Cholate Uptake Research Articles

Rhodococcus jostii Porin A (RjpA) Functions in Cholate Uptake

RjpA in Rhodococcus jostii is the ortholog of a channel-forming porin, MspA. Deletion of rjpA delayed growth of R. jostii on cholate but not on cholesterol. Eventual growth on cholate involved increased expression of other porins, namely, RjpB, RjpC, and RjpD. Porins appear essential for the uptake of bile acids by mycolic acid bacteria.

Functional elements associated with hepatic regeneration in living donors after right hepatic lobectomy

We quantified the rates of hepatic regeneration and functional recovery for 6 months after right hepatic lobectomy in living donors for liver transplantation. Twelve donors were studied pre-donation (baseline); 8 were retested at a mean ± SD of 11±3 days after donation (T1), 10 were retested at a mean of 91±9 days after donation (T2), and 10 were retested at a mean of 185±17 days after donation (T3). Liver and spleen volumes were measured with computed tomography (CT) and single-photon emission computed tomography (SPECT). Hepatic metabolism was assessed with caffeine and erythromycin, and hepatic blood flow (HBF) was assessed with cholates, galactose, and the perfused hepatic mass (PHM) by SPECT. The regeneration rates (mL kg(-1) of body weight day(-1)) by CT were 0.60±0.22 mL from the baseline to T1, 0.05±0.02 mL from T1 to T2, and 0.01±0.01 from T2 to T3; by SPECT they were 0.54±0.20, 0.04±0.01, and 0.01±0.02, respectively. At T3, the liver volumes were 84%±7% of the baseline according to CT and 92%±13% of the baseline according to SPECT. Changes in the hepatic metabolism did not achieve statistical significance. At T1, the unadjusted clearance ratios with respect to the baseline were 0.75±0.07 for intravenous cholate (P<0.001), 0.88±0.15 for galactose (P=0.07), 0.84±0.08 for PHM (P=0.002), and 0.83±0.19 for the estimated HBF (P=0.06). At T1, these ratios adjusted per liter of liver were up to 50% greater than the baseline values, suggesting recruitment of HBF by the regenerating liver. Increased cholate shunt, increased spleen volume, and decreased platelet count, were consistent with an altered portal circulation. In conclusion, initial hepatic regeneration is rapid, accounts for nearly two-thirds of total regeneration, and is associated with increases in HBF and cholate uptake. Right lobe donation alters the portal circulation of living donors, but the long-term clinical consequences, if there are any, are unknown.

Obstructive cholestasis down-regulates Oatp1 by TNFα signalling without affecting Oatp2 and Oatp4 expression and Sodium-independent bile salt uptake in rat liver

Background & Aims: Hepatic uptake of bile salts is mediated by sodium-dependent and sodium-independent transport systems. During extrahepatic cholestasis both function and expression of the Na+/taurocholate cotransporting polypeptide (Ntcp) are down-regulated. To test whether hepatic sodium-independent organic anion transporting polypeptides (Oatp1/Oatp1a1, Oatp2/Oatp1a4, Oatp4/Oatp1b2) are also affected by extrahepatic cholestasis, function and expression of all three Oatps have been determined in rats subjected to common bile duct ligation (CBDL; n=4) for up to 7 days with and without anti-cytokine pretreatment with etanercept and anakinra. Results: Protein expression of Oatp1 protein mass as determined by Western blotting remained unchanged after CBDL for 1 day, but then declined by 75±7% and 90±17%, respectively, after 3 and 7 days. In contrast Oatp2 and 4 protein expression was not affected by CBDL as compared to both untreated and sham-operated controls. After CBDL, Oatp1 steady-state mRNA as determined by Northern blotting was rapidly down-regulated by 68±21% of untreated controls (P<0.05) within 24 hours, and remained at similar levels at 3 and 7 days. Cytokine-inactivation studies with etanercept pretreatment demonstrated that TNFα dependent signals mediated the down-regulation of this transporter gene at both protein and mRNA level during obstructive cholestasis. Sodium-independent uptake of taurocholate and cholate into freshly isolated hepatocyte suspensions showed neither significant differences in Km nor Vmax values. Conclusions: These results indicate that sodium-independent transport of bile salts may be mediated by Oatp2 and 4 during biliary obstruction, since its expression remains unaffected and may compensate for loss of Oatp1 expression and function in cholestatic hepatocytes. However, continous bile salt uptake through maintained Oatp2 and 4 expression under these conditions could contribute to the progressive nature of obstructive liver damage.

Extrahepatic cholestasis downregulates Oatp1 by TNF‐α signalling without affecting Oatp2 and Oatp4 expression and sodium‐independent bile salt uptake in rat liver

Hepatic uptake of bile salts is mediated by sodium-dependent and sodium-independent transport systems. During extrahepatic cholestasis, both the function and the expression of the Na(+)/taurocholate cotransporting polypeptide (Ntcp) are downregulated. To test whether sodium-independent organic anion-transporting polypeptides are also affected by extrahepatic cholestasis, the function and expression of all three Oatps have been determined in common bile duct-ligated (CBDL) rats. Oatp1/Oatp1a1 protein mass remained unchanged after CBDL for 1 day, but then declined by 75+/-7% and 90+/-17%, respectively, after 3 and 7 days. In contrast, Oatp2/Oatp1a4 and Oatp4/Oatp1b2 protein expression was not affected by CBDL as compared with controls. After CBDL, Oatp1 mRNA was rapidly downregulated by 68+/-21% of untreated controls (P<0.05) within 24 h, and remained at similar levels at 3 and 7 days. Cytokine-inactivation studies with etanercept pretreatment demonstrated that TNF-alpha-dependent signals mediated the down-regulation of this transporter gene at both protein and mRNA levels during obstructive cholestasis. Sodium-independent uptake of taurocholate and cholate into freshly isolated hepatocyte suspensions showed neither significant differences in K(m) nor V(max) values. These results indicate that sodium-independent transport of bile salts may be mediated by Oatp2 and 4 during biliary obstruction, because its expression remains unaffected and may compensate for loss of Oatp1 expression and function in cholestatic hepatocytes.

Cyclopeptides ofLinum usitatissimum

Cyclolinopeptide A (CLA), a cyclic nonapeptide from linseed, possesses strong immunosuppressive and antimalarial activity along with the ability to inhibit cholate uptake into hepatocytes. The structure of the peptide was studied extensively in solution as well as in the solid state. It is postulated that both the Pro-Pro cis-amide bond and an 'edge-to-face' interaction between the aromatic rings of two adjacent Phe residues are important for biological activity. Structure-activity relationship studies of many linear and cyclic analogues of CLA suggest that the Pro-Xxx-Phe sequence and the flexibility of the peptide are important for the immunosuppressive activity.

Structural Consequences of Metal Complexation ofcyclo[Pro-Phe-Phe-Ala-Xaa]2 Decapeptides

The conformational features of both free and Ca2+-complexed cyclo[Pro-Phe-Phe-Ala-Xaa]2 (with Xaa= Glu(OtBu), Lys(CIZ), Leu, and Ala) in solution have been determined by NMR spectroscopy and extensive distance-geometry calculations. The decapeptides are conformationally homogeneous in solution and show common structural features in their free and complexed forms. The structures of the free form contain only trans peptide bonds and are topologically similar to the structure of gramicidin-S, folded up in two antiparallel extended structures, stabilized by interstrand hydrogen bonds, and closed at both ends by two beta-turns. In contrast, the Ca2+-complexed peptides present two cis peptide bonds and are generally similar to those observed for the metal-complexed forms of antamanide and related analogues, folded into a saddle shape with two beta-turns. The Glu(OtBu)-, Leu-, and Lys(ClZ)-containing peptides examined here maintain the biological activity of the cyclolinopeptide A in their ability to competitively inhibit cholate uptake. The natural antamanide and cyclolinopeptide A are both able to inhibit the uptake of bile salts into hepatocytes. They share the same postulated active sequence Pro-Phe-Phe. Based on our structural results, we conclude that the ability to adopt a global conformation, characterized by a clear amphipathic separation of hydrophobic and hydrophilic surfaces, is an important feature for the functioning of this class of peptides.

Hepatocellular transport proteins and their role in liver disease

TRANSPORT PROTEINS Basolateral transport systems Na-dependent bile salt uptake Uptake of bile salts into the liver was first characterized in experimental models such as the isolated perfused rat liver, isolated hepatocyte cultures and basolateral plasma membrane vesicles. These studies indicated that more than 80% of taurocholate uptake but less than 50% of cholate uptake into hepatocytes is sodiumdependent. Whereas unconjugated bile salts are uncharged molecules that can traverse membranes by passive nonionic diffusion, conjugation with glycine or taurine decreases their pKa values and necessitates the presence of a specific carrier protein for hepatocellular uptake. The chief uptake system for conjugated bile salts in mammalian liver was isolated by expression and molecular cloning strategies and has been called the Na + taurocholate cotransporting polypeptide (gene symbol: SLC10A1). Rat Ntcp consists of 362 amino acids with an apparent molecular mass of 51 kD and is expressed exclusively at the basolateral membrane of hepatocytes (Figure 1). Ntcp mediates sodium-dependent uptake of taurocholate and other bile salts when expressed in stably transfected COS-7, Chinese hamster ovary (CHO) and hepatoblastoma (HepG2) cells or in cRNA injected Xenopus laevis oocytes, with apparent Km values between 17-42 μmol/L. The only non-bile salt substrates that are transported by Ntcp are selected sulfated steroid conjugates such as estrone-3sulfate and dehydroepiandrosterone sulfate (DHEAS) . In human liver, NTCP represents a 349-amino acid protein. NTCP is structurally related to the intestinal bile salt transporter (IBAT), that also mediates the Nadependent uptake of bile saltsand that is expressed not only in ileum, but also in the kidney and in cholangiocytes. Na-dependent taurocholate uptake is reduced in experimental models of cholestasis such as bile duct l igat ion , endotoxinemia and par t ia l hepatectomy, is reduced in primary hepatocyte cultures and is absent in various hepatoma cell lines . These changes in hepatic Na dependent bile salt uptake correlate with expression levels of Ntcp. Thus, Ntcp mRNA and protein levels are decreased in bile duct ligation, endotoxinemia and ethinyl estradiol induced cholestasis. In patients with a diagnosis of extrahepatic biliary atresia and clinical evidence of cholestasis, NTCP mRNA levels are also decreased.

Solution and solid state structure of an aib-containing cyclodecapeptide inhibiting the cholate uptake in hepatocytes.

The conformational analysis of synthetic cyclodecapeptide c(Pro-Phe-phe-Aib-Leu)2 related to the cyclolinopeptide A, in the solid state and solution, has been carried out by x-ray diffraction and nmr spectroscopy. The structure of the monoclinic form obtained from methanol [a = 11.351 (5) A, b = 27.455 (2) A, c = 12.716 (8) A, beta = 99.65 (3) degrees; space group P2(1); Z = 2] shows the presence of six intramolecular NH...CO hydrogen bonds, with formation of four turns (three of type I and one of type III) and two C16 ring structures. All peptide units are trans. The solution structure, as found by nmr, indicates that, at room temperature, the peptide is conformationally homogeneous; the structure determined is perfectly symmetrical and topologically similar to that found in the solid state. The cyclodecapeptide exhibits similar biological activity to cyclolinopeptide A.

Hepatobiliary transport of hepatic 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors conjugated with bile acids

To obtain prodrugs with affinity to liver parenchymal cells, the hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors HR 780 and lovastatin (syn. mevinolin) were conjugated with the bile acids cholic acid, taurocholic acid, and glycocholic acid. Hepatic uptake and biliary excretion of the coupled drugs were investigated and compared with the noncoupled drugs. Studies were performed with livers of normal Wistar rats, and TR-/GT- Wistar rats with deficient drug excretion. The experiments showed that the parent drug HR 780 was slowly excreted into bile. In contrast, the excretion of the bile acid-conjugated HR 780 derivatives S 3554 (conjugated with cholate), S 3898 (conjugated with glycocholate), and S 4193 (conjugated with taurocholate) was rapid and very efficient in both groups of rat strains. The bile acid-conjugated HMG-CoA reductase inhibitors showed a 10 to 20 times higher affinity for the uptake systems of bile acids than the noncoupled parent drug compounds, and even higher affinities than the bile acids themselves. The cholate conjugate of HR 780 (compound S 3554) was shown to be a noncompetitive inhibitor of taurocholate uptake and a competitive inhibitor of sodium-independent cholate uptake (Ki = 1 mumol/L). Uptake of radiolabeled S 3554 into isolated rat hepatocytes was observed to be rapid, cell specific, saturable, energy dependent, and carrier mediated. However, the carrier for S 3554 uptake was found not to be the cloned Na(+)-dependent taurocholate cotransporting polypeptide Ntcp. Expression of this carrier cRNA in Xenopus laevis oocytes did not stimulate S 3554 uptake.

Hepatobiliary transport of hepatic 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors conjugated with bile acids

To obtain prodrugs with affinity to liver parenchymal cells, the hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors HR 780 and lovastatin (syn. mevinolin) were conjugated with the bile acids cholic acid, taurocholic acid, and glycocholic acid. Hepatic uptake and biliary excretion of the coupled drugs were investigated and compared with the noncoupled drugs. Studies were performed with livers of normal Wistar rats, and TR −/GT − Wistar rats with deficient drug excretion. The experiments showed that the parent drug HR 780 was slowly excreted into bile. In contrast, the excretion of the bile acid-conjugated HR 780 derivatives S 3554 (conjugated with cholate), S 3898 (conjugated with glycocholate), and S 4193 (conjugated with taurocholate) was rapid and very efficient in both groups of rat strains. The bile acid-conjugated HMG-CoA reductase inhibitors showed a 10 to 20 times higher affinity for the uptake systems of bile acids than the noncoupled parent drug compounds, and even higher affinities than the bile acids themselves. The cholate conjugate of HR 780 (compound S 3554) was shown to be a noncompetitive inhibitor of taurocholate uptake and a competitive inhibitor of sodium-independent cholate uptake (K 1 = 1 μmol/L). Uptake of radiolabeled S 3554 into isolated rat hepatocytes was observed to be rapid, cell specific, saturable, energy dependent, and carrier mediated. However, the carrier for S 3554 uptake was found not to be the cloned Na +-dependent taurocholate cotransporting polypeptide Ntcp. Expression of this carrier cRNA in Xenopus laevis oocytes did not stimulate S 3554 uptake.

Transport of dehydroepiandrosterone and dehydroepiandrosterone sulphate into rat hepatocytes

The purpose of the present study was to characterize the transport of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEAS) into hepatocytes at physiological and pharmacological concentrations. Hepatocytes were isolated from female Sprague-Dawley rats by collagenase perfusion. Uptake of [ 3H]DHEA and [ 3H]DHEAS at increasing concentrations (3.5 nM-100 μM) was measured by the rapid filtration technique at 30 s intervals up to 120 s. The uptake of DHEAS by hepatocytes was saturable ( K m = 17.0 μM; V max = 3.7 nmol/min/mg cell protein). In contrast, a specific saturable transport system for DHEA could not be detected in rat hepatocytes. It is suggested that DHEA enters the cell by diffusion. The uptake of DHEAS could be inhibited by antimycin A, carbonylcyanide- m-chlorophenylhydrazone, and dinitrophenol (inhibitors of the mitochondrial respiratory chain), by dinitrofluorobenzene and p-hydroxymercuribenzoate (NH 2- and SH-blockers, respectively), and by monensin (Na +-specific ionophore). No inhibition was seen in the presence of ouabain (inhibitor of Na +-K +-ATPase) and phalloidin (inhibitor of cholate transport and actin-blocker). Interestingly, DHEAS uptake was inhibited by bile acids (cholate, taurocholate and glycocholate). Conversely, [ 3H]cholate uptake was strongly inhibited by DHEAS, which indicates a competition for the same carrier. Replacement of sodium ion with choline markedly decreased uptake velocity at pharmacological DHEAS concentrations. The results suggest that DHEAS uptake is a saturable, energy-dependent, carrier-mediated, partially Na +-dependent process, and that DHEAS may be taken up via the multispecific bile acid transport system.

Identification and characterization of a basolateral dicarboxylate/cholate antiport system in rat hepatocytes.

The mechanisms and driving forces for the uptake of the unconjugated bile acid cholate were investigated both in cultured rat hepatocytes and in rat liver basolateral (sinusoidal) plasma membrane (BLPM) vesicles. Determination of initial uptake rates of [3H]cholate (0.1 microM) into cultured hepatocytes confirmed that the majority (75%) of the transmembrane transport was mediated by Na(+)-independent mechanisms. This portion of cholate uptake consisted of a pH-sensitive moiety representing nonionic diffusion, which may become quantitatively important at low pH and high cholate concentrations, as well as of a saturable (Michaelis constant 7.4 microM), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive transport moiety, suggesting the involvement of a carrier. This latter transport system was functionally characterized by 1) inhibition of cellular cholate uptake in the absence of extracellular sodium by the dicarboxylic acid alpha-ketoglutarate (alpha-KG; 1 mM) and by the organic anion p-aminohippurate (PAH; 1 mM); 2) stimulation of cellular cholate uptake by alpha-KG (10 microM) or PAH (1 mM) in the presence of an inwardly directed sodium gradient; 3) lack of sensitivity toward lithium in BLPM vesicles; 4) trans-stimulation of vesicular cholate uptake by alpha-KG or PAH, but not by benzoate; and 5) cis-inhibition of alpha-KG/alpha-KG self-exchange by extravesicular cholate (400 microM), PAH (5 mM), probenecid, or DIDS. Collectively, these data indicate the presence of a Na(+)-dicarboxylate cotransport-coupled organic anion exchanger in the hepatocyte basolateral plasma membrane that may be involved in cholate uptake in the liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholate uptake and conjugation by a porcine hepatocyte-based bioartificial liver (BAL): [formula omitted]∗, A.D. Moscioni, J. Rozga, E. Morsiani, A.A. Demetriou. Dept. of Surgery and Liver Support Unit, Cedars-Sinai Medical Center, Los Angeles, CA, USA

Cholate uptake and conjugation by a porcine hepatocyte-based bioartificial liver (BAL): [formula omitted]∗, A.D. Moscioni, J. Rozga, E. Morsiani, A.A. Demetriou. Dept. of Surgery and Liver Support Unit, Cedars-Sinai Medical Center, Los Angeles, CA, USA

Change of zonal bile acid processing after partial hepatectomy in the rat

The aim of this study was to analyze whether partial hepatectomy alters functional liver heterogeneity with respect to bile acid processing. One, 5 and 21 days after liver resection (≈8)% of liver mass) in male Sprague-Dawlye rats (300–400 g), isolated livers were perfused in either the antegrade or the retrograde direction, respectively, with 32 nmol cholate/min per g liver. Uptake, metabolism and biliary secretion kinetics were determined by bolus injection of 14C-cholate. Uptake and biliary recovery (within 30 mn) of cholate were >90% in all groups. One day postresection, liver mass had already doubled and it regenerated to over 80% 5 days after resection. Serum bile acid concentration increased rapidly, peaking 61 h after resection (176.7±28.5 μmol/l) (mean±SEM). Twenty-one days after resection it fell to control values (23.±3.8 μmol/l). T 25(T 50), the time (min) necessary to excrete 25% (50) of the bile acid load into bile, was strikingly different between periportal and pericentral cells of controls (1.8 vs 5.7 and 3.4 vs 8.1). Five days after resection this difference became smaller (1.4 vs 2.9 and 2.8 vs 5.5) due to accelerated biliary cholate secretion in pericentral cells. Pericentral cells of controls metabolized cholate more extensively to taurocholate (≈83%) and glycocholate (≈13%) than periportal cells of controls (65%, 10%), leading to a 5-fold higher proportion of unmetabolized cholate in periportal than pericentral cells (25% vs 5%). Five days after resection the percentage of taurocholate decreased significantly at the expense of an increased formation of glycocholate. Twenty-one days after resection, bile acid composition came to resemble that of controls. In conclusion, the results demosntrate a reduction of metabolic zonation with regard to bile acid processing after liver resection. Despite accelerated biliary bile acid secretion in pericentral cells of the regenerating liver, overall metabolism was not impaired as compared to controls. This is comparable to weaning rats where functional liver heterogeneity has not yet developed.

Cyclic somatostatin analogs bind specifically to pI 6.1 carboxylesterase of rat liver cells

The hydrophobic cyclohexapeptide cyclo(Phe-Thr-Lys-Trp-Phe- dPro) (008), an analog of somatostatin with retro sequence, was previously shown to competitively inhibit the uptake of cholate and taurocholate into isolated rat liver cells. Conversely, the competitive uptake inhibition of 008 into isolated rat hepatocytes by bile acids confirmed the observation of common binding and transport sites by bile acids and cyclosomatostatin. Furthermore the transport characteristics of 008 uptake revealed a significant and rapid binding to cell membranes. In this context it was of special interest to investigate the specificity of the binding component since specific binding of the substrate to membrane proteins could be responsible for the low K m of 008-transport. Therefore, the cyclohexapeptide 008 could be used as the ligand in affinity chromatography in order to isolate such binding proteins. The gel matrix used did not interact non-specifically with octylglucoside-solubilized proteins from isolated rat liver plasma membranes. In affinity chromatography of octylglucoside-solubilized plasma membranes, two dominant proteins with apparent molecular masses of 60 and 58 kDa bound specifically to the 008 ligand. When used as ligands in affinity chromatography, these membrane-associated 60 and 58 kDa proteins bound exclusively to aromatic cyclopeptides, e.g. cyclosomatostatin 008, but not to linear peptides or taurocholate derivatives. The amino acid sequences of tryptic digests of the 008-affinity-purified 58 kDa protein were identical to the sequence of a microsomal pI 6.1 carboxylesterase. Immunofluorescence of intact hepatocytes showed that this xenobiotic metabolizing enzyme is also located in sinusoidal rat liver plasma membranes and could therefore account for the extensive and specific binding of the cyclosomatostatin to sinusoidal plasma membranes of rat liver.

Uptake of taurocholic acid and cholic acid in isolated hepatocytes from rainbow trout

The uptake of the bile acids cholate (CHA) and taurocholate (TCHA) was studied in isolated hepatocytes from rainbow trout (Oncorhynchus mykiss). Both CHA and TCHA were taken up in a concentration- and temperature-dependent manner with optimum temperature at 15 degrees C and a strikingly efficient uptake even at low temperatures (0-5 degrees C). The total uptake was a combination of a saturable [Michaelis-Menten constant (Km) for CHA, 20 microM; Km for TCHA, 19 microM] and a nonsaturable component. The maximal uptake rate of the saturable component was 416 and 805 pmol.mg protein-1.min-1 for CHA and TCHA, respectively. The uptake of both bile acids was shown to be energy dependent, since it was inhibited by the metabolic inhibitors antimycin A, oligomycin and carbonyl cyanide m-chlorophenylhydrazone. The uptake was clearly Na+ independent, since isosmotic replacement of extracellular Na+ by Li+, choline, or K+ did not inhibit the uptake. Furthermore, it seemed to be independent of the presence of extracellular Cl-, since it was not inhibited by replacement of Cl- with sodium gluconate. On the whole, our results show that the hepatocellular uptake of bile acids in rainbow trout is mediated by a Na(+)-independent carrier system, with characteristics resembling the corresponding transport component in mammalian hepatocytes, but with high efficiency even at low temperatures.

Characteristics of bile salt uptake into skate hepatocytes

The substrate specificity for the transporter that mediates the hepatic uptake of organic anions in freshly isolated hepatocytes of the elasmobranch little skate (Raja erinacea) was determined for bile salts and bile alcohols. The Na(+)-independent transport system exhibits a substrate specificity, which is different from the specificity of Na(+)-dependent bile salt transport in mammals. Unconjugated and conjugated di- and tri-hydroxylated bile salts inhibit uptake of cholyltaurine and cholate competitively. Inhibition is significantly greater with unconjugated as opposed to glycine- or taurine-conjugated bile salts. However, the number of hydroxyl groups in the steroid moiety of the bile salts has only minor influences on the inhibition by the unconjugated bile salts. Since the transport system seems to represent an archaic organic-anion transport system, other anions, such as dicarboxylates, amino acids and sulphate, were also tested, but had no inhibitory effect on bile salt uptake. To clarify whether bile alcohols, the physiological solutes in skate bile, share this transport system, cholyltaurine transport was studied after addition of 5 beta-cholestane-3 beta,5 alpha,6 beta-triol, 5 alpha-cholestan-3 beta-ol and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. These bile alcohols inhibit cholyltaurine uptake non-competitively. In contrast, uptake of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol, which is Na(+)-independent, is not inhibited by cholyltaurine. The findings further characterize a Na(+)-independent organic-anion transport system in skate liver cells, which is not shared by bile alcohols and has preference for unconjugated lipophilic bile salts.

Deficiency of bile acid transport and synthesis in oval cells from carcinogen-fed rats

Freshly isolated oval cells, which we obtained from the livers of rats fed a choline-deficient/DL-ethionine-supplemented diet, did not transport bile acids. Compared with freshly isolated rat hepatocytes they took up only negligible amounts of [3H]taurocholate or [14C]cholate. The cells bound small amounts of radioactive bile acids. This portion of the total cell-associated radioactivity was enhanced on membrane permeabilization. In contrast to cultured liver parenchymal cells from untreated rats, no bile acid synthesis was detected in cultured oval cells. Cultured oval cells also lost the ability to conjugate exogenously added cholate (100 mumol/L) with taurine or glycine. However, when liver parenchymal cells were isolated from carcinogen-fed rats, bile acid uptake was diminished compared with that in hepatocytes from control animals. In particular, the maximum values of taurocholate and cholate uptake were decreased by 75% and 50%, respectively, whereas the Michaelis-Menten constant values were not altered. The study demonstrates that (a) oval cells lack typical liver parenchymal cell-specific properties such as bile acid uptake, bile acid synthesis and conjugation of bile acids with taurine/glycine and therefore do not contribute to bile acid dependent bile formation (b) proliferating in livers of rats fed a choline-deficient/DL-ethionine-supplemented diet are part of the bile duct epithelial cell compartment); and (c) bile acid uptake is reduced in liver parenchymal cells of rats fed a choline deficient/DL-ethionine-supplemented diet, and this effect is due to a decrease in transport capacity without a decrease in transport affinity.

Hepatoselective carrier-mediated sodium-independent uptake of pravastatin and pravastatin-lactone

Pravastatin and pravastatin-lactone are not taken up into extrahepatic cells such as fibroblasts, or hepatoma cells such as AS-30D ascites hepatoma cells or FAO cells. In contrast, pravastatin is taken up into isolated rat hepatocytes by a carrier mediated, saturable, temperature-dependent and energy-dependent mechanism. The kinetic parameters for the saturable uptake are K m 27 μ M, V max 537 pmol/ mg per min . The permeability coefficients were determined to be 9.829·10− 7 cm/s at 4°C, 1.76·10− 6 cm/s at 7°C, 3.85·10− 6 cm/s at 17°C and if5.82·10− 6 cm/s at 37°C. The activation energy is 60 KJ/mol for 100 μM pravastatin at 37°C. The Q 10 values are between 1.7 and 2.8. In the presence of metabolic inhibitors and in the absence of oxygen, transport is inhibited. Uptake of pravastatin is not dependent on an extracellular to intracellular sodium-gradient. Replacement of chloride by sulfate, nitrate, gluconate or thiocyanate significantly inhibits the uptake of pravastatin. Uptake is competitively inhibited by cholate and taurocholate in the presence and absence of sodium. Pravastatin, however, competitively inhibits the uptake of cholate and taurocholate only in the absence of sodium. In addition, pravastatin-lactone enters liver cells via an energy-dependent, carrier-mediated uptake system. For the saturable energy-dependent part of the hepatocellular uptake a K m value of 9 μM and a V max value of 621 pmol/mg per min was determined. The permeability coefficient of pravastatin-lactone uptake is calculated to be 5.41·10− 6 cm/s at 37°C. The uptake of pravastatin-lactone is competitively-noncompetitively inhibited by pravastatin and by lovastatin and vice versa. These results indicate that the hepatoselectivity of pravastatin is due to its carrier-mediated uptake into rat hepatocytes via a sodium-independent bile acid carrier. Pravastatin-lactone resembles pravastatin-sodium in its hepatoselectivity.

Differential effects of cyclosporin a on the transport of bile acids by human hepatocytes

Cyclosporin A (CsA) treatment has been reported to cause rises in serum bile acids both in humans and rats. It has also been shown to suppress bile flow in situ in rats and inhibit the transport of bile salts by rat hepatocytes. The purpose of this study was to examine the influence of CsA on uptake of radiolabelled cholate (CA), glycocholate (GC) and taurocholate (TC) by isolated human hepatocytes. CsA did not significantly change V max for CA uptake [0.23 ± 0.01 vs 0.25 ± 0.02 nmol/mg protein/min for control and CsA (10 μM), respectively], but significantly increased K m (37 ± 2 vs 86 ± 8 μM). Similarly, V max for TC uptake was not affected (0.51 ± 0.02 vs 0.67 ± 0.05 nmol/mg protein/min) while K m was significantly increased [46 ± 3 vs 109 ± 11 μM for control and CsA (10 μM), respectively). On the other hand, neither V max nor K m for GC uptake was affected by CsA. The data indicate a competitive pattern of inhibition induced by CsA on CA and TC uptake. Furthermore, CsA was found to cause a dose-related inhibition of accumulation of both cholate and taurocholate, but not GC accumulation. None of the concentrations of CsA showed a significant effect on the integrity of the human hepatocytes as assessed by ALT (alanine aminotransferase), AST (aspartate aminotransferase) and LDH (lactate dehydrogenase) release. The findings, in human hepatocytes, are generally consistent with the observations reported from rodent studies. They strongly support the contention that serum bile acid increases in CsA-treated patients are due to interference with the hepatocellular transport and accumulation of particular bile acids.