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Background/aims: Ntcp-mediated uptake of bile salts at the basolateral membrane of hepatocytes is required for maintenance of their enterohepatic circulation. Expression of Ntcp is reduced in various experimental models of cholestasis associated with increased plasma bile salt concentrations. Mdr2 P-glycoprotein-deficient mice lack biliary phospholipids and cholesterol but show unchanged biliary bile salt secretion and increased bile flow. These mice are evidently not cholestatic, but plasma bile salt concentrations are markedly increased. The aim of this study was to investigate the role of Ntcp in the elevated bile salt levels in mdr2 P-glycoprotein-deficient (-/-) mice., Methods: Plasma membranes were isolated from male wild-type (+/+) and mdr2 (-/-) mice for measurement of Na+-dependent taurocholate transport and assessment of Ntcp protein levels by Western blotting. Northern blot analysis and competitive reverse transcription-polymerase chain reaction were used to determine hepatic Ntcp mRNA levels., Results: Kinetic analysis showed a 2-fold decrease in the Vmax of Na+-dependent taurocholate transport, with an unaffected Km in (-/-) mice compared with (+/+) controls. Ntcp protein levels were 4-6-fold reduced in plasma membranes of (-/-) mice relative to sex-matched controls. Surprisingly, hepatic Ntcp mRNA levels were not significantly affected in the (-/-) mice., Conclusions: Elevated plasma bile salt levels in mdr2 P-glycoprotein-deficient mice in the absence of overt cholestasis are associated with reduced Ntcp expression and transport activity. This is due to posttranscriptional down-regulation of Ntcp.

Purpose: The objective was to investigate the ability of a glycosteroid (TC002) to increase the oral bioavailability of gentamicin., Methods: Admixtures of gentamicin and TC002 were administered to the rat ileum by injection and to dogs by ileal or jejunal externalized ports, or PO. Bioavailability of gentamicin was determined by HPLC. 3H-TC002 was injected via externalized cannulas into rat ileum or jejunum, or PO and its distribution and elimination was determined. The metabolism of TC002 in rats was evaluated by solid phase extraction and HPLC analysis of plasma, urine and feces following oral or intestinal administration., Results: The bioavailability of gentamicin was substantially increased in the presence of TC002 in both rats and dogs. The level of absorption was dependent on the concentration of TC002 and site of administration. Greatest absorption occurred following ileal orjejunal administration. TC002 was significantly more efficacious than sodium taurocholate, but similar in cytotoxicity. TC002 remained primarily in the GI tract following oral or intestinal administration and cleared rapidly from the body. It was only partly metabolized in the GI tract, but was rapidly and completely converted to its metabolite in plasma and urine., Conclusions: TC002 shows promise as a new drug transport agent for promoting intestinal absorption of polar molecules such as gentamicin.

Purpose: This study was designed to characterize taurocholate uptake properties in primary cultures of rat hepatocytes maintained under different matrix conditions., Methods: Hepatocytes isolated from male Wistar rats (230-280 g) were cultured on a simple collagen film, on a substratum of gelled collagen or between two layers of gelled collagen (sandwich configuration). Hepatocyte morphology, taurocholate uptake properties, and expression of the sinusoidal transport protein. Na+/taurocholate-cotransporting polypeptide (Ntcp) were examined in these cultures at day 0 and day 5., Results: By day 5, monolayer integrity had deteriorated in simple collagen cultures. In contrast, cell morphology was preserved in hepatocytes maintained in a sandwich configuration. At day 5, taurocholate accumulation at 5 min in hepatocytes cultured on a simple collagen film, on a substratum of gelled collagen, and in a sandwich configuration was approximately 13%, 20% and 35% of day-0 levels, respectively, and occurred predominately by a Na+-dependent mechanism. The initial taurocholate uptake rate vs. concentration (1-200 microM) profile was best described by a combined Michaelis-Menten and first-order function. In all cases, the estimated apparent Km values were comparable for day-0 and day-5 hepatocytes (3241 microM). In contrast, the Vmax values of hepatocytes cultured on a simple collagen film, on gelled collagen and in a sandwich configuration were approximately 5, 6 and 14% of the values at day 0, respectively; values for the first-order rate constant were 5-, 3- and 2-fold lower, respectively. Immunoblot analysis indicated that at day 5 Ntcp expression in hepatocytes cultured in a sandwich configuration was greater than in hepatocytes cultured on a simple collagen film., Conclusions: A collagen sandwich configuration reestablishes normal morphology and partially restores bile acid uptake properties in primary cultures of rat hepatocytes.

Turnover of cellular reduced glutathione (GSH) is accomplished predominantly by export into the extracellular space; however, the plasma membrane transport mechanisms that mediate GSH efflux are not well characterized. The present study examined GSH transport using secretory vesicles isolated from the sec6-4 mutant strain of Saccharomyces cerevisiae. In contrast with studies in mammalian membrane vesicles, GSH transport in yeast secretory vesicles was mediated largely by an ATP-dependent, low-affinity pathway (Km 19+/-5 mM). ATP-dependent [3H]GSH transport was cis-inhibited by substrates of the yeast YCF1 transporter, including sulphobromophthalein, glutathione S-conjugates and the alkaloid verapamil, and was competitively inhibited by S-(2, 4-dinitrophenyl)glutathione (DNP-SG). Similarly, GSH competitively inhibited ATP-dependent [3H]DNP-SG transport, with a Ki of 18+/-2 mM, but had no effect on ATP-dependent [3H]taurocholate transport. ATP-dependent GSH transport was not affected by either membrane potential or pH-gradient uncouplers, but was inhibited by 4, 4'-di-isothiocyanatostilbene-2,2'-disulphonate, probenecid and sulphinpyrazone, which are inhibitors of mrp1 and mrp2, mammalian homologues of the yeast YCF1 transporter. Western blot analysis of the secretory vesicle membrane fraction confirmed the presence of Ycf1p. These results provide the first direct evidence for low-affinity, ATP-dependent transport of GSH, and demonstrate that this ATP-dependent pathway displays kinetic characteristics similar to those of the yeast YCF1 transporter.

The mechanism for the efflux of taurocholic acid (TC) across the blood-brain barrier (BBB) was studied by examining the elimination of [3H]TC after microinjection into the cerebral cortex. The efflux of [3H]TC from the brain was saturable with a Vmax of 15.0 pmol/min/g brain and a Km value of 0.396 nmol/0.2 microl injectate. Efflux was inhibited by cholic acid (CA), a cationic cyclic octapeptide (octreotide; a somatostatin analogue) and an anionic cyclic pentapeptide (BQ-123; an endothelin receptor antagonist), with an IC50 value of 1.09 nmol/0.2 microl injectate, 1.12 nmol/0.2 microl injectate and 0.12 nmol/0.2 microl injectate, respectively. Probenecid (20 nmol/0.2 microl injectate), but not p-aminohippuric acid (10 nmol/0.2 microl injectate), inhibited the brain efflux of [3H]TC. In addition, elimination of [3H]BQ-123 after microinjection was saturable with a Vmax of 20.8 pmol/min/g brain and a Km of 2.92 nmol/0.2 microl injectate; it was also inhibited by TC with an IC50 value of 0.074 nmol/0.2 microl injectate. In contrast, no significant efflux of [14C]octreotide from the brain was observed until 60 min after microinjection. These results suggest that both TC and BQ-123 are transported from the brain to the circulating blood across the blood-brain barrier via specific mechanisms. Although mutual inhibition was observed between TC and BQ-123, kinetic analysis suggested that the two transport systems differ.

It has been proposed that the hepatocellular Na(+)-dependent bile salt uptake system exhibits a broad substrate specificity in intact hepatocytes. In contrast, recent expression studies in mammalian cell lines have suggested that the cloned rat liver Na(+)-taurocholate cotransporting polypeptide (Ntcp) may transport only taurocholate. To characterize its substrate specificity Ntcp was stably transfected into Chinese hamster ovary (CHO) cells. These cells exhibited saturable Na(+)-dependent uptake of [3H]taurocholate [Michaelis constant (K(m)) of approximately 34 microM] that was strongly inhibited by all major bile salts, estrone 3-sulfate, bumetanide, and cyclosporin A. Ntcp cRNA-injected Xenopus laevis oocytes and the transfected CHO cells exhibited saturable Na(+)-dependent uptake of [3H]taurochenodeoxycholate (Km of approximately 5 microM), [3H]tauroursodeoxycholate (Km of approximately 14 microM), and [14C]glycocholate (Km of approximately 27 microM). After induction of gene expression by sodium butyrate, Na(+)-dependent transport of [3H]estrone 3-sulfate (Km of approximately 27 microM) could also be detected in the transfected CHO cells. However, there was no detectable Na(+)-dependent uptake of [3H]bumetanide or [3H]cyclosporin A. These results show that the cloned Ntcp can mediate Na(+)-dependent uptake of all physiological bile salts as well as of the steroid conjugate estrone 3-sulfate. Hence, Ntcp is a multispecific transporter with preference for bile salts and other anionic steroidal compounds.

In vitro experiments using human liver tissue to study drug metabolism and transport are usually performed and interpreted without real consideration of the differences in procurement of the tissue, if it is obtained from different sources. Therefore, in this study the functionality of isolated hepatocytes and liver slices prepared either from healthy human liver tissue obtained from patients undergoing partial hepatectomy [livers from partial hepatectomy (PH-livers)] or from donor tissue remaining after reduced-size or split-liver transplantation [livers from transplantation (Tx-livers)] was compared. From each liver sample, both liver slices and hepatocytes were prepared and compared with respect to viability and drug disposition. The viability of hepatocytes was assessed by trypan blue exclusion, ATP content, and energy charge and that of liver slices by potassium retention. In both preparations phase I metabolism was studied using lidocaine and testosterone as substrates, whereas phase I and II metabolism was assessed with 7-ethoxycoumarin. The membrane transport capability of the hepatocytes was investigated by measuring the uptake of taurocholic acid. The hepatocytes from PH-livers and Tx-livers showed similar viabilities and functional capacities. Metabolism in cells and slices from Tx-livers was found to be quantitatively comparable. However, liver slices from PH-livers showed a significantly lower metabolic capacity, compared with cells from the same tissue. This may indicate that only some of the hepatocytes in the liver slices from PH-livers participate in the metabolism of the compounds studied and that a selection of healthy cells takes place during isolation of the hepatocytes. Our results imply that hepatocytes isolated from Tx-livers and PH-livers can be used in the same study without consideration of the procurement of the tissue. However, the procurement of the tissue may significantly influence the functions of liver slices; the liver slices prepared from PH-livers showed significantly lower metabolic function, compared with slices prepared from Tx-livers.

The sulfated bile alcohol scymnol sulfate (ScyS), 3 alpha,7 alpha,12 alpha,24 xi, 26,27-hexahydroxy-5 beta-cholestane-26(27)-sulfate, is the major bile salt in bile of an elasmobranch, the little skate. To investigate hepatic transport of bile alcohols in skate liver, [3H]ScyS and a potential precursor, 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestane (chtriol), were used as model compounds. Their transport into isolated hepatocytes was partially saturable, temperature sensitive, and Na+ independent. The uptake of ScyS was inhibited by cholyltaurine, and uptake of cholyltaurine was inhibited by ScyS in a competitive manner. In contrast, uptake of chtriol was not inhibited by cholyltaurine, suggesting separate transport systems. ScyS and chtriol showed a choleretic effect in isolated perfused livers. When ScyS was added to the perfusate of isolated perfused livers, > 25% was found in bile within 7 h. When chtriol was added to the perfusate, 10% of the dose was secreted into the bile mainly in the form of polar metabolites, whereas only nonmetabolized chtriol remained in the livers. The slow bile flow of 40-50 microliters/h and the high recovery in the liver suggest that metabolism may be the rate-limiting step in the hepatic elimination of chtriol. The major metabolites secreted into bile were identified by mass spectrometry and chromatography as scymnol and ScyS. To study the enterohepatic circulation, [3H]ScyS or [3H]chtriol was administered into the duodenum of free-swimming skates, and bile was collected through exteriorized indwelling cannulas over a 4-day period. More than 90% of the radioactivity was recovered from bile, indicating that there was a highly effective absorption in the intestinal epithelium, as well as specific transport mechanisms for hepatic uptake and biliary secretion of these compounds. This is the first direct demonstration of an enterohepatic circulation for a bile alcohol sulfate in fish liver.

Background & Aims: Chemotherapy of hepatocellular carcinomas is hampered by the insufficient accumulation of cytostatic drugs within the tumor cells. The aim of this study was to evaluate the feasibility of therapeutic strategies using antineoplastic agents coupled to bile acids., Methods: Expression of the Na(+)-taurocholate-cotransporting polypeptide (NTCP) was analyzed in six hepatocellular carcinomas and in nonmalignant liver tissue. Uptake of the cytostatic drug [3H]-chlorambucil-taurocholate (S2676) was measured in Xenopus laevis oocytes injected with total messenger RNA (mRNA) from the carcinomas or peritumor tissue or with complementary RNA encoding the NTCP or the organic anion-transporting polypeptide (OATP) of human liver., Results: Expression of hepatocellular carcinoma mRNA in oocytes resulted in mainly Na(+)-dependent uptake of chlorambucil-taurocholate. The level of NTCP mRNA in carcinomas amounted to 56% +/- 27% compared with peritumor tissue. Immunofluorescence studies confirmed the expression of NTCP on the surface of hepatocellular carcinoma cells. OATP expression, determined by immunoblotting, was similar in hepatocellular carcinomas and surrounding liver tissue (n = 3). NTCP mediated Na(+)-dependent uptake of chlorambucil-taurocholate (Michaelis constant, 11 mumol/L), whereas OATP mediated Na(+)-independent uptake., Conclusions: Hepatocellular carcinomas express the Na(+)-dependent bile acid transporter NTCP. Because NTCP mediates high-affinity uptake of chlorambucil-taurocholate, targeting of cytostatic bile acids to hepatocellular carcinomas could become a feasible therapeutic strategy.

The ileal Na+/bile acid cotransporter (IBAT) maintains the reabsorption of bile acids from the intestine in the enterohepatic circulation of bile acids. In the present study, we showed that S-8921 could dose-dependently inhibit the uptake of [3H] taurocholate in the COS7 cell line which constitutively expresses hamster IBAT. The IC50 value of S-8921 against 60 microM of [3H] taurocholate uptake was 66 +/- 8 microM and kinetic analysis revealed that the inhibition by 100 microM of S-8921 was a mixture of competitive and non-competitive types. In vivo administration of S-8921 by its incorporation into diet (0.001-0.1%) caused dose-dependent decrease of serum cholesterol concentrations accompanied by increased fecal excretion of bile acids in hamsters which were not loaded with cholesterol and bile acid. These data suggest that the inhibition of IBAT could decrease serum cholesterol in the non-cholesterol and -bile acid loaded normal condition.

Aim: It is possible to detect disturbances of bile acid absorption using a whole body counter after administration of Se-75 labelled bile acid analogues. We scrutinized the benefit of a modification of the test method., Methods: We investigated 77 patients with different forms of a gastrointestinal disease. After application of Se 75 homotaurocholic acid we measured patient-activity up to 7 days later including whole-body profile scans in the first 6 h., Results: The fractional retention after 7 days was between 20 and 67%. In cases of impaired absorption it was below 12%. Patients with liver diseases and after cholecystectomy (without bile acid resorption disturbance) showed normal values. Patients with Crohn's disease of the ileum or with intestinal ileac by-pass or with colestyramine treatment or with disturbance of vitamin B12-absorption or with cystic fibrosis showed a disturbance of bile acid absorption. The normal whole-body half-life was more than 2.8 days. The 24 and 72 h values were 62 and 31% in cases with normal absorption. Smaller values are signs of bile acid malabsorption. Impulse rates measured with the whole body counter are of an order of magnitude that allows to reduce the usually administered dose of 37 kBq to 9.25 kBq., Conclusion: This is an efficient method to detect disturbances of bile acid absorption. The usually administered activity of 37 kBq can be reduced to 9.25 kBq.

The cDNA for the rat liver organic anion-transporting polypeptide "oatp" has been shown to encode transport of bromosulfophthalein (BSP) and bile salts in Xenopus oocytes (E. Jacquemin, B. Hagenbuch, B. Stieger, A. W. Wolkoff, and P. J. Meier. Proc. Natl. Acad. Sci. USA 91: 133-137, 1994). Because oatp mRNA is expressed strongly in the kidney, we sought to determine whether renal oatp might play a role in the known secretion of a large variety of organic anions by the kidney. We transiently expressed a full-length oatp cDNA, cloned in pSPORT, in HeLa cell monolayers using the recombinant vaccinia virus vtf7-3. We tested an array of organic anions as candidate substrates by determining their ability to compete with tracer BSP for transport. HeLa cell monolayers transfected with the oatp cDNA transported tracer BSP and taurocholate at rates substantially higher than monolayers transfected with a control plasmid. Thus good expression can be obtained with the vaccinia-HeLa system using a standard plasmid cloning vector. BSP transport varied as a function of the medium albumin, ionic conditions, and pH in a fashion similar to that in Xenopus oocytes. Several organic anions known to be secreted by the classic secretory pathway, including p-aminohippurate (PAH), phenol red, and indigo carmine (10 microM) failed to inhibit oatp-mediated BSP transport. Direct testing using tracers revealed no oatp-mediated transport of sulfate, urate, PAH, several eicosanoids, or unconjugated or conjugated bilirubin. On the other hand, BSP transport was inhibited by approximately 50% by 10 microM corticosterone sulfate, spironolactone, and several other steroids. We conclude that the functional properties of oatp expressed in the HeLa cell/vaccinia transient expression system are comparable to those following expression in Xenopus oocytes and that steroids are likely to represent high-affinity endogenous oatp substrates. The latter hypothesis is addressed in greater detail in a companion paper.

Although substantial evidence indicates that estradiol-17 beta (E2) is conjugated to the glucuronide in the kidney and then excreted by a direct tubular secretory route and that the liver transports E2 glucuronides via carrier-mediated mechanisms, the transporters involved in these processes have not been identified. The so-called "organic anion-transporting polypeptide" (i.e., oatp) has a number of known substrates, including bromosulfophthalein (BSP) and taurocholic acid (TCA) (E. Jacquemin, B. Hagenbuch, B. Stieger, A. W. Wolkoff, and P. J. Meier. Proc. Natl. Acad. Sci. USA 91: 133-137, 1994). In a companion study, we determined that steroid hormones represent a class of hormones that interact strongly with oatp when the latter is transiently expressed in vitro. Here, we studied more extensively steroids and steroid anion conjugates as candidate oatp substrates. In HeLa cell monolayers transfected with a full-length oatp cDNA, [3H]estradiol 17 beta-D-glucuronide ([3H]E2-17G) was transported with a signal-to-noise ratio of 15:1 over that of monolayers transfected with a control plasmid. The affinity of oatp for [3H]E2-17G was significantly higher than that for TCA (K(m) of 3 microM vs. 27 microM, respectively). In contrast to E2-17G, unconjugated estradiol (E2) was not significantly transported by oatp. Several unconjugated steroids and anionic steroid conjugates were tested for their ability to compete with tracer E2-17G for oatp-mediated transport. Conjugation at the 17 or 3 position with the anion of a strong acid (sulfate) resulted in a greater degree of inhibition of tracer E2-17G transport than did conjugation at the 17 or 3 position with an uncharged group (acetate), suggesting that the strength of the negative charge at these positions is an important determinant of the affinity of a given steroid conjugate for oatp. We conclude that the preferred substrates for oatp are steroids with a strong 17- or 3-position anionic group. Since steroid sulfotransferases and glucuronosyltransferases are expressed in the proximal tubule, as is oatp, the transporter may serve as an apical exit pathway for steroids following their conjugation within the tubule cell.

The effects of bile duct ligation on biliary excretion of bile acids, glutathione, and lipids were studied in the rat. The bile duct of the rat was ligated for three days. The biliary bile acid excretion after bile duct cannulation was higher at first, but after 90 min became lower than that in the control rat. The bile flow in the bile duct-ligated rat was higher after bile duct cannulation and gradually decreased to the same level as in the control rat. Biliary glutathione excretion, which has been suggested to be a driving force for the bile acid-independent canalicular bile flow, was markedly decreased in the bile duct-ligated rat. The mannitol clearance was increased and the bile ductules showed proliferation in the bile duct-ligated rat, suggesting an increase in the ductular bile flow. Biliary excretion of lithocholate glucuronide was more markedly impaired than that of taurocholate. When taurocholate was infused at higher rates, which increases bile flow and biliary excretion of bile acid and lipids in the control rat, biliary bile acid and lipid excretion remained constant in the bile duct-ligated rat. These findings indicate that, in the bile duct-ligated rat, the ductular bile flow was increased and bile acid-independent canalicular bile flow was decreased and that, although the biliary excretion of bile acids was not as impaired as that of organic anions, the capacity of bile acid and lipid excretion was markedly decreased.

Purpose: Previously, we found that the biliary excretion of the 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040) glucuronide is severely impaired in Eisai hyperbilirubinemic rats (EHBR), while that of sulfate remains normal (Takenaka et al., J. Pharmacol. Exp. Ther., 274: 1362-1369, 1995). The purpose of the present study is to clarify the mechanisms for impairment of the biliary excretion of E3040 glucuronide in EHBR., Methods: We kinetically analyzed the disposition of the conjugates in the perfused liver at steady state. The uptake of the conjugates into the isolated canalicular membrane vesicles (CMVs) was also examined., Results: At steady state, the bile/liver unbound concentration ratios of the conjugates were 40-400 in both rat strains, indicating a highly concentrated process. The biliary excretion clearance (CLu,bile) of the glucuronide, defined for the unbound concentration in the liver, was decreased in EHBR to 1/30 of that in normal rats, whereas the CLu,bile of the sulfate was comparable between the two rat strains. In vitro, the transport of E3040 glucuronide into CMV prepared from SD rats exhibited the ATP dependency, whereas minimal effect of ATP was observed on the uptake of the glucuronide into CMV from EHBR. In contrast, the uptake of E3040 sulfate was comparable between SD rats and EHBR. Furthermore, ATP did not stimulate the uptake of sulfate into the CMVs., Conclusions: It was suggested (1) that the excretion of E3040 glucuronide across the bile canalicular membrane is mediated by the primary active transporter which is defective in EHBR and (2) that the bile canalicular transport system for E3040 sulfate is different from that for the glucuronide in that the former remains normal in EHBR.

Recently two different bile-acid carriers for the hepatocellular sodium-dependent uptake of taurocholate have been described. The first transport system was isolated and characterized by functional expression cloning in Xenopus laevis oocytes. The corresponding cDNA clone, named Ntcp for Na+/taurocholate co-transporting polypeptide, codes for a protein of 362 amino acids and shows no similarity to previously known sequences. The transport function of this carrier system is well documented by expression in Xenopus laevis oocytes and by transient and stably transfected cell lines. In addition, several lines of evidence implied that the well-known xenobiotic-metabolizing enzyme microsomal epoxide hydrolase (mEH, EC 3.3.2.3) is also able to mediate sinusoidal uptake of taurocholate. Furthermore, it was claimed that the same enzyme also mediates the uptake of the conjugated bile acid into the smooth endoplasmic reticulum (ER). No direct proof of the transport function of mEH by its heterologous expression has yet been published. In the present work we used a stable transfected cell line that expressed high levels of heterologous mEH for uptake studies of various bile acids and the loop diuretic bumetanide. The uptake of the conjugated bile acid taurocholate, of the non-conjugated bile acid cholate and of the organic anion bumetanide was measured in the transfected as well as in the non-transfected parental cell line. These organic anions represent the main substrates of the known transport systems for organic anions in the rat liver. The results show that the microsomal epoxide hydrolase is unable to transport taurocholate, cholate or bumetanide. Furthermore, Western-blot analysis revealed the expression of mEH in hepatoma tumor cell lines, which show no transport activity for these organic anions. These results show that it is unlikely that mEH can mediate the transport of these substrates.

The association of radiolabelled taurocholic acid with the solid fraction of a faecal fermentation mixture was measured. A human faecal inoculum was incubated with [24-14C]taurocholic acid and several non-starch polysaccharide sources (pectin, wheat bran, ispaghula (Plantago ovata) husk and seed), glucose or a substrate-free control. Portions of fermentation mixture were taken at 0, 3, 6, 21 and 24 h and centrifuged to acquire a supernatant fraction and a pellet containing the fermentation residue. 14C was measured in supernatant fractions and pellets at all time points. Volatile fatty acids (VFA) were measured at 0 and 24 h to confirm bacterial growth. Radioactivity in the pellet increased over time for all substrates. Glucose resulted in the greatest incorporation of taurocholic acid into the pellet, followed by pectin. At 24 h the proportion of the total radioactivity found in the pellet was 92% for glucose, 79% for pectin, 60% for wheat bran, 59% for ispaghula seed, 53% for ispaghula husk and 26% for the control (mean of duplicates). Glucose and pectin produced the greatest quantity of VFA at 24 h. VFA production was highly correlated with radioactivity in the pellet (r0.976, P < 0.005). These results suggest that the bile acid binding capacity of a faecal culture mixture may be strongly influenced by the fermentability of the available substrate and hence related to bacterial metabolic activity.

The absorption of lithocholate and its sulfate and glucuronide in rat jejunum and terminal ileum was studied. Tracer amounts of radiolabelled bile acids were administered to the ligated intestinal segments, and their absorption was monitored by biliary excretion through a bile duct catheter. Absorption of lithocholate was faster in the terminal ileum than in the jejunum. Although the sulfation reduced lithocholate absorption in the jejunum, it did not affect lithocholate absorption in the terminal ileum. This was due to the Na+-dependency of ileal absorption of lithocholate-sulfate assessed by perfusion studies. In contrast, the glucuronidation markedly reduced lithocholate absorption both in the jejunum and the terminal ileum. These findings indicate that the glucuronidation is more effective than sulfation in detoxifying lithocholate as far as the prevention of its intestinal absorption is concerned.

Background & Aims: Kupffer cell activation is hypothesized to play an etiopathogenic role in storage-related graft failure after liver transplantation. The aim of this study was to verify whether the elimination of Kupffer cells modifies the magnitude of cold ischemia/reperfusion injury of the liver., Methods: Rat Kupffer cells were eliminated by an intravenous injection of liposome-encapsulated dichloromethylene diphosphonate. Livers from control and treated rats were isolated and perfused before and after 24-hour cold ischemia in the University of Wisconsin solution (4 degrees C). Hepatocyte and sinusoidal endothelial cell functions were evaluated by taurocholate and hyaluronic acid elimination, respectively. Liver transplantation was also performed using control and treated donor livers stored under identical conditions., Results: Compared with baseline values, similar alterations were found in both groups after cold ischemia for hepatocyte function (intrahepatic resistance, bile secretion, lactate dehydrogenase release, oxygen consumption, and taurocholate intrinsic clearance) and for sinusoidal endothelial cell function (hyaluronic acid intrinsic clearance). The 10-day survival rate of animals undergoing transplantation was not different between the groups (6 of 15 vs. 4 of 15, control vs. treated donor livers, respectively)., Conclusions: The presence or absence of Kupffer cells does not modify the effect of 24-hour cold ischemia/reperfusion on the rat liver.

Olsalazine (OLZ), a relatively new form of 5-aminosalicylic acid (5-ASA), is being used for the treatment of colitis. A major side effect of olsalazine is diarrhea, reported in 12-25% of patients. One suggested mechanism for this side effect is enhanced ileal water and electrolyte secretion. We propose that OLZ may also inhibit ileal bile acid (BA) transport, resulting in choleretic diarrhea. This would result in excess BAs reaching the colon, with consequent BA-induced secretory diarrhea. Therefore, we studied the effect of OLZ on rat ileal absorption of taurocholate. BA uptake was determined in rat ileal segments, everted sacs, brush border membrane vesicles (BBMV), and Xenopus laevis oocytes. Segments and everted sacs were treated with 5 mM OLZ for 30 min prior to and throughout 10-min taurocholate (Tc) uptake. Terminal ileal BBMV were used to study the effect of OLZ on sodium-dependent bile acid uptake independent of cellular metabolism. Direct effects on the bile acid carrier were examined using Xenopus laevis oocytes expressing the cloned apical rat ileal BA transporter. In ileal segments 5 mM OLZ inhibited 10-min Tc uptake by 69.4 +/- 8.8% (P < 0.01) (N = 10 animals). Increasing concentrations of OLZ resulted in a dose-dependent inhibition of Tc uptake. Ten-minute Tc uptake with 0.5, 1.0, 2.0, 2.5, and 5 mM OLZ was inhibited by 13.5, 39.6, 49.7, and 70.5%, respectively. In BBMV, OLZ inhibited 45-sec Tc uptake in a dose-dependent manner but did not effect Na-dependent L-alanine uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Background: The effects of small intestine transplantation on enteric physiology are poorly understood. After orthotopic jejunoileal autotransplantation, dogs develop a severe watery diarrhea and lose up to 15% of their body weight. The cause of these changes has not been explained. Our aim was to determine the influence of jejunoileal autotransplantation on ileal absorption of water, electrolytes, and bile salts and the effects of proabsorptive and prosecretory agents on ileal transport., Methods: Seven dogs were studied before and at 2 and 8 weeks after in situ jejunoileal neural and lymphatic isolation (a model of small intestine autotransplantation). With a triple-lumen perfusion technique, net ileal fluxes of water, electrolytes, and bile salts were measured before and at 2 and 8 weeks after this model of jejunoileal autotransplantation. In addition, the effects of an intravenous infusion of vasoactive intestinal polypeptide (a prosecretory agent) and norepinephrine (a proabsorptive agent) on net transport were evaluated., Results: Dogs developed a profuse diarrhea after this model of autotransplantation. Ileal absorption of water and electrolytes decreased immediately (measured during operation), remained decreased for 2 weeks, and returned toward baseline by 8 weeks. A similar decrease in net flux of bile salts was shown at 2 weeks after transplantation and returned toward baseline by 8 weeks. The prosecretory response of vasoactive intestinal polypeptide on ileal fluxes of water and electrolytes was unchanged, whereas the proabsorptive response to norepinephrine increased after this model of autotransplantation., Conclusions: Jejunoileal autotransplantation decreases ileal absorption of water, electrolytes, and bile salts. The profuse watery diarrhea observed in dogs after small intestine autotransplantation may be a secretory and/or a bile salt-induced diarrhea related to the effects of jejunoileal denervation.

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)

Isolated, vascularly perfused, rat duodenojejunum and ileum preparations were used to study the transport parameters of cholyltaurine (C-tau; taurocholate). After a bolus administration of C-tau containing trace amounts of [3H]C-tau into the lumen of the isolated vascularly perfused duodenojejunum, the rate of appearance of C-tau in the portal effluent was dose-dependent over the range 5-20 mM. At a concentration of 20 mM, the rate of absorption was constant over time and amounted to 1.25 nmol.min-1.cm-1; 2.6% of the luminal load of C-tau was recovered in the portal effluent over the 40-min experimental period. Intra-arterial infusion of ouabain (10(-3)M) did not modify significantly the absorption rate of C-tau. The C-tau absorption from the lumen of the isolated vascularly perfused ileum was 7-fold higher than that from the duodenojejunum. Total absorption of C-tau was dose-dependent over the range 1-20 mM and the estimated Km and Tmax of the absorptive area of the rat ileum were 11.5 mM and 17.7 nmol.min-1.cm-1, respectively. Intraarterial infusion of ouabain reduced by 84% the recovery of C-tau in the portal effluent. In conclusion, the absorption parameters of bile acids in the duodenojejunum and in the ileum of the ex vivo rat intestinal preparations are consistent with a passive diffusion process in the proximal small intestine and an active transport in the ileum. The isolated vascularly perfused duodenojejunum and ileum preparations are therefore appropriate models for studying bile acid absorption process and the coupling with the associated local metabolic, motor, secretory and vascular events.

Ursodeoxycholic acid (UDCA) is a nontoxic bile acid currently used in the treatment of different cholestatic diseases. This bile acid is also considered to be a 'hypercholeretic bile acid'. The effects of daily administration of UDCA on bile flow and the bile acid secretion rate in man are not fully known. The aim of this study was to explore the effects by UDCA on bile flow and its different functions. The biliary clearances of polyethylene glycol 900 (PEG 900) and mannitol were measured simultaneously in patients with an indwelling T tube after cholecystectomy. One group (n = 6) were given UDCA (10 mg/kg/day) for at least 14 days before the investigation and a control group (n = 8) received no bile acid treatment. The bile secretion was studied in acute experiments where bile was drained for 6 h. The bile plasma ratio for PEG 900 was 31 and that for mannitol was 0.7 which corresponds well with the results obtained in different animal species. The relationship between bile flow and the bile salt secretion rate, as expressed by linear regression analysis, showed no difference between the 2 groups (control y = 0.22 + 0.012x, r = 0.61, p < 0.001; UDCA y = 0.20 + 0.016x, r = 0.88, p < 0.001). The relationship between the biliary clearance of PEG 900 and the bile salt secretion rate, as expressed with linear regression analysis, showed a significantly steeper slope for the UDCA-treated patients (control y = 8.1 + 0.34x, r = 0.41, p < 0.05; UDCA y = 5 + 1.58x, r = 0.78, p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Background/aims: The liver loses protein during fasting. This study sought to determine if hepatic protein loss during fasting selectively preserves functions important to survival such as uptake of fatty acids, which are major energy substrates in that condition., Methods: Initial [3H]oleate uptake and efflux rates in hepatocytes from starved (for 48 hours) and fed male rats were measured in media containing 250 mumol/L albumin at oleate/albumin ratios of 0.2:1-2:1. Uptake rates of sulfobromophthalein, taurocholate, and glucose were also determined., Results: Initial oleate uptake rate was saturable with respect to unbound oleate concentration. Maximum initial velocity expressed per cell number did not differ between fasted and fed animals, but measured cell volume and estimated surface area were decreased in starved vs. fed hepatocytes (921 +/- 21 vs. 1623 +/- 58 microns2, respectively; P < 0.001). Consequently, when expressed per surface area, maximum initial velocity was greater in starved cells (17 +/- 3 vs. 10 +/- 2 [pmol.min-1.micron2] x 10(-7); P < 0.02). Expressed similarly, oleate efflux was also greater from starved hepatocytes and was inhibited by an antibody to plasma membrane fatty acid binding protein (FABPpm). FABPpm concentration per unit area of plasma membrane also increased in starved hepatocytes (P < 0.05). By contrast, uptake rates of sulfobromophthalein, taurocholate, and glucose by starved hepatocytes were decreased when expressed per cell number and unchanged per unit area., Conclusions: During fasting, the hepatocellular uptake mechanism for oleate is selectively preserved compared with those for sulfobromophthalein, taurocholate, or glucose.

The existence of transporters for bile acids (BA) in liver and intestine has been well documented, but information is still needed as to their respective transport capacity. In the present investigation, we compared the hepatic and intestinal transport rates for BA, using perfused livers and intestines. The livers and intestines were separately perfused and dose-response curves (0.25-10 mM) for tauroursodeoxycholate, taurocholate and taurodeoxycholate were obtained. The intestinal and mesenteric concentration and bile acid pattern were also evaluated in six non-fasting rabbits. Taurocholic, tauroursodeoxycholic and taurodeoxycholic acid ileal absorption showed saturation kinetics in the intestine as in the liver; the maximal uptake velocity for each bile acid in the liver was tenfold higher than the respective maximal transport velocity in the intestine; the Km values obtained in the liver were of the same order of magnitude, i.e. in the millimolar range. Taurocholic, tauroursodeoxycholic and taurodeoxycholic acid transport differences in the liver paralleled those in the intestine. Although the intestine was not homogeneously filled, the bile acid concentration in the ileal content fell into the range of the Km for the three studied bile acids, while the portal blood total bile acid concentration was inferior to the observed Kms of liver uptake. Therefore, both the hepatic and intestinal systems do not operate at their maximal transport rates at the prevailing concentrations in portal blood and luminal content, and the hepatic transport occurs at its highest efficiency (below the Km values) in physiological conditions.

Background/aims: Hyperbilirubinemia associated with sepsis is frequently observed in humans. In this study, an experimental rat model was developed to study bilirubin metabolism and transport during endotoxemia., Methods: Rats were injected intravenously with a single bolus of lipopolysaccharide (1 mg/kg); after 18 hours, the liver was removed for single-pass perfusion. Unconjugated bilirubin, bilirubin ditaurate (125 nmol/min), and/or taurocholate (1.5 mumol/min) were infused. Rate constants for uptake were determined from the disappearance of a bolus of bilirubin ditaurate in a recirculating perfusion., Results: In endotoxemic livers, biliary excretion of bilirubin-glucuronides was reduced by 49% (2.04 +/- 0.2 and 3.99 +/- 0.24 nmol.min-1.g liver-1). Similar results were obtained with bilirubin ditaurate, indicating that the reduced transport is not caused by a reduced conjugation capacity. The rate constant of sinusoidal uptake was significantly reduced during endotoxemia (0.191 +/- 0.034 vs. 0.090 +/- 0.035, respectively). Secretion of taurocholate into bile was also reduced (92 +/- 22 vs. 127 +/- 10 nmol.min-1.g liver-1)., Conclusions: In endotoxemic rats, biliary clearance of bilirubin and taurocholate is substantially decreased, suggesting that decreased output of bilirubin-glucuronides is not caused by impaired conjugation but by a reduction in transport.

The enterohepatic circulation of bile salts may be substrate dependent. We hypothesize that decreased intestinal delivery of bile salts results in downregulation of ileal and hepatocyte bile salt transport in the biliary-diverted rat. Maximal velocity (Vmax) of taurocholate transport by ileal brush-border membrane (BBM) vesicles was downregulated in the bile-diverted animals by 45.5% (559.9 +/- 57.8 pmol.mg protein-1.min-1 in bile-diverted rats vs. 1,026.6 +/- 170.9 pmol.mg protein-1.min-1 in shams). Similarly, taurocholate transport Vmax by hepatocyte basolateral membrane (BLM) was downregulated by 37.8% (2.62 +/- 0.18 pmol.mg protein-1.min-1 in bile-diverted rats vs. 6.93 +/- 0.41 pmol.mg protein-1.min-1 in shams). Cholesterol content (mumol/mg protein) of the membranes was increased in both BBM (0.478 +/- 0.055 vs. 0.272 +/- 0.029) and BLM (0.410 +/- 0.052 vs. 0.294 +/- 0.044) in diverted rats compared with shams. Fluorescence anisotropy was significantly higher in diverted animals compared with shams for both BBM (0.2333 +/- 0.001 vs. 0.2120 +/- 0.004) and BLM (0.1524 +/- 0.002 vs. 0.1426 +/- 0.005). We conclude that biliary diversion in the rat leads to downregulation of both ileal BBM and hepatocyte BLM taurocholate transport. Alterations in transporter expression caused by diversion may, in part, be mediated by changes in membrane lipid composition or fluidity.

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.

Background/aims: Liver microcirculation in cirrhosis is characterized by development of intrahepatic shunts and capillarization of sinusoids secondary to cell necrosis and deposition of new collagen, resulting in both decreased drug elimination and increased vascular resistance with portal hypertension. The aim of this study was to examine the effects of increased portal blood flow on hepatic microcirculation and drug elimination in 13 perfused livers from cirrhotic rats., Methods: Intrahepatic resistance was assessed under basal conditions (21.2 +/- 0.3 mL/min) and 1 hour after doubling the flow (41.6 +/- 1.0 mL/min). A multiple indicator dilution technique was used at both flow rates to measure sinusoidal volume, albumin and sucrose extravascular volumes, and cellular water volume. Hepatic elimination of labeled taurocholate and propranolol was also measured, and the recovery of 15-microns microspheres was used to evaluate large intrahepatic shunts., Results: After doubling the flow, intrahepatic resistance decreased by 31%. Sinusoidal and extravascular volume increased significantly without a change in microsphere recovery. However, there was a marked increase in taurocholate and propranolol elimination by cirrhotic livers. Moreover, during high flow, significant correlations were found between changes in albumin extravascular volume and taurocholate and propranolol elimination., Conclusions: Increased portal blood flow in cirrhotic rats induces a decrease in intrahepatic resistance without changes in intrahepatic shunting and improves drug elimination by the liver without deleterious effects on hepatocyte viability.

A human liver plasma membrane model for the evaluation of the specific binding and transport processes of drugs presenting high hepatic clearance such as vinca alkaloids was developed. Uptake of the two structural antitumor analogs, navelbine (NVB) and vincristine (VCR), which exhibit wide variabilities in their respective pharmacokinetic parameters and antitumor spectra, was investigated. The high yield, the enzymatic profile and the retention of physiologic transport capacities, as demonstrated by taurocholate uptake, revealed that this membrane preparation was well suited for studies of hepatic drug transport systems. For both drugs two distinct processes were observed: mainly membrane binding and transport. NVB was found to bind to the membrane vesicles more intensively than VCR, but the transport processes were almost identical. However only NVB uptake seems to involve Na(+)-dependent processes. These significant differences may be related to the respective lipophilicity of the drugs. The more lipophilic molecule (NVB) presents the highest uptake, which is presumably at the origin of its greatest distribution volume in vivo.

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

A cDNA for the rat liver sodium-dependent bile acid cotransporter was expressed in COS-7 cells to study the functional properties of the translated protein in a mammalian cell line. A 1.2-kb insert was ligated into a pMAMneo vector and transiently transfected using electroporation. After optimal conditions were established, the transiently transfected COS cells were screened with fluorescent-conjugated labeled bile acids for evidence of expression of the cotransporter after 48 h. The uptake of [3H]taurocholate ([3H]TC) was then determined in cells transfected with or without the bile acid insert. Progressive uptake of [3H]TC (0.45 microM) was observed for 30 min in the presence of sodium. In contrast, no uptake of [3H]TC was observed in the absence of sodium, in nontransfected COS cells, or in COS cells transfected with the empty plasmid. Kinetic studies revealed a Michaelis constant (Km) of 29 microM, essentially identical to the Km of this cotransporter described in intact rat hepatocytes and membrane vesicles. Uptake of [3H]TC (5.0 microM) at 5 min (n = 3-6) was inhibited by 100 microM taurochenodeoxycholic acid (81%), tauroursodeoxycholic acid (77%), cholic acid (55%), chenodeoxycholic acid (74%), and ursodeoxycholic acid (56%) but not by 100 microM taurodehydrocholate, 1 mM probenecid, or 100 microM bilirubin. In contrast, bumetanide (500 microM) inhibited [3H]TC uptake by 52%. These studies indicate that the isolated cDNA codes for a physiological bile acid transporter present in rat hepatocytes and that posttranslational factors present in mammalian cells may not be as important in defining properties of this cotransport system.

We have recently shown (D. Sorrentino, R.B. Robinson, C.-L. Kiang, and P.D. Berk. J. Clin. Invest. 84: 1325-1333, 1989) that, in a variety of isolated cell types, the uptake of oleate at physiological albumin concentrations is consistent with traditional pharmacokinetic theory (i.e., driven by unbound ligand). Lower albumin concentrations were associated with a deviant uptake pattern for which alternative theories have been proposed. Whether other classes of organic anions exhibit similar behavior is unknown. Therefore, we examined the effect of albumin on uptake of two widely studied organic anions, sulfobromophthalein (BSP) and taurocholate. Initial uptake velocity of [35S]BSP and [3H]taurocholate by isolated hepatocytes was studied employing a fixed albumin concentration and ligand-to-albumin molar ratios from 0.01:1 to 2:1 for taurocholate and 0.031:1 to 0.75:1 for BSP. In other experiments, albumin and ligand were altered in parallel, keeping their molar ratio constant. Unbound taurocholate concentrations were measured directly by equilibrium dialysis; unbound BSP concentrations were calculated from published data (K.J. Baker and S.E. Bradley. J. Clin. Invest. 45: 281-287, 1966). At 600 microM albumin, uptake of both ligands was a function of the unbound ligand concentration. At low ligand-to-albumin molar ratios and consequent unbound ligand concentrations this relationship was linear; over the entire range of unbound ligand concentrations studied, both ligands exhibited Michaelis-Menten kinetics, with definable maximal velocity and Michaelis constant values. At low albumin concentrations, the relationships between uptake and unbound ligand were unchanged for taurocholate; however, BSP exhibited altered kinetics similar to those observed with oleate. Nontraditional uptake kinetics at low albumin concentrations appear to correlate with very high affinity for albumin.

The effects of trichloroethylene (TRI) on bile acid transport in isolated rat hepatocytes have been studied using doses ranging from 0.5 to 4.0 microliters/flask and a 20-min equilibration period. It was found that TRI caused a dose-related suppression of initial rates of uptake of cholic acid (CA) and taurocholic acid (TC) with no significant effect on enzyme leakage and intracellular potassium ion contents. Accumulation over 30 min for each of those two bile acids was also inhibited. A noncompetitive inhibition of bile acid uptake was shown as indicated by a decrease in maximum velocity (Vmax) and unchanged Michaelis constant (Km). Thirty minutes after cessation of TRI exposure in vitro the uptake of bile acids had gradually returned to normal levels. No significant interference of efflux was found in cells preloaded with either CA or TC. After dosing rats with 1 mmol/kg TRI in vivo the inhibition of uptake of CA and TC by subsequently isolated hepatocytes was not detected until 4 hr. By 16 hr uptake had returned to normal. The accumulation of bile acids was also suppressed at 4 and 8 hr. The inhibition of uptake after in vivo treatment was also noncompetitive. The data are consistent with the reversible increase of serum bile acids (SBA) in experimental animals after exposure to TRI. Furthermore, they support the contention that it is an interference with bile acid uptake, rather than actual cell damage, that is responsible for TRI-induced increases in SBA. Thus, the changes in SBA seem to be the result of interference with a physiological process rather than an event associated with significant pathological consequences.

This study aimed at examining the mechanisms participating in excessive faecal bile acid loss in cystic fibrosis. The study was designed to define the relation between faecal fat and faecal bile acid loss in patients with and without cystic fibrosis related liver disease; to assess terminal ileal bile acid absorption by a seven day whole body retention of selenium labelled homotaurocholic acid (SeHCAT); and to determine if small intestinal bacterial overgrowth contributes to faecal bile acid loss. The study population comprised 40 patients (27 men; median age 18 years) with cystic fibrosis (n = 8) and without (n = 32) liver disease and eight control subjects. Faecal bile acid excretion was significantly higher in cystic fibrosis patients without liver disease compared with control subjects (mean (SEM) 21.5 (2.4) and 7.3 (1.2) micromoles/kg/24 hours respectively; p < 0.01) and patients with liver disease (7.9 (1.3) micromoles/kg/24 hours; p < 0.01). No correlation was found between faecal fat (g fat/24 hours) and faecal bile acid (micromoles 24 hours) excretion. Eight (33%) of cystic fibrosis patients had seven day SeHCAT retention < 10% (normal retention > 20%). SeHCAT retention in cystic fibrosis patients with liver disease was comparable with control subjects (30.0 (SEM) 8.3% v 36.8 (5.9)%; p = NS) while SeHCAT retention in cystic fibrosis patients who did not have liver disease was significantly reduced (19.9 (3.8); p < 0.05). Although evidence of small bowel bacterial overgrowth was present in 40% of patients no relation was found between breath hydrogen excretion, faecal fat, and faecal bile acid loss. The results are consistent with the presence of an abnormality in terminal ideal function in patients with cystic fibrosis who do not have liver disease and that a defect in the ileal absorption of bile acids may be a contributory factor to excessive faecal bile acid loss. Faecal bile acid loss in cystic fibrosis is unrelated to the presence of intraluminal fat or intestinal bacterial overgrowth.

The importance of membrane voltage in uptake of bile salts into hepatocytes is not known. Electrogenicity of the primary bile salt transport process, Na-bile salt cotransport, has been difficult to determine because the large K and Cl conductances of the sinusoidal membrane (GK and GCl, respectively) obscure any transport associated currents. In the present study hepatocytes were treated to reduce these membrane conductances and electrogenic entry of taurocholate and glycocholate was demonstrated. Intracellular voltage and resistance changes resulting from bile salt transport were measured in hepatocytes in which GK and GCl were blocked by impalement with Na acetate microelectrodes and external exposure to quinine (400 microM). This increased the cell input resistance from 153 +/- 17 to 230 +/- 17 M omega (n = 14, P < 0.001). Under these conditions, exposure to 100 microM of taurocholate or glycocholate produced Na-dependent depolarizations of 3.0 +/- 0.5 and 4.2 +/- 0.8 mV, respectively. These correspond to transport currents of 13.9 and 7.6 pA/cell, which are comparable to those predicted from known [3H]taurocholate uptake rates if one positive charge enters the cell with each bile salt molecule. Although uptake of these two bile salts was electrogenic, this was not the case for all bile salts. Na-dependent transport of taurodehydrocholate, which occurs at similar rates to that for taurocholate, produced no voltage change. The unconjugated bile salts cholate and ursodeoxycholate also produced no measurable voltage or resistance changes. In conclusion, Na-dependent uptake of taurocholate and glycocholate is electrogenic, whereas uptake of taurodehydrocholate, ursodeoxycholate, and cholate is predominantly electroneutral.(ABSTRACT TRUNCATED AT 250 WORDS)

Bile acids are efficiently recovered from the intestinal lumen by a Na(+)-dependent transport process that is localized in the ileal enterocyte brush-border membrane. To establish a cell culture model for this process, we examined the Na+ dependence of cholyltaurine (C-tau; taurocholate) transport across monolayers of differentiated Caco-2 cells grown on permeable filter inserts. Transport of [3H]C-tau was Na+ dependent (> 20-fold stimulation), saturable, and time linear for at least 60 min. The apparent Michaelis constant of [3H]C-tau transport was approximately 65 microM, and the maximal transport rate was approximately 800 pmol.min-1.mg protein-1. Transport of [3H]C-tau in the apical-to-basolateral direction was 17-fold greater than transport in the reverse direction. Lowered incubation temperature, various metabolic inhibitors, and various unlabeled bile acids inhibited [3H]C-tau transport. Caco-2 cells thus transport bile acids in a manner similar to that described for ileal brush-border membrane vesicles and isolated ileal enterocytes and are therefore an appropriate model for studying the molecular basis of ileal bile acid transport.

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.

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

Total parenteral nutrition is associated with decreased primary bile acid output. This may result from reduced synthesis or decreased absorption of primary bile acids in the enterohepatic circulation. Ileal atrophy occurs with total parenteral nutrition, and we postulated that absorption of primary bile acids is reduced. Ileal absorption of taurocholate was investigated in miniswine on total parenteral nutrition and this was compared to orally fed animals. Following cholecystectomy and bile duct cannulation, 20 cm, of ileum was perfused with taurocholate. Since miniswine have no significant endogenous taurocholate secretion, the measured taurocholate output reflected ileal absorption. To examine uptake and secretion of taurocholate by the liver, pigs on total parenteral nutrition and controls were infused with intravenous taurocholate (0.5 to 2.5 mumole/kg/min), and bile acid output was determined. Baseline bile acid output was decreased in animals on total parenteral nutrition, yet the response to intravenous taurocholate was similar to controls. When taurocholate was perfused in the ileum, taurocholate output was markedly different. Taurocholate output (pmole/cm ileum/min) at the taurocholate perfusate concentrations of 3 and 20 mM was, respectively, 65 +/- 14 and 183 +/- 63 for controls and 18 +/- 3 and 63 +/- 17 for miniswine on total parenteral nutrition. The latter group's output was significantly lower, at P < 0.05. Both groups of pigs had equal increases in bile acid output with intravenous infusion of taurocholate, suggesting normal hepatic uptake and secretion. Ileal perfusion with taurocholate, however, resulted in decreased taurocholate output with total parenteral nutrition due to decreased ileal absorption of bile acids.(ABSTRACT TRUNCATED AT 250 WORDS)

In the present study, the transport of taurocholate by brush border membrane vesicles (BBMV), prepared from the proximal and distal half of the small intestine of cats was investigated. Uptake of taurocholate (0.01 mmol/l) into BBMV from the distal small intestine was clearly enhanced in the presence of an inwardly directed initial Na+ gradient compared to choline+ gradient conditions, whereas uptake by BBMV from the proximal half of the small intestine was not substantially different between the two incubation conditions. Calculated uptake of taurocholate at a 10s incubation period was mainly due to transfer of taurocholate into the vesicular lumen rather than to extensive binding of taurocholate to the membranes. Evaluation of the kinetics of taurocholate transport across the brush border membrane of the distal half of the small intestine under Na+ gradient conditions revealed a saturable Na(+)-dependent component and a diffusive component with the subsequent apparent parameters: Vmax (maximal transport velocity) = 0.59 nmol/mg protein.10s, KM (affinity constant) = 0.12 mmol/l, D (diffusion constant) = 2.0 microliters/mg protein.10s. The results indicate Na+/taurocholate co-transport across the brush border membrane of the distal half of the small intestine in cats. Thus, the carrier-mediated intestinal bile salt absorption in the cat appears to be similar as described in other species.

Recent studies have indicated that exercise causes alterations in the biotransformation of some xenobiotics and the clearances of antipyrine and [14C]aminopyrine. The present study has investigated whether chronic voluntary physical activity alters hepatobiliary excretory function by comparing the clearance and biliary excretion of model substrates for each of four carrier-mediated transport systems (anion, uncharged, bile acid and cation; n = 8 for each chemical in each group) in active and inactive female rats. The active rats had access to running wheels and voluntarily ran 11.2 +/- 0.68 km/day. The active rats were fed ad libitum, and ate 37% more food than weight-matched, restricted-fed sedentary control rats. Basal bile flow was 34% higher in active rats than in inactive rats, and excretion of bile acids, cholesterol and phospholipid were also increased. The biliary excretion and biliary clearance of the anion, indocyanine green, were elevated in active rats, although total clearance and serum concentrations were not different due to decreased non-biliary clearance. Serum elimination and total clearance of the uncharged substrate, ouabain, were elevated in the active rats, due entirely to increased nonbiliary clearance. Total clearance of the bile acid, taurocholate, was higher in active rats due to an increased biliary clearance. In contrast, there were no differences in either the biliary excretion or clearances of the cation, procainamide ethobromide, between the two groups of rats. Finally, no differences in volume of distribution or elimination half-life were noted between inactive and active rats for any of the substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

To examine whether the relatively selective inhibition of hepatic cholesterol synthesis by the hydrophilic 3-hydroxyl-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor pravastatin in vivo may be due to the existence of a specific uptake mechanism in the liver, the uptake by isolated rat hepatocytes was investigated. The uptake was composed of a saturable component [Michaelis constant (Km) 29 microM, maximal uptake rate 546 pmol.min-1.mg-1] and nonspecific diffusion (nonspecific uptake clearance 1.6 microliters.min-1.mg-1), inhibited by hypothermia, metabolic inhibitors, sulfhydryl-modifying reagents, and inhibitor of anion exchanger, whereas replacement of Na+ by choline+ or Cl- by gluconate- did not alter the uptake. Competitive inhibition was observed by a more highly lipophilic HMG-CoA reductase inhibitor simvastatin (open acid form), dibromosulfophthalein, cholate, and taurocholate. Pravastatin inhibited Na(+)-independent taurocholate uptake with an inhibition constant comparable with the Km value of pravastatin itself. Furthermore, the hepatic permeability clearance in vivo obtained with intact rats was comparable with that in vitro, indicating that the carrier-mediated active transport system we demonstrated in vitro is responsible for the hepatic uptake in vivo. These findings demonstrated that the hepatic uptake of pravastatin occurs via a carrier-mediated active transport mechanism utilizing the so-called multispecific anion transporter, which is common with the Na(+)-independent bile acid uptake system, and that this is one of the mechanisms for its selective inhibition of hepatic cholesterol synthesis in vivo.

The uptake of taurocholate into brush border membrane vesicles prepared from human full term placenta was studied using a rapid filtration technique. The taurocholate uptake into brush border membrane vesicles was sensitive to extravesicular osmolarity, and pre-incubation of the brush border membrane vesicles with the taurocholate increased the uptake of taurocholate into the brush border membrane vesicles. These findings indicate that the uptake of taurocholate by brush border membrane vesicles represents transport into vesicles. The uptake of taurocholate into vesicles was not dependent on Na+ electrochemical gradient (extravesicular > intravesicular). But this uptake was markedly increased when the intravesicular space was rendered electrically more positive by the use of lowly permeant anions or valinomycin-induced K+ diffusion membrane potentials. These findings indicate that the taurocholate transport into brush border membrane vesicles was dependent on membrane potential. The initial rate of taurocholate transport into brush border membrane vesicles exhibited saturation kinetics with respect to the taurocholate concentration, an apparent Km of 67 microM and Vmax of 0.30 nmol/mg protein/20 sec were calculated.

Background: Developmental changes in passive bile salt absorption may alter the enterohepatic circulation., Methods: 14-, 21-, and 40-day-old anesthetized male Sprague-Dawley rats were studied. Jejunum and ileum were isolated, cannulated, and injected or perfused with a taurocholate, [3H]taurocholate, and nonabsorbable marker solution. Bile was collected., Results: Using bolus injection, jejunal taurocholate absorption rates and total taurocholate absorption were nonsaturable, linearly related to taurocholate dose, and decreased from 14 days (1.62 nmol.cm-1.min-1) to 21 days (1.05 nmol.cm-1.min-1) and 40 days (0.54 nmol.cm-1.min-1). While total taurocholate absorption decreased (14 days, 52.4%; 21 days, 43.7%; 40 days, 30.5%), hepatic taurocholate clearance increased (14 days, 18.2%; 21 days, 23.7%; 40 days, 37.3%). Hepatic taurocholate clearance was saturated only at 14 days. Using jejunal perfusion, total taurocholate absorption (14 days, 62.0%; 21 days, 43.1%; 40 days, 45.3%) and taurocholate absorption rate decreased with age (14 days, 941.13 nmol.cm-2.min-1 per micromole of taurocholate; 21 days, 411.28 nmol.cm-2.min-1 per micromole of taurocholate; 40 days, 334.50 nmol.cm-2.min-1 per micromole of taurocholate)., Conclusions: Passive jejunal bile salt absorption and decreased hepatic bile salt clearance could account for the low intraluminal and high serum bile salt levels observed in immature animals and in human neonates.

Although bile acid malabsorption (BAM) in post-cholecystectomy diarrhea (PCD) is a well-known clinical condition, its true etiopathogenetic role is not entirely clear. The SeHCAT (23-selena-25-homotaurocholic acid) test, a simple and reliable BAM test, was performed in 33 cholecystectomized patients, 26 with chronic diarrhea. The test revealed a marked degree of BAM in 25/26 cases. Cholestyramine in doses of 2-12 g/day was effective in 23/25, ineffective in two, and was not tolerated in one patient. When treatment was suspended, diarrhea recurred in nine, whereas bowel habit remained regular in 60%, with brief sporadic episodes of diarrhea in the other cases. The SeHCAT test was repeated in 11 cases after cholestyramine treatment interruption, and revealed the normalization of parameters in two patients and an improvement in three. We conclude that BAM is an important etiopathogenetic factor in PCD that responds favorably to cholestyramine. In 60% of the cases, it resolved diarrhea definitively, although without eliminating BAM in all cases: this suggests that existence of other factors associated with BAM. The SeHCAT test is essential for a differential diagnosis between PCD and the irritable bowel syndrome.

The availability of the gamma-labelled bile acid 75SeHCAT, that allows a non-invasive assessment of the enterohepatic circulation of bile acids, has prompted in the last 10 years the implementation of several studies involving wide series of normal subjects and patients with various organic and functional bowel disorders. The clinical indications for performing a SeHCAT test have been clearly defined: the test can identify with high accuracy, in the setting of the irritable bowel syndrome, the patients with bile acid malabsorption that can be confidently and successfully treated with cholestyramine; it can also assess whether, and to what extent, the diarrhoea presenting in patients with intestinal organic disorders is due to bile acid malabsorption, permitting an optimal therapeutic strategy to be designed. The parameters of the hepatic handling of SeHCAT after bolus intravenous administration have been characterized in normals, and studies on various chronic hepatic disorders are now in progress. Interesting results are emerging from studies performed in patients with chronic non-obstructive cholestatic disease, where a specific defect in the excretion rate of SeHCAT is present: these studies may cast more light on the abnormalities of bile secretion and on the mechanism of action of drugs used to treat this condition, forming the rationale for the use of intravenous SeHCAT for hepatobiliary dynamic scintigraphy as a sophisticated liver function test. In conclusion, the SeHCAT test has become an important diagnostic tool for the gastroenterologist studying the diarrhoea, and awaits more studies to be used also by the hepatologist. The relatively long physical half-life of 75Se (180 days), preventing a wider use of the test, could theoretically be overcome by the synthesis of a similar gamma-labelled bile acid with a shorter half-life.

Hepatocyte dysfunction eventually results in the loss of canalicular bile formation. Without canalicular flow, intestinal bile acid may originate from plasma by reverse transport. Anhepatic rats with preserved intestinal function permit evaluation of such transport. In the present study, plasma taurocholate clearance was markedly decreased in anhepatic rats. The relative proportion of free cholate increased with time. Peripheral tissues contained virtually only cleared taurocholate, but the intestinal contents were mainly free cholate. This indicates the intestinal contents as the source of the plasma cholate and shows an equilibrium between intestinal and plasma bile acid even without bile flow. The enteral administration of an anion exchange resin to anhepatic rats increased intestinal bile acid recovery and decreased the bile acid recovery in tissue. Plasma bile acid concentration was decreased and fractional loss increased threefold, confirming the anhepatic plasma-intestine bile acid equilibrium. However, the enhanced plasma clearance produced by the resin was less than 1% of the fractional loss found in the intact rat. These data show a very limited bile acid flux between intestine and plasma without bile flow, which could be modestly influenced by an intestinal bile acid sequestrant.