ATP-dependent Taurocholate Transport Research Articles

Activity of the Bile Salt Export Pump (ABCB11) Is Critically Dependent on Canalicular Membrane Cholesterol Content

Mutations in ATP8B1 cause severe inherited liver disease. The disease is characterized by impaired biliary bile salt excretion (cholestasis), but the mechanism whereby impaired ATP8B1 function results in cholestasis is poorly understood. ATP8B1 is a type 4 P-type ATPase and is a flippase for phosphatidylserine. Atp8b1-deficient mice display a dramatic increase in the biliary extraction of cholesterol from the canalicular (apical) membrane of the hepatocyte. Here we studied the hypothesis that disproportionate cholesterol extraction from the canalicular membrane impairs the activity of the bile salt transporter, ABCB11, and as a consequence causes cholestasis. Using single pass liver perfusions, we show that not only ABCB11-mediated transport but also Abcc2-mediated transport were reduced at least 4-fold in Atp8b1 deficiency. We show that canalicular membranes of cholestatic Atp8b1-deficient mice have a dramatically reduced cholesterol to phospholipid ratio, i.e. 0.75 +/- 0.24 versus 2.03 +/- 0.71 for wild type. In vitro depletion of cholesterol from mouse liver plasma membranes using methyl-beta-cyclodextrin demonstrated a near linear relation between cholesterol content of the membranes and ATP-dependent taurocholate transport. Abcc2-mediated transport activity was not affected up to 30% of membrane cholesterol depletion but declined to negligible levels at 70% of membrane cholesterol depletion. These effects were reversible as cholesterol repletion of the liver membranes completely restored Abcb11- and Abcc2-mediated transport. Our data demonstrate that membrane cholesterol content is a critical determinant of ABCB11/ABCC2 transport activity, provide an explanation for the etiology of ATP8B1 disease, and suggest a novel mechanism protecting the canalicular membrane against luminal bile salt overload.

Effect of thiazolidinediones on bile acid transport in rat liver

The thiazolidinedione derivatives, troglitazone, rosiglitazone, and pioglitazone, are novel insulin-sensitizing drugs that are useful in the treatment of type 2 diabetes. However, hepatotoxicity associated with troglitazone led to its withdrawal from the market in March 2000. In view of case reports of hepatotoxicity from rosiglitazone and pioglitazone, it is unclear whether thiazolidinediones as a class are associated with hepatotoxicity. Although the mechanism of troglitazone-associated hepatotoxicity has not been elucidated, troglitazone and its major metabolite, troglitazone sulfate, competitively inhibit adenosine triphosphate (ATP)-dependent taurocholate transport in isolated rat canalicular liver plasma membrane vesicles mediated by the canalicular bile salt export pump (Bsep). These results suggest that cholestasis may be a factor in troglitazone-associated hepatotoxicity. To determine whether this effect is 1) limited to canalicular bile acid transport and 2) is specific to troglitazone, the effect of troglitazone, rosiglitazone, and ciglitazone on bile acid transport was examined in rat basolateral (blLPM) and canalicular (cLPM) liver plasma membrane vesicles. In cLPM vesicles, troglitazone, rosiglitazone, and ciglitazone (100 μM) all significantly inhibited ATP-dependent taurocholate transport. In blLPM vesicles, these three thiazolidinediones also significantly inhibited Na +-dependent taurocholate transport. Inhibition of bile acid transport was concentration dependent and competitive in both cLPM and blLPM vesicles. In conclusion, these findings are consistent with a class effect by thiazolidinediones on hepatic bile acid transport. If hepatotoxicity is associated with this effect, then hepatotoxicity is not limited to troglitazone. Alternatively, if hepatotoxicity is limited to troglitazone, other mechanisms are responsible for its reported hepatotoxicity.

Troglitazone-induced intrahepatic cholestasis by an interference with the hepatobiliary export of bile acids in male and female rats. Correlation with the gender difference in troglitazone sulfate formation and the inhibition of the canalicular bile salt export pump (Bsep) by troglitazone and troglitazone sulfate

Troglitazone is a thiazolidinedione insulin sensitizer drug for the treatment of type 2 non-insulin-dependent diabetes mellitus (NIDDM). Based on an increasing number of reports on troglitazone-associated liver toxicity, the cholestatic potential of troglitazone and its major metabolite troglitazone sulfate has been investigated. In isolated perfused rat livers troglitazone (10 μM) reduced the bile flow by 25% (female) to 50% (male) within 60 min. After single intravenous administrations of troglitazone to rats of both genders, rapid and dose-dependent increases in the plasma bile acid concentrations were observed, with male rats being more sensitive than female rats. In male rat liver tissue fivefold higher troglitazone sulfate levels were measured as compared to female rat liver tissue. This difference was due to the formation rate of troglitazone sulfate, which was four times faster in cytosolic fractions of male rat liver as compared to female rat liver (Clint=132 and 35 μl min −1 mg −1, respectively). Troglitazone sulfate strongly inhibited the ATP-dependent taurocholate transport mediated by the canalicular bile salt export pump (Bsep) in isolated canalicular rat liver plasma membrane preparations of both genders (IC 50 value of 0.4–0.6 μM), while troglitazone was 10 times less potent (IC 50 values of 3.9 μM). This high Bsep inhibition potential and the efficient formation and accumulation of troglitazone sulfate in liver tissue, suggested that troglitazone sulfate was mainly responsible for the interaction with the hepatobiliary export of bile acids at the level of the canalicular Bsep in rats. Such an interaction might lead potentially also in man to a troglitazone-induced intrahepatic cholestasis, potentially contributing to the formation of troglitazone-induced liver injuries.

Bile salt export pump is highly conserved during vertebrate evolution and its expression is inhibited by PFIC type II mutations.

Bile secretion is a fundamental function of the liver of all vertebrates and is generated by ATP-dependent transport proteins at the canalicular membrane of hepatocytes, particularly by the bile salt export pump BSEP. To determine the evolutionary origin and structure-function relationship of this transport mechanism, a liver cDNA library from the marine skate Raja erinacea, a 200 million-year-old vertebrate, was screened for BSEP orthologues. A full-length clone was isolated that encodes for 1,348 amino acids and shares 68.5% identity to human BSEP. Northern blot analysis revealed a 5-kb transcript only in skate liver. Expression of skate Bsep in Sf9 cells demonstrated a sixfold stimulation of ATP-dependent taurocholate transport compared with controls, with a Michaelis-Menten constant of 15 microM, which is comparable to rat Bsep. Sequences at the site of published mutations in human BSEP are also conserved in skate Bsep. When two of these mutations were introduced into the skate Bsep cDNA, this resulted in defective expression of the mutant proteins in Sf9 cells. These studies demonstrate that Bsep is a liver-specific ATP-dependent export pump that is highly conserved throughout evolution and provide insights into critical determinants for the function of this transporter in higher vertebrates.

Cholestatic Potential of Troglitazone as a Possible Factor Contributing to Troglitazone-Induced Hepatotoxicity: In Vivo and in Vitro Interaction at the Canalicular Bile Salt Export Pump (Bsep) in the Rat

Troglitazone is a thiazolidinedione insulin sensitizer drug for the treatment of type 2 non-insulin-dependent diabetes mellitus (NIDDM). Based on an increasing number of reports on troglitazone-associated liver toxicity, the cholestatic potential of troglitazone has been investigated. Rapid and dose-dependent increases in the plasma bile acid concentrations were observed in rats after a single intravenous administration of troglitazone. A radiolabeled taurocholic acid tracer accumulated in liver tissue, indicating an interference with the hepatobiliary export of bile acids. In isolated canalicular rat liver plasma membrane preparations, troglitazone competitively inhibited the ATP-dependent taurocholate transport (apparent K(i) value, 1.3 microM), mediated by the canalicular bile salt export pump (Bsep). Troglitazone sulfate, the main troglitazone metabolite eliminated into bile, also showed competitive Bsep inhibition with an apparent K(i) value of 0.23 microM. A comparable inhibition was observed for both compounds in canalicular plasma membrane vesicles prepared from Mrp2-deficient (TR(-)) rats, suggesting a direct (cis-) inhibition of Bsep by troglitazone and troglitazone sulfate. A high accumulation potential was observed for troglitazone sulfate in rat liver tissue, indicating that the hepatobiliary export of this conjugated metabolite might represent a rate-limiting step in the overall elimination process of troglitazone. This accumulation in combination with the high Bsep inhibition potential suggested that mainly troglitazone sulfate was responsible for the interaction with the hepatobiliary export of bile acids at the level of the canalicular Bsep in rats. Such an interaction might lead to a troglitazone-induced intrahepatic cholestasis in humans as well, contributing to the formation of a troglitazone-induced liver toxicity.

Drug- and estrogen-induced cholestasis through inhibition of the hepatocellular bile salt export pump (Bsep) of rat liver

Background & Aims: Drug-induced cholestasis is a frequent form of acquired liver disease. To elucidate the molecular pathogenesis of drug-induced cholestasis, we investigated the effects of prototypic cholestatic drugs on the canalicular bile salt export pump (Bsep) of rat liver. Methods: Vesicles were isolated from Bsep-, Mrp2-, and Bsep/Mrp2-expressing Sf9 cells. Canalicular plasma membrane (cLPM) vesicles from rat liver and Sf9 cell vesicles were used to study adenosine triphosphate (ATP)-dependent solute uptake by a rapid filtration technique. Results: Bsep-expressing Sf9 cell vesicles showed ATP-dependent transport of numerous monoanionic bile salts with similar Michaelis constant values as in cLPM vesicles, whereas several known substrates of the multispecific organic anion transporter Mrp2 were not transported by Bsep. Cyclosporin A, rifamycin SV, rifampicin, and glibenclamide cis-inhibited Bsep-mediated bile salt transport to similar extents as ATP-dependent taurocholate transport in cLPM vesicles. In contrast, the cholestatic estrogen metabolite estradiol-17β-glucuronide inhibited ATP-dependent taurocholate transport only in normal cLPM and in Bsep/Mrp2-coexpressing Sf9 cell vesicles, but not in Mrp2-deficient cLPM or in selectively Bsep-expressing Sf9 cell vesicles, indicating that it trans-inhibits Bsep only after its secretion into bile canaliculi by Mrp2. Conclusions: These results provide a molecular basis for previous in vivo observations and identify Bsep as an important target for induction of drug- and estrogen-induced cholestasis in mammalian liver. GASTROENTEROLOGY 2000;118:422-430

The Sister of P-glycoprotein Represents the Canalicular Bile Salt Export Pump of Mammalian Liver

Canalicular secretion of bile salts is a vital function of the vertebrate liver, yet the molecular identity of the involved ATP-dependent carrier protein has not been elucidated. We cloned the full-length cDNA of the sister of P-glycoprotein (spgp; Mr approximately 160,000) of rat liver and demonstrated that it functions as an ATP-dependent bile salt transporter in cRNA injected Xenopus laevis oocytes and in vesicles isolated from transfected Sf9 cells. The latter demonstrated a 5-fold stimulation of ATP-dependent taurocholate transport as compared with controls. This spgp-mediated taurocholate transport was stimulated solely by ATP, was inhibited by vanadate, and exhibited saturability with increasing concentrations of taurocholate (Km approximately 5 microM). Furthermore, spgp-mediated transport rates of various bile salts followed the same order of magnitude as ATP-dependent transport in canalicular rat liver plasma membrane vesicles, i.e. taurochenodeoxycholate > tauroursodeoxycholate = taurocholate > glycocholate = cholate. Tissue distribution assessed by Northern blotting revealed predominant, if not exclusive, expression of spgp in the liver, where it was further localized to the canalicular microvilli and to subcanalicular vesicles of the hepatocytes by in situ immunofluorescence and immunogold labeling studies. These results indicate that the sister of P-glycoprotein is the major canalicular bile salt export pump of mammalian liver.

Evidence for an ATP-dependent bile acid transport protein other than the canalicular liver ecto-ATPase in rats

BACKGROUND & AIMS: Canalicular secretion is rate limiting in overall blood-to-bile transport of bile acids. Studies using transfected cells have implicated the canalicular ecto-adenosine triphosphatase (ecto- ATPase) in adenosine triphosphate (ATP)-dependent bile acid transport. However, the structural features of this ecto-ATPase are not those anticipated for an in-to-out ATP-dependent transporter. The aim of this study was to explore the possible existence of an ATP-dependent bile acid transport mechanism distinct from ecto-ATPase. METHODS: Bile acid transport activity and ecto-ATPase expression were analyzed in primary rat hepatocytes, rat hepatoma HTC cells, and specially adapted HTC (HTC- R) cells using plasma membrane vesicles and Northern blot, slot blot, ribonuclease protection assay, and Western blot analyses. RESULTS: Plasma membranes isolated from HTC-R cells exhibited ATP-dependent taurocholate transport, which was many-fold greater than that in HTC cells. Hepatocytes showed the highest transport rates. Protein and RNA analyses showed very low expression of ecto-ATPase in HTC and HTC-R cells compared with hepatocytes. There was no difference between the two cell types at both the RNA and protein level. CONCLUSIONS: These findings show the presence in HTC-R cells and, apparently in hepatocytes, of one or more proteins other than the ecto-ATPase that mediate ATP-dependent transport of bile acids. (Gastroenterology 1997 Jul;113(1):249-54)

Prolactin increases ATP-dependent taurocholate transport in canalicular plasma membrane from rat liver.

The taurocholate (TC) maximal secretory rate (SRm) in the isolated perfused liver is increased in postpartum rats and ovariectomized rats treated with ovine prolactin (oPRL). The present studies were designed to characterize the mechanism(s) by which oPRL increases TC transport in the liver. oPRL (300 micrograms/day i.v. for 7 days) increased the SRm 1.6-fold from 185 to 364 nmol.min-1.mg protein-1 in the perfused rat liver and the maximal rate of transport for ATP-dependent transport 1.7-fold from 66 to 109 nmol.min-1.mg protein-1 in canalicular liver plasma membrane (cLPM) vesicles without changing the Michaelis constant (5-6 microM). The oPRL-mediated increases in biliary excretion in the perfused liver and ATP-dependent TC transport in cLPM vesicles were significantly inhibited by cycloheximide treatment (2 mg/kg). oPRL (300 micrograms/day iv for 7 days) increased expression of Ca(2+)-Mg(2+)-ecto-adenosinetriphosphatase mRNA sixfold and increased protein expression two- to threefold, but had no effect on the expression of P-glycoprotein (mdr1b and mdr2) mRNA. Thus the increase in ATP-dependent transport in cLPM vesicles due to oPRL treatment accounts for the increased TC SRm in the perfused liver. The oPRL-mediated increased TC transport may be associated with increased expression of proteins related to bile acid transport.

Structure-specific inhibition by bile acids of adenosine triphosphate—dependent taurocholate transport in rat canalicular membrane vesicles

The adenosine triphosphate (ATP)-dependent transport system is a major determinant of canalicular bile acid secretion. The system transports bile acids and neither organic cations nor non—bile acid organic anions, such as glucuronides or glutathione adducts. To define the structural specificity of the ATP-dependent system, the authors examined the ability of various bile acids to inhibit ATP-dependent taurocholate transport by rat liver canalicular membrane vesicles. Only bile acids with a negative charge inhibited transport, which was unaffected by side chain length. Conjugated, but not unconjugated, mono- and di-hydroxy bile acids inhibited transport. The presence of 7α- and 12α-hydroxylation also influenced inhibition of ATP-dependent taurocholate transport. Inhibition of transport by bile acids was kinetically competitive. These results suggest that the canalicular ATP-dependent bile acid transport system depends on bile acid side chain charge, conjugation, and hydroxylation.

Structure-specific inhibition by bile acids of adenosine triphosphate—dependent taurocholate transport in rat canalicular membrane vesicles

The adenosine triphosphate (ATP)-dependent transport system is a major determinant of canalicular bile acid secretion. The system transports bile acids and neither organic cations nor non-bile acid organic anions, such as glucuronides or glutathione adducts. To define the structural specificity of the ATP-dependent system, the authors examined the ability of various bile acids to inhibit ATP-dependent taurocholate transport by rat liver canalicular membrane vesicles. Only bile acids with a negative charge inhibited transport, which was unaffected by side chain length. Conjugated, but not unconjugated, mono- and di-hydroxy bile acids inhibited transport. The presence of 7 alpha- and 12 alpha-hydroxylation also influenced inhibition of ATP-dependent taurocholate transport. Inhibition of transport by bile acids was kinetically competitive. These results suggest that the canalicular ATP-dependent bile acid transport system depends on bile acid side chain charge, conjugation, and hydroxylation.

Hepatocellular transport of bile acids. Evidence for distinct subcellular localizations of electrogenic and ATP-dependent taurocholate transport in rat hepatocytes.

To investigate whether electrogenic and ATP-dependent taurocholate transport activities are both mediated by the same bile acid-transporting polypeptide in rat liver, we further purified isolated canalicular membrane vesicles by free flow electrophoresis. Removal of most of the contaminating endoplasmic reticulum resulted in a complete loss of electrogenic taurocholate transport from an ecto-ATPase-enriched canalicular membrane subfraction. In contrast, ATP-dependent taurocholate transport remained associated with both an ecto-ATPase-enriched and an ecto-ATPase-free canalicular membrane subfraction. Microsomes containing 64% of total endoplasmic reticulum exhibited saturable electrogenic (Km approximately 270 microM), but no ATP-dependent taurocholate uptake. Golgi membrane vesicles were devoid of any taurocholate transport activity. These results indicate that electrogenic taurocholate transport resides entirely in the endoplasmic reticulum, whereas ATP-dependent bile acid transport is an intrinsic function of the canalicular membrane as well as of a so far unidentified intracellular membrane bound compartment. Hence, the two transport activities are most probably mediated by two different bile acid transporting polypeptides. Furthermore, the finding of ATP-dependent taurocholate transport in virtually ecto-ATPase-free vesicles argues against the concept of primary active bile acid transport being exclusively mediated by the canalicular ecto-ATPase.

ATP-dependent export pumps and their inhibition by cyclosporins

Cyclosporins are potent tools to inhibit several primary-active, ATP-dependent export carriers. This has been demonstrated in membrane vesicle transport assays for CsA and for its non-immunosuppressive analog PSC 833. Inhibition in the low micromolar and in the nanomolar concentration range is shown for the three distinct ATP-dependent export carriers in the liver canalicular membrane mediating the secretion into bile of leukotrienes (LTC 4, other cysteinyl leukotrienes, and related conjugates), bile salts (taurocholate), and amphiphilic, mostly cationic substances (daunorubicin and other P-glycoprotein substrates). Competitive inhibition by cyclosporins is most potent for ATP-dependent taurocholate transport with K i values of 0.2 and 0.6 μ m for CsA and PSC 833, respectively. This inhibition is in agreement with in vivo studies in the rat demonstrating a block at the canalicular membrane in the hepatobiliary elimination of labeled taurocholate. The data suggest that cholestasis, as a side effect during CsA therapy, is largely due to inhibition of the ATP-dependent bile salt export carrier in the canalicular membrane. Inhibition by cyclosporins is less effective with respect to ATP-dependent leukotriene transport, both during biosynthetic release from mastocytoma cells and during hepatobiliary excretion. The K i values for the former were 4.5 and 30 μ m, and the K m/ K i ratios only 0.015 and 0.002 for CsA and PSC 833, respectively. Distinct transporters are inhibited by the cyclosporins with different potency and structurally modified cyclosporins may serve to induce preferential inhibition of a selected transporter. This is illustrated by the inhibition of the multidrug export carrier with daunorubicin as substrate using PSC 833 as inhibitor with a K i value of 0.3 μ m in an in vitro membrane transport system.

Ethinylestradiol treatment induces multiple canalicular membrane transport alterations in rat liver.

We investigated the effects of 17 alpha-ethinylestradiol treatment of rats on various transport functions in isolated basolateral and canalicular liver plasma membrane vesicles. Both membrane subfractions were purified to a similar degree from control and cholestatic livers. Although moderate membrane lipid alterations were predominantly observed in basolateral vesicles, no change in basolateral Na+/K(+)-ATPase activity was found. Furthermore, while Na(+)-dependent taurocholate uptake was decreased by approximately 40% in basolateral vesicles, the maximal velocity of ATP-dependent taurocholate transport was decreased by 63% in canalicular membranes. In contrast, only minimal changes or no changes at all were observed for electrogenic taurocholate transport in "cholestatic" canalicular membranes and total microsomes, respectively. However, canalicular vesicles from cholestatic livers also exhibited marked reductions in ATP-dependent transport of S-(2,4-dinitrophenyl)glutathione and in Na(+)-dependent uptake of adenosine, while in the same vesicles HCO3-/SO4- exchange and Na+/glycine cotransport activities were markedly stimulated. These data show that in addition to the previously demonstrated sinusoidal transport abnormalities ethinylestradiol-induced cholestasis is also associated with multiple canalicular membrane transport alterations in rat liver. Hence, functional transport alterations at both polar surface domains might ultimately be responsible for the inhibitory effects of estrogens on the organic anion excretory capacity and on bile formation in rat liver.

Inhibition by cyclosporin A of Adenosine triphosphate-dependent transport from the hepatocyte into bile

Background: Immunosuppressive treatment with cyclosporin A may be associated with impaired hepatobiliary elimination of bile salts and with cholestasis. Inhibition by cyclosporin A of the primary-active adenosine triphosphate (ATP)-dependent transport systems responsible for excretion of bile salts and cysteinyl leukotrienes across the hepatocyte canalicular membrane into bile may explain the cholestatic side effect. Methods: ATP-dependent transport of bile salt and of cysteinyl leukotrienes was studied in human liver plasma membrane vesicles and additionally in rat liver plasma membrane vesicles enriched in canalicular membranes. Results: Inhibition of ATP-dependent taurocholate transport in human liver by 50% was measured at 3 μmol/L cyclosporin A and at 4 μmol/L fujimycin. Kinetic analyses in rat liver indicated non-competitive inhibition by cyclosporin A with respect to ATP and competitive inhibition with respect to taurocholate with inhibition constant (Ki) values of 1.0 and 0.3 μmol/L, respectively. Conclusions: The ATP-dependent export carriers for bile salts and cysteinyl leukotrienes in the hepatocyte canalicular membrane are novel targets for inhibitory side effects of cyclosporin A. Inhibition of ATP-dependent bile salt transport may induce cholestasis.

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

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.

ATP-dependent taurocholate transport by rat liver canalicular membrane vesicles

We conducted an experimental study to examine the possibility that ATP is involved in the mechanism by which bile acids are excreted through the liver canalicular membrane in opposing the concentration gradient. Canalicular membrane vesicles were purified from the livers of Sprague-Dawley rats, and the uptake of tritiated sodium taurocholate into canalicular membrane vesicles was determined by rapid filtration technique. Vesicle-associated sodium taurocholate was increased in the presence of ATP and ATP-regenerating system. This was also observed at a voltage-clamped condition. ATP-dependent uptake into the osmotically reactive intravesicular space was saturated with increasing concentrations of sodium taurocholate (Km = 47 μmol/L, Vmax = 270 pmoles/mg protein · 20s). ATP-dependent uptake increased to the point of saturation when the sodium taurocholate concentration was 50 μmol/L and the ATP concentration was increased from 0 to 1 mmol/L (Km = 64 μmol/L). Among the several nucleotides used, ATP was a potent stimulator of transport, whereas a nonhydrolyzable analogue (i.e., adenosine 5′-[β,γ-imino]triphosphate) showed no effect. In addition, ATP-dependent transport was inhibited by vanadate in a dose-dependent manner. From these results it was concluded that the primary active transport of sodium taurocholate is present in hepatocellular canalicular membranes. This transport is directly dependent on ATP, and hydrolysis of γ-phosphate of ATP is required. (Hepatology 1991;14:655-659.)

ATP-dependent transport of taurocholate across the hepatocyte canalicular membrane mediated by a 110-kDa glycoprotein binding ATP and bile salt.

Direct photoaffinity labeling of liver plasma membrane subfractions enriched in sinusoidal and canalicular membranes using [35S]adenosine 5'-O-(thiotriphosphate) ([35S]ATP gamma S) allows the identification of ATP-binding proteins in these domains. Comparative photoaffinity labeling with [35S]ATP gamma S and with the photolabile bile salt derivative (7,7-azo-3 alpha, 12 alpha-dihydroxy-5 beta-[3 beta-3H]-cholan-24-oyl-2'- aminoethanesulfonate 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 [3H] 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 gp110 into liposomes resulted in an ATP-dependent uptake of [3H]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.