Vectorial transport of bile salts across MDCK cells expressing both rat Na+-taurocholate cotransporting polypeptide and rat bile salt export pump

Bile salts are predominantly taken up by hepatocytes via the basolateral Na(+)-taurocholate cotransporting polypeptide (NTCP/SLC10A1) and secreted into the bile by the bile salt export pump (BSEP/ABCB11). In the present study, we transfected rat Ntcp and rat Bsep into polarized Madin-Darby canine kidney cells and characterized the transport properties of these cells for eight bile salts. Immunohistochemical staining demonstrated that Ntcp was expressed at the basolateral domains, whereas Bsep was expressed at the apical domains. Basal-to-apical transport of taurocholate across the monolayer expressing only Ntcp and that coexpressing Ntcp/Bsep was observed, whereas the flux across the monolayer of control and Bsep-expressing cells was symmetrical. Basal-to-apical transport of taurocholate across Ntcp/Bsep-coexpressing monolayers was significantly higher than that across monolayers expressing only Ntcp. Kinetic analysis of this vectorial transport of taurocholate gave an apparent K(m) value of 13.9 +/- 4.7 microM for cells expressing Ntcp alone, which is comparable with 22.2 +/- 4.5 microM for cells expressing both Ntcp and Bsep and V(max) values of 15.8 +/- 4.2 and 60.8 +/- 9.0 pmol.min(-1).mg protein(-1) for Ntcp alone and Ntcp and Bsep-coexpressing cells, respectively. Transcellular transport of cholate, glycocholate, taurochenodeoxycholate, chenodeoxycholate, glycochenodeoxycholate, tauroursodeoxycholate, ursodeoxycholate, and glycoursodeoxycholate, but not that of lithocholate was also observed across the double transfectant. This double-expressing system can be used as a model to clarify vectorial transport of bile salts across hepatocytes under physiological conditions.     

The order of hepatic cytotoxicity of bile salts in vitro does not agree with that examined in vivo in rats

Using an enzyme release from isolated rat hepatocytes incubated with a bile salt as a marker, the cytotoxic order of bile salts was found to be taurochenodeoxycholate, glycochenodeoxycholate greater than tauroursodeoxycholate, glycoursodeoxycholate, cholate greater than taurocholate, glycocholate. Thus, the cytotoxicity of conjugates of ursodeoxycholate was greater than that of conjugates of cholate. However, these data do not agree with the order of cytotoxicity of these bile salts previously studied in vivo by the authors which demonstrated the least cytotoxic nature of conjugates of ursodeoxycholate.     

Transport by vesicles of glycine- and taurine-conjugated bile salts and taurolithocholate 3-sulfate: a comparison of human BSEP with rat Bsep

The bile salt export pump (BSEP) of hepatocyte secretes conjugated bile salts across the canalicular membrane in an ATP-dependent manner. The biliary bile salts of human differ from those of rat in containing a greater proportion of glycine conjugates and taurolithocholate 3-sulfate (TLC-S). In the present study, the transport properties of hBSEP and rBsep were investigated using membrane vesicles from HEK293 cells infected with recombinant adenoviruses containing hBSEP or rBsep cDNA. ATP-dependent uptake of radiolabeled glycine-, taurine-conjugated bile salts, and [(3)H]cholate was observed when hBSEP or rBsep was expressed. Comparison of initial uptake rates indicated that for both transporters, taurine-conjugated bile salts were transported more rapidly than glycine-conjugated bile salts, however, hBSEP transported glycine conjugates to an extent that was approximately 2-fold greater than rBsep. In addition, [(3)H]TLC-S was significantly transported by hBSEP, and hardly transported by rBsep. The mean K(m) value for the uptake of [(3)H]TLC-S by hBSEP was 9.5+/-1.5 microM, a value similar to that for hMRP2 (8.2+/-1.3 microM). In conclusion, both hBSEP and rBsep transport taurine-conjugated bile salts better than glycine-conjugated bile salts, but hBSEP transports glycine conjugates to a greater extent as compared to rBsep. TLC-S, which is present in human bile but not rodent bile, is more avidly transported by hBSEP compared with rBsep.     

Bile-salt inhibition of sodium ion-coupled D-glucose and L-alanine accumulation by brush-border-membrane vesicles from hamster jejunum.

The effects of bile salts on Na+-coupled accumulation of D-glucose and L-alanine by brush-border-membrane vesicles isolated from hamster jejunum were investigated. The approximate percentage inhibition of Na+-coupled D-glucose accumulation produced by various bile salts at a concentration of 1 mM were: deoxycholate and chenodeoxycholate, 60%; glycine and taurine conjugates of deoxycholate and chenodeoxycholate, 40--50%; lithocholate, 45%; cholate and its glycine and taurine conjugates, less than 10%. Inhibition of Na+-coupled accumulation of D-glucose was rapid, reversible and not due to dissolution of the vesicles. Na+-coupled accumulation of L-alanine was also inhibited by deoxycholate. Deoxycholate but not cholate enhanced (1) the rate of Na+ influx, (2) the rate of influx of D-glucose and L-alanine in the absence of a Na+ gradient and (3) the rate of efflux of D-glucose and L-alanine from vesicles preloaded with this sugar or amino acid. Deoxycholate-stimulated efflux of D-glucose was not blocked by phlorizin, which completely prevented efflux in the absence of this bile salt. These results suggest that selected bile salts inhibit Na+-coupled accumulation of D-glucose and L-alanine by enhancing the rate of dissipation of the Na+ gradient required for substrate accumulation. In addition, bile salts may also decrease D-glucose and L-alanine accumulation by increasing the rate of efflux of these substrates across the brush-border plasma membrane.     

Fluorescent choleretic and cholestatic bile salts take different paths across the hepatocyte: transcytosis of glycolithocholate leads to an extensive redistribution of annexin II

We have used fluorescent derivatives of the choleretic bile salts cholate and chenodeoxycholate, the cholestatic salt lithocholate, and the therapeutic agent ursodeoxycholate to visualize distinct routes of transport across the hepatocyte and delivery to the canalicular vacuole of isolated hepatocyte couplets. The cholate and chenodeoxycholate derivatives produced homogeneous intracellular fluorescence and were rapidly transported to the vacuole, while the lithocholate analogue accumulated more slowly in the canalicular vacuole and gave rise to punctate fluorescence within the cell. Fluorescent ursodeoxycholate showed punctate intracellular fluorescence against a high uniform background indicating use of both pathways. Inhibition of vesicular transport by treatment with colchicine and Brefeldin A had no effect on the uptake of any of the compounds used, but it dramatically impaired delivery of both the lithocholate and the ursodeoxycholate derivatives to the canalicular vacuole. We conclude that while the chenodeoxycholate and cholate analogues traverse the hepatocyte by a cytoplasmic route, lithocholate and ursodeoxycholate analogues are transported by vesicle-mediated transcytosis. Treatment of couplets with glycine derivatives of lithocholate and ursodeoxycholate, but not cholate or chenodeoxycholate, led to a marked relocalization of annexin II, which initially became concentrated at the basolateral membrane, then moved to a perinuclear distribution and finally to the apical membrane as the incubation progressed. This suggests that lithocholate and ursodeoxycholate treatment leads to a rapid induction of transcytosis and that annexin II exchange occurs upon membrane fusion at all stages of the hepatocyte transcytotic pathway. These results indicate that isolated hepatocyte couplets may provide an inducible model system for the study of vesicle-mediated transcytosis.     

Choleretic effects of differently structured bile acids in the guinea pig

The effects of 10 differently structured bile acids on bile flow and composition were studied in anesthetized, bile duct-cannulated guinea pigs. At the infusion rates of 2 and 4 mumole/min/kg, all bile acids produced choleresis. The most potent was chenodeoxycholate, which increased bile flow by an average of 31.25 microliters/mumole of bile acids excreted in bile. The weakest choleretic was tauroursodeoxycholate (11.02 mu/mumole). When the choleretic activity was plotted against bile acid hydrophobicity (high-performance liquid chromatography retention factor, obtained from the literature), linearity was observed with similarly conjugated bile acids. The order of potency was deoxycholate greater than chenodeoxycholate greater than cholate greater than ursodeoxycholate, both for the glycine and taurine conjugates, and for the unconjugated bile acids as well. Conjugation was also important, and the rank ordering for the choleretic activity (unconjugated bile acids greater than glycine-conjugates greater than taurine-conjugates) was the same as that for the hydrophobicity. When the choleretic activity was plotted against bile acid micellar aggregation number (in 0.15 M NaCl at 36 degrees C, obtained from the literature), a linear, direct relationship was observed. All bile acids produced similar effects on bile electrolyte concentrations: both bicarbonate and chloride slightly declined during choleresis, whereas bile acid concentrations increased. These studies suggest that, in the guinea pig the differing choleretic activities of differently structured bile acids are not due to their forming micelles in bile of different sizes; either the more hydrophobic bile acids form vesicles, whereas the more hydrophilic form micelles; or bile acids produce choleresis, in part or exclusively, by stimulating an additional secretory mechanism, possibly an inorganic ion pump; or both.     

Physical and biological properties of fluorescent dansylated bile salt derivatives: the role of steroid ring hydroxylation

The hydroxyl groups of bile salts play a major role in determining their physical properties and physiologic behavior. To date, no fluorescent bile salt derivatives have been prepared which permit evaluation of the functional role of the steroid ring. We have prepared five fluorescent cholanoyl derivatives using a dansyl-ethylene diamine precursor linked to the sulfonyl group of taurine; N-(5-dimethylamino-1-naphthalenesulfonyl)-N'-(2-aminoethanesulf onyl)- ethylenediamine. The fluorescent dansyl-taurine was conjugated to the carboxyl group of free bile acids, enabling the labeling of the series: dehydrocholate, ursodeoxycholate, cholate, chenodeoxycholate and deoxycholate. Despite a systematic hydrophobic shift compared with the native bile salts (aqueous solubility and water:octanol partitioning), the influence of steroid ring hydroxylation was retained, with the dehydrocholate and cholate derivatives more water soluble than the dihydroxy derivatives. Similarly, the sequence of HPLC mobilities, reflecting relative hydrophilicity, was identical in the dansyl-taurine derivatives and the native taurine-conjugated bile salts. Cellular uptake of all five steroid derivatives was rapid, and partial inhibition of [3H]taurocholate uptake was observed in isolated hepatocytes. Rates of biliary excretion of the dansylated derivatives by the isolated perfused rat liver correlated closely with hydrophilicity. Collectively, these findings indicate that the influence of the hydroxyl groups is retained in this series of dansylated steroids, and that hydroxylation is a key determinant of their hepatocellular transport and biliary excretion. These fluorescent bile salt derivatives may thus serve as unique probes for investigating structure-function relationships in hepatic processing of steroid-based compounds.     

Taurocholate is more potent than cholate in suppression of bile salt synthesis in the rat

Synthesis of bile salts is regulated through negative feedback inhibition by bile salts returning to the liver. Individual bile salts have not been distinguished with regard to inhibitory potential. We assessed inhibition of bile salt synthesis by either cholate or its taurine conjugate in bile fistula rats. After allowing synthesis to maximize, baseline synthesis was determined by measuring bile salt output in four consecutive 6-hr periods. Next, sodium cholate (+[(14)C]cholate) or taurocholate (+[(14)C]taurocholate) was infused into the jugular vein for 36 hr and bile was collected in 6-hr aliquots. Hepatic flux of exogenous bile salt was determined by measuring output of radioactivity in bile divided by specific activity of the infusate. Synthesis was determined during the last four 6-hr periods of infusion by subtracting exogenous bile salt secretion from the total bile salt output. Thirteen studies using cholate and 13 using taurocholate were performed. Hepatic flux of infused bile salt varied from 1 to 12 micro mol/100 g per rat per hr. Percent suppression of synthesis varied directly with hepatic flux of exogenous bile salt for both cholate and taurocholate in a linear fashion (r = 0.66, P < 0.01 and r = 0.87, P < 0.0005, respectively). Slope of the taurocholate line was 7.82 (% suppression/ micro mol per 100 g per hr), while slope of the cholate line was 3.66 (P < 0.05), indicating that taurocholate was approximately twice as potent as cholate in suppression of synthesis. At fluxes of 10-12 micro mol/100 g per hr, taurocholate suppressed synthesis 84 +/- 8 (SEM) % while cholate suppressed synthesis only 42 +/- 12% (P < 0.02). The x-intercept of the taurocholate line was 0.65 ( micro mol/100 g per hr), while that of the cholate line was -1.01 (NS) suggesting that the threshold for initial suppression of synthesis did not differ for these two bile salts. We conclude that taurocholate is a more effective inhibitor of hepatic bile salt synthesis than cholate, and that intestinal deconjugation of bile salts may play a role in the regulation of synthesis.-Pries, J. M., A. Gustafson, D. Wiegand, and W. C. Duane. Taurocholate is more potent than cholate in suppression of bile salt synthesis in the rat.     

Solubilization of lipids from hamster bile-canalicular and contiguous membranes and from human erythrocyte membranes by conjugated bile salts.

We have demonstrated in vitro the efficacy of the taurine-conjugated dihydroxy bile salts deoxycholate and chenodeoxycholate in solubilizing both cholesterol and phospholipid from hamster liver bile-canalicular and contiguous membranes and from human erythrocyte membrane. On the other hand, the dihydroxy bile salt ursodeoxycholate and the trihydroxy bile salt cholate solubilize much less lipid. The lipid solubilization by the four bile salts correlated well with their hydrophobicity: glycochenodeoxycolate, which is more hydrophobic than the tauro derivative, also solubilized more lipid. All the dihydroxy bile salts have a threshold concentration above which lipid solubilization increases rapidly; this correlates approximately with the critical micellar concentration. The non-micelle-forming bile salt dehydrocholate solubilized no lipid at all up to 32 mM. All the dihydroxy bile acids are much more efficient at solubilizing phospholipid than cholesterol. Cholate does not show such a pronounced discrimination. Lipid solubilization by chenodeoxycholate was essentially complete within 1 min, whereas that by cholate was linear up to 5 min. Maximal lipid solubilization with chenodeoxycholate occurred at 8-12 mM; solubilization by cholate was linear up to 32 mM. Ursodeoxycholate was the only dihydroxy bile salt which was able to solubilize phospholipid (although not cholesterol) below the critical micellar concentration. This similarity between cholate and ursodeoxycholate may reflect their ability to form a more extensive liquid-crystal system. Membrane specificity was demonstrated only inasmuch as the lower the cholesterol/phospholipid ratio in the membrane, the greater the fractional solubilization of cholesterol by bile salts, i.e. the total amount of cholesterol solubilized depended only on the bile-salt concentration. On the other hand, the total amount of phospholipid solubilized decreased with increasing cholesterol/phospholipid ratio in the membrane.     

Precipitation and 13C-NMR relaxation enhancement measurements of the interactions of bile acids with synthetic cationic bile acid derivatives, and with spin labelled fatty acids.

In an investigation of novel potential bile acid sequestrants, the affinities of the sodium salts of the glycine and taurine conjugates of naturally occurring bile acids (cholate, deoxycholate, chenodeoxycholate and lithocholate) for several cationic ammonium bile acid derivatives have been investigated by measurements of the extent to which the derivatives are able to precipitate the bile acids. This is roughly proportional to the lipophilicity of the interacting species. Thus, amino and ammonium derivatives of cholic acid do not precipitate taurocholate or glycocholate to any great extent, whereas ammonium derivatives of deoxycholate and lithocholate are much more effective. To complement the precipitation measurements, high resolution 13C-NMR has been applied to investigate the weaker interactions between the ammonium cholate derivative and glycocholate, glycodeoxycholate and glycochenodeoxycholate. Addition of either of the latter two bile acids to the cationic ammonium compound results in considerable broadening of the 13C resonances of both species, indicating the formation of relatively rigid structures. In addition, we have used T2 relaxation enhancement induced by spin-labelled fatty acids to examine the mechanism of interaction with bile acids of amphiphilic anions, which might compete with bile acids for sites on bile acid sequestrants. Low concentrations of 16-DOXY L-Stearate dramatically broaden the 13C-NMR resonances of deoxycholate carbons 19, 18 and 7 in particular, while 5-DOXY L-Stearate exerts much less specific effects. These results have been incorporated into a snapshot model of bile acid-fatty acid interactions.     

Substrate specificities of rat oatp1 and ntcp: implications for hepatic organic anion uptake

Transport of a series of 3H-radiolabeled C23, C24, and C27 bile acid derivatives was compared and contrasted in HeLa cell lines stably transfected with rat Na+/taurocholate cotransporting polypeptide (ntcp) or organic anion transporting polypeptide 1 (oatp1) in which expression was under regulation of a zinc-inducible promoter. Similar uptake patterns were observed for both ntcp and oatp1, except that unconjugated hyodeoxycholate was a substrate of oatp1 but not ntcp. Conjugated bile acids were transported better than nonconjugated bile acids, and the configuration of the hydroxyl groups (alpha or beta) had little influence on uptake. Although cholic and 23 norcholic acids were transported by ntcp and oatp1, other unconjugated bile acids (chenodeoxycholic, ursodeoxycholic) were not. In contrast to ntcp, oatp1-mediated uptake of the trihydroxy bile acids taurocholate and glycocholate was four- to eightfold below that of the corresponding dihydroxy conjugates. Ntcp mediated high affinity, sodium-dependent transport of [35S]sulfobromophthalein with a Km similar to that of oatp1-mediated transport of [35S]sulfobromophthalein (Km = 3.7 vs. 3.3 muM, respectively). In addition, for both transporters, uptake of sulfobromophthalein and taurocholic acid showed mutual competitive inhibition. These results indicate that the substrate specificity of ntcp is considerably broader than previously suspected and caution the extrapolation of transport data obtained in vitro to physiological function in vivo.     

Biological properties of the 2-fluoro-beta-alanine conjugates of cholic acid and chenodeoxycholic acid in the isolated perfused rat liver

The isolated perfused rat liver was used to examine the hepatic extraction, biliary secretion and effect on bile flow of the 2-fluoro-beta-alanine conjugates of cholic acid and chenodeoxycholic acid. The naturally occurring taurine and glycine conjugates of these bile acids were used for comparisons. The 2-fluoro-beta-alanine conjugates were extracted by the liver to a similar extent as the taurine and glycine conjugates. The biliary secretion rate and increase in bile flow were similar for all the cholic acid conjugates. On the other hand, the maximal biliary secretion rate of the 2-fluoro-beta-alanine conjugate of chenodeoxycholate was similar to that of the glycochenodeoxycholate, but 47% lower than that of taurochenodeoxycholate. In addition, the 2-fluoro-beta-alanine conjugate of chenodeoxycholate produced a decrease in bile flow that was comparable to that observed with the glycochenodeoxycholate (54% vs. 74%), but which was greater than that produced by the taurochenodeoxycholate (12%). In summary, these data demonstrate that the biological properties of the 2-fluoro-beta-alanine conjugates of cholic acid and chenodeoxycholic acid are not markedly different from those of the naturally occurring taurine and glycine conjugates. These data also suggest that the amino acid moiety can influence the biliary secretion and cholestatic properties of chenodeoxycholic acid conjugates.     

Modulation of gamma-glutamyl transpeptidase activity by bile acids

The free bile acids (cholate, chenodeoxycholate, and deoxycholate) stimulate the hydrolysis and transpeptidation reactions catalyzed by gamma-glutamyl transpeptidase, while their glycine and taurine conjugates inhibit both reactions. Kinetic studies using D-gamma-glutamyl-p-nitroanilide as gamma-glutamyl donor indicate that the free bile acids decrease the Km for hydrolysis and increase the Vmax; transpeptidation is similarly activated. The conjugated bile acids increase the Km and Vmax of hydrolysis and decrease both of these for transpeptidation. This mixed type of modulation has also been shown to occur with hippurate and maleate (Thompson, G.A., and Meister, A. (1980) J. Biol. Chem. 255, 2109-2113). Glycine conjugates are substantially stronger inhibitors than the taurine conjugates. The results with free cholate indicate the presence of an activator binding domain on the enzyme with minimal overlap on the substrate binding sites. In contrast, the conjugated bile acids, like maleate and hippurate, may overlap on the substrate binding sites. The results suggest a potential feedback role for bile ductule gamma-glutamyl transpeptidase, in which free bile acids activate the enzyme to catabolize biliary glutathione and thus increase the pool of amino acid precursors required for conjugation (glycine directly and taurine through cysteine oxidation). Conjugated bile acids would have the reverse effect by inhibiting ductule gamma-glutamyl transpeptidase.     

12alpha-Hydroxysteroid dehydrogenase from Clostridium group P strain C48-50 ATCC No. 29733: partial purification and characterization.

The growth of Clostridium group P strain C48-50 [an anaerobe that contains 12alpha-hydroxysteroid dehydrogenase (12alpha-HSDH) in the absence of other dehydrogenases active upon bile salts] is greatly enhanced by the addition of 2.0% d-fructose or d-glucose to the growth medium. Other sugars were less effective. The production of NADP-dependent 12alpha-HSDH paralleled the growth of the organism which was optimal at 72 hr. Growth (and enzyme production) were suppressed by the addition of bile salt to the medium; the order of suppression was deoxycholate > chenodeoxycholate > cholate; 1 mM of either of the dihydroxy-bile salts inhibited 96% of the growth and 100% of the enzyme production. Kinetic studies on cell-free preparations of 12alpha-HSDH revealed a pH optimum of 7.8 with greater linearity of NADP evolution with time occurring only at more alkaline pH values (9-10). Lineweaver-Burke plots revealed Michaelis constant (K(m)) values in the range of 3-5 x 10(-4) M for deoxycholate and its glycine and taurine conjugates, while higher values were found for cholate and conjugates (K(m) value for taurocholate was 3 x 10(-3) M). Although there was no activity with NAD, 12alpha-HSDH was shown to bind onto both NAD- and NADP-Sepharose columns, with stronger binding on the latter. The enzyme was purified 20-fold by NAD-Sepharose chromatography. The molecular weight was estimated at 100,000 by Sephadex G-200 and a series of molecular weight markers. Substrate specificity studies showed that a variety of bile salts containing 12alpha-OH groups reacted; notably, the 3alpha-sulfates of cholate and deoxycholate were nonsubstrates.-Macdonald, I. A., J. F. Jellett and D. E. Mahony. 12alpha-Hydroxysteroid dehydrogenase from Clostridium Group P strain C48-50 #29733: partial purification and characterization.     

Bile salt excretion in skate liver is mediated by a functional analog of Bsep/Spgp, the bile salt export pump

Biliary secretion of bile salts in mammals is mediated in part by the liver-specific ATP-dependent canalicular membrane protein Bsep/Spgp, a member of the ATP-binding cassette superfamily. We examined whether a similar transport activity exists in the liver of the evolutionarily primitive marine fish Raja erinacea, the little skate, which synthesizes mainly sulfated bile alcohols rather than bile salts. Western blot analysis of skate liver plasma membranes using antiserum raised against rat liver Bsep/Spgp demonstrated a dominant protein band with an apparent molecular mass of 210 kDa, a size larger than that in rat liver canalicular membranes, approximately 160 kDa. Immunofluorescent localization with anti-Bsep/Spgp in isolated, polarized skate hepatocyte clusters revealed positive staining of the bile canaliculi, consistent with its selective apical localization in mammalian liver. Functional characterization of putative ATP-dependent canalicular bile salt transport activity was assessed in skate liver plasma membrane vesicles, with [(3)H]taurocholate as the substrate. [(3)H]taurocholate uptake into the vesicles was mediated by ATP-dependent and -independent mechanisms. The ATP-dependent component was saturable, with a Michaelis-Menten constant (K(m)) for taurocholate of 40+/-7 microM and a K(m) for ATP of 0.6+/-0.1 mM, and was competitively inhibited by scymnol sulfate (inhibition constant of 23 microM), the major bile salt in skate bile. ATP-dependent uptake of taurocholate into vesicles was inhibited by known substrates and inhibitors of Bsep/Spgp, including other bile salts and bile salt derivatives, but not by inhibitors of the multidrug resistance protein-1 or the canalicular multidrug resistance-associated protein, indicating a distinct transport mechanism. These findings provide functional and structural evidence for a Bsep/Spgp-like protein in the canalicular membrane of the skate liver. This transporter is expressed early in vertebrate evolution and transports both bile salts and bile alcohols.     

Quantitative estimation of the hydrophilic-hydrophobic balance of mixed bile salt solutions

This paper describes the derivation of a bile salt monomeric hydrophobicity index that quantitatively defines the composite hydrophilic-hydrophobic balance of a mixture of bile salts. The index is based on the logarithms of bile salt capacity factors determined using reversed phase high performance liquid chromatography (HPLC) (stationary phase octadecyl silane; mobile phase methanol-water 70:30 w/w, ionic strength 0.15). It has been standardized arbitrarily to set indices of taurocholate and taurolithocholate to 0 and 1, respectively. Indices of tauroursodeoxycholate, taurohyodeoxycholate, taurochenodeoxycholate, and taurodeoxycholate were found to be -0.47, -0.35, +0.46, and +0.59, respectively. Whereas capacity factors and hydrophobicity indices of taurine-conjugated bile salts were constant for pH 2.8-9.0, the hydrophilic-hydrophobic balance of glycine-conjugated and unconjugated bile salts was strongly influenced by pH. At alkaline pH (greater than 8.5), hydrophobicity indices of fully ionized unconjugated (n = 4) and glycine-conjugated (n = 6) bile salts differed by only 0.14 +/- 0.02 and 0.05 +/- 0.01, respectively, from those of the corresponding taurine conjugates. At acid pH (less than 3.5) the hydrophobicity indices of four unconjugated bile acids (protonated form) exceeded those of the corresponding salts (ionized form) by 0.76 +/- 0.04; indices of six glycine-conjugated bile acids exceeded those of the corresponding salts by only 0.26 +/- 0.03. Capacity factors of the salt forms of cholate and its conjugates increased dramatically with increasing ionic strength of the mobile phase; retention of the protonated forms (cholic and glycocholic acids) was only minimally influenced by ionic strength. Thus the difference in hydrophilic-hydrophobic balance between a bile acid and its corresponding salt decreases with increasing ionic strength. Examples are given of calculation of hydrophobicity indices for biliary bile salts (fully ionized) from four species under conditions of intact enterohepatic circulation. Mean values, from least to most hydrophobic, were: rat (-0.31) less than dog (0.11) less than hamster (0.22) less than human (0.32). This study provides a rational basis for calculating the hydrophilic-hydrophobic balance of mixed bile salt solutions over a broad range of pH.     

Localization of the Sodium-Taurocholate cotransporting polypeptide in membrane rafts and modulation of its activity by cholesterol in vitro

BACKGROUND: The relevance of discrete localization of hepatobiliary transporters in specific membrane microdomains is not well known. AIM: To determine whether the Na+/taurocholate cotransporting polypeptide (Ntcp), the main hepatic sinusoidal bile salt transporter, is localized in specific membrane microdomains. METHODS: Presence of Ntcp in membrane rafts obtained from mouse liver was studied by immunoblotting and immunofluorescence. HEK-293 cells stably transfected with rat Ntcp were used for in vitro studies. Expression, localization and function of Ntcp in these cells were assessed by immunoblotting, immunofluorescence and biotinylation studies and Na+ -dependent taurocholate uptake assays, respectively. The effect of cholesterol depletion/repletion assays on Ntcp function was also investigated. RESULTS: Ntcp localized primarily to membrane rafts in in vivo studies and localized partially in membrane rafts in transfected HEK-293 cells. In these cells, membrane cholesterol depletion resulted in a shift of Ntcp localization into non-membrane rafts, which correlated with a 2.5-fold increase in taurocholate transport. Cholesterol repletion shifted back part of Ntcp into membrane rafts, and normalized taurocholate transport to values similar to control cells. CONCLUSION: Ntcp localizes in membrane rafts and its localization and function are regulated by membrane cholesterol content. This may serve as a novel regulatory mechanism of bile salt transport in liver.     

Degradation of the sodium taurocholate cotransporting polypeptide (NTCP) by the ubiquitin-proteasome system

The sodium taurocholate cotransporting polypeptide (Ntcp, Slc10a1) is the major uptake system for bile acids into liver cells. This study investigated the degradation of rat Ntcp and human NTCP by the ubiquitin-proteasome system (UPS). In stably transfected HepG2 cells, rat Ntcp was complex-glycosylated and localized at the plasma membrane. Inhibition of proteasomes by MG-132 or lactacystin led to the accumulation of intracellular Ntcp, a process dependent on de novo protein synthesis. Intracellular Ntcp was core-glycosylated, indicating an endoplasmic reticulum (ER) origin. Core-glycosylated Ntcp was found in cytosolic, detergent-insoluble deposits with characteristics of aggresomes: they co-localized with ubiquitin at the microtubule organization center and Ntcp from these deposits was polyubiquitinated. Transient transfections of Ntcp/NTCP induced intracellular deposits that co-localized with ubiquitin, even in the absence of proteasome inhibitors. Similarly, in livers of patients with progressive familial intrahepatic cholestasis, NTCP could be detected co-localized with ubiquitin in hepatocytes. We conclude that maturing Ntcp/NTCP is degraded by the ubiquitin-proteasome system at the level of ER-associated degradation (ERAD). An imbalance in the synthesis and degradation of NTCP at the level of the ER or alterations in the ERAD machinery might be the cause of intracellular NTCP deposits in transient transfections and in cholestatic livers.     

Basolateral and canalicular transport of xenobiotics in the hepatocyte: A review

The molecular and functional characterization of severalproteins involved in the uptake and excretion of xenobioticsand endogenous compounds in the hepatocyte has been achievedthrough intensive research conducted in the past few years.These studies have lead to the identification of specificmembrane transporters located in the basolateral andcanalicular membrane domains of the hepatocyte. The organicanion-transporting polypeptide (OATP), present in thebasolateral membrane of the hepatocyte, is responsible for thetranslocation of xenobiotics from the sinusoidal space into thehepatocyte. Once inside the cell, unconjugated neutral, anionicand cationic xenobiotics can be secreted into bile by themultidrug-resistance P-glycoprotein 1 (MDR1). Conjugatedxenobiotics (e.g. glucuronides and glutathione conjugates) aresecreted into bile by the canalicular multispecific organicanion transporter (cMOAT). Other transporters play keyphysiological roles, including the basolateral uptake of bilesalts (sodium-taurocholate cotransporter, NTCP) and thesecretion into bile of conjugated and unconjugated bile salts(bile salt export pump, BSEP) and phospholipids (MDR2).Experimental approaches used to investigate the role of thebasolateral and canalicular transporters in the hepatocyte haveincluded both in vivo and in vitro models. Animalmodels lacking canalicular transporters include the`hyperbilirubinemic' rats (Groningen-Yellow (GY), Eisaihyperbilirubinemic (EHB) and TR<sup>-</sup> rats), which aredeficient in the cMOAT protein, and `knock-out' mice, lackingeither the MDR1 or MDR2 transporter. Although no animal modelsare currently available for the study of basolateraltransporters, their function has been conveniently investigatedthrough heterologous expression in Xenopus laevis oocytesand also with basolateral membrane vesicles isolated fromhepatocytes. The total number of basolateral and canaliculartransport proteins present in the hepatocyte is still unknown,but current knowledge indicates that there are at least fourpresent in the basolateral membrane and five in the canaliculardomain. The present review focuses on the current knowledgeabout the most relevant hepatocyte transporters involved in theuptake of foreign and endogenous compounds from the sinusoidalspace and in their active secretion into bile. The first partof the review deals with the basolateral (sinusoidal) transportof organic anions, and the major basolateral transporters (e.g.NTCP, OATP) are described here, both in terms of their knownbiochemistry and physiology. In the second part of the review,the canalicular (apical) transport of organic anions isdiscussed and the biochemistry and physiological role of MDR1,MDR2, cMOAT and BSEP is described in detail. The concludingremarks point out areas of research that need to be addressedin order to answer important questions that still remainunanswered in this important field of study.     

Hydroxyl/bile acid exchange. A new mechanism for the uphill transport of cholate by basolateral liver plasma membrane vesicles

In order to characterize the driving forces for the concentrative uptake of unconjugated bile acids by the hepatocyte, the effects of pH gradients on the uptake of [3H]cholate by rat basolateral liver plasma membrane vesicles were studied. In the presence of an outwardly directed hydroxyl gradient (pH 6.0 outside and pH 7.5 inside the vesicle), cholate uptake was markedly stimulated and the bile acid was transiently accumulated at a concentration 1.5- to 2-fold higher than at equilibrium ("overshoot"). In the absence of a pH gradient (pH 6.0 or 7.5 both inside and outside the vesicle), uptake was relatively slower and no overshoot was seen. Reductions in the magnitude of the transmembrane pH gradient were associated with slower initial uptake rates and smaller overshoots. Cholate uptake under pH gradient conditions was inhibited by furosemide and bumetanide but not by 4, 4'-diisothiocyano-2,2'-disulfonic stilbene (SITS), 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (DIDS), or probenecid. In the absence of a pH gradient, an inside-positive valinomycin-induced K+ diffusion potential caused a slight increase in cholate uptake which was insensitive to furosemide. Moreover, in the presence of an outwardly directed hydroxyl gradient, uphill cholate transport was observed even under voltage clamped conditions. These findings suggest that pH gradient-driven cholate uptake was not due to associated electrical potentials. Despite an identical pKa to that of cholate, an outwardly directed hydroxyl gradient did not drive uphill transport of three other unconjugated bile acids (deoxycholate, chenodeoxycholate, ursodeoxycholate), suggesting that a non-ionic diffusion mechanism cannot account for uphill cholate transport. In canalicular vesicles, although cholate uptake was relatively faster in the presence of a pH gradient than in the absence of a gradient, peak uptake was only slightly above that found at equilibrium under voltage clamped conditions. These findings suggest a specific carrier on the basolateral membrane of the hepatocyte which mediates hydroxyl/cholate exchange (or H+-cholate co-transport). A model for uphill cholate transport is discussed in which the Na+ pump would ultimately drive Na+/H+ exchange which in turn would drive hydroxyl/cholate exchange.