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Background and Aims: Cholestasis is characterized by intrahepatic accumulation of bile constituents, including bile acids (BAs), which promote liver damage. The apical sodium-dependent BA transporter (ASBT) plays an important role in BA reabsorption and signaling in ileum, bile ducts, and kidneys. Our aim was to investigate the pharmacokinetics and pharmacological activity of A3907, an oral and systemically available ASBT inhibitor in experimental mouse models of cholestasis. In addition, the tolerability, pharmacokinetics, and pharmacodynamics of A3907 were examined in healthy humans., Approach and Results: A3907 was a potent and selective ASBT inhibitor in vitro. In rodents, orally administered A3907 distributed to the ASBT-expressing organs, that is, ileum, liver, and kidneys, and dose dependently increased fecal BA excretion. A3907 improved biochemical, histological, and molecular markers of liver and bile duct injury in Mdr2-/- mice and also had direct protective effects on rat cholangiocytes exposed to cytotoxic BA concentrations in vitro . In bile duct ligated mice, A3907 increased urinary BA elimination, reduced serum BA levels, and prevented body weight loss, while improving markers of liver injury. A3907 was well tolerated and demonstrated target engagement in healthy volunteers. Plasma exposure of A3907 in humans was within the range of systemic concentrations that achieved therapeutic efficacy in mouse., Conclusions: The systemic ASBT inhibitor A3907 improved experimental cholestatic disease by targeting ASBT function at the intestinal, liver, and kidney levels, resulting in marked clearance of circulating BAs and liver protection. A3907 is well tolerated in humans, supporting further clinical development for the treatment of cholestatic liver diseases., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Mutations of the bile salt export pump (BSEP) or the multidrug resistance P-glycoprotein 3 (MDR3) are linked to impaired bile salt homeostasis and lead to progressive familial intrahepatic cholestasis (PFIC)-2 and -3, respectively. The regulation of bile salt transporters in PFIC is not known. Expression of hepatobiliary transporters in livers of ten patients with a PFIC phenotype was studied by quantitative reverse transcription polymerase chain reaction, Western blotting, and immunofluorescence microscopy. PFIC was diagnosed by clinical and laboratory findings. All patients could be assigned to PFIC-2 or PFIC-3 by the use of BSEP- and MDR3-specific antibodies and by MDR3 gene-sequencing. Whereas in all PFIC-2 patients, BSEP immunoreactivity was absent from the canalicular membrane, in three PFIC-3 livers, canalicular MDR3 immunoreactivity was detectable. Serum bile salts were elevated to 276 +/- 233 and to 221 +/- 109 micromol/L in PFIC-2 and PFIC-3, respectively. Organic anion transporting polypeptide OATP1B1, OATP1B3, and MRP2 mRNA and protein levels were reduced, whereas sodium taurocholate cotransporting polypeptide (NTCP) was only reduced at the protein level, suggesting a posttranscriptional NTCP regulation. Whereas MRP3 mRNA and protein were not significantly altered, MRP4 messenger RNA and protein were significantly increased in PFIC. In conclusion, PFIC-2 may be reliably diagnosed by immunofluorescence, whereas the diagnosis of PFIC-3 requires gene-sequencing. Several mechanisms may contribute to elevated plasma bile salts in PFIC: reduced bile salt uptake via NTCP, OATP1B1, and OATP1B3, decreased BSEP-dependent secretion into bile, and increased transport back into plasma by MRP4. Upregulation of MRP4, but not of MRP3, might represent an important escape mechanism for bile salt extrusion in PFIC.

Cholestatic liver injury is associated not only with accumulation of bile acids but also with activation of proinflammatory cytokines. Common bile duct ligation (CBDL) induces sustained downregulation of the Na(+)/taurocholate cotransporter (Ntcp) in rodent liver. Although repression of Ntcp during endotoxemia is cytokine mediated, it is unclear whether inflammatory cytokines contribute to this downregulation in obstructive cholestasis. Cytokine inactivation in CBDL rats and mice was either performed directly with tumor necrosis factor alpha (etanercept) or interleukin 1 beta inactivation (anakinra/AMG 719) or indirectly Kupffer cell depletion via intraperitoneal administration of liposome-encapsulated dichloromethylene bisphosphonate. Protein and messenger RNA (mRNA) expression of Ntcp and short heterodimer partner (SHP) were analyzed via Western and Northern blotting. Key regulators of Ntcp (hepatocyte nuclear factor 1 alpha [HNF-1alpha], HNF-4alpha, retinoid X receptor alpha [RXRalpha]:retinoic acid receptor alpha [RARalpha]) were studied via electrophoretic mobility shift analysis and nuclear Western blot analysis. Both methods of cytokine inactivation failed to maintain Ntcp protein or mRNA expression within 3 days after CBDL in either rats or mice (20%-40% of sham controls), while SHP mRNA expression increased three- to five-fold. Decreased nuclear HNF-1alpha and HNF-4alpha protein levels (45% and 60% of sham controls, respectively) and HNF-1alpha binding activity (32% of sham controls) were not restored during cytokine inactivation after CBDL, indicating cytokine-independent mechanisms of Ntcp regulation. RXRalpha:RARalpha binding remained unchanged in all experimental conditions. In conclusion, during obstructive cholestasis accumulating bile acids per se, without major contribution of cytokines, leads to downregulation of Ntcp via repression of HNF-1alpha and HNF-4alpha.

Ursodeoxycholic acid (UDCA) is used in the treatment of cholestatic liver diseases, but its mechanism of action is not yet well defined. The aim of this study was to explore the protective mechanisms of the taurine-conjugate of UDCA (tauroursodeoxycholic acid [TUDCA]) against glycochenodeoxycholic acid (GCDCA)-induced apoptosis in primary cultures of rat hepatocytes. Hepatocytes were exposed to GCDCA, TUDCA, the glyco-conjugate of UDCA (GUDCA), and TCDCA. The phosphatidylinositol-3 kinase pathway (PI3K) and nuclear factor-kappaB were inhibited using LY 294002 and adenoviral overexpression of dominant-negative IkappaB, respectively. The role of p38 and extracellular signal-regulated protein kinase mitogen-activated protein kinase (MAPK) pathways were investigated using the inhibitors SB 203580 and U0 126 and Western blot analysis. Transcription was blocked by actinomycin-D. Apoptosis was determined by measuring caspase-3, -9, and -8 activity using fluorimetric enzyme detection, Western blot analysis, immunocytochemistry, and nuclear morphological analysis. Our results demonstrated that uptake of GCDCA is needed for apoptosis induction. TUDCA, but not TCDCA and GUDCA, rapidly inhibited, but did not delay, apoptosis at all time points tested. However, the protective effect of TUDCA was independent of its inhibition of caspase-8. Up to 6 hours of preincubation with TUDCA before addition of GCDCA clearly decreased GCDCA-induced apoptosis. At up to 1.5 hours after exposure with GCDCA, the addition of TUDCA was still protective. This protection was dependent on activation of p38, ERK MAPK, and PI3K pathways, but independent of competition on the cell membrane, NF-kappaB activation, and transcription. In conclusion, TUDCA contributes to the protection against GCDCA-induced mitochondria-controlled apoptosis by activating survival pathways.

Hepatobiliary transporters are down-regulated in toxic and cholestatic liver injury. Cytokines such as tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta) are attributed to mediate this regulation, but their particular contribution in vivo is still unknown. Thus, we studied the molecular mechanisms by which Ntcp, Oatp1, Oatp2, and Mrp2 are regulated by proinflammatory cytokines during liver injury. Rats were injected intraperitoneally with either carbon tetrachloride or endotoxin. Inactivation of TNF-alpha and IL-1 beta was achieved by repetitive intraperitoneal injection of etanercept and anakinra, respectively. Messenger RNA (mRNA) levels of transporters and binding activities as well as nuclear protein levels of Ntcp, Oatp2, and Mrp2 transactivators were determined 20 to 24 hours later. In contrast to IL-1 beta, TNF-alpha inactivation alone fully prevented down-regulation of Ntcp, Oatp1, and Oatp2 mRNA as well as reduced binding activity of hepatocyte nuclear factor 1 (HNF-1) in CCl(4)-induced toxic injury. In endotoxemia, down-regulation of Mrp2, and partially in case of Ntcp, could be prevented by IL-1 beta but not TNF-alpha blockade. However, inactivation of either cytokine led to preservation of HNF1 and partially of retinoid X receptor/retinoic acid receptor (RXR/RAR) binding activity. No effect of anticytokines was seen on pregnane X receptor (PXR) and constitutive androstane receptor (CAR) binding activity as well as nuclear protein mass. In conclusion, TNF-alpha represents the master cytokine responsible for HNF1-dependent down-regulation of Ntcp, Oatp1, and Oatp2 in CCl(4)-induced toxic liver injury. IL-1 beta predominates in a complex signaling network of Ntcp and Mrp2 regulation in cholestatic liver injury. In contrast to in vitro studies, HNF1 and RXR/RAR-independent mechanisms appear to be more important in regulation of Mrp2 and Ntcp gene expression in endotoxemia.

Hepatic uptake of organic anions, including bile salts, is mediated by members of the organic anion-transporting polypeptide (Oatp) family. In rat liver, Oatp1 (Slc21a1), Oatp2 (Slc21a5), and Oatp4 (Slca10) are expressed at the basolateral membrane of hepatocytes and may be differentially regulated under pathophysiologic conditions such as cholestasis. The aim of this study was to determine the effects of cholic acid (CA) and ursodeoxycholic acid (UDCA) on the expression of Oatp4 compared with Ntcp, Oatp1, and Oatp2. Wistar rats were fed with CA (0.5%) or both CA (0.5%) and UDCA (0.25%) for 3 weeks. Oatp expression was studied by Northern and Western blot analysis as well as immunofluorescence analysis. Transport function was compared measuring biliary secretion of (14)C-CA and (14)C-taurocholic acid (TCA). In CA-fed animals, biliary secretion of (14)C-CA and (14)C-TCA was markedly delayed over 40 minutes compared with controls. Accordingly, Oatp4 protein was significantly down-regulated in CA-fed animals together with Oatp1 and Ntcp. Cofeeding of CA plus UDCA prevented the impairment of (14)C-CA and (14)C-TCA secretion and the down-regulation of Oatp4. Oatp4 messenger RNA (mRNA) levels did not differ significantly between bile salt-fed groups, suggesting a posttranscriptional effect of CA on Oatp4 expression. In contrast to Oatp1 and Oatp4, Oatp2 protein expression was increased by CA feeding, indicating a differential regulation of Oatp transporters. In conclusion, we show that CA feeding may cause cholestasis associated with a posttranscriptional down-regulation of Oatp4. UDCA may prevent impairment of hepatic function by restoring hepatic transporter expression.

To maintain ongoing vectorial bile secretion, hepatocytes localize distinct transport systems at their basolateral and canalicular membrane domains. Here we compare the expression of the basolateral Na(+)-taurocholate cotransporter (Ntcp) and organic anion transporting polypeptides 1 and 2 (Oatp1, Oatp2) and the canalicular bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2) in primary cultured rat hepatocytes. During 72 hours of culturing time the messenger RNA (mRNA) and protein levels of Ntcp and Oatp1 decreased in parallel to 2% to 7% of initial values at 3 hours. Although Oatp2 mRNA exhibited a similar down-regulation to 4%, Oatp2 protein and function were maintained at 25% to 47% of initial values. Furthermore, Bsep and Mrp2 protein levels were maintained at about 50%, while the Mrp2 mRNA showed a transient up-regulation to 154% at 24 and 48 hours. Also, induction of Mrp1 mRNA and protein was observed starting after 24 hours. These results indicate transcriptional down-regulation or decreased mRNA stability of Ntcp and Oatp1, transcriptional and posttranslational regulation of Oatp2, Bsep, and Mrp2 and transcriptional up-regulation of Mrp1 in primary cultured hepatocytes. Furthermore, and most importantly, the observed changes in transporter expression closely resemble the altered transporter phenotypes of cholestatic and proliferating hepatocytes in vivo, thus indicating that primary cultured hepatocytes acquire a cholestatic phenotype, and that the transporter expression might be a suitable differentiation marker for maintenance of hepatocytes in vitro.

We characterized expression and activity of the bile salt transporters Na(+)/taurocholate (TC) cotransporting polypeptide (Ntcp), and bile salt export pump (Bsep), and the expression of organic anion transporting polypeptides 1 and 2 (Oatp1 and 2) and multidrug resistance associated protein-2 (Mrp2) in pregnancy and throughout lactation in rats. The V(max) for Na(+)/TC cotransport in basolateral liver plasma membrane was increased 1.7-fold in 2 days postpartum relative to control and pregnant rats. This correlated well with an increase in Ntcp messenger RNA (mRNA) and a 2-fold increase in Ntcp protein. Ntcp mRNA remained significantly elevated until 14 days postpartum but had begun to decline by 21 days postpartum. The maximal secretory rate (nmol/min/g liver) for TC in the single pass isolated perfused liver was also increased by 10%, 31%, and 24% at 2, 14, and 21 days postpartum and correlated with increased expression of Ntcp and Bsep mRNA and protein. Infusion of ovine prolactin (oPRL) to ovariectomized rats increased expression of both Ntcp and Bsep mRNA and protein. These data indicate a coordinate increased expression of bile salt transporters postpartum and by PRL. Mrp2 mRNA was stable in pregnancy and postpartum, whereas Mrp2 protein expression decreased significantly in pregnancy, but returned to control levels postpartum. Organic anion transporting polypeptide 2 (Oatp2) mRNA was decreased in pregnancy and increased postpartum, but changes in Oatp2 protein were not significant. Oatp1 mRNA and protein were unchanged in pregnancy and postpartum.

The Na+-taurocholate cotransport polypeptide (ntcp) is the primary transporter for the uptake of bile acids in the liver. The second messenger adenosine 3':5'-cyclic monophosphate (cAMP) rapidly increases ntcp protein concentration in the plasma membrane, yet the mechanism is unknown. To investigate this, HepG2 cells were transiently transfected with a carboxy-terminal-tagged green fluorescence protein (GFP) conjugate of ntcp, and then examined by confocal video microscopy. Transporter activity was directly assayed with 3H-taurocholic acid (TC) scintigraphy. ntcp-GFP targeted to the plasma membrane in transfected cells, and the conjugate protein transported 3H-TC as effectively as unmodified rat ntcp. Stimulation of ntcp-GFP cells with cAMP increased GFP fluorescence in the plasma membrane by 40% (P <.0001) within 2.5 minutes and by 55% within 10 minutes. Similarly, cAMP increased transport of bile acids by 30%. Cytochalasin D, an inhibitor of microfilaments, did not prevent ntcp-GFP from targeting to the plasma membrane, but completely abolished the increase in GFP fluorescence seen in response to cAMP. In contrast, the microtubule inhibitor, nocodazole, prevented development of membrane fluorescence in 48 (96%) of 50 cells. Cells regained plasma membrane fluorescence within 2 hours after nocodazole removal. These findings suggest that targeting of ntcp to the plasma membrane consists of 2 steps: 1) delivery of ntcp to the region of the plasma membrane via microtubules; and 2) insertion of ntcp into the plasma membrane, in a microfilament- and cAMP-sensitive fashion.

We have previously shown that cholestasis and bile acids inhibit 2', 5' oligoadenylate synthetase (OAS) activity in the liver and in primary hepatocyte cultures. Here, we assessed the influence of bile acids on interferon (IFN) pathway activation in three hepatoma cell lines. In HepG2 cells, bile acids (100-200 micromol/L) inhibited IFN-induced 2',5' OAS activity to an extent depending on their surface activity index. In Western blot analysis, IFN-induced expression of two major antiviral proteins, MxA and OAS p100, was reduced by 54% +/- 8% and 44% +/- 12%, respectively, when cells were preincubated for 4 hours with 100 micromol/L chenodeoxycholic acid (CDCA). In the same conditions, CDCA did not modify the IFN-induced signal transducers and activators of transcription (STAT)s tyrosine phosphorylation. In contrast, it reduced IFN-induced MxA promoter activity by 60%. The inhibitory effect of CDCA was not mediated by a 4beta-phorbol 12beta-myristate 13alpha-acetate (PMA)-sensitive protein kinase C (PKC)-dependent pathway. Finally, using CHO cells stably expressing a functional human bile acid carrier (Na+-dependent taurocholate cotransporting polypeptide [NTCP]), we found that bile acid inhibition of the IFN pathway occurred in the range of more physiological concentrations (12-50 micromol/L). In summary, our results provide strong evidence that bile acids inhibit the induction of proteins involved in the antiviral activity of IFN. This might partly explain the lack of responsiveness to IFN therapy in some patients with advanced chronic viral liver diseases.

Na+/taurocholate (Na+/TC) cotransport in hepatocytes is mediated primarily by Na+/TC cotransporting polypeptide (Ntcp), and cyclic adenosine monophosphate (cAMP) stimulates Na+/TC cotransport by inducing translocation of Ntcp to the plasma membrane. The aim of the present study was to determine if Ntcp is a phosphoprotein and if cAMP alters Ntcp phosphorylation. Freshly prepared hepatocytes from rat livers were incubated with carrier-free 32PO4 for 2 hours, followed by incubation with 10 micromol/L 8-chlorophenylthio adenosin 3':5'-cyclic monophosphate (CPT-cAMP) for 15 minutes. Subcellular fractions isolated from 32P-labeled hepatocytes were subjected to immunoprecipitation using Ntcp antibody, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography to determine if Ntcp is phosphorylated. Ntcp immunoprecipitated from plasma membranes isolated from nonlabeled hepatocytes was subjected to immunoblot analysis using anti-phosphoserine, anti-phosphothreonine, or anti-phosphotyrosine antibody to determine whether Ntcp is a serine, threonine, or tyrosine phosphoprotein. Hepatocytes were loaded with bis-(2-amino-5-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid (MAPTA), a Ca2+ buffering agent, and the effect of CPT-cAMP on TC uptake, cytosolic [Ca2+], and ntcp phosphorylation and translocation was determined. In addition, the effect of cAMP on protein phosphatases 1 and 2A (PP1/2A) was determined in homogenates and plasma membranes obtained from CPT-cAMP-treated hepatocytes. Phosphorylation study showed that phosphorylated Ntcp is detectable in plasma membranes, and cAMP treatment resulted in dephosphorylation of Ntcp. Immunoblot analysis with phosphoamino antibodies revealed that Ntcp is a serine/threonine, and not a tyrosine, phosphoprotein, and cAMP inhibited both serine and threonine phosphorylation. In MAPTA-loaded hepatocytes, CPT-cAMP failed to stimulate TC uptake, failed to increase cytosolic [Ca2+], and failed to induce translocation and dephosphorylation of Ntcp. cAMP did not alter the activity of PP1/2A in either homogenates or in plasma membranes. Taken together, these results suggest that Ntcp is a serine/threonine phosphoprotein and is dephosphorylated by cAMP treatment. Activation of PP1/2A is not involved in cAMP-mediated dephosphorylation of Ntcp. Both translocation and dephosphorylation of Ntcp may be involved in the regulation of hepatic Na+/TC cotransport.

The regulatory responses of bile acid (BA) transport in the terminal ileum to perturbations in BA homeostasis are complex, and conflicting results have been reported by different investigators. These studies were designed to examine the response of this system to a reduction in ileal bile salt concentrations at both a functional and molecular level. Common bile duct ligation (BDL) or feeding of a novel bile acid-binding compound, GT31-104HB, for 7 days were used to reduce ileal apical membrane bile salt flux. Apical bile acid transport function was assessed by examining sodium-dependent uptake of [3H]-taurocholate (TC) into brush border membrane vesicles (BBMV). Expression of the apical sodium-dependent bile acid transporter (ASBT) and the ileal lipid-binding protein (ILBP) were assessed by Western blotting with quantitation using [125I]-labeled secondary antibody and a phosphorimager. Neither common BDL nor intestinal sequestration of BA led to a change in ileal bile acid transport function or the expression of the ASBT or the ILBP. These results indicate that a reduction in presentation of bile salts to the apical surface of the terminal ileum does not modulate the expression of the genes involved in their transport.

To assess the effects of cholestasis during pregnancy on fetal and neonatal mRNA expression, protein mass, and function of the Na+/taurocholate cotransporting polypeptide (Ntcp), common bile duct ligation (BDL) was performed in pregnant rats on day 14 of pregnancy (maternal cholestasis [MC] group), and livers were harvested at days 20 and 21 of fetal life, as well as at days 4, 7, 14, 21, and 28 after birth. Sham-operated rats and their litters were used as controls. Ntcp steady-state mRNA levels, protein mass, and function were determined by Northern blotting, immunoblotting, and taurocholate (TC) transport studies in isolated short-term cultured hepatocytes, respectively. In addition, protein mass and function of the organic anion transporting polypeptide (Oatp1), another sinusoidal bile acid transporter, were studied at 4 weeks of age. The majority of pregnant cholestatic rats (94%) were able to carry pregnancy to term. Body and liver weights of the offspring from the MC group were lower than those from sham-operated animals at all postnatal time points. Ntcp steady-state mRNA levels, protein mass, and function were unaffected by MC. The ontogenic pattern of expression was identical in offspring from MC and controls with detection of the Ntcp mRNA at day 21 of fetal life. There was a significant increase in mRNA postnatally, reaching adult levels by 7 days of age. Protein mass and function of Ntcp as well as of Oatp1 were similar in offspring from MC and control groups at 4 weeks of age. In conclusion, maternal obstructive cholestasis during the last third of pregnancy does not affect the fetal/neonatal expression of the basolateral bile acid transporters, Ntcp and Oatp1. This suggests that the impaired bile acid excretion described in this experimental model is not related to altered uptake of bile acids in the affected neonate.

To test the hypothesis that impaired activity of the bile canalicular organic anion transporting system mrp2 (cmoat) is a key event in the etiology of 17alpha-ethinylestradiol (EE)-induced intrahepatic cholestasis in rats, EE (5 mg/kg subcutaneously daily) was administered to male normal Wistar (NW) and mrp2-deficient Groningen Yellow/Transport-deficient Wistar (GY/TR-) rats. Elevated plasma bilirubin levels in GY/TR- rats increased upon EE-treatment from 65 +/- 8.4 micromol/L to 183 +/- 22.7 micromol/L within 3 days, whereas bilirubin levels remained unaffected in NW rats. Biliary bilirubin secretion was 1.5-fold increased in NW rats but remained unaltered in GY/TR- rats. Plasma bile salt concentrations remained unchanged in both strains, although hepatic levels of the sinusoidal Na+-taurocholate cotransporting protein (ntcp) were markedly reduced. Biliary secretion of endogenous bile salt was not affected in either strain. A clear reduction of mrp2 levels in liver plasma membranes of NW rats was found after 3 days of treatment. The bile salt-independent fraction of bile flow (BSIF) was reduced from 2.6 to 2.0 microL/min/100 g body weight in NW rats with a concomitant 62% reduction of biliary glutathione secretion. The absence of mrp2 and biliary glutathione in GY/TR- rats did not prevent induction of EE-cholestasis; a similar absolute reduction of BSIF, i.e., from 1.1 to 0.6 microL/min/100 g bodyweight, was found in these animals. EE treatment caused a reduction of the maximal biliary secretory rate (S(RM)) of the mrp2 substrate, dibromosulphthalein (DBSP), from 1,040 to 695 nmol/min/100 g body weight (-38%) in NW rats and from 615 to 327 nmol/min/100 g body weight (-46%) in GY/TR- rats. These results demonstrate that inhibition of mrp2 activity and/or biliary glutathione secretion is not the main cause of EE-induced cholestasis in rats. The data indicate that alternative pathways exist for the biliary secretion of bilirubin and related organic anions that are also affected by EE.

Although bile salts are toxic to the liver at high plasma concentrations, the effects of physiological concentrations of bile salts on normal hepatic function are poorly understood. We examined the effect of taurocholate (TC) on the basolateral uptake of [3H]TC in WIF-B cells, a hybrid cell line stably exhibiting in vitro the structural and functional polarity of hepatocytes. Cells were grown in the absence or presence of TC (50 micromol/L) over 12 days, and then incubated with [3H]TC concentrations ranging from 1 to 250 micromol/L. For both control and TC-grown cells, uptake of [3H]TC was linear over 2 minutes. In control cells, the Km for [3H]TC Na+-dependent uptake over 1 minute was 6 +/- 5 micromol/L, and the Vmax was 45 +/- 6 pmol TC/mg protein/min (+/- SEM). TC-grown cells exhibited no significant change in Km but showed a doubling of Vmax to 87 +/- 6 pmol TC/mg protein/min (P < .005). In both control and TC-grown cells, maximal uptake of [3H]TC occurred following 10 to 12 days in culture, with TC-grown cells consistently showing greater rates of [3H]TC uptake from 4 to 14 days in culture. Western blots immunostained for the basolateral Na+-dependent plasma membrane protein, ntcp, revealed the appropriate approximately 50-kd band in control and TC-grown cells, and confocal immunofluorescence microscopy demonstrated staining along the basolateral plasma membrane. Northern blots hybridized with a cDNA probe directed against ntcp indicated a modest TC-induced increase in mRNA levels. Reverse-transcriptase polymerase chain reaction (RT-PCR) using RNA isolated from WIF-B cells and oligonucleotide primers specific for rat ntcp or human NTCP transcripts revealed only the presence of the rat ntcp transcript. We conclude that bile salts, at concentrations normally found in mammalian portal blood, may be capable of promoting enhanced hepatocellular bile salt uptake via an increase in basolateral Na+-dependent plasma membrane transport capacity.

The Na+-dependent bile salt uptake systems Ntcp (rodents) and NTCP (human), and the Na+-independent organic anion transporters oatpl (rat) and OATP (human) mediate sinusoidal uptake of a variety of amphipathic organic compounds into hepatocytes. Their properties indicate that an overall hepatic clearance of albumin-bound compounds is mediated by a limited number of multispecific transporters with partially overlapping substrate specificities.

The recent cloning of a human sodium-dependent bile acid transporter (NTCP) permits analysis of its expression in human liver disease and investigation of potential primary defects in its expression. NTCP from normal human liver (NHL) was first characterized in detail. Northern blotting of RNA from NHL revealed a 1.8-kb NTCP transcript. Western blotting of crude NHL plasma membranes using a carboxyterminal antipeptide antibody showed that NTCP is a 39-kd polypeptide that is N-glycosylated to a final molecular weight of 56 kd. Indirect immunofluorescent analysis of NHL sections indicated that the NTCP protein is expressed on the basolateral surface of hepatocytes. We hypothesized that the clinical phenotype of a defect in NTCP might be hypercholanemia in the relative absence of liver disease. Accordingly, the coding region of the NTCP gene of two children with this phenotype was sequenced after reverse transcription/polymerase chain reaction (RT/PCR) amplification. No primary defects in the deduced NTCP amino acid sequence were found. Despite the extremely high serum bile salt levels (235 and 126 micromol/L) in these two patients, NTCP messenger RNA (mRNA) and protein expression were quantitatively normal, in contrast to the published observations in a rat model of cholestasis secondary to common bile duct ligation. Hepatic steady-state NTCP mRNA levels in a group of 23 pre- and postportoenterostomy biliary atresia patients were inversely related to total bilirubin, indicating that extrahepatic bile duct obstruction leads to down-regulation of NTCP mRNA levels, similar to that observed in rat common bile duct ligation. Therefore the lack of down-regulation in the two patients with hypercholanemia indicates that elevated serum bile salts are not sufficient to down-regulate NTCP expression, these two patients have abnormal responses to hypercholanemia, or these two patients have a defect in a gene other than NTCP that influences hepatic clearance of bile salts.

Expression and function of the hepatic Na+/taurocholate cotransporter (ntcp) are down-regulated in several models of experimental cholestasis. To test whether retention and/or depletion of biliary constituents are involved in ntcp regulation, ntcp expression was quantified in several animal models with altered levels of these constituents. In choledochocaval fistula rats (CCF) (retention model), ntcp mRNA expression specifically declined after 1 and 3 days by 76 +/- 4% (P < .005) and 31 +/- 9% (P < .05), respectively, returning to control levels by 7 days. However, protein expression as assessed by Western blotting remained unchanged for up to 7 days of CCF. In rats with bile fistulas (depletion model) for 0.5, 1, 2, 4, and 7 days, both ntcp protein and mRNA expression remained unaltered. Infusion of either taurocholate or taurochenodeoxycholate for 12 hours also did not effect ntcp mRNA expression in intact animals, probably because of its inability to increase serum and intrahepatic bile acid levels. In rats with selective bile duct ligation (SBDL), ntcp mRNA levels were down-regulated by 40 +/- 10% (P < .05) only after 12 and 24 hours in ligated lobes, and mRNA levels returned to control values in these lobes after 2 and 4 days. ntcp mRNA expression remained unchanged in the nonobstructed lobes at any time. When data from CCF and SBDL rats were combined, serum bile acids correlated linearly with ntcp mRNA (r = .62, P < .0005) over a 0 to 110-micromol/L range. Our results indicate that ntcp is constitutively expressed and remains uneffected by either depletion or increased flux of biliary constituents. However, retention of biliary constituents results in rapid down-regulation of ntcp mRNA, consistent with the concept that hepatocytes may be protected from bile acid toxicity during cholestasis by this mechanism.

The liver recovers bile acids from the portal circulation primarily via an active process that is dependent on sodium ions. Hepatocytes lose the ability to transport bile acids in culture, and, in liver-derived permanent cell lines, this ability is severely reduced or absent. To study the importance of bile acids in regulating liver-specific functions (e.g., cellular bile acid and cholesterol metabolism), we have re-established active bile acid transport in cultured cells. The complementary DNA (cDNA) encoding the rat sodium/taurocholate cotransporting polypeptide (ntcp) was placed under the control of a cytomegalovirus promoter and transfected into the rat hepatoma cell line, McArdle RH-7777. Transfected cells were screened for the ability to take up [3H]-taurocholate. Clones that displayed the ability to take up taurocholate were expanded (designated McNtcp) and further characterized. The apparent Michaelis constant (Km) for taurocholate uptake was similar among the different clones. The observed maximum velocity (Vmax), however, differed and was positively correlated with the abundance of recombinant ntcp messenger RNA (mRNA). The highest level of taurocholate uptake activity observed in McNtcp cells was comparable with that of freshly isolated hepatocytes. Efflux of accumulated taurocholate from McNtcp cells proceeded in a manner similar to primary hepatocytes, indicating that McArdle RH-7777 cells have retained the ability to secrete bile acids. Moreover, taurocholate uptake in McNtcp cells was inhibited by other bile acid species. Based on the observed kinetic parameters, the reconstituted McArdle RH-7777 cells mimic the ability of primary hepatocytes to transport bile acids.

Bile acids are taken up into human liver by Na+-dependent and Na+-independent transport mechanisms. In hepatocarcinogenesis, numerous liver-specific functions are lost and the uptake of organic anions is markedly reduced. We have investigated the molecular and functional derangements of bile acid transport in the human hepatoblastoma cell line HepG2. Uptake of [3H]-taurocholic acid was saturable and entirely Na+ independent, with the kinetic characteristics of the human liver organic anion transporting polypeptide (OATP). OATP, but not the Na+-dependent bile acid transporter (Na+-taurocholate-cotransporting polypeptide [NTCP]), was detectable by reverse-transcription polymerase chain reaction (RT-PCR) analysis of HepG2 RNA. The level of OATP expression in HepG2 cells was determined by Northern blot analysis and was found to be 40% in comparison with normal liver. Transfection of an OATP-derived phosphorothioate (PTO)-antisense oligonucleotide into HepG2 cells resulted in 77% inhibition of temperature-dependent bile acid uptake. Injection of HepG2 messenger RNA (mRNA) into Xenopus laevis oocytes significantly stimulated Na+-independent taurocholate uptake, indicating the expression of a bile acid transport protein. We conclude that bile acid uptake into human hepatoblastoma HepG2 cells is mediated by the multi-specific organic anion transporting polypeptide OATP. Therapeutic strategies employing bile acid-derived cytostatic agents for the treatment of hepatocellular carcinomas may therefore depend upon the expression of the Na+-independent bile acid transporter OATP in hepatic malignancies.

We investigated the molecular mechanism underlying the progressive loss of Na(+)-dependent bile salt uptake in primary cultured rat hepatocytes. A specific cDNA probe was used to quantitate the levels of mRNA encoding the Na(+)-taurocholate-cotransporting polypeptide at various culture times. Hepatocytes were cultured on collagen in the presence of insulin (10(-7) mol/L), dexamethasone (10(-7) mol/L) and 10% fetal calf serum for up to 72 hr. During this time period the dissociation constant of Na(+)-dependent taurocholate uptake remained stable (19 to 39 mumol/L), whereas the maximum velocity values decreased from 100% at 3 hr to 55%, 22% and 4% at 24, 48 and 72 hr, respectively. Concomitantly the levels of the Na(+)-taurocholate-cotransporting polypeptide mRNA also decreased from 100% at 3 hr to 41%, 24% and 4% at the later time points. In contrast, Northern hybridization with complementary DNA probes for three common housekeeping gene products revealed a 1.8- to 3.4-fold increase in the levels of mRNA encoding the alpha-subunit of the Na+K(+)-ATPase, beta-actin and glycerol-3-phosphate dehydrogenase. These data indicate that the loss of Na(+)-dependent bile salt uptake in primary cultures of rat hepatocytes is caused by decreased levels of its specific mRNA. Hence the studies further confirm that without specific measures (primary) cultured rat hepatocytes reverse their liver-specific phenotype to a more fetal pattern of gene expression.