High-affinity efflux transport system for glutathione conjugates on the luminal membrane of a mouse brain capillary endothelial cell line (MBEC4)

Cumulative evidence suggests that several organic anions are excreted from the brain to the blood across the blood-brain barrier. in the present study , we carried out a kinetic investigation of the transport activity in MBEC4 , an immortalized cell line established from BALB/c mouse cerebral microves sel endothelial cells. The presence of an efflux system in intact cells was examined by using monochlorobimane (MCB), which is conjugated with glutath ione intracellularly to produce glutathione bimane (GS-B). The efflux of GS -B was inhibited by ATP depletion and also by 1-chloro-2,4-dinitrobenzne, a precursor of 2,4-dinitrophenyl-S-glutathione, in a concentration-dependent manner. Using this MBEC4 monolayer, we investigated the direction of this transport activity. Although the efflux of GS-B was observed on both lumina l and abluminal sides of MBEC4 monolayer, the profile differed for the two sides with respect to the concentration dependence of MCB; the analysis sug gested the presence of high-affinity transport system on the luminal side. To investigate the mechanism for the transport, we examined the ATP-depende nt uptake of GS-B into the membrane vesicles prepared from MBEC4. ATP-depen dent uptake systems with high (K-m = 35 nM) and low (K-m = 14 mu M) affinit ies were identified. These results suggested that this high-affinity transp ort system of glutathione conjugates is expressed on the luminal side of th e blood-brain barrier and is involved in the detoxification of xenobiotics.