Substrate specificity of the rat liver Na+-bile salt cotransporter inXenopus laevis oocytes and in CHO cells

Abstract

It has been proposed that the hepatocellular Na+-dependent bile salt uptake system exhibits a broad substrate specificity in intact hepatocytes. In contrast, recent expression studies in mammalian cell lines have suggested that the cloned rat liver Na+-taurocholate cotransporting polypeptide (Ntcp) may transport only taurocholate. To characterize its substrate specificity Ntcp was stably transfected into Chinese hamster ovary (CHO) cells. These cells exhibited saturable Na+-dependent uptake of [3H]taurocholate [Michaelis constant ( K m) of ∼34 μM] that was strongly inhibited by all major bile salts, estrone 3-sulfate, bumetanide, and cyclosporin A. Ntcp cRNA-injected Xenopus laevis oocytes and the transfected CHO cells exhibited saturable Na+-dependent uptake of [3H]taurochenodeoxycholate ( K m of ∼5 μM), [3H]tauroursodeoxycholate ( K m of ∼14 μM), and [14C]glycocholate ( K m of ∼27 μM). After induction of gene expression by sodium butyrate, Na+-dependent transport of [3H]estrone 3-sulfate ( K m of ∼27 μM) could also be detected in the transfected CHO cells. However, there was no detectable Na+-dependent uptake of [3H]bumetanide or [3H]cyclosporin A. These results show that the cloned Ntcp can mediate Na+-dependent uptake of all physiological bile salts as well as of the steroid conjugate estrone 3-sulfate. Hence, Ntcp is a multispecific transporter with preference for bile salts and other anionic steroidal compounds.