Tolbutamide uptake via pH- and membrane-potential-dependent transport mechanism in mouse brain capillary endothelial cell line

The purpose of this study was to investigate the transport mechanism of tolbutamide across the blood-brain barrier (BBB) using MBEC4 cells as an in vitro BBB model. METHODS: The BBB transport of tolbutamide was studied by using a mouse brain capillary endothelial cell line, MBEC4, cultured on dishes with their luminal membrane facing the culture medium. RESULTS: The uptake of 14C]tolbutamide by MBEC4 cells was dependent on temperature and energy. The uptake coefficient of 14C]tolbutamide increased markedly with decreasing pH of the external medium from neutral to acidic. Valinomycin and replacement of chloride with sulfate or gluconate significantly increased the initial uptake of 14C]tolbutamide, while replacement with nitrate significantly decreased it. The uptake was significantly reduced by a proton ionophore, FCCP, and an anion-exchange inhibitor, DIDS. The initial uptake of 14C]tolbutamide was saturable with Kt of 0.61+/-0.03 mM (pH 7.4) and 1.76+/-0.19 mM (pH 6.5). At pH 6.5, the initial uptake of 14C]tolbutamide was significantly reduced by several sulfa drugs, salicylic acid, valproic acid and probenecid, and was competitively inhibited by sulfaphenazole (Ki=3.47+/-0.50 mM) and valproic acid (Ki=2.29+/-0.43 mM). CONCLUSION: These observations indicate the existence of a pH- and membrane-potential-dependent anion exchange and/or proton-cotransport system(s) for concentrative uptake of tolbutamide and sulfa drugs in MBEC4 cells.