ursodoxicoltaurine and Cholestasis

Bile acids (BAs) are key molecules in generating bile flow, which is an essential function of the liver. In the last decades, there have been great advances in the understanding of BA physiology, and new insights have emerged regarding the role of BAs in determining cell damage and death in several liver diseases. This new knowledge has helped to better delineate the pathophysiology of cholestasis and the adaptive responses of hepatocytes to cholestatic liver injury as well as of the mechanisms of injury of biliary epithelia. In this context, therapeutic approaches for liver diseases using hydrophilic BA (i.e., ursodeoxycholic acid, tauroursodeoxycholic, and, more recently, norursodeoxycholic acid), have been revamped. In the present review, we summarize current experimental and clinical data regarding these BAs and its role in the treatment of certain liver diseases.

Topics: Bile Acids and Salts; Cholestasis; Humans; Liver; Liver Diseases; Taurochenodeoxycholic Acid; Ursodeoxycholic Acid

Topics: 1-Naphthylisothiocyanate; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 11; Bile Acids and Salts; Cholagogues and Choleretics; Cholestasis; Liver; Mice; Receptors, Cytoplasmic and Nuclear

Cholestasis is a pathophysiologic syndrome with limited therapeutic options. Tauroursodeoxycholic acid (TUDCA) has been employed to treat hepatobiliary disorders and is as effective as UDCA in alleviating cholestatic liver disease in clinical trials. Until now, TUDCA's mechanism of action toward cholestasis remains unclear. In the present study, cholestasis was induced with a cholic acid (CA)-supplemented diet or α-naphthyl isothiocyanate (ANIT) gavage in wild-type and Farnesoid X Receptor (FXR) deficient mice, using obeticholic acid (OCA) as control. The effects of TUDCA on liver histological changes, transaminase level, bile acid composition, hepatocyte death, expression of Fxr and nuclear factor erythroid 2-related factor 2 (Nrf2) and target genes, as well as apoptotic signaling pathways, were investigated. Treating CA-fed mice with TUDCA markedly alleviated liver injury, attenuated bile acids retention in liver and plasma, increased Fxr and Nrf2 nuclear levels and modulated the expression of targets regulating synthesis and transportation of bile acids, including BSEP, MRP2, NTCP and CYP7A1. TUDCA, but not OCA, activated Nrf2 signaling and exerted protective effects against cholestatic liver injury in Fxr-/- mice fed with CA. Furthermore, in both mice with CA- and ANIT-induced cholestasis, TUDCA decreased expression of GRP78 and CCAAT/enhancer-binding protein homologous protein (CHOP), reduced death receptor 5 (DR5) transcription, caspase-8 activation, and BID cleavage, and subsequently inhibited activation of executioner caspases and apoptosis in liver. We confirmed that TUDCA protected against cholestatic liver injury by alleviating BAs burden of dually activating hepatic Fxr and Nrf2. Moreover, inhibiting CHOP-DR5-caspase-8 pathway contributed to the anti-apoptotic effect of TUDCA in cholestasis.

Topics: Animals; Bile Acids and Salts; Caspase 8; Cholestasis; Liver; Mice; NF-E2-Related Factor 2

The role of endoplasmic reticulum stress and the unfolded protein response (UPR) in cholestatic liver disease and fibrosis is not fully unraveled. Tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid, has been shown to reduce endoplasmic reticulum (ER) stress and counteract apoptosis in different pathologies. We aimed to investigate the therapeutic potential of TUDCA in experimental secondary biliary liver fibrosis in mice, induced by common bile duct ligation. The kinetics of the hepatic UPR and apoptosis during the development of biliary fibrosis was studied by measuring markers at six different timepoints post-surgery by qPCR and Western blot. Next, we investigated the therapeutic potential of TUDCA, 10 mg/kg/day in drinking water, on liver damage (AST/ALT levels) and fibrosis (Sirius red-staining), in both a preventive and therapeutic setting. Common bile duct ligation resulted in the increased protein expression of CCAAT/enhancer-binding protein homologous protein (CHOP) at all timepoints, along with upregulation of pro-apoptotic caspase 3 and 12, tumor necrosis factor receptor superfamily, member 1A (TNFRsf1a) and Fas-Associated protein with Death Domain (FADD) expression. Treatment with TUDCA led to a significant reduction of liver fibrosis, accompanied by a slight reduction of liver damage, decreased hepatic protein expression of CHOP and reduced gene and protein expression of pro-apoptotic markers. These data indicate that TUDCA exerts a beneficial effect on liver fibrosis in a model of cholestatic liver disease, and suggest that this effect might, at least in part, be attributed to decreased hepatic UPR signaling and apoptotic cell death.

Topics: Animals; Apoptosis; Biliary Tract; Biliary Tract Diseases; Blotting, Western; Caspase 12; Caspase 3; Cholagogues and Choleretics; Cholestasis; Disease Models, Animal; Fibrosis; Gene Expression; Liver; Liver Cirrhosis; Male; Mice; Reverse Transcriptase Polymerase Chain Reaction; Taurochenodeoxycholic Acid; Transcription Factor CHOP; Tumor Necrosis Factor-alpha; Unfolded Protein Response

The bile salt export pump (BSEP), encoded by the abcb11 gene, is the major canalicular transporter of bile acids from the hepatocyte. BSEP malfunction in humans causes bile acid retention and progressive liver injury, ultimately leading to end-stage liver failure. The natural, hydrophilic, bile acid ursodeoxycholic acid (UDCA) is efficacious in the treatment of cholestatic conditions, such as primary biliary cirrhosis and cholestasis of pregnancy. The beneficial effects of UDCA include promoting bile flow, reducing hepatic inflammation, preventing apoptosis, and maintaining mitochondrial integrity in hepatocytes. However, the role of BSEP in mediating UDCA efficacy is not known. Here, we used abcb11 knockout mice (abcb11-/-) to test the effects of acute and chronic UDCA administration on biliary secretion, bile acid composition, liver histology, and liver gene expression. Acutely infused UDCA, or its taurine conjugate (TUDC), was taken up by the liver but retained, with negligible biliary output, in abcb11-/- mice. Feeding UDCA to abcb11-/- mice led to weight loss, retention of bile acids, elevated liver enzymes, and histological damage to the liver. Semiquantitative RT-PCR showed that genes encoding Mdr1a and Mdr1b (canalicular) as well as Mrp4 (basolateral) transporters were upregulated in abcb11-/- mice. We concluded that infusion of UDCA and TUDC failed to induce bile flow in abcb11-/- mice. UDCA fed to abcb11-/- mice caused liver damage and the appearance of biliary tetra- and penta-hydroxy bile acids. Supplementation with UDCA in the absence of Bsep caused adverse effects in abcb11-/- mice.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 11; ATP-Binding Cassette Sub-Family B Member 4; ATP-Binding Cassette Transporters; Bile Canaliculi; Biological Transport; Cholestasis; Diet; Disease Models, Animal; Gene Expression Regulation; Infusions, Intravenous; Liver; Mice; Mice, Knockout; Multidrug Resistance-Associated Proteins; RNA, Messenger; Taurochenodeoxycholic Acid; Time Factors; Ursodeoxycholic Acid

Cholestatic liver injury following extra- or intrahepatic bile duct obstruction causes nonparenchymal cell proliferation and matrix deposition leading to end-stage liver disease and cirrhosis. In cholestatic conditions, nitric oxide (NO) is mainly produced by a hepatocyte-inducible NO synthase (iNOS) as a result of enhanced inflow of endotoxins to the liver and also by accumulation of bile salts in hepatocytes and subsequent hepatocellular injury. This study was aimed to investigate the role of NO and S-nitrosothiol (SNO) homeostasis in the development of hepatocellular injury during cholestasis induced by bile duct ligation (BDL) in rats. Male Wistar rats (200-250 g) were divided into four groups (n=10 each), including sham-operated (SO), bile duct-ligated (BDL), tauroursodeoxycholic acid (TUDCA, 50 mg/kg) and S-methylisothiourea (SMT, 25 mg/kg) treated. After 7 days, BDL rats showed elevated serum levels of gamma-glutamiltranspeptidase, aspartate aminotransferase, alanine aminotransferase, LDH, and bilirubin, bile duct proliferation and fibrosis, compared with the SO group. TUDCA treatment did not significantly alter these parameters, but the iNOS inhibitor SMT ameliorated hepatocellular injury, as shown by lower levels of circulating hepatic enzymes and bilirubin, and a decreased grade of bile duct proliferation and fibrosis. Both TUDCA and SMT treatments reversed Mrp2 canalicular pump expression to control levels. However, only SMT treatment significantly lowered the increased levels of plasma NO and S-nitrosation (S-nitrosylation) of liver proteins in BDL rats. Moreover, BDL resulted in a reduction of the S-nitrosoglutathione reductase (GSNOR/Adh5) enzymatic activity and a downregulation of the GSNOR/Adh5 mRNA expression that was reverted by SMT, but not TUDCA, treatment. A total of 25 liver proteins, including S-adenosyl methionine synthetase, betaine-homocysteine S-methyltransferase, Hsp90 and protein disulfide isomerase, were found to be S-nitrosated in BDL rats. In conclusion, the inhibition of NO production during induced cholestasis ameliorates hepatocellular injury. This effect is in part mediated by the improvement of cell proficiency in maintaining SNO homeostasis.

Topics: Aldehyde Oxidoreductases; Animals; ATP-Binding Cassette Transporters; Bile Ducts; Cholagogues and Choleretics; Cholestasis; Down-Regulation; Enzyme Inhibitors; Homeostasis; Isothiuronium; Ligation; Liver; Male; Nitric Oxide; Nitrosation; Proteins; Rats; Rats, Wistar; RNA, Messenger; S-Nitrosothiols; Taurochenodeoxycholic Acid

Ursodeoxycholic acid (UDCA) exerts anticholestatic effects in part by protein kinase C (PKC)-dependent mechanisms. Its taurine conjugate, TUDCA, is a cPKC alpha agonist. We tested whether protein kinase A (PKA) might contribute to the anticholestatic action of TUDCA via cooperative cPKC alpha-/PKA-dependent mechanisms in taurolithocholic acid (TLCA)-induced cholestasis.. In perfused rat liver, bile flow was determined gravimetrically, organic anion secretion spectrophotometrically, lactate dehydrogenase (LDH) release enzymatically, cAMP response-element binding protein (CREB) phosphorylation by immunoblotting, and cAMP by immunoassay. PKC/PKA inhibitors were tested radiochemically. In vitro phosphorylation of the conjugate export pump, Mrp2/Abcc2, was studied in rat hepatocytes and human Hep-G2 hepatoma cells.. In livers treated with TLCA (10 micromol/l)+TUDCA (25 micromol/l), combined inhibition of cPKC by the cPKC-selective inhibitor Gö6976 (100 nmol/l) or the non-selective PKC inhibitor staurosporine (10 nmol/l) and of PKA by H89 (100 nmol/l) reduced bile flow by 36% (p<0.05) and 48% (p<0.01), and secretion of the Mrp2/Abcc2 substrate, 2,4-dinitrophenyl-S-glutathione, by 31% (p<0.05) and 41% (p<0.01), respectively; bile flow was unaffected in control livers or livers treated with TUDCA only or TLCA+taurocholic acid. Inhibition of cPKC or PKA alone did not affect the anticholestatic action of TUDCA. Hepatic cAMP levels and CREB phosphorylation as readout of PKA activity were unaffected by the bile acids tested, suggesting a permissive effect of PKA for the anticholestatic action of TUDCA. Rat and human hepatocellular Mrp2 were phosphorylated by phorbol ester pretreatment and recombinant cPKC alpha, nPKC epsilon, and PKA, respectively, in a staurosporine-sensitive manner.. UDCA conjugates exert their anticholestatic action in bile acid-induced cholestasis in part via cooperative post-translational cPKC alpha-/PKA-dependent mechanisms. Hepatocellular Mrp2 may be one target of bile acid-induced kinase activation.

Topics: Animals; Cholagogues and Choleretics; Cholestasis; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Humans; Liver; Male; Multidrug Resistance-Associated Protein 2; Protein Kinase C-alpha; Protein Kinase Inhibitors; Rats; Taurochenodeoxycholic Acid

Ursodeoxycholic acid (UDCA) is used in the therapy of cholestatic liver diseases. Apoptosis induced by toxic bile acids plays an important role in the pathogenesis of liver injury during cholestasis and appears to be mediated by the human transcription factor AP-1. We aimed to study if TUDCA can decrease taurolitholic acid (TLCA)-induced apoptosis by modulating AP-1. TLCA (20 microM) upregulated AP-1 proteins cFos (26-fold) and JunB (11-fold) as determined by quantitative real-time PCR in HepG2-Ntcp hepatoma cells. AP-1 transcriptional activity increased by 300% after exposure to TLCA. cFos and JunB expression as well as AP-1 transcriptional activity were unaffected by TUDCA (75 microM). However, TUDCA significantly decreased TLCA-induced upregulation of cFos and JunB. Furthermore, TUDCA inhibited TLCA-induced AP-1 transcriptional activity and reduced TLCA-induced apoptosis. These data suggest that reversal of bile acid-induced AP-1 activation may be relevant for the antiapoptotic effect of TUDCA in liver cells.

Topics: Apoptosis; Base Sequence; Bile Acids and Salts; Cells, Cultured; Cholagogues and Choleretics; Cholestasis; Humans; Liver Diseases; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Taurochenodeoxycholic Acid; Taurolithocholic Acid; Transcription Factor AP-1

Cholestasis, induced by liver ischemia-reperfusion injury (IRI), is characterized by dilatation of bile canaliculi and loss of microvilli. Tauroursodeoxycholic acid (TUDCA) is an anti-cholestatic agent, modulating protein kinase C (PKC) alpha pathway. PKC reduces ischemic damage in several organs, its isoform alpha modulates ezrin, a key protein in the maintenance of cell lamellipoidal extensions. We evaluated the effects of TUDCA on cholestasis, canalicular changes and PKCalpha-ezrin expression in a rat model of liver IRI. Livers flushed and stored with Belzer solution or Belzer + 10 mm TUDCA (4 degrees C for 6 h) were reperfused (37 degrees C with O(2)) with Krebs-Ringer bicarbonate + 2.5 micromol/min of Taurocholate or TUDCA. Bile was harvested for bile flow assessment. Liver tissue was employed for Electron Microscopy (EM) and for PKCalpha and ezrin immunoblot and immunofluorescence. The same experiments were conducted with the PKCalpha inhibitor Go-6976. TUDCA-treated livers showed increased bile flow (0.25+/-0.17 vs. 0.042+/-0.02 microl/min/g liver, P<0.05) and better preservation of microvilli and bile canalicular area at EM. These effects were associated with increased PKCalpha and ezrin expression (P=0.03 and P=0.04 vs. control respectively), as also confirmed by immunofluorescence data. PKCalpha inhibition abolished these TUDCA effects. TUDCA administration during IRI reduces cholestasis and canalicular damage in the liver modulating PKCalpha-ezrin pathway.

Topics: Animals; Bile; Bile Canaliculi; Carbazoles; Cholagogues and Choleretics; Cholestasis; Cytoskeletal Proteins; Enzyme Inhibitors; L-Lactate Dehydrogenase; Liver; Male; Microscopy, Electron, Scanning; Protein Kinase C-alpha; Rats; Rats, Wistar; Reperfusion Injury; Taurochenodeoxycholic Acid

Ursodeoxycholic acid exerts anticholestatic effects in chronic cholestatic liver disease in humans as well as in experimental animal models of cholestasis. Its taurine conjugate, TUDCA, was recently shown to stimulate insertion of the apical conjugate export pump, Mrp2 (ABCC2), into canalicular membranes of rat hepatocytes made cholestatic by exposure to taurolithocholic acid (TLCA). The aim of this immunoelectronmicroscopic study was to test whether TLCA and TUDCA modulate the canalicular density of the other key apical transporter, the bile salt export pump, Bsep (ABCB11), in a similar way. Immunoelectronmicroscopic analysis of Bsep density on canalicular membranes, microvilli, and pericanalicular area of hepatocytes was performed in rat liver tissue prepared after liver perfusion with bile acids or carrier medium only. TLCA (10 micromol/l for 50 min) decreased Bsep density in canalicular membranes to 31% of controls (P<0.05) when bile flow was reduced to 35% of controls (P<0.05). Concomitantly, Bsep density in a 1 microm pericanalicular zone increased to 202% (P<0.05) indicating effective retrieval of Bsep from the canalicular membrane induced by TLCA. Coadministration of TUDCA (25 micromol/l) led to a 3.2-fold increase of Bsep density in canalicular membranes equal to control liver (P<0.05 vs TLCA) in association with a 3.8-fold increase of bile flow (P<0.05 vs TLCA). Stimulation of apical membrane insertion of key transporters like the bile salt export pump, Bsep, and-as previously shown-the conjugate export pump, Mrp2, may contribute to the anticholestatic action of UDCA amides in cholestatic conditions.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 11; ATP-Binding Cassette Transporters; Bile; Bile Canaliculi; Cholestasis; Liver; Microscopy, Immunoelectron; Multidrug Resistance-Associated Protein 2; Rats; Taurochenodeoxycholic Acid

Chronic cholestatic liver diseases are characterized by impaired balance between proliferation and death of cholangiocytes, as well as vanishing of bile ducts and liver failure. Ursodeoxycholic acid (UDCA) is a bile acid widely used for the therapy of cholangiopathies. However, little is known of the cytoprotective effects of UDCA on cholangiocytes. Therefore, UDCA and its taurine conjugate tauroursodeoxycholic acid (TUDCA) were administered in vivo to rats simultaneously subjected to bile duct ligation and vagotomy, a model that induces cholestasis and loss of bile ducts by apoptosis of cholangiocytes. Because these two bile acids act through Ca2+ signaling, animals were also treated with BAPTA/AM (an intracellular Ca2+ chelator) or Gö6976 (a Ca2+-dependent protein kinase C-alpha inhibitor). The administration of UDCA or TUDCA prevented the induction of apoptosis and the loss of proliferative and functional responses observed in the bile duct ligation-vagotomized rats. These effects were neutralized by the simultaneous administration of BAPTA/AM or Gö6976. UDCA and TUDCA enhanced intracellular Ca2+ and IP3 levels, together with increased phosphorylation of protein kinase C-alpha. Parallel changes were observed regarding the activation of the MAPK and PI3K pathways, changes that were abolished by addition of BAPTA/AM or Gö6976. These studies provide information that may improve the response of cholangiopathies to medical therapy.

Topics: Animals; Apoptosis; Bile Ducts; Calcium; Cell Proliferation; Cholestasis; Cytoprotection; Disease Models, Animal; Egtazic Acid; Enzyme Activation; Epithelium; Ligation; Male; Mitogen-Activated Protein Kinase Kinases; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinase C-alpha; Rats; Rats, Inbred F344; Signal Transduction; Taurochenodeoxycholic Acid; Ursodeoxycholic Acid; Vagotomy

Ethinylestradiol (EE) induces intrahepatic cholestasis in experimental animals being its derivative, ethinylestradiol 17beta-glucuronide, a presumed mediator of this effect. To test whether glucuronidation is a relevant step in the pathogenesis of cholestasis induced by EE (5 mg/kg b.wt. s.c. for 5 consecutive days), the effect of simultaneous administration of galactosamine (200 mg/kg b.wt. i.p.) on biliary secretory function was studied. A single injection of this same dose of galactosamine was able to decrease hepatic UDP-glucuronic acid (UDP-GA) levels by 85% and excretion of EE-17beta-glucuronide after administration of a tracer dose of [3H]EE by 40%. Uridine (0.9 g/kg b.wt. i.p.) coadministration reverted the effect of galactosamine on hepatic UDP-GA levels and restored the excretion of [3H]EE-17beta-glucuronide. When administered for 5 days, galactosamine itself did not alter any of the serum markers of liver injury studied (aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase) or biliary secretory function. When coadministered with EE, galactosamine partially prevented the impairment induced by this estrogen in total bile flow, the bile-salt-independent fraction of bile flow, basal bile salt secretion, and the secretory rate maximum of tauroursodeoxycholate. Uridine coadministration partially prevented galactosamine from exerting its anticholestatic effects. In conclusion, galactosamine administration partially prevented EE-induced cholestasis by a mechanism involving decreased UDP-GA availability for subsequent formation of EE 17beta-glucuronide. The evidence thus supports the hypothesis that EE 17beta-glucuronide is involved in the pathogenesis of EE cholestasis.

Topics: Animals; Bile; Cholestasis; Ethinyl Estradiol; Galactosamine; Liver; Male; Rats; Rats, Wistar; Taurochenodeoxycholic Acid; Uridine; Uridine Diphosphate Glucuronic Acid

Topics: Animals; Cell Communication; Cholestasis; Gap Junctions; Liver; Mice; Taurochenodeoxycholic Acid

To examine the extent and mechanisms of apoptosis in cholestatic liver injury and to explore the role of the transcription factor nuclear factor-kappa B in protection against bile acid-induced apoptosis.. Cholestatic liver injury was induced by bile duct ligation in Wistar rats. Furthermore, primary cultures of rat hepatocytes were exposed to glycochenodeoxycholic acid (GCDCA), tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA) and to cytokines. Apoptosis was determined by TUNEL-staining, active caspase-3 staining, activation of caspase-8, -9 and -3.. Limited hepatocyte apoptosis and an increased expression of NF-kappaB-regulated anti-apoptotic genes A1 and cIAP2 were detected in cholestatic rat livers. Bcl-2 expression was restricted to bile duct epithelium. In contrast to TCDCA and TUDCA, GCDCA induced apoptosis in a Fas-associated protein with death domain (FADD)-independent pathway in hepatocytes. Although bile acids do not activate NF-kappaB, NF-kappaB activation by cytokines (induced during cholestasis) protected against GCDCA-induced apoptosis in vitro by upregulating A1 and cIAP2.. GCDCA induces apoptosis in a mitochondria-controlled pathway in which caspase-8 is activated in a FADD-independent manner. However, bile acid-induced apoptosis in cholestasis is limited. This could be explained by cytokine-induced activation of NF-kappaB-regulated anti-apoptotic genes like A1 and cIAP2.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Carrier Proteins; Cells, Cultured; Cholestasis; Cytokines; Disease Models, Animal; Fas-Associated Death Domain Protein; Gene Expression; Glycochenodeoxycholic Acid; Hepatocytes; Male; NF-kappa B; Rats; Rats, Wistar; Specific Pathogen-Free Organisms; Taurochenodeoxycholic Acid

Accumulating bile acids (BA) trigger cholangiocyte proliferation in chronic cholestasis. The aim of this study was to determine if ursodeoxycholate (UDCA) or tauroursodeoxycholate (TUDCA) chronic feeding prevents the increased cholangiocyte growth and secretion in bile duct-ligated (BDL) rats, if UDCA and TUDCA effects are associated with increased cholangiocyte apoptosis, and to determine if this inhibition is dependent on increased intracellular Ca(2+) ([Ca(2+)](i)) and activation of protein kinase C (PKC) alpha. Immediately after BDL, rats were fed UDCA or TUDCA (both 275 micromol/d) for 1 week. We determined the number of bile ducts in liver sections, cholangiocyte proliferation (by measurement of H(3) histone and proliferating cellular nuclear antigen in isolated cholangiocytes), and ductal secretion. In purified cholangiocytes from 1-week BDL rats, we evaluated if UDCA and TUDCA directly inhibit cholangiocyte proliferation and secretin-stimulated adenosine 3', 5'-monophosphate levels. We determined if UDCA and TUDCA activate PKC, increase [Ca(2+)](i), and alter the apical BA transporter (ABAT) expression in cholangiocytes. UDCA and TUDCA inhibited in vivo the cholangiocyte proliferation, secretion, and ABAT expression. In vitro UDCA and TUDCA inhibition of cholangiocyte growth and secretion required increased [Ca(2+)](i) and PKC alpha. In conclusion, activation of Ca(2+)-dependent PKC alpha is required for UDCA and TUDCA inhibition of cholangiocyte growth and secretion. Reduced cholangiocyte ABAT may decrease endogenous BA stimulation of cholangiocyte growth and secretion.

Topics: Animals; Apoptosis; Bile Acids and Salts; Bile Ducts; Calcium; Carrier Proteins; Cell Division; Cholagogues and Choleretics; Cholestasis; Down-Regulation; Hepatitis; Hydroxysteroid Dehydrogenases; Isoenzymes; Ligation; Liver; Male; Membrane Glycoproteins; Organ Size; Protein Kinase C; Protein Kinase C-alpha; Rats; Rats, Inbred F344; Taurochenodeoxycholic Acid; Ursodeoxycholic Acid

Tauroursodeoxycholate (TUDC) provides partial protection against taurolithocholate (TLC) induced cholestasis, possibly by inducing a signalling cascade activating protein kinase C (PKC). The potential protective effects of beta muricholic acid (beta-MC), another 7-beta-hydroxylated bile salt, have not previously been studied in TLC cholestasis.. To study the effect of beta-MC on TLC induced cholestasis and also to investigate further the effects of agents affecting intracellular signalling, notably DBcAMP (a cell permeable cAMP analogue) and several protein kinase inhibitors.. Functional studies were carried out analysing the proportion of hepatocyte couplets able to accumulate the fluorescent bile acid analogue cholyl-lysyl-fluorescein (CLF) into their sealed canalicular vacuole (cVA of CLF assay).. It was found that both beta-MC and DBcAMP were as effective as TUDC in protecting against TLC induced cholestasis. The PKC inhibitors staurosporin and H7 but not the specific protein kinase A (PKA) inhibitor KT5720 abolished the protective effects of TUDC and beta-MC. BAPTA/AM, a chelator of intracellular Ca(2+), significantly decreased the protective effect of both bile salts, and that of DBcAMP. PKC and PKA inhibitors had no effect on protection with DBcAMP.. Beta-MC was as effective as TUDC in protecting against TLC cholestasis. Mobilisation of Ca(2+) and activation of PKC, but not of PKA, are involved in the anticholestatic effect of the two 7-beta-hydroxylated bile salts. The hepatoprotective effects of DBcAMP involved Ca(2+) mobilisation, but not PKC or PKA activation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Analysis of Variance; Animals; Bucladesine; Calcium; Carbazoles; Chelating Agents; Cholagogues and Choleretics; Cholestasis; Cholic Acids; Cyclic AMP-Dependent Protein Kinases; Egtazic Acid; Enzyme Activation; Enzyme Inhibitors; Indoles; Liver; Male; Protein Kinase C; Pyrroles; Rats; Signal Transduction; Staurosporine; Taurochenodeoxycholic Acid; Taurolithocholic Acid

S-adenosyl-L-methionine (SAMe) and tauroursodeoxycholate (TUDC) exert an additive ameliorating effect on taurolithocholate (TLC)-induced cholestasis. The aims were to investigate the protective effect of SAMe on 17beta-estradiol-glucuronide (17betaEG) cholestasis and to find out whether SAMe and TUDC may exert an additive, ameliorating effect.. Hepatocyte couplet function was assessed by canalicular vacuolar accumulation (cVA) of cholyllysylfluorescein (CLF). Cells were co-treated with 17betaEG and SAMe, TUDC, or both (protection study), or treated with 17betaEG and then with SAMe, TUDC or both (reversion study) before CLF uptake. Couplets were also co-treated with SAMe and dehydroepiandrosterone (DHEA), a competitive substrate for the sulfotransferase involved in 17betaEG detoxification. The effects of 17betaEG, SAMe and TUDC were also examined on intracellular distribution of F-actin.. Both SAMe and TUDC significantly protected against, and reversed, 17betaEG-induced cholestasis, but their effects were not additive. DHEA abolished the protective effect of SAMe. 17BetaEG did not affect the uptake of CLF into hepatocytes at the concentrations used, and also, it did not affect the intracellular distribution of F-actin.. 17BetaEG does not affect the uptake of CLF into hepatocytes. SAMe and TUDC protect and reverse 17betaEG-induced cholestasis, but without an additive effect. Protection by SAMe may involve facilitating the sulfation of 17betaEG.

Topics: Actins; Animals; Biological Transport, Active; Cholestasis; Cholic Acids; Dehydroepiandrosterone; Estradiol; Fluoresceins; Hepatocytes; In Vitro Techniques; Male; Rats; Rats, Wistar; S-Adenosylmethionine; Taurochenodeoxycholic Acid

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.

Topics: Animals; Anions; Bile; Bile Canaliculi; Cell Membrane; Cholagogues and Choleretics; Cholestasis; Glutathione; Horseradish Peroxidase; Isoenzymes; L-Lactate Dehydrogenase; Male; Membranes; Microscopy, Immunoelectron; Mitochondrial Proteins; Protein Kinase C; Rats; Rats, Sprague-Dawley; Ribosomal Proteins; Saccharomyces cerevisiae Proteins; Taurochenodeoxycholic Acid; Tissue Distribution

The monohydroxy bile acid, taurolithocholate (TLC), causes cholestasis in vivo and in isolated perfused livers. It is also cholestatic in vitro and, in this study using isolated rat hepatocyte couplets, causes a reduction of the accumulation of (fluorescent) bile acid in the canalicular vacuoles (cVA) of this polarized cell preparation. The hepatoprotective bile acid, tauroursodeoxycholate (TUDCA), partially protects against the action of TLC when added at the same time. It also partially reverses the cholestatic effect if added after the cells have been exposed to TLC. A second hepatoprotective compound, S-adenosyl-L-methionine (SAMe) also not only partially protects against the action of TLC when added at the same time, but it too is able to partially reverse the cholestatic effect. Neither hepatoprotective agent is fully effective alone, but their effects are additive. In combination, a full restoration of cVA is observed in moderate cholestasis, but not in severe cholestasis. We discuss briefly some possible mechanisms involved in the additive mode of action of both hepatoprotective compounds. In summary, we show for the first time that SAMe and TUDCA can exert an additive effect in the amelioration of TLC-induced cholestasis in isolated rat hepatocyte couplets. This finding may be of possible clinical relevance.

Topics: Animals; Bile Acids and Salts; Bile Canaliculi; Cholestasis; Fluorescent Dyes; Male; Rats; Rats, Wistar; S-Adenosylmethionine; Taurochenodeoxycholic Acid; Taurolithocholic Acid; Vacuoles

The prevention of the hepatotoxic effects produced by intravenous infusion of taurochenodeoxycholic acid (TCDCA) by coinfusion with taurohyodeoxycholic acid (THDCA) was evaluated in bile fistula rats; the hepatoprotective effects of the latter were also compared with those of tauroursodeoxycholic acid (TUDCA). Rats infused with TCDCA at a dose of 8 micromol/min/kg showed reduced bile flow and calcium secretion, as well as increased biliary release of alkaline phosphatase (AP) and lactate dehydrogenase (LDH). This was associated with a very low biliary secretion rate of TCDCA (approximately 1 micromol/min/kg). Simultaneous infusion of THDCA or TUDCA at the same dose preserved bile flow and almost totally abolished the pathological leakage of the two enzymes into bile. The effect was slightly more potent for THDCA. The maximum secretion rate of TCDCA increased to the highest value (8 micromol/min/kg) when coinfused with either of the two hepatoprotective bile acids (BA), which were efficiently and completely secreted in the bile, without metabolism. Calcium output was also restored and phospholipid (PL) secretion increased with respect to the control saline infusion. This increase was higher in the THDCA study. These data show that THDCA is highly effective in the prevention of hepatotoxicity induced by intravenous infusion of TCDCA by facilitating its biliary secretion and reducing its hepatic residence time; this was associated with selective stimulation of PL biliary secretion.

Topics: Alkaline Phosphatase; Animals; Calcium; Cholagogues and Choleretics; Cholestasis; Injections, Intravenous; L-Lactate Dehydrogenase; Liver; Phospholipids; Rats; Taurochenodeoxycholic Acid; Taurocholic Acid; Taurodeoxycholic Acid

The regulation of cAMP synthesis by hormones and bile acids is altered in isolated hamster hepatocytes 2 days after bile duct ligation (BDL) [Y. Matsuzaki, B. Bouscarel, M. Le, S. Ceryak, T. W. Gettys, J. Shoda, and H. Fromm. Am. J. Physiol. 273 (Gastrointest. Liver Physiol. 36): G164-G174, 1997]. Therefore, studies were undertaken to elucidate the mechanism(s) responsible for this impaired modulation of cAMP formation. Hepatocytes were isolated 48 h after either a sham operation or BDL. Both preparations were equally devoid of cholangiocyte contamination. Although the basal cAMP level was not affected after BDL, the ability of glucagon to maximally stimulate cAMP synthesis was decreased by approximately 40%. This decreased glucagon effect after BDL was not due to alteration of the total glucagon receptor expression. However, this effect was associated with a parallel 50% decreased expression of the small stimulatory G protein alpha-subunit (GsalphaS). The expression of either the large subunit (GsalphaL) or the common beta-subunit remained unchanged. The expression of Gialpha2 and Gialpha3 was also decreased by 25 and 46%, respectively, and was associated with the failure of ANG II to inhibit stimulated cAMP formation. Therefore, alterations of the expression of GsalphaS and Galphai are, at least in part, responsible for the attenuated hormonal regulation of cAMP synthesis. Because cAMP has been reported to stimulate both bile acid uptake and secretion, impairment of cAMP synthesis and bile acid uptake may represent an initial hepatocellular defense mechanism during cholestasis.

Topics: Angiotensin II; Animals; Cholestasis; Colforsin; Common Bile Duct; Cricetinae; Cyclic AMP; Glucagon; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Keratins; Ligation; Liver; Male; Mesocricetus; Receptors, Glucagon; Taurochenodeoxycholic Acid; Ursodeoxycholic Acid

Tauroursodeoxycholic acid (TUDCA) is of potential benefit in cholestatic disorders. However, the effect of TUDCA on hepatic ischemia-reperfusion injury is unknown. We studied this subject with particular regard to its roles in hepatic calcium mobilization. Three doses of TUDCA were used with continuous intravenous infusion (1.0, 0.1, and 0.01 micromol/kg body weight/min). At 3 hr after 1 hr of ischemia and reperfusion in 70% rat liver, high-dose TUDCA reduced hepatic reperfused injury according to biochemical and histological findings and significantly increased bile flow after reperfusion. It significantly increased tissue calcium content and serum calcium concentration after reperfusion. Furthermore, it also enhanced biliary calcium concentration and total output during reperfusion. In conclusion, TUDCA has a salutary effect on ischemia-reperfusion injury of the liver. However, it is still unclear how the calcium mobilization induced by TUDCA is associated with the hepatoprotection against ischemia-reperfusion injury.

Topics: Animals; Bile; Calcium; Cholestasis; Liver; Liver Diseases; Male; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Taurochenodeoxycholic Acid

Bile acids exert cellular and molecular effects in the liver, but little is known about tissue concentrations. The aim of this study was to characterize bile acid composition in human and rat liver tissue and hepatocyte nuclei and examine the effects of experimental cholestasis and bile acid administration.. Bile acids were measured by gas chromatography-mass spectrometry.. Liver tissue concentrations of sham-operated rats were 130.8 +/- 21.3 nmol/g, representing 2%-4% of the bile acid pool; cholic and delta 22-beta-muricholic acids were the major bile acids identified. Concentrations increased 7-8-fold with bile duct ligation; deoxycholate and hyodeoxycholate disappeared. Lithocholate concentrations were higher in ligated rats (6.4 +/- 0.4 vs. 3.9 +/- 0.5 nmol/g for sham-operated). Total bile acid concentrations in human liver tissue were 61.6 +/- 29.7 nmol/g and comprised mainly chenodeoxycholic and cholic acids. Concentrations were higher during ursodeoxycholate or tauroursodeoxycholate administration (157.2 +/- 45.6 and 161.6 +/- 43.4 nmol/g, respectively), and liver tissue was enriched 30% in ursodeoxycholate at the expense of hydrophobic bile acids. Bile acids were identified in rat hepatic nuclei (50-110 pmol/4 x 10(7) nuclei), accounting for < 0.1% of liver tissue levels.. Human and rat liver tissue bile acid concentrations are low, increase with bile acid administration or bile duct ligation, and account for only a small fraction of the bile acid pool.

Topics: Adult; Animals; Bile Acids and Salts; Cell Nucleus; Cholestasis; Humans; Liver; Male; Middle Aged; Rats; Rats, Sprague-Dawley; Taurochenodeoxycholic Acid; Ursodeoxycholic Acid

The effect of tauro-beta-muricholate (beta MC-tau) and tauro-alpha-muricholate (alpha MC-tau) on oestradiol-17 beta-glucuronide (E217G)-induced cholestasis was compared with that of tauroursodeoxycholate (UDC-tau) in rats. Like UDC-tau, alpha MC-tau and beta MC-tau infused at the rate of 0.2 mumol/min per 100 g bodyweight (BW) completely inhibited the cholestasis induced by E217G infused at the rate of 0.06 mumol/min per 100 g BW for 20 min. These findings indicate that beta MC-tau and alpha MC-tau are useful in protecting against various types of experimental cholestasis, as well as against bile acid-induced cholestasis.

Topics: Animals; Cholestasis; Estradiol; Isomerism; Male; Rats; Rats, Sprague-Dawley; Taurochenodeoxycholic Acid; Taurocholic Acid

Estradiol-17 beta-D-glucuronide (E-17G), a metabolite of natural estrogen, is well known to cause intrahepatic cholestasis in humans. We therefore investigated the effect of sodium tauroursodeoxycholate (T-UDCA), on E-17G-induced cholestasis in female rats.. For the evaluation of the drug, animals given E-17G (10 mumol/kg) were divided into three groups, and T-UDCA was administered intravenously at various doses after E-17G treatment.. T-UDCA significantly prevented a marked reduction of bile flow in E-17G-treated rats in all experimental schedules. Furthermore, T-UDCA significantly increased in the biliary E-17G excretion rate at an early stage after E-17G treatment in rats. However, this drug caused no significant change in the biliary excretion rate of estradiol-3-sulfate-17 beta-D-glucuronide (E-3S-17G), which is identified as the major biliary metabolite with E-17G throughout the recovery periods.. These results suggest that T-UDCA can improve E-17G induced acute cholestasis by rapidly increasing the biliary E-17G excretion rate. Thus our finding may provide a useful approach for attempts to prevent drug-induced acute cholestasis in humans.

Topics: Animals; Bile; Bile Acids and Salts; Cholestasis; Estradiol; Female; Rats; Rats, Sprague-Dawley; Taurochenodeoxycholic Acid; Ursodeoxycholic Acid

We investigated the effect of sodium tauroursodeoxycholate (UR-906) on cholestasis in common bile duct-ligated rats in comparison with the effect of dehydrocholic acid. UR-906 (30-180 mumol/kg) and dehydrocholic acid (180 mumol/kg) were intravenously given once daily for consecutive 20 days in rats and the common bile duct was ligated for the last 10 days. On the next day after the last test drug administration, serum biochemical and plasma hemostatic variables were determined. UR-906 significantly ameliorated the elevation of serum cholesterol, phospholipid, bilirubin and bile acid concentrations in bile duct-ligated rats. UR-906 significantly suppressed the prolongation of plasma prothrombin time and activated partial thromboplastin time. Furthermore, UR-906 significantly suppressed the decreases in plasma coagulation factor II and X activities. However, dehydrocholic acid did not cause significant changes in any of the variables examined in this model. These results suggest that UR-906 has a beneficial effect against cholestasis induced by bile duct ligation in rats and that this drug may be useful in the treatment of clinical cholestatic disorders.

Topics: Animals; Bile Acids and Salts; Bile Ducts; Bilirubin; Cholagogues and Choleretics; Cholestasis; Cholesterol; Dehydrocholic Acid; Disease Models, Animal; Factor X; Hemostasis; Ligation; Male; Partial Thromboplastin Time; Phospholipids; Prothrombin; Prothrombin Time; Rats; Rats, Wistar; Taurochenodeoxycholic Acid

It has been suggested that vesicular transport of bile acids in hepatocytes occurs, especially at high-dose loads.. The effect was studied of colchicine, a vesicular transport inhibitor, on lithocholate-3-O-glucuronide-induced cholestasis in rats. Cholestasis was induced by an intravenous infusion of lithocholate-3-O-glucoronide at the rate of 0.1 mumol.min-1.100 g-1 for 40 min.. Colchicine treatment almost completely inhibited cholestasis and increased biliary excretion of lithocholate-3-O-glucoronide, whereas lumicolchicine had no effect. Treatment with vinblastine, another vesicular transport inhibitor, also reduced the cholestasis. Colchicine did not affect biliary excretion of taurocholate infused at the rate of 0.3 mumol.min-1.100 g-1 for 40 min, but markedly inhibited its biliary excretion when infused at the rate of 1.5 mumol.min-1.100 g-1 for 40 min. Colchicine had no effect on biliary excretion of tauroursodeoxycholate (1.5 mumol.min-1.100 g-1 for 40 min), lithocholate-3-sulfate (0.3 mumol.min-1.100 g-1 for 40 min), or a trace amount of lithocholate-3-O-glucuronide.. These findings indicate that lithocholate-3-O-glucoronide-induced cholestasis is caused by its increased access to the vesicular transport pathway, possibly beyond the capacity of the transport by the cytosolic binders, and that the transport of lithocholate-3-O-glucoronide via the vesicular pathway induces cholestasis. Furthermore, the contribution of the vesicular pathway to hepatic transport may be different among bile acids, and lithocholate-3-O-glucuronide seems to have higher accessibility to this transport system.

Topics: Animals; Bile; Cholestasis; Colchicine; Glucuronates; Lithocholic Acid; Male; Rats; Rats, Sprague-Dawley; Taurochenodeoxycholic Acid; Taurocholic Acid; Vinblastine

Taurochenodeoxycholate (TCDC) (or taurocholate, TC) excessively i.v. infused in rats causes an acute cholestasis accompanied by an excessive excretion of various proteins (lactate dehydrogenase, LDH, albumin, etc.) into the bile. This cholestasis was initially found to be effectively prevented by a simultaneous infusion of tauroursodeoxycholate (TUDC). Later this property was found to be shared by glycoursodeoxycholate (GUDC) and tauro (and glyco) alpha and beta-muricholate (MC) all known to be relatively hydrophilic. The extent of the preventative effect appears to be comparable for taurine and glycine conjugates of all three bile salts (UDC, alpha-MC and beta-MC). An albumin leakage into the bile enhanced by TCDC infusion appears to be mainly from albumin in the serum, since i.v. injected 125I-human serum albumin excretion into the bile paralled the rat albumin excretion. Despite very drastic biochemical abnormalities induced by TCDC infusion, morphological correlates in the liver are scarce both from light and electron microscopic examinations, the only correlate with biochemical parameters being a sporadic necrosis of hepatocytes, especially in the periportal areas. Although there is not sufficient morphological evidence, it appears that TCDC infusion causes a direct communication between serum and bile leading to a rapid leakage of large molecules such as albumin and even gamma-globulin. Conjugates of hydrophilic bile salts such as UDC, alpha-MC and beta-MC efficiently prevent such bile abnormalities but their hydrophilicity is not the sole determinant of this property since a more hydrophilic bile salt such as taurodehydrocholate does not possess this property. The underlying mechanism(s) for this protective property remains uncertain.

Topics: Albumins; Animals; Bile; Bile Acids and Salts; Cholestasis; Cholic Acids; L-Lactate Dehydrogenase; Liver; Male; Necrosis; Rats; Rats, Wistar; Taurochenodeoxycholic Acid

Tauroursodeoxycholic acid is known to have a hepatoprotective action in cholestatic disorders. We evaluated whether oral pretreatment with tauroursodeoxycholic acid could protect the liver from ischemia-reperfusion injury, with particular regard to its effect on bile flow and biliary calcium excretion.. A 1-hour in vivo ischemia-reperfusion model of 70% of the lobes of rat liver was used. Animals were divided into six groups (each group; n = 8); a non-ischemia sham group (CS), a control group without bile acids (CON), and 4 bile acid groups; 10 mg/kg and 50 mg/kg (U10, U50), taurocholic acid 10 mg/kg (CA10) and tauroursodeoxycholic acid 10 mg/kg (CD10). Bile acids were given orally for 7 days before operation.. Three hours after reperfusion, oral bile acid pretreatment failed to reduce the hepatic ischemia-reperfusion injury biochemically, but histological improvement was observed in the tauroursodeoxycholic acid groups. After reperfusion, tauroursodeoxycholic acid significantly increased bile flow from the ischemic liver, and also significantly increased serum calcium concentration. Although tauroursodeoxycholic acid did not change biliary calcium concentration, it significantly enhanced total biliary calcium output during reperfusion.. Thus, tauroursodeoxycholic acid inhibited tissue calcium accumulation and enhanced sinusoidal and biliary calcium output during hepatic ischemia-reperfusion. However, it is still unclear if calcium mobilization is part of the protective mechanisms of tauroursodeoxycholic acid in ischemia-reperfusion injury of the liver.

Topics: Analysis of Variance; Animals; Bile Acids and Salts; Calcium; Cholestasis; Evaluation Studies as Topic; Isomerism; Liver; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Stimulation, Chemical; Taurochenodeoxycholic Acid

Cyclosporin A is an essential immunosuppressive drug, but it is potentially toxic to the kidney and liver. Ursodeoxycholic acid, a hydrophilic bile acid, has been reported to improve cholestasis in liver disease in man. The purpose of this work was to examine whether tauroursodeoxycholate could reduce cyclosporin A-induced hepatic or renal injuries in the rat. After randomization into three groups (N = 8), rats received daily for 17 days: cyclosporin A intraperitoneally alone (30 mg/kg) or cyclosporin A intraperitoneally and tauroursodeoxycholate (60 mg/kg) by gavage; control received the cyclosporin A excipient. Under tauroursodeoxycholate, cholestatic parameters (bile flow, bile salt secretion, serum bile salts, serum bilirubin) improved significantly without affecting cyclosporin A blood levels, and excretion of the drug and its metabolites in bile increased by 47%. Serum creatinine levels were better preserved, although not significantly. These results show that tauroursodeoxycholate prevents cyclosporin A-induced cholestasis in long-term treatment in rats, possibly by facilitating the drug elimination in bile.

Topics: Animals; Cholestasis; Cyclosporine; Isomerism; Male; Random Allocation; Rats; Rats, Sprague-Dawley; Taurochenodeoxycholic Acid

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.

Topics: Albumins; Animals; Bile; Cholestasis; Isomerism; L-Lactate Dehydrogenase; Liver; Male; Rats; Rats, Wistar; Taurochenodeoxycholic Acid; Taurocholic Acid

To assess the effects of tauroursodeoxycholic acid (TUDCA) on bile excretory function, we examined whether TUDCA modulates vesicular exocytosis in the isolated perfused liver of normal rats in the presence of high (1.9 mM) or low (0.19 mM) extracellular Ca++ and in cholestatic rats 24 h after bile duct ligation. In addition, the effects of TUDCA on Ca++ homeostasis were compared in normal and in cholestatic hepatocytes. In the isolated perfused rat liver, TUDCA (25 microM) stimulated a sustained increase in the biliary excretion of horseradish peroxidase, a marker of the vesicular pathway, in the presence of high, but not low extracellular Ca++ or in the cholestatic liver. In contrast, TUDCA stimulated bile flow to the same extent regardless of the concentration of extracellular Ca++ or the presence of cholestasis. In indo-1-loaded hepatocytes, basal cytosolic free Ca++ ([Ca++]i) levels were not different between normal and cholestatic cells. However, in cholestatic cells [Ca++]i increases induced by TUDCA (10 microM) and its 7 alpha-OH epimer taurochenodeoxycholic acid (50 microM) were reduced to 22% and 26%, respectively, compared to normal cells. The impairment of TUDCA-induced [Ca++]i increase in cholestatic cells could be mimicked by exposing normal cells to low extracellular Ca++ (21%) or to the Ca++ channel blocker NiCl2 (23%). These data indicate that (a) dihydroxy bile acid-induced Ca++ entry may be of functional importance in the regulation of hepatocellular vesicular exocytosis, and (b) this Ca++ entry mechanism across the plasma membrane is impaired in cholestatic hepatocytes. We speculate that the beneficial effect of ursodeoxycholic acid in cholestatic liver diseases may be related to the Ca+(+)-dependent stimulation of vesicular exocytosis by its conjugate.

Topics: Acetylglucosaminidase; Animals; Bile; Biomarkers; Calcium; Cells, Cultured; Cholestasis; Cytosol; Exocytosis; Extracellular Space; Horseradish Peroxidase; Kinetics; Liver; Lysosomes; Male; Phenylephrine; Rats; Rats, Sprague-Dawley; Reference Values; Taurochenodeoxycholic Acid; Taurocholic Acid; Vasopressins

Rats which were taurine-deprived through beta-alanine administration and untreated rats were used to elucidate the mechanism of hepatoprotective effects of ursodeoxycholate (UDC). Animals were infused with taurochenodeoxycholate (TCDC, 0.4 mumol.min-1.100 g-1) alone or in combination with tauroursodeoxycholate (TUDC), or with UDC (both 0.6 mumol.min-1.100 g-1) for 2 h. Ursodeoxycholate as well as TUDC prevented severe cholestasis and liver damage induced by TCDC infusion in both untreated and taurine-deprived rat groups. In untreated rats, however, UDC was less effective in hepatoprotection than TUDC as indicated by sequential changes in biliary LDH output during the period of 30 to 120 min (P < 0.05). In rats receiving UDC and TCDC, total biliary output of LDH for 2 h was significantly higher in taurine-deprived rats than that in the control (73.40 +/- 10.10 vs 41.14 +/- 12.56: P < 0.05), suggesting that the difference became greater upon taurine deprivation. In contrast, in rats receiving TUDC and TCDC, the protective effect was comparable for the taurine-deprived and untreated rats. When the animals were infused with UDC and TCDC, taurine-deprived rats exhibited a biliary excretion rate for TUDC half that of control rats (P < 0.05). Furthermore, a highly significant correlation was observed between the biliary excretion rate of TUDC and biliary output of LDH (r = -0.886, P < 0.0001). These results suggest that UDC conjugates, especially TUDC, and not UDC may play a major role in the prevention of cholestasis and liver cell damage caused by TCDC infusion.

Topics: Alkaline Phosphatase; Animals; Bile; Bile Acids and Salts; Cholestasis; L-Lactate Dehydrogenase; Liver; Male; Rats; Rats, Wistar; Taurochenodeoxycholic Acid; Ursodeoxycholic Acid

The mechanism of lithocholate-3-O-glucuronide-induced cholestasis is unknown. In this study, we investigated the cholestatic effects of this agent in a congenital hyperbilirubinemic rat, EHBR. We also studied the effects of ursodeoxycholate-3-O-glucuronide and tauroursodeoxycholate on lithocholate-3-O-glucuronide-induced cholestasis in rats. Lithocholate-3-O-glucuronide, administered at the rate of 0.1 mumol/min/100 g for 40 min, a cholestatic dose in control rats, failed to cause cholestasis in EHBR, and biliary lithocholate-3-O-glucuronide excretion was delayed. Biliary concentrations of this agent did not correlate with the severity of cholestasis. Both tauroursodeoxycholate and ursodeoxycholate-3-O-glucuronide, infused at the rate of 0.2 mumol/min/100 g for 120 min, completely inhibited cholestasis induced by lithocholate-3-O-glucuronide administered at the rate of 0.1 mumol/min/100 g for 40 min. Only tauroursodeoxycholate enhanced biliary lithocholate-3-O-glucuronide excretion. These findings indicate that lithocholate-3-O-glucuronide-induced cholestasis is induced by damage at the level of the bile canalicular membrane. Ursodeoxycholate-3-O-glucuronide inhibits this cholestasis, possibly by inhibiting the access of lithocholate-3-O-glucuronide to the bile canalicular membrane.

Topics: Animals; Bile; Cholestasis; Glucuronates; Hyperbilirubinemia, Hereditary; Lithocholic Acid; Liver; Male; Rats; Rats, Sprague-Dawley; Taurochenodeoxycholic Acid; Ursodeoxycholic Acid

The effect of the co-infusion of ursodeoxycholate and its taurine conjugate, 3-O-glucuronide and 3,7-disulfate on estradiol-17 beta-glucuronide-induced cholestasis was examined. Estradiol-17 beta-glucuronide was intravenously administered to bile-drained rats at a rate of 0.075 mumol/min/100 g for 20 min. Co-infusion of ursodeoxycholate and its conjugates was simultaneously begun at a rate of 0.2 mumol/min/100 g and continued for 120 min. Ursodeoxycholate failed to improve and tauroursodeoxycholate only partially improved estradiol-17 beta-glucuronide-induced cholestasis between 20 and 40 min, although both bile acids increased bile flow after 80 min. Tauroursodeoxycholate increased biliary estradiol-17 beta-glucuronide excretion. Ursodeoxycholate-3-O-glucuronide completely inhibited cholestasis induced by estradiol-17 beta-glucuronide without changing biliary estradiol-17 beta-glucuronide excretion. Although ursodeoxycholate-3,7-disulfate had only a minor effect on cholestasis, it increased biliary excretion of estradiol-17 beta-glucuronide. In the Eizai hyperbilirubinuria rat (EHBR), a hyperbilirubinemic mutant Sprague-Dawley rat, the same dose of estradiol-17 beta-glucuronide failed to induce cholestasis with a marked delay in biliary excretion of estradiol-17 beta-glucuronide. In summary, ursodeoxycholate-3-O-glucuronide is more effective than tauroursodeoxycholate in inhibiting estradiol-17 beta-glucuronide-induced cholestasis and ursodoexycholate-3,7-disulfate had little effect. However, the unexpected effects of ursodeoxycholate-3-O-glucuronide and 3,7-disulfate on excretion of estradiol-17 beta-glucuronide suggest that the interaction of these anions at the canalicular membrane is complicated, with interaction occurring at more than two pathways of the biliary excretion of these anions.

Topics: Animals; Bile; Cholagogues and Choleretics; Cholestasis; Estradiol; Hyperbilirubinemia, Hereditary; Isomerism; Male; Rats; Rats, Mutant Strains; Taurochenodeoxycholic Acid; Ursodeoxycholic Acid

Topics: Animals; Bile; Cholagogues and Choleretics; Cholestasis; Humans; Liver Cirrhosis, Biliary; Rats; Taurochenodeoxycholic Acid

In order to cast light on the anti-cholestatic and cytoprotective properties of ursodeoxycholic acid (UDCA), intrahepatic transport and secretion of bile salts and biliary phospholipids were investigated by using isolated perfused livers from colchicine-pretreated rats. Administration of taurocholic acid (TCA) after colchicine pretreatment induced marked cholestasis. Tauroursodeoxycholic acid (TUDCA) treatment, in contrast, was associated with maintenance of bile flow, with excretion rates of bile acids and phospholipids similar to those in control animals. Furthermore, TCA-induced cholestasis in colchicine-treated rat livers was clearly decreased by co-administration of TUDCA. Although simultaneous addition of UDCA also showed slight improvement, with or without taurine pre-treatment, biliary bile-salt analysis also showed that cholestasis was markedly remitted as the excretion of taurine-conjugated UDCA was increased. The results suggest that the cytoprotective and anti-cholestatic effects of TUDCA may be linked to action at the intrahepatocyte level, represented by mild detergent effects on organelle lipids and preservation of intracellular transport even under microtubule-dysfunctional conditions. In addition, it was indicated that cytoprotective effects of UDCA may also be exerted after its conjugation with taurine inside hepatocytes.

Topics: Animals; Aspartate Aminotransferases; Bile; Cholestasis; Colchicine; gamma-Glutamyltransferase; Male; Microtubules; Rats; Rats, Inbred Strains; Secretory Rate; Taurochenodeoxycholic Acid; Taurocholic Acid; Ursodeoxycholic Acid

The present study has demonstrated that tauroursodeoxycholate (TUDC), but not taurocholate, can reverse chlorpromazine (CPZ)-induced cholestasis in the isolated perfused rat liver. At an infusion rate of 1.5 mumol/min, TUDC led to restoration of bile flow in the perfused rat liver made cholestatic by the addition of 250 microM CPZ. This reversal was accompanied by an increased excretion of CPZ and its metabolites. A higher infusion rate of 5.0 mumols TUDC/min, however, led to only a transient increase in bile flow and to no increase in CPZ excretion. In contrast to the effects of TUDC, infusion of taurocholate led to an exacerbation of CPZ-induced cholestasis. The differences in the efficacy of the two bile salts may be due to their relative detergent (hydrophobic) properties.

Topics: Animals; Bile; Chlorpromazine; Cholestasis; In Vitro Techniques; Isomerism; Kinetics; Liver; Male; Perfusion; Rats; Rats, Inbred Strains; Taurochenodeoxycholic Acid; Taurocholic Acid; Time Factors

Chlorpromazine at a concentration of 250 microM and estradiol-17 beta-D-glucuronide at 17.5 microM on infusion led to a sharp reduction in bile flow by the in vitro perfused rat liver. This was accompanied by fragmentation and a loss of canalicular microvilli, dilatation of canaliculi, and thickening of pericanalicular ectoplasm. Less prominent were the smooth endoplasmic reticulum dilatation, lysosomal lamination, and the appearance of amorphous bile in hepatocyte cytoplasm. The bile flow and electron microscopy appearance were restored to normal by infusion of tauroursodeoxycholate in a concentration of 5 mumols/min for the estradiol-17 beta-D-glucuronide-induced cholestasis and 1.5 mumol/min for the chlorpromazine-induced cholestasis. Changes in ultrastructure paralleled changes in bile flow. These observations demonstrate the feasibility of electron microscopy studies on the perfused liver, and the rapidity with which cholestatic changes appear.

Topics: Animals; Bile; Chlorpromazine; Cholestasis; Estradiol; In Vitro Techniques; Kinetics; Liver; Microscopy, Electron; Perfusion; Rats; Taurochenodeoxycholic Acid; Time Factors

Cholestasis and enhanced biliary leakage of proteins such as lactate dehydrogenase (LDH) and albumin are known to be induced by infusions of relatively toxic bile salts such as taurocholate (TC) and taurochenodeoxycholate (TCDC). Tauroursodeoxycholate (TUDC) was previously shown to prevent these bile abnormalities when simultaneously infused (1-5). In the present study, we examined whether tauro beta-muricholate (T beta-MC) has a similar effect. The enhanced biliary excretion of LDH and albumin induced by the infusion of TCDC at a rate of 0.4 mumol/min/100 g was markedly prevented by the simultaneous infusion of T beta-MC or TUDC at a rate one-fourth that of TCDC. Increased LDH level in plasma and hemolysis caused by the infusion of TCDC were also reduced by either T beta-MC or TUDC. These results indicate that T beta-MC has a preventive effect on TCDC-induced hepatobiliary changes, which is as efficient as that of TUDC as shown previously, suggesting that the 7 beta-hydroxy group is important for this hepatoprotective effect. Furthermore, our results suggest that beta-muricholic acid may also have clinical value since current reports demonstrate a beneficial effect of ursodeoxycholic acid on a variety of cholestatic conditions, including primary biliary cirrhosis.

Topics: Animals; Bile; Cholestasis; Cholic Acids; L-Lactate Dehydrogenase; Liver; Male; Rats; Rats, Inbred Strains; Taurochenodeoxycholic Acid

This study was designed to test the hypothesis that increasing the infusion rate of bile salts could overcome drug-induced cholestasis. Cholestasis was induced by administration of 17.5 mumol/L estradiol-17 beta-D-glucuronide during the infusion of taurocholate, tauroursodeoxycholate or dehydrocholate at 20 nmol/min/gm liver. After 30 min, a bolus of 10 mumol of the bile salts was added to the perfusate, and the infusion rate of each bile salt was increased. Taurocholate at a rate of 62 or 125 nmol/min/gm liver, caused a prompt dose-dependent increase of the depressed bile flow and bile salt excretion. A higher rate of taurocholate infusion (180 nmol/min/gm liver) was less effective than either the 62 or 125 rate in increasing bile flow. Infusion of tauroursodeoxycholate at 250 or 390 nmol/min/gm liver also led to a dose-dependent recovery. Further increase of tauroursodeoxycholate infusion rate of 580 nmol/min/gm liver did not provide any additional recovery in bile flow. Dehydrocholate, at rates of 62 or 125 nmol/min/gm liver, gave only a slight enhancement of bile flow. Both taurocholate and tauroursodeoxycholate caused a marked removal of the estradiol-17 beta-D-glucuronide, which had accumulated in the liver. At lower taurocholate infusion rates, the estradiol-17 beta-D-glucuronide was excreted mainly in the bile. At the highest rate, however, biliary excretion of estradiol-17 beta-D-glucuronide declined significantly, and a marked back-efflux of the estrogen into the perfusate was noted. In contrast, tauroursodeoxycholate led to enhanced biliary estradiol-17 beta-D-glucuronide excretion at all increased tauroursodeoxycholate infusion rates and to only a small increase in back-efflux of estradiol-17 beta-D-glucuronide at the two highest tauroursodeoxycholate infusion rates.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Bile; Bile Acids and Salts; Biological Transport; Cholagogues and Choleretics; Cholestasis; Dehydrocholic Acid; Estradiol; Male; Rats; Rats, Inbred Strains; Sucrose; Taurochenodeoxycholic Acid; Taurocholic Acid

Ursodeoxycholate has been advocated for the treatment of cholestatic liver diseases. The coinfusion of tauroursodeoxycholate with taurolithocholate in the perfused rat liver completely prevented the decrease of bile flow and the increase of oxygen uptake found with taurolithocholate only. Bile flow and bile salt secretion were increased with the coinfusion of both bile acids as compared with the infusion of tauroursodeoxycholate only (+4.30 microliters/g liver per 30 min) with 16 and 32 mumol/l tauroursodeoxycholate (+1.55 microliters/g liver per 30 min with 80 and 160 mumol/l). Morphological examination revealed a 50% decrease of the number of necrotic cells in the periportal area. Tauroursodeoxycholate did not inhibit the uptake of taurolithocholate, but increased its transcellular passage and biotransformation. Thus, tauroursodeoxycholate prevents taurolithocholate-induced cholestasis and liver cell toxicity probably by an intracellular mechanism.

Topics: Animals; Chenodeoxycholic Acid; Cholestasis; Lithocholic Acid; Liver; Rats; Taurochenodeoxycholic Acid; Taurolithocholic Acid

Biliary excretion of various proteins (5'-nucleotidase, alkaline phosphatase, lactate dehydrogenase, and albumin) was investigated in pentobarbital sodium-anesthetized rats infused with different bile salts [taurocholate (TC), taurochenodeoxycholate (TCDC), and tauroursodeoxycholate (TUDC)]. A TCDC infusion at 0.4 mumol . min-1 . 100 g body wt-1 caused much higher increases in the biliary excretion of these proteins compared with the respective values in rats that received an infusion of TC at a threefold higher rate (1.2 mumol . min-1 . 100 g body wt-1). In contrast, a TUDC infusion at 1.8 mumol . min-1 . 100 g body wt-1 showed the minimum effect on these protein leakages. A combined infusion of TCDC (0.4 mumol . min-1 . 100 g-1) and TUDC (0.6 mumol . min-1 . 100 g-1) resulted in drastic (8- to 20-fold) decreases in excretion of these enzymes and albumin compared with respective values in rats infused with TCDC alone. Similar preventive effects were observed with the addition of TUDC to the infusion of TC (1.2 mumol . min-1 . 100 g-1). These results suggest that the hepatic cytotoxic effects of TC and TCDC can be prevented by the simultaneous infusion of TUDC in rats.

Topics: 5'-Nucleotidase; Albumins; Alkaline Phosphatase; Animals; Bile; Bile Acids and Salts; Chenodeoxycholic Acid; Cholestasis; Isomerism; L-Lactate Dehydrogenase; Male; Nucleotidases; Rats; Rats, Inbred Strains; Taurochenodeoxycholic Acid; Taurocholic Acid; Time Factors

A combined infusion of taurocholate (TC) and glycoursodeoxycholate (GU) resulted in a longer choleretic period and a significantly higher excretion of TC compared with the infusion of TC alone, as has been previously observed for the combined infusion of tauroursodeoxycholate (TU) and TC in the rat. It was concluded that GU is as effective as TU in preventing TC induced cholestasis in this species.

Topics: Animals; Bile; Bile Acids and Salts; Chenodeoxycholic Acid; Cholestasis; Deoxycholic Acid; Drug Evaluation, Preclinical; Rats; Secretory Rate; Taurochenodeoxycholic Acid; Taurocholic Acid; Ursodeoxycholic Acid

The nature of transport pathway(s) for the biliary excretion of taurocholate and tauroursodeoxycholate was examined by comparing the biliary transport maximum (Tm) value for taurocholate during the infusion of taurocholate alone with that of taurocholate combined with tauroursodeoxycholate. The combined infusion of tauroursodeoxycholate resulted in an appreciable excretion of tauroursodeoxycholate while the excretion rate of taurocholate was not reduced in comparison with the Tm value of taurocholate alone. Furthermore, the Tm state of taurocholate was maintained for a much longer period with the simultaneous infusion of tauroursodeoxycholate than by the infusion of taurocholate alone. The cholestasis usually produced by the excess infusion of taurocholate was also prevented when tauroursodeoxycholate was simultaneously infused. Since plasma taurocholate concentration was not significantly different from the two rat groups, the results suggest the presence of the facilitative interaction of tauroursodeoxycholate with the taurocholate excretion.

Topics: Animals; Bile Acids and Salts; Chenodeoxycholic Acid; Cholestasis; Kinetics; Male; Rats; Rats, Inbred WF; Taurochenodeoxycholic Acid; Taurocholic Acid