Vol. 286, Issue 2, 1043-1050, August 1998

Contribution of Sodium Taurocholate Co-Transporting Polypeptide to the Uptake of Its Possible Substrates Into Rat Hepatocytes¹

Hirokazu Kouzuki, Hiroshi Suzuki, Kousei Ito, Rui Ohashi and Yuichi Sugiyama

Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

As one of the Na⁺-dependent transporters responsible for the hepatic uptake of ligands, sodium taurocholate (TC) co-transporting polypeptide (NTCP) has been cloned from rat liver and its substrate specificity has been clarified by examining the inhibition of TC uptake mediated by NTCP. The contribution of NTCP to the Na⁺-dependent uptake of ligands into rat hepatocytes, however, still needs to be clarified. To determine the contribution of NTCP, we examined the uptake of ligands into primary cultured hepatocytes (cultured for 4 h) and into COS-7 cells, transiently expressing NTCP, and normalized the uptake of ligands with TC as a reference compound. Western Blot analysis indicated that NTCP was glycosylated much less extensively in the transfected COS-7 cells, although the expression level was comparable for the cultured hepatocytes and transfectant. Kinetic parameters for the Na⁺-dependent uptake of TC were similar for the cultured hepatocytes and NTCP-transfected COS-7 cells (K_m = 17.7 vs. 17.4 µM; V_max = 1.63 vs. 1.45 nmol/min/mg protein). Glycocholic acid and cholic acid were taken up by NTCP-transfected COS-7 cells. The contribution of NTCP to the Na⁺-dependent uptake of glycocholic acid into rat hepatocytes was approximately 80%, whereas that of cholic acid was 40%. In addition, the analysis indicated that the contribution of NTCP to the Na⁺-dependent uptake of several ligands (ouabain, ibuprofen, glutathione-conjugate of bromosulfophthalein, glucuronide- and sulfate-conjugates of 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole) was negligible. Thus, this is a convenient method to determine the contribution of NTCP to the uptake of ligands into hepatocytes. It is also suggested that multiple transport mechanisms are responsible for the Na⁺-dependent uptake of organic anions into hepatocytes.