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GSH transport in human cerebrovascular endothelial cells and human astrocytes: evidence for luminal localization of Na+-dependent GSH transport in HCEC1Portions of this work were presented at the annual meeting of the Federation of the American Societies of Experimental Biology, April 17–21, 1999, Washington, DC [FASEB J. 13(4), 571.4A (1999)].1

2000; Elsevier BV; Volume: 852; Issue: 2 Linguagem: Inglês

10.1016/s0006-8993(99)02184-8

1872-6240

Ram Kannan, Rita Chakrabarti, Diana Tang, K.J Kim, Neil Kaplowitz,

Amino Acid Enzymes and Metabolism

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 γ-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 Ω 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 0.05). 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.