THE PEPTIDE-BASED THROMBIN INHIBITOR CRC-220 IS A NEW SUBSTRATE OF THE BASOLATERAL RAT-LIVER ORGANIC ANION-TRANSPORTING POLYPEPTIDE

The peptidomimetic thrombin inhibitor CRC 220, nyl-L-aspartyl-D-4-amid inophenylalanyl-piperidide, is taken up into isolated rat hepatocytes through active, carrier-mediated transport. This uptake is inhibited b y bile acids. Functional expression in Xenopus laevis oocytes was perf ormed to identify the transport system responsible for the hepatocellu lar CRC 220 uptake. Injection of poly(A)(+)RNA in X. laevis oocytes re sulted in a two- to three-times higher uptake of CRC 220, compared wit h uninjected or water-injected control oocytes. Taurocholate (200 mu m ol/L) inhibited this uptake completely. No uptake of the peptidomimeti c thrombin inhibitor was observed, when X. laevis oocytes were injecte d with complementary RNA (cRNA) encoding either the cloned rat liver N a+-dependent taurocholate transporter Ntcp, the renal oligopeptide car rier rhaPT or the intestinal oligopeptide transporter PepT1. However, after injection of cRNA of the cloned rat liver Na+-independent organi c anion transporting polypeptide oatp, a specific and saturable CRC 22 0 uptake was observed (Michaelis-Menten constant 29.5 mu mol/L). Cis-i nhibition with known oatp-substrates, e.g., 20 mu mol/L Bromsulphalein (R) (BSP), 2007 mu mol/L taurocholate and 2007 mu mol/L cholate, occur red in oatp-expressing X. laevis oocytes, whereas substrates of the tw o peptide carriers as well as dipeptide- and single-amino acid constit uents of the thrombin inhibitor itself lacked any significant inhibito ry effects. These data show that the modified dipeptide CRC 220 is a h ighly selective substrate of the organic anion transporting polypeptid e oatp in the basolateral plasma membrane of rat hepatocytes.