Atp–Dependent Canalicular Bile Acid Transport: Another Piece of the Puzzle

Abstract

ABSTRPlCT The secretion of bile by the liver is primarily determined by the ability of the hepatocyte to transport bile acids into the bile canaliculus. A carrier–mediated process for the transport of taurocholate, the major bile acid in humans and rats, was previously demonstrated in canalicular membrane vesicles from rat liver. This process is driven by an outside–positive membrane potential that is, however, insufficient to explain the large bile acid concentration gradient between the hepatocyte and bile. In this study, we describe an ATP–dependent transport system for taurocholate in inside–out canalicular membrane vesicles from rat liver. The transport system is saturable, temperature–dependent, osmotically sensitive, specifically requires ATP, and does not function in sinusoidal membrane vesicles and right side–out canalicular membrane vesicles. Transport was inhibited by other bile acids but not by substrates for the previously demonstrated ATP–dependent canalicular transport systems for organic cations or nonbile acid organic anions. Defects in ATP–dependent canalicular transport of bile acids may contribute to reduced bile secretion (cholestasis) in various developmental, inheritable, and acquired disorders. Direct photoaffinity labeling of liver plasma membrane subfractions enriched in sinusoidal and canalicular membranes using [35S]adenosine 5′–O–(thiotriphosphate) ([35S]ATPγS) allows the identification of ATP–binding proteins in these domains. Comparative photoaffinity labeling with [35S]ATPγS and with the photolabile bile salt derivative (7,7–azo–3α,12α–dihydroxy–5β–[3β–H]–cholan–24–oyl)–2′–aminethanesulfonate followed by immunoprecipitation with a monoclonal antibody (Be 9.2) revealed the identity of the ATP–binding and the bile salt–binding canalicular membrane glycoprotein with the apparent Mr of 110,000 (gp110). The isoelectric point of this glycoprotein was 3.7. Transport of bile salt was studied in vesicles enriched in canalicular and sinusoidal liver membranes. Incubation of canalicular membrane vesicles with [H] taurocholate in the presence of ATP resulted in an uptake of the bile salt into the vesicles which was sensitive to vanadate. ATP–dependent taurocholate transport was also observed in membrane vesicles from mutant rats deficient in the ATP–dependent transport of cysteinyl leukotrienes and related amphiphilic anions. Substrates of the P–glycoprotein (gp170), such as verapamil and doxorubicin, did not interfere with the ATP–dependent transport of taurocholate. Reconstitution of purified gp 110 into liposomes resulted in an ATP–dependent uptake of [H]taurocholate. These results demonstrate that gp110 functions as carrier in the ATP–dependent transport of bile salts from the hepatocyte into bile. This export carrier is distinct from hitherto characterized ATP–dependent transport systems.