Impaired Bile Flow Research Articles

The farnesoid X receptor controls gene expression in a ligand- and promoter-selective fashion.

Farnesoid X receptor (FXR) is a nuclear receptor for bile acids. Ligand activated-FXR regulates transcription of genes to allow feedback control of bile acid synthesis and secretion. There are five major bile acids in humans. We have previously demonstrated that lithocholate acts as an FXR antagonist, and here we show that the other four bile acids, chenodeoxycholate (CDCA), deoxycholate (DCA), cholate (CA), and ursodeoxycholate (UDCA), act as selective FXR agonists in a gene-specific fashion. In an in vitro coactivator association assay, CDCA fully activated FXR, whereas CA partially activated FXR and DCA and UDCA had negligible activities. Similar results were also obtained from a glutathione S-transferase pull-down assay in which only CDCA and the synthetic FXR agonist GW4064 significantly increased the interaction of SRC-1 with FXR. In FXR transactivation assays with a bile salt export pump (BSEP) promoter-driven luciferase construct, bile acids showed distinct abilities to activate the BSEP promoter: CDCA, DCA, CA, and UDCA increased luciferase activity by 25-, 20-, 18-, and 8-fold, respectively. Consistently, CDCA increased BSEP mRNA by 750-fold in HepG2 cells, whereas DCA, CA, and UDCA induced BSEP mRNA by 250-, 75-, and 15-fold, respectively. Despite the partial induction of BSEP mRNA, CA, DCA, and UDCA effectively repressed expression of cholesterol 7alpha-hydroxylase, another FXR target. We further showed that all four bile acids significantly increased FXR protein, suggesting the existence of an auto-regulatory loop in FXR signaling pathways. In conclusion, these results suggest that the binding of each bile acid results in a different FXR conformations, which in turn differentially regulates expression of individual FXR targets.

Genetic cholestasis, causes and consequences for hepatobiliary transport.

Bile salts take part in an efficient enterohepatic circulation in which most of the secreted bile salts are reclaimed by absorption in the terminal ileum. In the liver, the sodium-dependent taurocholate transporter at the basolateral (sinusoidal) membrane and the bile salt export pump at the canalicular membrane mediate hepatic uptake and hepatobiliary secretion of bile salts. Canalicular secretion is the driving force for the enterohepatic cycling of bile salts and most genetic diseases are caused by defects of canalicular secretion. Impairment of bile flow leads to adaptive changes in the expression of transporter proteins and enzymes of the cytochrome P-450 system involved in the metabolism of cholesterol and bile acids. Bile salts act as ligands for transcription factors. As such, they stimulate or inhibit the transcription of genes encoding transporters and enzymes involved in their own metabolism. Together these changes appear to serve mainly a hepatoprotective function. Progressive familial intrahepatic cholestasis (PFIC) results from mutations in various genes encoding hepatobiliary transport proteins. Mutations in the FIC1 gene cause relapsing or permanent cholestasis. The relapsing type of cholestasis is called benign recurrent intrahepatic cholestasis, the permanent type of cholestasis PFIC type 1. PFIC type 2 results from mutations in the bile salt export pump (BSEP) gene. This is associated with permanent cholestasis since birth. Serum gamma-glutamyltransferase (gamma-GT) activity is low to normal in PFIC types 1 and 2. Bile diversion procedures, causing a decreased bile salt pool, have a beneficial effect in a number of patients with these diseases. However, liver transplantation is often necessary. PFIC type 3 is caused by mutations in the MDR3 gene. MDR3 is a phospholipid translocator in the canalicular membrane. Because of the inability to secrete phospholipids, patients with PFIC type 3 produce bile acid-rich toxic bile that damages the intrahepatic bile ducts. Serum gamma-GT activity is elevated in these patients. Ursodeoxycholic acid therapy is useful for patients with a partial defect. Liver transplantation is a more definitive therapy for these patients.

Taurolithocholic Acid Exerts Cholestatic Effects via Phosphatidylinositol 3-Kinase-dependent Mechanisms in Perfused Rat Livers and Rat Hepatocyte Couplets

Taurolithocholic acid (TLCA) is a potent cholestatic agent. Our recent work suggested that TLCA impairs hepatobiliary exocytosis, insertion of transport proteins into apical hepatocyte membranes, and bile flow by protein kinase Cepsilon (PKCepsilon)-dependent mechanisms. Products of phosphatidylinositol 3-kinases (PI3K) stimulate PKCepsilon. We studied the role of PI3K for TLCA-induced cholestasis in isolated perfused rat liver (IPRL) and isolated rat hepatocyte couplets (IRHC). In IPRL, TLCA (10 micromol/liter) impaired bile flow by 51%, biliary secretion of horseradish peroxidase, a marker of vesicular exocytosis, by 46%, and the Mrp2 substrate, 2,4-dinitrophenyl-S-glutathione, by 95% and stimulated PI3K-dependent protein kinase B, a marker of PI3K activity, by 154% and PKCepsilon membrane binding by 23%. In IRHC, TLCA (2.5 micromol/liter) impaired canalicular secretion of the fluorescent bile acid, cholylglycylamido fluorescein, by 50%. The selective PI3K inhibitor, wortmannin (100 nmol/liter), and the anticholestatic bile acid tauroursodeoxycholic acid (TUDCA, 25 micromol/liter) independently and additively reversed the effects of TLCA on bile flow, exocytosis, organic anion secretion, PI3K-dependent protein kinase B activity, and PKCepsilon membrane binding in IPRL. Wortmannin also reversed impaired bile acid secretion in IRHC. These data strongly suggest that TLCA exerts cholestatic effects by PI3K- and PKCepsilon-dependent mechanisms that are reversed by tauroursodeoxycholic acid in a PI3K-independent way.

Cholestasis

In contrast with urine formation, bile flow is not dependent on hydrostatic forces, but driven by osmotic pressure of solutes secreted across the apical membrane of hepatocytes and bile duct...

Estradiol-17beta-D-glucuronide induces endocytic internalization of Bsep in rats.

Endocytic internalization of the multidrug resistance-associated protein 2 (Mrp2) was previously suggested to be involved in estradiol-17beta-D-glucuronide (E217G)-induced cholestasis. Here we evaluated in the rat whether a similar phenomenon occurs with the bile salt export pump (Bsep) and the ability of DBcAMP to prevent it. E217G (15 micromol/kg i.v.) impaired bile salt (BS) output and induced Bsep internalization, as assessed by confocal microscopy and Western blotting. Neither cholestasis nor Bsep internalization occurred in TR- rats lacking Mrp2. DBcAMP (20 micromol/kg i.v.) partially prevented the decrease in bile flow and BS output and substantially prevented E217G-induced Bsep internalization. In hepatocyte couplets, E217G (50 microM) diminished canalicular accumulation of a fluorescent BS and decreased Bsep-associated fluorescence in the canalicular membrane; DBcAMP (10 microM) fully prevented both effects. In conclusion, our results suggest that changes in Bsep localization are involved in E217G-induced impairment of bile flow and BS transport and that DBcAMP prevents this effect by stimulating insertion of canalicular transporter-containing vesicles. Mrp2 is required for E217G to induce its harmful effect.

Homocysteine Prevents Total Parenteral Nutrition (TPN)–Induced Cholestasis Without Changes in Hepatic Oxidative Stress in the Rat

The role of oxidative stress in total parenteral nutrition (TPN)-associated cholestasis with liver glutathione depletion was recently shown. The aims of this study were to test the appearance of cholestasis and oxidative stress during TPN, and the hypothesis that reducing oxidative stress with a precursor of glutathione (GSH), homocysteine, would restore bile flow. Three groups of rats (weight, 179-278 g) were studied: 1) D/aa group received dextrose and amino acids (3.4 g/d); 2) D/aa/L group received the same amount of amino acids, and lipids were added on an equicaloric basis (50 kcal/d) with a lowered amount of dextrose; and 3) a control group, which received dextrose perfusion and had free access to chow. A subgroup of D/aa/L rats (n = 6) received a TPN solution containing homocysteine. After 5 days of TPN, bile was collected during 2 hours. In liver homogenates, GSH, thiobarbituric acid reactive substances (TBARS), and carbonyl content of proteins (Prot-CO) were measured to test the level of oxidative stress and hepatic lipid and protein oxidation. After TPN, bile flow was significantly lower in the D/aa group than in the control group. Addition of lipids further decreased bile flow. Addition of homocysteine to TPN with lipids significantly increased bile flow. Aspartate aminotransferase increased significantly in both TPN groups compared with the control group. gamma-Glutamyl transpeptidase was not different among TPN groups. An increased hepatic lipid oxidation was demonstrated by TBARS level in both TPN groups when compared with the control group. However, the liver GSH contents were not different. Protein oxidation was also significantly increased by TPN. The addition of homocysteine to TPN solution increased bile flow without liver injury or changes of lipid and protein oxidation. This study shows that TPN administered to rats induces a decrease of bile flow and an oxidative stress but that the two changes are not directly correlated. Addition of lipids further impairs bile flow but does not increase the occurrence of liver injury. Consequently, it seems more likely that TPN primarily induces a cholestatic effect that in turn induces an oxidative stress rather than inducing an oxidative stress that leads to cholestasis. However, an association of both mechanisms is not totally excluded.

Homocysteine Prevents Total Parenteral Nutrition (TPN)–Induced Cholestasis Without Changes in Hepatic Oxidative Stress in the Rat

ABSTRACTBackgroundThe role of oxidative stress in total parenteral nutrition (TPN)–associated cholestasis with liver glutathione depletion was recently shown. The aims of this study were to test the appearance of cholestasis and oxidative stress during TPN, and the hypothesis that reducing oxidative stress with a precursor of glutathione (GSH), homocysteine, would restore bile flow.MethodsThree groups of rats (weight, 179–278 g) were studied: 1) D/aa group received dextrose and amino acids (3.4 g/d); 2) D/aa/L group received the same amount of amino acids, and lipids were added on an equicaloric basis (50 kcal/d) with a lowered amount of dextrose; and 3) a control group, which received dextrose perfusion and had free access to chow. A subgroup of D/aa/L rats (n = 6) received a TPN solution containing homocysteine. After 5 days of TPN, bile was collected during 2 hours. In liver homogenates, GSH, thiobarbituric acid reactive substances (TBARS), and carbonyl content of proteins (Prot‐CO) were measured to test the level of oxidative stress and hepatic lipid and protein oxidation.ResultsAfter TPN, bile flow was significantly lower in the D/aa group than in the control group. Addition of lipids further decreased bile flow. Addition of homocysteine to TPN with lipids significantly increased bile flow. Aspartate aminotransferase increased significantly in both TPN groups compared with the control group. γ‐Glutamyl transpeptidase was not different among TPN groups. An increased hepatic lipid oxidation was demonstrated by TBARS level in both TPN groups when compared with the control group. However, the liver GSH contents were not different. Protein oxidation was also significantly increased by TPN. The addition of homocysteine to TPN solution increased bile flow without liver injury or changes of lipid and protein oxidation.DiscussionThis study shows that TPN administered to rats induces a decrease of bile flow and an oxidative stress but that the two changes are not directly correlated. Addition of lipids further impairs bile flow but does not increase the occurrence of liver injury. Consequently, it seems more likely that TPN primarily induces a cholestatic effect that in turn induces an oxidative stress rather than inducing an oxidative stress that leads to cholestasis. However, an association of both mechanisms is not totally excluded.

Biliary inorganic phosphate as a tool for assessing cold preservation-reperfusion injury: a study in the isolated perfused rat liver model.

Ischemia-reperfusion injury is a major cause of early graft dysfunction after liver transplantation. The bile flow has been suggested as an index of ischemic damage, and severely impaired bile flow seems to be predictive of poor survival in experimental studies. Looking for injury markers, biliary inorganic phosphate has the potential of being a useful endogenous marker of diminished hepatobiliary function because this anion is excreted in the bile by a paracellular pathway and it can detect changes in permeability. The goal of this study was to evaluate the effects of cold preservation-reperfusion of the liver on bile flow and bile inorganic phosphate and their relationship with storage-related graft failure. The isolated and perfused rat liver was used to evaluate the injury for ischemia-reperfusion. The intrahepatic resistance, lactate dehydrogenase release, and potassium and biliary inorganic phosphate concentration were used to estimate viability and function of freshly isolated or cold-preserved livers. The intrahepatic resistance and the bile flow were consistent and significantly decreased throughout the perfusion time in relation to the increment in storage. Inorganic phosphate is more concentrated in bile from preserved livers, showing an alteration in paracellular pathway, confirmed by the biliary excretion of horseradish peroxidase. After preservation, concentration and excretion of the paracellular marker were increased during the first peak. The second peak appears earlier in preserved livers (10 minutes) with a different shape but without changes in concentration. In conclusion, inorganic phosphate in bile shows changes in paracellular permeability as occurs in livers after 48 hours of cold preservation.

Lithocholic Acid Decreases Expression of Bile Salt Export Pump through Farnesoid X Receptor Antagonist Activity

Bile salt export pump (BSEP) is a major bile acid transporter in the liver. Mutations in BSEP result in progressive intrahepatic cholestasis, a severe liver disease that impairs bile flow and causes irreversible liver damage. BSEP is a target for inhibition and down-regulation by drugs and abnormal bile salt metabolites, and such inhibition and down-regulation may result in bile acid retention and intrahepatic cholestasis. In this study, we quantitatively analyzed the regulation of BSEP expression by FXR ligands in primary human hepatocytes and HepG2 cells. We demonstrate that BSEP expression is dramatically regulated by ligands of the nuclear receptor farnesoid X receptor (FXR). Both the endogenous FXR agonist chenodeoxycholate (CDCA) and synthetic FXR ligand GW4064 effectively increased BSEP mRNA in both cell types. This up-regulation was readily detectable at as early as 3 h, and the ligand potency for BSEP regulation correlates with the intrinsic activity on FXR. These results suggest BSEP as a direct target of FXR and support the recent report that the BSEP promoter is transactivated by FXR. In contrast to CDCA and GW4064, lithocholate (LCA), a hydrophobic bile acid and a potent inducer of cholestasis, strongly decreased BSEP expression. Previous studies did not identify LCA as an FXR antagonist ligand in cells, but we show here that LCA is an FXR antagonist with partial agonist activity in cells. In an in vitro co-activator association assay, LCA decreased CDCA- and GW4064-induced FXR activation with an IC(50) of 1 microm. In HepG2 cells, LCA also effectively antagonized GW4064-enhanced FXR transactivation. These data suggest that the toxic and cholestatic effect of LCA in animals may result from its down-regulation of BSEP through FXR. Taken together, these observations indicate that FXR plays an important role in BSEP gene expression and that FXR ligands may be potential therapeutic drugs for intrahepatic cholestasis.

Peptide Mimetic HIV Protease Inhibitors Are Ligands for the Orphan Receptor SXR

The orphan nuclear receptor SXR coordinately regulates drug clearance in response to a wide variety of xenobiotic compounds. This signaling system protects the body from exposure to toxic compounds; however, it can also pose a severe barrier to drug therapy. We now demonstrate that the human immunodeficiency virus (HIV) protease inhibitor ritonavir binds SXR and activates its target genes. This represents an example of a commonly used therapeutic agent that effectively activates SXR. We also show that other protease inhibitors are weaker (saquinavir) or unable to activate SXR (nelfinavir, indinavir) thus defining analogs that fail to induce SXR-regulated clearance pathways. Interestingly, HIV protease inhibitors are distinct from previously known SXR ligands in that they are peptide mimetic compounds. This expands the ligand specificity of SXR to include this unique chemical class whose pharmaceutical significance is expanding. Finally, we show that SXR ligands activate expression of multiple resistance protein 2, a critical regulator of bile flow and biliary drug excretion. These findings have important implications for the role of SXR in regulating drug clearance and hepatic disorders associated with impaired bile flow.

Tauroursodeoxycholic acid inserts the apical conjugate export pump, Mrp2, into canalicular membranes and stimulates organic anion secretion by protein kinase C-dependent mechanisms in cholestatic rat liver.

Ursodeoxycholic acid (UDCA) exerts anticholestatic effects by undefined mechanisms. Previous work suggested that UDCA stimulates biliary exocytosis via Ca(++)- and protein kinase C (PKC)-dependent mechanisms. Therefore, the effect of taurine-conjugated UDCA (TUDCA) was studied in the experimental model of taurolithocholic acid (TLCA)-induced cholestasis on bile flow, hepatobiliary exocytosis, distribution of PKC isoforms, and density of the apical conjugate export pump, Mrp2, in canalicular membranes. Isolated perfused rat livers were preloaded with horseradish peroxidase (HRP), a marker of vesicular exocytosis, and were perfused with bile acids or dimethylsulfoxide (control) only. PKC isoform distribution and membrane density of Mrp2 were studied using immunoblotting and immunoelectron-microscopic techniques. Biliary secretion of the Mrp2 substrate, 2,4-dinitrophenyl-S-glutathione (GS-DNP), was studied in the presence or absence of the PKC inhibitor, bisindolylmaleimide I (BIM-I; 1 micromol/L). TLCA (10 micromol/L) impaired bile flow by 51%; biliary secretion of HRP and GS-DNP by 46% and 95%, respectively; membrane binding of the Ca(++)-sensitive alpha-isoform of PKC by 32%; and density of Mrp2 in the canalicular membrane by 79%. TUDCA (25 micromol/L) reversed the effects of TLCA on bile flow, secretion of HRP and GS-DNP, and distribution of alpha-PKC. TUDCA reduced membrane binding of epsilon-PKC and increased Mrp2 density 4-fold in canalicular membranes of cholestatic hepatocytes. BIM-I inhibited the effect of TUDCA on GS-DNP secretion in cholestatic livers by 49% without affecting secretion in controls. In conclusion, TUDCA may enhance the secretory capacity of cholestatic hepatocytes by stimulation of exocytosis and insertion of transport proteins into apical membranes via PKC-dependent mechanisms.

The pathophysiology of cholestasis with special reference to primary biliary cirrhosis

Cholestasis in primary biliary cirrhosis results from impairment of bile flow either by reduced transport at the level of the canaliculi or by disturbed bile flow through damaged intrahepatic bile ductules. Whatever its cause, the expression of hepatic transport proteins will be affected. In cholestatic rats: the expression of the multispecific organic anion transporter mrp2 is decreased; the bile salt export pump bsep and the phospholipid transporter mdr2 are less affected; the carrier protein for hepatic uptake of bile salts ntcp is sharply down-regulated; Mrp3, a basolateral ATP-dependent transporter for glucuronides and bile salts, is upregulated. Thus, bile salts that cannot exit the hepatocyte because of the cholestasis are effectively removed across the basolateral membrane.These may be adaptive responses in defence against overloading of hepatocytes with cytotoxic bile salts. These responses show that the expression of hepatic transporter proteins is highly regulated. This occurs by transcriptional and post-transcriptional mechanisms.Primary biliary cirrhosis starts as a disease of the small intrahepatic bile ducts and therefore the experimental evidence for ‘cross-talk’ between hepatocytes and cholangiocytes is of great interest for this disease and needs to be further investigated. New insights in bile physiology may enable the development of new therapies for cholestatic liver diseases as primary biliary cirrhosis.

LOW SERUM BETA-CAROTENE CONCENTRATIONS ARE COMMON IN CHILDREN WITH CHOLESTATIC LIVER DISEASE

80 Although prior studies have shown that beta-carotene is poorly absorbed in other malabsorptive states (such as cystic fibrosis), there are no reports of serum beta-carotene concentrations in children with cholestatic liver diseases. Since beta-carotene can function as an antioxidant and oxidant stress in the liver may be involved in cholestatic liver injury, low concentrations of beta-carotene may be clinically important in cholestasis. We hypothesized that children with liver diseases associated with impaired bile flow would have significant malabsorption of beta-carotene and low serum concentrations compared to children with non-cholestatic liver diseases. Serum samples were obtained from 41 children, ages 6 months or older, with chronic liver disease, including 14 with biliary atresia, 8 with other cholestatic liver diseases (Alagille syndrome, PFIC, PSC, neonatal hepatitis), and 19 with non-cholestatic liver diseases (alpha-1 antitrypsin deficiency, congenital hepatic fibrosis, glycogen storage disease, cryptogenic cirrhosis). All children were receiving foods containing carotenoids at the time of evaluation. Serum was protected from light, stored frozen and then analyzed for beta-carotene by high performance liquid chromatography. Deficient serum concentrations of beta-carotene were defined as 0.05 µg/ml or lower, based on large population studies. Values in Table are mean ± SEM.TABLEConclusions: Deficient serum beta-carotene concentrations were present in a high proportion of children with chronic cholestatic disorders and less frequently in non-cholestatic liver disease. The possible consequences of these low concentrations on oxidant hepatic injury and strategies for repleting beta-carotene in cholestasis are under investigation.

Induction of the mitochondrial permeability transition as a mechanism of liver injury during cholestasis: a potential role for mitochondrial proteases

As part of this thematic series on mitochondria in cell death, we would like to review our data on: (1) the role of the mitochondrial permeability transition (MPT) in hepatocyte necrosis during cholestasis; and (2) the concept that endogenous mitochondrial protease activity may lead to the MPT. Many chronic human liver diseases are characterized by cholestasis, an impairment in bile flow. During cholestasis an accumulation of toxic hydrophobic bile salts in the hepatocyte causes necrosis. We tested the hypothesis that toxic hydrophobic bile salt, glycochenodeoxycholate (GCDC), causes hepatocyte necrosis by inducing the MPT. GCDC induces a rapid, cyclosporin A-sensitive MPT. The hydrophilic bile salt, ursodeoxycholate (UDCA), prevents the GCDC-induced MPT and hepatocyte necrosis providing an explanation for its beneficial effect in human liver disease. We have also demonstrated that the calcium-dependent MPT is associated with an increase in calpain-like protease activity and inhibited by calpain inhibitors. In an experimental model of cholestasis, mitochondrial calpain-like protease activity increases 1.6-fold. We propose for the first time that activation of mitochondrial proteases may initiate the MPT and cell necrosis during cholestasis.

Choleretic activity of Gentiana lutea ssp. symphyandra in rats

Effects of an ethanolic extract prepared from G. lutea ssp. symphyandra roots on the bile production and liver in rats were investigated. Bile flows of rats which were treated by a single i.p. dose of CCl(4) 24 h prior to experiments were measured after the cannulation of bile duct under urethane anaesthesia. After an equilibration period of 1 h, the lyophilized extract were administered intraduodenally (500 mg/kg i.p.), while control animals received physiological saline only. To monitor the effect of multiple dose therapy, rats received the same dose of G. lutea ssp. symphyandra extract for 3 days (2 days prior to CCl(4) administration) and their bile flows were measured after the cannulation. In all groups, bile samples were collected for 3 h with 15 min intervals. After the completion of bile flow experiment, rat livers were removed and put in neutral formaldehyde solution (10%) for the histological examination. According to results obtained, multiple dose treatment of rats with the plant extract normalized the decreased bile flow due CCl(4), whereas single dose therapy was ineffective on the impaired bile flow. These data indicate that the extract prepared from Gentiana lutea ssp. symphyandra roots has a hepatoprotective activity.

Ischemic Cholangitis

Ischemia-induced bile duct lesions have been collectively labeled as ischemic cholangitis. The biliary epithelium is dependent on arterial blood flow, unlike the hepatic parenchyma with its dual arterial and portal venous blood supply. As such, the biliary epithelium is susceptible to injury when arterial blood flow is compromised. This compromise can occur at the level of the major, named hepatic artery branches or at the microscopic, peribiliary capillary plexus level. Typically, ischemic cholangitis manifests as segmental strictures and cholangiectases with resultant mechanical impairment of bile flow and, occasionally, secondary infection of the biliary system. Ischemic cholangitis after liver transplantation is becoming an important problem and likely is attributable to numerous factors. Hepatic arterial infusion of chemotherapy and systemic vasculitis are other causes of ischemic cholangitis. The role of ischemia in other chronic biliary and ductopenic diseases remains speculative.

Expression, distribution, and activity of Na+,K+-ATPase in normal and cholestatic rat liver.

Hepatocellular Na+,K+-ATPase is an important driving force for bile secretion and has been localized to the basolateral plasma membrane domain. Cholestasis or impaired bile flow is known to modulate the expression, domain specificity, and activity of various transport systems involved in bile secretion. This study examined Na+, K+-ATPase after ethinylestradiol (EE) treatment and after bile duct ligation (BDL), two rat models of cholestasis. It applied quantitative immunoblotting, biochemical and cytochemical determination of enzyme activity, and immunocytochemistry to the same livers. The data showed a good correlation among the results of the different methods. Neither EE nor BDL induced alterations in the subcellular distribution of Na+,K+-ATPase, which was found in the basolateral but not in the canalicular (apical) plasma membrane domain. Protein expression and enzyme activity showed a small (approximately 10%) decrease after EE treatment and a similar increase after BDL. These modest changes could not be detected by immunofluorescence, immuno EM, or cytochemistry. The data, therefore, demonstrate that Na+,K+-ATPase is only slightly affected by EE and BDL. They suggest that other components of the bile secretory apparatus that take effect downstream of the primary basolateral driving force may play a more prominent role in the pathogenesis of cholestasis.

A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis.

Cholestasis, or impaired bile flow, is an important but poorly understood manifestation of liver disease. Two clinically distinct forms of inherited cholestasis, benign recurrent intrahepatic cholestasis (BRIC) and progressive familial intrahepatic cholestasis type 1 (PFIC1), were previously mapped to 18q21. Haplotype analysis narrowed the candidate region for both diseases to the same interval of less than 1 cM, in which we identified a gene mutated in BRIC and PFIC1 patients. This gene (called FIC1) is the first identified human member of a recently described subfamily of P-type ATPases; ATP-dependent aminophospholipid transport is the previously described function of members of this subfamily. FIC1 is expressed in several epithelial tissues and, surprisingly, more strongly in small intestine than in liver. Its protein product is likely to play an essential role in enterohepatic circulation of bile acids; further characterization of FIC1 will facilitate understanding of normal bile formation and cholestasis.

Hepatic canalicular membrane 5: Expression and localization of the conjugate export pump encoded by the MRP2 (cMRP/cMOAT) gene in liver.

The liver converts endogenous and xenobiotic lipophilic compounds into anionic conjugates with glutathione, glucuronate, or sulfate. These conjugates are transported across the canalicular (apical) membrane into bile by a 190 kDa membrane glycoprotein that has been cloned recently. This apical conjugate-transporting ATPase has been termed canalicular multidrug resistance protein (cMRP) because of the similarity in substrate specificity and sequence with the multidrug resistance protein (MRP1), canalicular multispecific organic anion transporter (cMOAT), or multidrug resistance protein 2 (MRP2). The amino acid sequence identity of human MRP2 and MRP1 is 49%. MRP2 is predominantly expressed in hepatocytes and localized to apical membrane domains. MRP2 is not expressed in the human Dubin-Johnson syndrome, which is therefore associated with an inherited deficiency in the secretion of amphiphilic anionic conjugates into the bile. The rat homolog Mrp2 is absent in two mutant strains of rats with different point mutations in the corresponding gene. These mutant rats are hyperbilirubinemic and deficient in the ATP-dependent transport of conjugates from hepatocytes into bile. Impairment of bile flow (cholestasis) can be associated with a down-regulation of the expression of the conjugate export pump, and MRP2 contributes to bile flow as an important driving force.

Total parenteral nutrition impairs bile flow and alters bile composition in newborn piglet.

Cholestatic liver disease complicates total parenteral nutrition (TPN) in premature neonates. We investigated TPN-induced liver disease in the newborn piglet, hypothesizing that: (1) TPN impairs bile flow by reducing the bile acid-dependent (BADF) and the bile acid-independent component of bile flow (BAIF); and (2) TPN changes bile composition. For three weeks, eight piglets received TPN and nine piglets were fed milk. Basal bile flow was measured and bile composition analyzed for bile acids, cholesterol (C), phospholipids (PL), and PL fatty acids. Bile flow was also measured after stimulation with 20, 50, and 100 mu/kg taurocholic acid (TCA). Liver histology and bilirubin content were examined. Basal bile flow was reduced from 11.6 +/- 1.2 microliters/g liver/10 min in orally fed animals to 1.6 +/- 0.4 microliters/g liver/10 min in the TPN group. The stimulated bile flow in the TPN group did not respond to TCA and was lower than in the orally fed animals at each TCA dose. Both BADF and BAIF were significantly lower in the TPN group. Bile acid secretion was less than 50% of control values and total C and PL secretions were less than 5% of control. Liver and serum bilirubin were elevated in the TPN group. The newborn piglet is a valid model to study TPN-induced cholestasis, characterized by decreased bile acid secretion, impaired BADF and BAIF, and reduced bile flow stimulation after intravenous TCA.