGT). Transendothelial electrical resistance (TEER) and uptake of [35S]GSH from luminal and abluminal fluids of HCEC were measured in Na+-containing and Na+-free (choline chloride) buffers using cells grown on gelatin-coated membrane filters. TEER of HCEC monolayers in regular medium was 40.1 +/- 8.0 ohms cm2. Human astrocyte-conditioned medium (ACM) caused no change in TEER, but increased GGT activity approximately threefold when measured in cell lysates. Luminal and abluminal GSH uptake increased in a time-dependent fashion and were not affected by inhibition of GGT activity with acivicin. Sodium dependency was only observed for luminal uptake (Na+-containing 2.41 +/- 0.15 vs. Na+-free 0.96 +/- 0.03 pmol/30 min/million cells, p < 0.001) but not for abluminal uptake (1.02 +/- 0.13 vs. 1.11 +/- 09, p > 0.05). Apparent efflux via the luminal membrane was lower in the presence of sodium as compared to that without sodium, further suggesting that a Na+-dependent uptake process for GSH is operative at this membrane. GSH uptake and efflux were also demonstrated in neonatal rat and fetal human astrocytes, both exhibiting partial Na+-dependency of uptake. In conclusion, our results show for the first time, that HCEC and astrocytes take up GSH by both Na+-dependent and -independent mechanisms. The Na+-dependent GSH transport process in HCEC appears to be localized to luminal plasma membranes of HCEC.
Kannan
R
R
Department of Medicine, University of Southern California, Los Angeles 90033, USA. kannan@hsc.usc.edu
Chakrabarti
R
R
Tang
D
D
Kim
K J
KJ
Kaplowitz
N
N
D000818:Animals; D000831:Animals, Newborn; D000975:Antioxidants / Q000378:metabolism / Q000493:pharmacokinetics; D001253:Astrocytes / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D001812:Blood-Brain Barrier / Q000187:drug effects / Q000502:physiology*; D001921:Brain / Q000098:blood supply / Q000166:cytology; D002352:Carrier Proteins / Q000737:chemistry / Q000502:physiology; D002461:Cell Line, Transformed; D002560:Cerebrovascular Circulation; D017077:Culture Media, Conditioned / Q000494:pharmacology; D017097:Electric Impedance; D004730:Endothelium, Vascular / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005333:Fetus / Q000166:cytology; D005978:Glutathione / Q000096:biosynthesis / Q000493:pharmacokinetics*; D006801:Humans; D007555:Isoxazoles / Q000494:pharmacology; D026901:Membrane Transport Proteins; D017434:Protein Structure, Tertiary; D051381:Rats; D012964:Sodium / Q000494:pharmacology*
D000975:Antioxidants; D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D004791:Enzyme Inhibitors; D007555:Isoxazoles; D026901:Membrane Transport Proteins; C086860:glutathione transporter; D012964:Sodium; D005978:Glutathione; C006303:acivicin