Rat liver canalicular membrane vesicles contain an ATP-dependent bile acid transport system.

Abstract

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. Taurocholate is the major bile acid in humans and rats. Its uptake from plasma by hepatocytes utilizes a Na' cotransport system that requires Na',K+-ATPase for generation of a Na' gradient (1, 2). Two pathways for intracellular bile acid transport have been demonstrated. One involves microtubule-dependent vesicular transport from the Golgi apparatus to the bile canaliculus (3, 4). The other is the major physiologic pathway and involves direct transport to the canaliculus, possibly by cytosolic binding proteins (5). The secretion of bile acids into the canaliculus is a major determinant ofbile flow. Although the contents ofthe bile canaliculus have never been measured due to its inaccessibility, studies ofductal bile suggest that there is a large bile acid concentration gradient between the hepatocyte and canalicular contents (6, 7). Previous studies using vesicles selectively derived from the canalicular domain of hepatocyte plasma membrane revealed carrier-mediated transport of taurocholate and other bile acids that is driven by an outside-positive membrane potential (8, 9). However, the membrane potential is insufficient to explain the concentration gradients of bile acids that are postulated to occur across the canalicular membrane (6).We have described (10, 11) two distinct ATP-dependent transport systems in the canalicular membrane. One system utilizes P-glycoprotein, the product of the multidrugresistance gene, and transports mainly hydrophobic cations, such as daunomycin (10). The carrier protein for the second system has not been purified but its substrates are nonbile acid organic anions, such as bilirubin diglucuronide, oxidized glutathione (GSSG), and glutathione S-conjugates (11-13).The latter transport system is defective in TR-mutant rats, which have a phenotype that resembles the defect in patients with Dubin-Johnson syndrome (11,14). While studying these transport processes, an ATP-dependent transport of bile acids in canalicular membrane vesicles (CMVs) was detected and has been now characterized. (GSBSP) and dinitrophenyl glutathione (GSDNP) were synthesized nonenzymatically (16,17). Rabbit polyclonal antibody against the extracellular domain of -glutamyltransferase (y-GT) was kindly provided by Masayasu Inoue (Kumamoto University Medical School, Kumamoto, Japan). Bilirubin diglucuronide, which was purified by HPLC (18), was kindly provided by N. Roy-Cho...

Involvement of microtubules in the swelling-induced stimulation of transcellular taurocholate transport in perfused rat liver

“…The present findings support the previous speculation that cell swelling leads to a rapid insertion into the canalicular membrane of intracellularly stored bile-acid transporter molecules Haussinger et al, 1992a), which have been identified recently as primary active, i.e. ATP-dependent, transporters (Adachi et al, 1991;Muller et al, 1991;Nishida et al, 1991;Stieger et al, 1992). The currently available evidence for such a cell-volume control of short-term bile-acid-transporter insertion/retrieval in the canalicular membrane is as follows.…”

Familial intrahepatic cholestasis 1: Studies of localization and function

“…10,16 Inside-out-oriented vesicles were separated from right-side-out-oriented vesicles by antibody-induced density perturbation through a continuous 20% to 50% sucrose gradient as described previously. 10,16,20,21 Briefly, CMV were incubated with anti-␥-glutamyl transpeptidase antiserum that recognizes the extracellular domain of ␥-glutamyl transpeptidase. After 20-minute incubation at 37°C and subsequently at 4°C, anti-rabbit IgG antiserum was added.…”

“…Vesicles were recovered from the lowand high-density fractions. 10,16,20,21 Both vesicle preparations were tested for leucine-aminopeptidase (LAP) activity in the presence and absence of detergent. LAP is a canalicular ecto-peptidase.…”

The canalicular multispecific organic anion transporter and conjugated hyperbilirubinemia in rat and man

“…Transport studies in canalicular, inside-out membrane vesicles from rat liver demonstrated strongly increased uptake of taurocholate in the presence of ATP [22][23][24]. In addition, ATP-dependent taurocholate uptake by these canalicular membrane vesicles was competitively inhibited by other bile salts including glycocholate [24,25]; it has since been demonstrated that the electrogenic taurocholate transport in canalicular membrane vesicles was probably caused by a contamination of the canalicular membrane fraction with endoplasmic reticulum mem- branes [26]. Further purification of the canalicular membrane fraction revealed a complete loss of electrogenic taurocholate transport in the enriched canalicular fraction, while the ATP-dependent taurocholate transport remained.…”