Taurocholate Efflux Research Articles

An anion exchanger mediates bile acid transport across the placental microvillous membrane

Microvillous membrane vesicles from the term human placental syncytiotrophoblast were used to characterize further the properties of a transport mechanism for bile acids. Taurocholate (TC) uptake into an osmotically reactive intravesicular space was temperature dependent and independent of sodium. TC uptake (2 microM) was markedly inhibited by 250 microM taurine and glycine-conjugated cholate and chenodeoxycholate and unconjugated cholate but not by chenodeoxycholate, deoxycholate, etianic acid, bromosulfophthalein, pyruvate, lactate, alanine, or taurine. The initial rate of TC uptake was inhibited significantly by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) but was not inhibited significantly by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, amiloride, or furosemide. Preincubation of vesicles with DIDS in the presence of TC partially blocked the action of the inhibitor. Efflux of 5 microM TC from membrane vesicles was stimulated by the presence of 50 microM TC in the incubation media. Basal as well as transstimulated TC efflux was inhibited by DIDS. The initial rate of TC influx followed saturation kinetics with an apparent Michaelis constant of 112 +/- 23 microM and maximal velocity of 2.01 +/- 0.19 nmol.mg protein-1.min-1. When the transmembrane electrical potential difference across the brush-border membrane vesicles was altered by external anion replacement or by valinomycin-induced K+ diffusion potentials, TC uptake was not significantly affected. DIDS-sensitive TC uptake was stimulated two-to threefold by an outwardly directed hydroxyl gradient (pH 7.7in/5.5out) compared with TC influx under pH-equilibrated conditions (pH 7.7in/7.7out). These studies are consistent with an electroneutral anion-exchange mechanism that mediates transfer of conjugated bile acids across the microvillous membrane of the syncytiotrophoblast.

Characterization of endogenous carrier-mediated taurocholate efflux from Xenopus laevis oocytes.

Taurocholate efflux was studied in Xenopus laevis oocytes and is consistent with a carrier-mediated process. This carrier can be competitively inhibited and trans stimulated by glycocholate. Transport is also trans stimulated by taurochenodeoxycholate and S-hexylglutathione, but not taurolithocholate or daunomycin, reflecting a range of specificity including substrates of both the hepatic canalicular bile acid transporter and the multispecific organic anion transporter. In addition, ATP added to the outside of the oocyte results in an increase in the maximal velocity of this transport process. The physiologic function of this endogenous carrier is not known, but it may act as a generalized system for the efflux of potentially toxic organic anions.

Utilization of ATP-depleted cells in the analysis of taurocholate uptake by isolated rat hepatocytes

The usefulness of ATP-depleted rat hepatocytes in transport studies was examined. ATP-depleted hepatocytes were prepared by incubating cell suspensions with 30 microM rotenone. In ATP-depleted hepatocytes, plasma membrane permeability was increased and mitochondrial membrane potential decreased, while both intracellular volume and pH remained normal. Furthermore, in the presence of valinomycin, the initial uptake rates of 3H-tetraphenyl phosphonium (TPP+) with varied medium concentrations of potassium were predicted according to the Goldman-Hodgkin-Katz equation, which demonstrated that a potassium diffusion potential could be produced in this system. Using the thus-characterized ATP-depleted cells, the uptake mechanism of taurocholate was investigated. In the presence of an inwardly directed Na gradient, the taurocholate uptake was markedly stimulated and bile acid was transiently accumulated at a concentration 3-times higher than at equilibrium ('overshoot') in ATP-depleted cells. No overshoot was observed in viable cells, however, which suggests that in ATP-depleted cells the Na gradient, a driving force for taurocholate uptake, decreased with time. In both viable and ATP-depleted cells, the relationship between medium concentrations of Na and the Na-dependent initial uptake rate were sigmoidal, and the Hill coefficients were close to 2. The Na-dependent initial uptake rate of taurocholate was stimulated by a valinomycin-induced inside negative potassium-diffusion potential in ATP-depleted cells, and the movement of a 'one plus' (as a net) charge was revealed by fitting the data to the Goldman-Hodgkin-Katz equation. These results support the hypothesis that sodium-coupled hepatic uptake of taurocholate occuthrough an electrogenic process with the stoichiometry of 2 Na: 1 taurocholate, although this issue is controversial. In the presence of an outwardly directed sodium gradient, efflux of taurocholate from ATP-depleted cells was not stimulated. Consequently, the physiological transport vector of taurocholate from blood to cell is not only due to the direction of the sodium gradient (blood to cell) but also to membraneous orientation of transport carriers. In conclusion, kinetic analysis using ATP-depleted hepatocytes allowed the formulation of a new approach to clarify the as yet unresolved issues concerning transport stoichiometry and the mechanism for vectorial transport of taurocholate.

Bicarbonate-induced activation of taurocholate transport across the basal plasma membrane of human term trophoblast

The efflux of [14C]taurocholate from previously loaded vesicles, obtained from basal plasma membrane of human trophoblast, was studied. Apparent Km (620 microM) and Vmax (1.79 nmol.min-1.mg protein-1) values were similar to those found in influx experiments (Marin et al., Gastroenterology 99: 1431-1438, 1990). Transmembrane gradients of both bicarbonate (100 mM) and unlabeled taurocholate (0.5 mM) accelerated [14C]taurocholate efflux. The bicarbonate-induced effect was not abolished by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and K(+)-valinomycin voltage clamp. Neither was it mimicked by 5,5'-dimethyloxazolidine 2,4-dione (DMO) or by other organic (taurine, glycine, lactate, or acetate) or inorganic (Cl-, SCN-, HPO24-, or SO24-) anions, and it was not sensitive to carbonic anhydrase inhibitors. No effect of bicarbonate was observed either in the absence of gradient or in the presence of a cis-directed gradient. Bicarbonate-induced transstimulation was related to an increase in the value for the apparent Vmax (+30%). Study of the stoichiometry suggests that the most probable coupling ratio is one, bicarbonate: taurocholate. In summary, these results provide evidence for the existence of a bicarbonate-driven anion exchange in the basal plasma membrane of the human term placental trophoblast.

Kinetics of Taurocholate Efflux From Freshly Isolated Suspensions and Primary Cultures of Rat Hepatocytes

Isolated rat hepatocytes have been advocated as a model to study aspects of mechanism of chemical-induced interference with biliary excretory function. Some technical problems do exist in studying efflux, such as the reuptake of the previously excreted substrate. Another concern is the loss of liver-specific functions in hepatocytes with continuing time in culture. It is important to address such technical aspects and to determine whether the process of efflux is compromised in primary cultures of hepatocytes. In the presence of Na+ the apparent efflux of taurocholate from hepatocytes was shown to be significantly confounded by reuptake of substrate. The unidirectional efflux was best demonstrated in buffer where choline replaced Na+. A comparison of efflux kinetics for cultured cells to those in suspension showed that both apparent affinity for transport carriers and transport capacity were greater in the former. The simple diffusion component for efflux increased with the time in culture, but affinity for transport carriers and transport capacity remained unchanged over 6 to 24 hr. However, it was not possible to determine meaningful kinetic constants after 24 hr in culture because the uptake of taurocholate was so low. Primary cultured hepatocytes may therefore be of limited value in the study of efflux of bile salts in the longer term, mainly because of the inability of cells to take up and accumulate a sufficiently high level of bile salts.

Receptor-mediated stimulation of taurocholate efflux from the rat hepatocyte and the ex vivo perfused rat liver

The peptide hormone, arginine-vasopressin([Arg 8]vasopressin, AVP), stimulates efflux of the bile salts taurocholate and glycocholate from the rat hepatocyte in suspension via its association with the V 1 receptor on the hepatic cell membrane. At a concentration ratio of 5:1 (antagonist to hormone), the V 1 vasopressin antagonist, (dCH 2) 5Tyr(Me)AVP, inhibits the vasopressin induced efflux of taurocholate by approximately 82%, and of glycocholate, by approximately 85%. In contrast, the V 2 antagonist (d(CH 2) 5[D-Ile 2,Ala 4]AVP, does not interfere with the stimulation of taurocholate and glycocholate efflux by vasopressin. In the isolated perfused rat liver, vasopressin (5 × 10 −10 M) causes an immediate increase of 55 ± 12% over baseline in [ 14C]taurocholate secretion and a corresponding increase in bile flow. A more gradual and prolonged increase in [ 14C]taurocholate secretion, reflecting an increased biliary concentration of [ 14C]taurocholate, is observed beginning 6 min after vasopressin, reaching a plateau of 23 ± 12% over baseline by 14 min and returning to baseline by 30 min. The mean rate of 14C secretion during the 30 min following administration of vasopressin (non-steady state) is increased by 14.3 ± 6.4% over pre-infusion steady-state baseline (P < 0.05). Prior administration of the V 1 receptor antagonist d(CH 2) 5Tyr(Me)AVP attenuates these effects of vasopressin. The combination of these in vitro and in vivo findings suggest that vasopressin may play a role in regulating bile salt efflux. Furthermore, these studies in the isolated hepatocyte and the intact liver may provide a unique approach for defining biochemical changes associated with bile salt transport from the hepatic cell.

Identification and comparsion of bile acid-binding polypeptides in ileal basolateral membrane

Bile acid-binding polypeptides were examined using basolateral membrane vesicles and enterocytes isolated from rat ileum. The uptake of a photolabile taurocholate derivative, (7,7,-azo-3 alpha,12 alpha-dihydroxy-5 beta[3 beta-3H]cholan-24-oyl)-2- aminoethanesulfonate,7,7-azo-TC, in ileal vesicles preloaded with paraaminohippurate (PAH) was stimulated with respect to uptake in unpreloaded vesicles. The PAH-transstimulated uptake of 7,7-azo-TC was inhibited by taurocholate and vice versa. Irradiation of membrane vesicles in the presence of 7,7-azo-TC irreversibly inhibited PAH-transstimulated taurocholate uptake. Photoaffinity labeling of basolateral membrane vesicles directly with [3H] 7,7-azo-TC and separation of proteins by SDS-PAGE revealed incorporation of radioactivity into several polypeptides. Photoaffinity labeling of vesicles in the presence of taurocholate inhibited the labeling of 54,000 and 59,000 mol. wt. polypeptides. The efflux of taurocholate from ileal enterocytes was cis-inhibited by 7,7-azo-TC and transstimulated by PAH. Irradiation of enterocytes in the presence of 7,7-azo-TC inhibited taurocholate efflux greater than the presence of 7,7-azo-TC in the dark. When enterocytes that were irradiated in the presence of [3H] 7,7-azo-TC were fractionated and the resultant basolateral membrane fraction was subjected to SDS-PAGE, incorporation of radioactivity into the 54,000 and 59,000 mol. wt. polypeptides was seen. In contrast, when the brush-border membrane fraction was subjected to SDS-PAGE, greatest incorporation of radioactivity was seen in the previously described 99,000 mol. wt. polypeptide. These studies suggest that 7,7-azo-TC shared transporters with natural bile acid and identified polypeptides that may be involved in bile acid transport across the basolateral membrane and differ from that seen in the brush-border membrane of the ileal epithelial cell.

Effects of chlorpromazine on taurocholate transport in isolated rat hepatocytes

Chlorpromazine has been shown to have no effect on the uptake of the endogenous bile salt substrate, taurocholate, by isolated rat hepatocytes. It has been shown, however, to inhibit directly release of taurocholate from pre-loaded cells over extended incubation. However, there was no inhibition of the efflux process per se as shown by similar initial rates of taurocholate efflux in the presence or absence of Chlorpromazine. Pretreatment of rats with chlorpromazine (100 μmoles/kg) resulted in no change in the ability to transport (that is, accumulate or secrete) taurocholate by hepatocytes isolated 2, 24, 36, 48, or 60 hr later. The data indicate that, if a direct effect on bile acid transport is important in chlorpromazine induced biliary dysfunction, then it involves release rather than uptake at the cell membrane. However, as efflux itself is not inhibited chlorpromazine may interfere with release of taurocholate from intracellular sites.

Inhibition of Taurocholate and Ouabain Transport in Isolated Rat Hepatocytes by Cyclosporin A

The use of cyclosporin A in transplantation procedures has been reported to cause hepatotoxicity as evidenced by elevated serum bilirubin and bile salt levels. However, these biochemical abnormalities could also result from interference with hepatic transport processes. This possibility was investigated in the present study in which the effect of cyclosporin A on transport processes was examined in isolated rat liver cells. Taurocholate, ouabain, and α-aminoisobutyric acid were selected as compounds known to enter liver cells by distinct active transport systems and cadmium was selected as a substance taken up by a combination of simple and facilitated diffusion. Cyclosporin A was found to cause a dose-related inhibition of both taurocholate and ouabain uptake. On the other hand, the uptake of α-aminoisobutyric acid and of cadmium were unaffected by cyclosporin A. These findings indicate a substrate-specific effect of cyclosporin A rather than a general effect on cellular transport. Efflux of taurocholate from preloaded hepatocytes was also inhibited by cyclosporin A. Cyclosporin A caused a decrease in maximum velocity for ouabain uptake with no change in Km. Kinetic analysis for both uptake and efflux of taurocholate showed an unchanged maximum velocity and an increased Km. The data indicate that the ability of liver cells to take up and release bile acids is impaired in the presence of cyclosporin A. These findings provide a possible explanation for the finding of increased serum bile acids during cyclosporin A therapy and suggest that hepatic clearance of other compounds could also be impaired.

Stimulation of taurocholate and glycocholate efflux from the rat hepatocyte by arginine vasopressin.

Vasopressin induces alterations in the transmembrane distribution of the bile salts taurocholate and glycocholate but not of cholate, chenodeoxycholate, or chenodeoxycholate derivatives in isolated rat hepatocytes in suspension. Studies were conducted to define the specific transport events modulated by vasopressin. Unidirectional uptake of cholate, taurocholate, and glycocholate, monitored within a 15-s time frame, is not altered by vasopressin. Km values for cholate, taurocholate, and glycocholate influx were found to be 57, 12, and 26 microM, respectively. Vmax values for influx of cholate, taurocholate, and glycocholate were 1.7, 1.8, and 2.4 nmol.mg protein-1.min-1, respectively. At half-maximal effective concentrations, arginine vasopressin increases unidirectional efflux of taurocholate by 34% (EC50 = 1.5 X 10(-9) M) and glycocholate by 17% (EC50 = 5 X 10(-9) M). In the presence of 0.05 microM arginine vasopressin, the apparent Km value for taurocholate efflux decreases from 1.57 mM (control) to 0.94 mM; for glycocholate, the apparent Km decreases from 5.19 mM (control) to 3.22 mM. Vasopressin does not significantly change the Vmax values for taurocholate and glycocholate efflux (0.40 and 1.05 nmol.mg protein-1.min-1, respectively). These studies suggest that modulation of bile salt efflux by vasopressin may be utilized to probe bile salt transport pathways in the rat hepatocyte.

Effects of chlorpromazine on Na+-K+-ATPase pumping and solute transport in rat hepatocytes

Inhibition of Na+-K+-ATPase and sodium-dependent bile acid transport has been suggested as a mechanism for the cholestasis produced by certain drugs such as chlorpromazine. We examined the effects of chlorpromazine (and in selected studies, two of its metabolites) on Na+-K+-ATPase cation pumping (ouabain-suppressible 86Rb uptake), exchangeable intracellular sodium content, membrane potential (assessed by 36Cl- distribution), and sodium-dependent transport of taurocholate and alanine in primary cultures of rat hepatocytes. Chlorpromazine (10-300 microM), 7,8-dihydroxychlorpromazine (10-300 microM), and ouabain (0.1-2 mM), but not chlorpromazine sulfoxide, produced a concentration-dependent decrease in Na+-K+-ATPase cation pumping and an increase in intracellular sodium content. Chlorpromazine (100 microM) and ouabain (0.75 mM) also modestly decreased hepatocyte membrane potential. In further studies, chlorpromazine (75 and 100 microM) and ouabain (0.1, 0.5, and 0.75 mM) decreased initial sodium-dependent uptake rates of taurocholate and alanine by 18-63%. Although the steady-state intracellular content of alanine was decreased 25-53% by both agents, chlorpromazine increased the steady-state content of taurocholate by 171% and decreased taurocholate efflux, apparently related to partitioning of taurocholate into a large, slowly turning over intracellular pool. These studies provide direct evidence that chlorpromazine inhibits Na+-K+-ATPase cation pumping in intact cells and that partial inhibition of Na+-K+-ATPase cation pumping is associated with a reduction of both the electrochemical sodium gradient and sodium-dependent solute transport. These effects of chlorpromazine may contribute to chlorpromazine-induced cholestasis in animals and humans.

Isolation and characterization of the putative canalicular bile salt transport system of rat liver.

Through labeling with the sodium salt of the photolabile bile salt derivative (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-[3 beta-3H]cholan-24-oyl)- 2-aminoethanesulfonic acid, a bile salt-binding polypeptide with an apparent molecular weight of 100,000 was identified in isolated canalicular but not basolateral (sinusoidal) rat liver plasma membranes. This labeled polypeptide was isolated from octyl glucoside-solubilized canalicular membranes by DEAE-cellulose and subsequent wheat germ lectin Sepharose chromatography. The purified protein still contained covalently incorporated radioactive bile salt derivative and exhibited a single band with an apparent molecular weight of 100,000 on sodium dodecyl sulfate-gels. Antibodies were raised in rabbits and their monospecificity toward this canalicular polypeptide demonstrated by immunoblot analysis. No cross-reactivity was found with basolateral membrane proteins. The antibodies inhibited taurocholate uptake into isolated canalicular but not basolateral membrane vesicles. In addition, the antibodies also decreased efflux of taurocholate from canalicular vesicles. If the canalicular bile salt-binding polypeptide was immunoprecipitated from Triton X-100-solubilized canalicular membranes and subsequently deglycosylated with trifluoromethanesulfonic acid, the apparent molecular weight was decreased from 100,000 to 48,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). These studies confirm previous results in intact liver tissue and strongly indicate that a canalicular specific glycoprotein with an apparent molecular weight of 100,000 is directly involved in canalicular excretion of bile salts.

Inhibition of taurocholate efflux from rat hepatic canalicular membrane vesicles by glutathione disulfide

In right-side out rat hepatic canalicular membrane vesicles glutathione disulfide (GSSG) inhibited the efflux of taurocholate approx. 70% in the presence or approx. 55% in the absence of a valinomycin-mediated K + diffusion potential; maximal inhibition occurred at 5 mM GSSG. The inhibition by GSSG was abolished by dithioerythritol. Neither dithioerythritol alone nor GSH inhibited taurocholate efflux. S-(2,4-Dinitrophenyl) glutathione and N-ethylmaleimide showed intermediate inhibitory effects.

Properties of the canalicular bile acid transport system in rat liver.

4,4-Di-isothiocyanostilbene-2,2'-disulphonic acid inhibition of taurocholate efflux from canalicular vesicles was used to demonstrate that potential driven and 'carrier'-mediated canalicular excretion of taurocholate occur via a common, rather than two separate, pathways. This electrogenic canalicular bile acid 'carrier' preferentially transports trihydroxylated and conjugated dihydroxylated bile acids, but not the unphysiological oxo bile acids, and possibly extends its substrate specificity to other amphipathic molecules such as sulphobromophthalein.

Effects of ethinyl estradiol on substrate uptake and efflux by isolated rat hepatocytes

Ethinyl estradiol is a well documented, predictable cholestatic agent. The direct effects of this drug on hepatocellular uptake and efflux have been investigated. Uptake of taurocholate and ouabain, which are both actively transported, was inhibited, while uptake of cadmium, which occurs by a combination of facilitated and simple diffusion, was unaffected. The V max for taurocholate uptake was not altered by the presence of ethinyl estradiol, whereas the K m was increased, suggesting a reduced affinity of receptor for taurocholate. Efflux of taurocholate from pre-loaded cells remained unchanged in the presence of ethinyl estradiol. The data are not entirely consistent with an interference of Na +, K +-ATP ase by ethinyl estradiol being an important site of action. The observations may help to explain the mechanism of biliary dysfunction induced by ethinyl estradiol.

Mechanisms of taurocholate transport in canalicular and basolateral rat liver plasma membrane vesicles. Evidence for an electrogenic canalicular organic anion carrier.

The driving forces for taurocholate transport were determined in highly purified canalicular (cLPM) and basolateral rat liver plasma membrane (LPM) vesicles. Alanine transport was also examined for comparison. Inwardly directed Na+ but not K+ gradients transiently stimulated [3H]taurocholate (1 microM) and [3H]alanine (0.2 mM) uptake into basolateral LPM 3-4- fold above their respective equilibrium values (overshoots). Na+ also stimulated [3H]taurocholate countertransport and tracer exchange in basolateral LPM whereas valinomycin-induced inside negative K+ diffusion potentials stimulated alanine uptake but had no effect on taurocholate uptake. In contrast, in the "right-side out" oriented cLPM vesicles, [3H]taurocholate countertransport and tracer exchange were not dependent on Na+. Efflux of [3H]taurocholate from cLPM was also independent of Na+ and could be trans-stimulated by extra-vesicular taurocholate. Furthermore, an inside negative valinomycin-mediated K+ diffusion potential inhibited taurocholate uptake into and stimulated taurocholate efflux from the cLPM vesicles. These studies provide direct evidence for a "carrier mediated" and potential-sensitive conductive pathway for the canalicular excretion of taurocholate. In addition, they confirm the presence of a possibly electroneutral Na+-taurocholate cotransport system in basolateral membranes of the hepatocyte.

Excretion of taurocholate from isolated hepatocytes.

Efflux of taurocholate from isolated rat hepatocytes was studied to characterize the mechanism of bile acid secretion. Cells were incubated with taurocholate for 15 min. The amount of the intracellularly accumulated bile acid was directly related to the concentration in the medium. Transfer of the loaded cells from the incubation medium to a medium without taurocholate led to taurocholate efflux. Efflux was saturable, its activation energy amounted to 12 kcal/mol (50 kJ). It was strongly inhibited by the metabolic inhibitor antimycin A and to a lesser extend by the uncoupler carbonylcyanide-m-chlorophenylhydrazone. Dinitrofluorobenzene and mersalyl, reagents which react with amino acids, inhibited efflux by about 30% when applied at concentrations of 50 muM. Ouabain increased the rate of efflux. The observations indicate that secretory functions are maintained in isolated liver cells.

Investigations on the sodium dependence of bile acid fluxes in the isolated perfused rat liver

At [Na +] 0 = 118 mM the concentrative transfer of cholic and taurocholic acid from the perfusate into the isolated rat liver displays saturation kinetics (taurocholate: V = 299 nmol · min −1 · g −1, K m = 61 μM: V = 327 nmol · min −1 · g−1, K m = 436 μM). Perfusion with an isotonic sodium-free medium did not change the feature of a carrier-mediated transport but did markedly reduce V without affecting K m (taurocholate: V = 65 nmol · min −1 · g −1, K m = 78 μM; cholate: V = 104 nmol · min −1 · g −1, K m = 354 μM). It was experimentally assured that the observed reduction of bile salt uptake was not a consequence of regurgitation of bile salts or due to an excessive intracellular accumulation during cholestasis in the sodium-free state. The rate of taurocholate efflux is very low when compared with the rapid rate of the uptake. A stimulatory action of extracellular sodium on this pathway was also observed. Inhibition of the (Na + + K +-ATPase) by 1 mM ouabain resulted in a decrease of bile salt uptake. Activation of the enzyme by potassium readmission to a K +-deprived liver enhanced bile salt uptake. The immediate response to alteration of the enzyme activity suggests a close association of a fraction of bile acid active transport with the sodium pump.