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Unlabelled: Progressive familial intrahepatic cholestasis type 2 (PFIC2) is caused by a mutation in the bile salt export pump (BSEP/ABCB11) gene. We previously reported that E297G and D482G BSEP, which are frequently found mutations in European patients, result in impaired membrane trafficking, whereas both mutants retain their transport function. The dysfunctional localization is probably attributable to the retention of BSEP in endoplasmic reticulum (ER) followed by proteasomal degradation. Because sodium 4-phenylbutyrate (4PBA) has been shown to restore the reduced cell surface expression of mutated plasma membrane proteins, in the current study, we investigated the effect of 4PBA treatment on E297G and D482G BSEP. Transcellular transport and cell surface biotinylation studies using Madin-Darby canine kidney (MDCK) II cells demonstrated that 4PBA treatment increased functional cell surface expression of wild-type (WT), E297G, and D482G BSEP. The prolonged half-life of cell surface-resident BSEP with 4PBA treatment was responsible for this result. Moreover, treatment of Sprague-Dawley rats with 4PBA resulted in an increase in BSEP expression at the canalicular membrane, which was accompanied by an increase in the biliary excretion of [(3)H]taurocholic acid (TC)., Conclusion: 4PBA treatment with a clinically achievable concentration enhances the cell surface expression and the transport capacity of WT, E297G, and D482G BSEP in MDCK II cells, and also induces functional BSEP expression at the canalicular membrane and bile acid transport via canalicular membrane in vivo. 4PBA is a potential pharmacological agent for treating not only PFIC2 patients with E297G and D482G mutations but also other cholestatic patients, in whom the BSEP expression at the canalicular membrane is reduced.

The role of the cholangiocyte apical Na(+)-dependent bile acid transporter (ASBT) in bile formation is unknown. Bile acid absorption by bile ducts results in cholehepatic shunting, a pathway that amplifies the canalicular osmotic effects of bile acids. We tested in isolated cholangiocytes if secretin enhances ASBT translocation to the apical membrane from latent preexisting intracellular stores. In vivo, in bile duct-ligated rats, we tested if increased ASBT activity (induced by secretin pretreatment) results in cholehepatic shunting of bile acids. We determined the increment in taurocholate-dependent bile flow and biliary lipid secretion and taurocholate (TC) biliary transit time during high ASBT activity. Secretin stimulated colchicine-sensitive ASBT translocation to the cholangiocyte plasma membrane and (3)H-TC uptake in purified cholangiocytes. Consistent with increased ASBT promoting cholehepatic shunting, with secretin pretreatment, we found TC induced greater-than-expected biliary lipid secretion and bile flow and there was a prolongation of the TC biliary transit time. Colchicine ablated secretin pretreatment-dependent bile acid-induced choleresis, increased biliary lipid secretion, and the prolongation of the TC biliary transit. In conclusion, secretin stimulates cholehepatic shunting of conjugated bile acids and is associated with increased cholangiocyte apical membrane ASBT. Bile acid transport by cholangiocyte ASBT can contribute to hepatobiliary secretion in vivo.

The liver is the major source of reduced glutathione (GSH) in blood plasma. The transport protein mediating the efflux of GSH across the basolateral membrane of human hepatocytes has not been identified so far. In this study we have localized the multidrug resistance protein 4 (MRP4; ABCC4) to the basolateral membrane of human, rat, and mouse hepatocytes and human hepatoma HepG2 cells. Recombinant human MRP4, expressed in V79 hamster fibroblasts and studied in membrane vesicles, mediated ATP-dependent cotransport of GSH or S-methyl-glutathione together with cholyltaurine, cholylglycine, or cholate. Several monoanionic bile salts and the quinoline derivative MK571 were potent inhibitors of this unidirectional transport. The K(m) values were 2.7 mmol/L for GSH and 1.2 mmol/L for the nonreducing S-methyl-glutathione in the presence of 5 micromol/L cholyltaurine, and 3.8 micromol/L for cholyltaurine in the presence of 5 mmol/L S-methyl-glutathione. Transport of bile salts by MRP4 was negligible in the absence of ATP or without S-methyl-glutathione. These findings identify a novel pathway for the efflux of GSH across the basolateral hepatocyte membrane into blood where it may serve as an antioxidant and as a source of cysteine for other organs. Moreover, MRP4-mediated bile salt transport across the basolateral membrane may function as an overflow pathway during impaired bile salt secretion across the canalicular membrane into bile. In conclusion, MRP4 can mediate the efflux of GSH from hepatocytes into blood by cotransport with monoanionic bile salts.

Four animal models were used to quantitatively evaluate hepatic alterations in this study: (1) a carbon tetrachloride control group (phenobarbital treatment only), (2) a CCl(4)-treated group (phenobarbital with CCl(4) treatment), (3) an alcohol-treated group (liquid diet with alcohol treatment), and (4) a pair-fed alcohol control group (liquid diet only). At the end of induction, single-pass perfused livers were used to conduct multiple indicator dilution (MID) studies. Hepatic spaces (vascular space, extravascular albumin space, extravascular sucrose space, and cellular distribution volume) and water hepatocyte permeability/surface area product were estimated from nonlinear regression of outflow concentration versus time profile data. The hepatic extraction ratio of (3)H-taurocholate was determined by the nonparametric moments method. Livers were then dissected for histopathologic analyses (e.g., fibrosis index, number of fenestrae). In these 4 models, CCl(4)-treated rats were found to have the smallest vascular space, extravascular albumin space, (3)H-taurocholate extraction, and water hepatocyte permeability/surface area product but the largest extravascular sucrose space and cellular distribution volume. In addition, a linear relationship was found to exist between histopathologic analyses (fibrosis index or number of fenestrae) and hepatic spaces. The hepatic extraction ratio of (3)H-taurocholate and water hepatocyte permeability/surface area product also correlated to the severity of fibrosis as defined by the fibrosis index. In conclusion, the multiple indicator dilution data obtained from the in situ perfused rat liver can be directly related to histopathologic analyses.

Male Wistar rats were infused intravenously with taurochenodeoxycholate (0.4 mumol/min/100 gm) alone (group A) or with one of the three bile salts (tauroursodeoxycholate [group B], tauro beta-muricholate [group C] or tauro alpha-muricholate [group D]) at a rate of 0.2 mumol/min/100/gm for 1 hr. One-hour bile flow and bile salt excretion rates were significantly lower in group A than in the other three coinfused (B, C, D) groups. Biliary 1-hr outputs of lactate dehydrogenase and albumin in the bile, on the other hand, were significantly higher in group A than in the other groups. Plasma concentrations of lactate dehydrogenase at the time of killing (1 hr) were two to three times higher in group A than in the other groups. Although tauro alpha-muricholate does not possess a 7 beta-hydroxy group, the 6 beta-hydroxy group that tauro alpha-muricholate possesses thus appears to be as effective as a 7 beta-hydroxy group in reducing the liver damage caused by toxic bile salts such as taurochenodeoxycholate. The so-called hepatoprotective effects of tauroursodeoxycholate and tauro beta-muricholate found in previous studies may require explanation(s) other than the presence of a 7 beta-hydroxy group in their molecular structures.

The purpose of this study was to provide further insight into the mechanism of bilirubin diglucuronide excretion through the hepatocyte canalicular membrane by investigating the uptake of (3H)bilirubin diglucuronide by purified canalicular membrane vesicles of rat liver. The uptake was analyzed by a rapid filtration technique. The difference between vesicle-associated (3H)bilirubin diglucuronide at 37 degrees C and at 0 degree C during the initial 1 min was regarded as uptake. Twenty second uptake was saturated by increasing the (3H)bilirubin diglucuronide concentration at a vesicle-inside-directed 100 mmol/L KCl gradient (Km = 75 mumol/L, Vmax = 320 pmol/mg protein.20 sec at 37 degrees C). No sodium dependency was observed. When canalicular membrane vesicles were preincubated with nonlabeled bilirubin diglucuronide, the uptake increased 1.3-fold (transstimulation). Vesicle-inside-positive potential induced by valinomycin and potassium caused a 1.4-fold increase in the uptake. When Cl- was replaced by equivalent ion concentrations of SO4(2-), HCO3-, NO3- and SCN-, the uptake was 78%, 244%, 68% and 50%, respectively, and specific stimulation by HCO3- was observed (Km = 75 mumol/L, Vmax = 700 pmol/mg protein.20 sec at a vesicle-inside-directed 100 mmol/L KHCO3 gradient at 37 degrees C). The uptake was inhibited in a dose-dependent manner by the addition of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. The uptake was ATP independent. From these results, it was concluded that bilirubin diglucuronide transport through the canalicular membrane is carrier mediated, electrogenic and stimulated by HCO3-.

Nonbile salt cholephiles and bile salts are two classes of organic anions that are efficiently taken up and excreted by the liver. Recent evidence suggests that a microtubular system-dependent, colchicine-sensitive transcellular pathway may transport both classes of these ligands. The relationship of this pathway to flux rates, however, remains unclear. Some structural evidence suggests an important role for a Golgi-associated vesicular system. Monensin, like colchicine, is a perturbing agent that is believed to target primarily Golgi and related organelles. The effects of a minimal effective dose of both colchicine (0.06 mg to 0.12 mg/100 gm body wt) and monensin (0.6 mg/100 gm body wt) were examined in the isolated perfused rat liver in a single-pass mode. The nonbile salt cholephile, phenol red, was studied at two doses: 1 nmol and 5 mumol. Sodium taurocholate was studied at three doses: 2 nmol, 1 mumol and 5 mumol. Colchicine affected the transcellular transport for both classes of organic anions equally. Partially inhibitory effects on both anions occurred only at high ligand flux rates. In contrast, monensin greatly impaired the transport of nonbile salt cholephiles but had no influence on transcellular bile salt flux. We conclude that the monensin effect appears to define a distinct transcellular transport pathway for each of the two classes of organic anions.

We used compartmental modeling to describe taurocholate transport by isolated rat liver cells in suspension. Cells are preincubated in the presence of unlabeled taurocholate. When a steady-state for taurocholate is reached, radiolabeled taurocholate is added to the medium and its exchange kinetics between the medium and the cells are followed over time. Because the studies are performed under steady-state conditions, the kinetics can be described by linear compartmental models. We found a closed two-compartment model sufficient to describe the steady-state transport data. Simulations reveal that if the pools of free and bound intracellular taurocholate exchange rapidly, the cells will behave as a single, kinetically homogeneous compartment and intracellular events will not influence the exchange kinetics of taurocholate between the medium and the cells. The two-compartment model was used to study the concentration dependence of taurocholate transport by isolated cells. Steady-state transport rates and taurocholate concentrations in the medium and the cells were calculated using the model equations. Taurocholate influx, accumulation and efflux processes were studied simultaneously by examining the relationship between appropriate combinations of these variables. Application of this approach to study the inhibition of taurocholate transport by taurochenodeoxycholate is illustrated. In conclusion, this method provides a complementary approach to initial rate studies, which are generally used to investigate bile acid transport by isolated cells.

Trihydroxy bile acids with differing nonsterol chain length and charge were synthesized to define the effect of these parameters on the ability to competitively inhibit the Na(+)-dependent uptake of 14C taurocholate into isolated rat hepatocytes. Compounds with long side chains (greater than or equal to 0.8 nm) beyond carbon-17 of the sterol nucleus and carrying a negative charge or no charge were potent inhibitors. Introduction of a positive charge into the side chain weakened inhibition. When the length of the chain beyond carbon-17 fell below about 0.7 nm, charge still influenced inhibitory potency, but the effect was reversed and positively-charged chains yielded slightly greater inhibition than negatively-charged chains. From these results one may postulate a positively-charged cell surface domain extending outward from a point about 0.7 nm from the sterol nucleus receptor region. Up to about 0.7 nm from the sterol nucleus receptor region one might postulate a negative cell surface charge to account for the weaker inhibitory potency of compounds with short negatively-charged chains. Nonetheless, a short chain, regardless of charge, weakened inhibition, suggesting that a long negatively-charged side chain is necessary to orient the sterol moiety for optimal receptor fit. These data confirm that the Na(+)-dependent taurocholate transport site is sensitive to alterations of side chain charge and length and emphasize the importance of structure when designing bile acid analogs to probe taurocholate transport mechanisms.

The hepatic extraction of unconjugated and taurine-conjugated bile acids, provided with different hydrophilicity values, has been measured in the perfused rat liver, in order to evaluate the role of the bile acid structure and bile acid hydrophilicity on their uptake by the liver. Ursocholic, cholic, ursodeoxycholic and chenodeoxycholic acids, free and taurine-conjugated, were injected into the portal vein in dose response studies, using a nonrecirculating perfusion system. For all of the bile acids, the uptake process showed saturation. In addition, a nonsaturable component was apparent in bile acids provided with the lowest hydrophilicity values, as expressed by the lowest values of the water to octanol partition coefficient. The maximum uptake velocity increased with increasing values of the partition coefficient, which in turn were associated with 7-OH alpha to beta epimerization, the presence of 12-OH in alpha position and taurine conjugation. The ratio of maximum uptake velocity to Km (Km being the half-saturation constant) appeared to be markedly increased by taurine conjugation and by 7-OH alpha to beta epimerization, whereas it was reduced by the presence of 12-OH in alpha position.