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 shortterm cultured rat hepatocytes and their effect on BSP uptake was determined. These investigations revealed that BSP uptake requires external Cl-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 40C. This revealed an gM-M; 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. (J. Clin. Invest. 1991Invest. . 87:1496Invest. -1502

A 55-kD organic anion binding protein (OABP) was identified previously in liver cell plasma membrane sinusoidal subfractions. Although this protein was localized to the surface of hepatocytes by immunofluorescence, immunoblot analysis revealed reactivity toward both plasma membrane and mitochondrial fractions. To clarify these findings, an immunoreactive clone from a rat liver cDNA expression library was isolated, the 1,500-base pair cDNA insert was sequenced, and the corresponding j-galactosidase fusion protein was expressed and purified. The resulting sequence corresponded to that of the rat mitochondrial Fl-adenosine triphosphatase (Fl-ATPase) ,-subunit. This protein and OABP are of similar size and are mutually immunologically cross-reactive. That the antigen was present on the cell surface as well as in mitochondria was suggested from studies of immunoprecipitation after cell-surface iodination, and light-and electron-microscopic immunocytochemistry. Photoaffinity labeling of bovine FI-ATPase with high-specific-activity V5Slsulfobromophthalein revealed binding only to the #-subunit. Hepatocyte uptake of bilirubin and sulfobromophthalein requires cellular ATP and mitochondria also transport these organic anions, which at high doses inhibit respiration. The presence of an organic anion binding site on the F,-ATPase #-subunit suggests that it may play a role in these processes. (J. Clin. Invest. 1990. 86:220-227.)

In previous studies, we identified a 55 kD organic anion-binding protein in liver cell sinusoidal plasma membrane subfractions. Other investigators identified another 55 kD bromosulfophthalein/bilirubin binding protein on the surface of rat hepatocytes and HepG2 cells and suggested that this protein served as a transporter for these ligands. In this study, transport of 35S-sulfobromophthalein by the human hepatoma cell line, HepG2, was quantified in the presence and absence of bovine serum albumin to further clarify the possible function of these plasma membrane binding proteins. In contrast to results in normal rat hepatocytes, virtually no uptake of 35S-sulfobromophthalein by HepG2 cells in the presence of bovine serum albumin was found. In the absence of albumin, HepG2 cells expressed temperature-dependent uptake of 35S-sulfobromophthalein. However, the high-affinity Cl(-)-dependent sulfobromophthalein transport that characterizes normal rat hepatocytes was absent, as indicated by an approximately 95-fold lower affinity and 170-fold higher capacity of HepG2 cells for sulfobromophthalein compared with previous results with rat hepatocytes. These results suggest that 55 kD sulfobromophthalein/bilirubin-binding protein on the liver cell surface differs from organic anion-binding protein and is not responsible for sulfobromophthalein extraction in the presence of albumin, although it may play some role in lower affinity transport by cells. Immunoblot analysis and metabolic labeling of HepG2 cells demonstrated synthesis of organic anion-binding protein. However, light microscopic immunocytochemistry and immunoprecipitation of surface iodinated rat hepatocytes and HepG2 cells with antibody to a recombinant organic anion-binding protein fusion protein indicated absence of organic anion-binding protein on the surface of HepG2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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