Vol. 288, Issue 1, 198-203, January 1999

High-Affinity Efflux Transport System for Glutathione Conjugates on the Luminal Membrane of a Mouse Brain Capillary Endothelial Cell Line (MBEC4)¹

Masashi Homma, Hiroshi Suzuki, Hiroyuki Kusuhara, Mikihiko Naito, Takashi Tsuruo and Yuichi Sugiyama

Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan (M.H., H.S., H.K., Y.S.); and Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo, Japan (M.N., T.T.)

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 microvessel endothelial cells. The presence of an efflux system in intact cells was examined by using monochlorobimane (MCB), which is conjugated with glutathione 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 luminal and abluminal sides of MBEC4 monolayer, the profile differed for the two sides with respect to the concentration dependence of MCB; the analysis suggested the presence of high-affinity transport system on the luminal side. To investigate the mechanism for the transport, we examined the ATP-dependent uptake of GS-B into the membrane vesicles prepared from MBEC4. ATP-dependent uptake systems with high (K_m = 35 nM) and low (K_m = 14 µM) affinities were identified. These results suggested that this high-affinity transport system of glutathione conjugates is expressed on the luminal side of the blood-brain barrier and is involved in the detoxification of xenobiotics.