Role of chloride and intracellular pH on the activity of the rat hepatocyte organic anion transporter

Previous studies in cultured rat hepatocytes revealed that initial uptake of sulfobromophthalein (BSP) was markedly reduced upon removal of Cl^- from the medium. In the present study, unidirectional Cl^- gradients were established in short-term cultured rat hepatocytes and their effect on BSP uptake was determined. These investigations revealed that BSP uptake requires external CT^- and is not stimulated by unidirectional Cl^- gradients, suggesting that BSP transport is not coupled to Cl^- transport. In contrast, BSP transport is stimulated by an inside-to-outside OH^- gradient, consistent with OH^- exchange or H⁺ cotransport. As the presence of Cl^- is essential for but not directly coupled to BSP transport, binding of ³⁵S-BSP to hepatocytes was determined at 4°C. This revealed an ∼ 10-fold higher affinity of cells for BSP in the presence as compared to the absence of Cl^- (K_a = 3.2±0.8 vs. 0.42±0.09 μM^-1; P < 0.02). Affinity of BSP for albumin was Cl^--independent, and was ∼ 10% of its affinity for cells in the presence of Cl^-. These results indicate that extracellular Cl^- modulates the affinity of BSP for its hepatocyte transporter.