Stimulation of bile duct epithelial secretion by glybenclamide in normal and cholestatic rat liver

Cholestasis is a cardinal complication of liver disease, but most treatments are merely supportive. Here we report that the sulfonylurea glybenclamide potently stimulates bile flow and bicarbonate excretion in the isolated perfused rat liver. Video-microscopic studies of isolated hepatocyte couplets and isolated bile duct segments show that this stimulatory effect occurs at the level of the bile duct epithelium, rather than through hepatocytes. Measurements of cAMP, cytosolic pH, and Ca2+ in isolated bile duct cells suggest that glybenclamide directly activates Na+-K+-2Cl- cotransport, rather than other transporters or conventional second-messenger systems that link to secretory pathways in these cells. Finally, studies in livers from rats with endotoxin- or estrogen-induced cholestasis show that glybenclamide retains its stimulatory effects on bile flow and bicarbonate excretion even under these conditions. These findings suggest that bile duct epithelia may represent an important new therapeutic target for treatment of cholestatic disorders.     

Hepatoprotection in ethinylestradiol-treated rats is provided by tauroursodeoxycholic acid, but not by ursodeoxycholic acid

Ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA) have been suggested as potential treatments for drug-induced cholestasis. It was therefore decided to study the effects of administration of UDCA or TUDCA on individual serum bile acid concentration, conventional liver tests and associated hepatic ultrastructural changes in ethinylestradiol-treated (EE) rats mg/kg per day). Control rats were treated s.c. with propylene glycol. EE-treated rats were randomly assigned to receive daily i.p. injections of placebo, TUDCA or UDCA. Four rats in each group were treated for 4 consecutive days, and a second four for 14 days. After 4 days of treatment, the serum levels of cholic acid and taurocholic acid were significantly increased in EE-treated rats. None of the conventional liver tests were significantly different among the four groups. After 14 days of treatment the serum levels of cholic acid, chenodeoxycholic acid, glycocholic acid, glycochenodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid, bilirubin, alkaline phosphatase and gamma glutamyltransferase were significantly raised in EE and EE plus UDCA treated rats. EE plus TUDCA treated rats, however, had no significant changes in these individual serum bile acids or conventional liver tests. The ultrastructure of livers from EE plus TUDCA treated rats was similar to those of controls. On the other hand, EE and EE plus UDCA rats both showed a significant reduction in sinusoidal microvilli. These results show that treatment of rats for 4 days with EE induces significant rises in the serum concentrations of two individual bile acids and that TUDCA protects against this.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of S-adenosyl-L-methionine and dilinoleoylphosphatidylcholine on liver lipid composition and ethanol hepatotoxicity in isolated perfused rat liver

We investigated whether S-adenosyl-L-methionine (SAMe), dilinoleoylphosphatidylcholine (DLPC), or SAMe + DLPC influence liver lipid composition as well as acute ethanol hepatotoxicity in the isolated perfused rat liver (IPRL). SAMe (25 mg/kg intramuscularly three times a day) was administered for five consecutive days, while DLPC was administered intraperitoneally for five days. The liver was then isolated, perfused with taurocholate to stabilize bile secretion, and exposed to 0.5% ethanol for 70 min. SAMe, without changing total phospholipid (PL) content, induced an increase in the phosphatidylcholine/phosphatidylethanolamine (PC/PE) molar ratio in both liver homogenate and microsomes and a significant enrichment of 16:0-20:4 and 18:0-20:4 PC molecular species. DLPC induced a significant enrichment of PL in liver homogenate and microsomes due to a contemporary increase in PC and PE. The PC enrichment specifically involved 16:0-20:4 and 18:0-20:4 PC molecular species besides the HPLC peak containing the administered 18:2-18:2 PC species. DLPC + SAMe increased the concentration of PC in liver homogenate and microsomes due to a specific enrichment of 16:0-22:6, 16:0-20:4, and 18:0-20:4 PC molecular species, and the HPLC peak containing the administered 18:2-18:2 PC species. Ethanol acute exposure in the control IPRLs for 70 min induced a depletion of cholesterol in both liver homogenate and microsomes without significant changes in the composition of PL classes and PC molecular species. SAMe, DLPC, or SAMe + DLPC counteracted the cholesterol depletion induced by ethanol, indicating that phospholipid changes promoted by these treatments all induce a major resistance of liver membranes to the effect of ethanol. Ethanol administration in control IPRLs induced a fivefold increase of AST and LDH release in the perfusate, depletion of glutathione in homogenates and mitochondria, decreased oxygen liver consumption, and inhibition of bile flow. These effects of ethanol were significantly antagonized by SAMe. In contrast, DLPC alone only minimally attenuated enzyme release in the perfusate and the inhibitory effect of ethanol on bile flow, but it failed to influence the depletion of total and mitochondrial glutathione or the depressed oxygen consumption induced by ethanol. DLPC, administered together with SAMe, added nothing to the protective effect of SAMe against ethanol hepatotoxicity and cholestasis. In conclusion, this study demonstrates that both SAMe and DLPC induced marked modifications in the lipid composition of liver membranes with a similar enrichment of polyunsaturated PC molecular species. Only SAMe, however, significantly protected against the hepatotoxic and cholestatic effect of acute ethanol administration, an effect associated with maintained normal glutathione mitochondrial levels and oxygen liver consumption. This indicates that the protective effect of SAMe against ethanol toxicity is linked to multiple mechanisms, the maintenance of glutathione levels probably being one of the most important.     

Intravenous ursodeoxycholic acid reduces cholestasis in parenterally fed newborn piglets

BACKGROUND & AIMS: Cholestasis complicates total parenteral nutrition (TPN) in preterm infants. Ursodeoxycholic acid (UDCA) is used for several cholestatic problems. The hypothesis of this study was that intravenous UDCA prevents TPN-induced cholestasis by (1) maintaining normal basal and stimulated bile flow, (2) altering bile composition, and (3) changing hepatocyte membrane composition and Na+,K(+)-adenosine triphosphatase (ATPase) activity. METHODS: Three groups of piglets were studied: group 1 received sow's milk, groups 2 and 3 received TPN, and group 3 also received 100 mumol.kg-1.day-1 UDCA intravenously. After 3 weeks, basal and stimulated bile flow were measured. Cholesterol, bile acids, phospholipids, and phospholipid fatty acids were analyzed in bile, and fluidity, phospholipid fatty acid composition, and Na+,K(+)-ATPase were analyzed in hepatocyte membranes. RESULTS: Bile acid secretion and basal and stimulated bile flow were similar in control and UDCA-treated animals but reduced to < 50% in the TPN group. Bile acid-dependent and -independent bile flow were lower in the TPN group. UDCA did not normalize abnormalities in TPN-induced bile composition. Sinusoidal but not canalicular membrane fluidity was different in TPN than in control and UDCA-treated animals. UDCA also increased Na+,K(+)-ATPase activity. Bile and membrane phospholipid fatty acids reflected dietary fatty acids. CONCLUSIONS: Intravenous UDCA improves bile flow and reduces bilirubin levels in the serum and liver in piglets with TPN-induced cholestasis.     

Effect of sodium tauroursodeoxycholate (UR-906) on liver dysfunction in bile duct-ligated rats

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.     

Acute ethanol hepatotoxicity is modulated by bile salt hydrophilic-hydrophobic properties

The influence of the hydrophobic-hydrophilic properties of bile salts (BS) on acute ethanol hepatotoxicity was investigated. Bile flow, biliary BS secretion and enzyme (LDH,AST) release in the perfusate were measured before and after exposure to low (0.1%) or high (1%) doses of ethanol in in vitro isolated livers perfused with 1 microM/min taurocholate (TCA), tauroursodeoxycholate (TUDCA) or taurodeoxycholate (TDCA). Ethanol promotes a rapid decrease of basal bile flow and BS secretion in TCA-perfused livers [-28% of basal values with 0.1% (N = 6), and -35% with 1% ethanol (N = 6)]. Bile flow and BS secretion were minimally decreased by ethanol in livers perfused with a hydrophilic BS (TUDCA) [-8% decrease of basal values with 0.1% ethanol (N = 6), and -10% with 1% ethanol (N = 9); p < 0.02 vs TCA-perfused livers]. In contrast, when livers were perfused with a hydrophobic BS (TDCA), ethanol showed a higher cholestatic effect than either TCA- or TUDCA-perfused livers. Enzyme release in the perfusate was not modified by 0.1% ethanol, while 1% ethanol promoted a 4-5 fold increase in LDH and AST release in the perfusate of TCA-perfused livers with respect to a mere 2-fold increase in TUDCA-perfused livers and a 6-7 fold increase in TDCA perfused livers (p < 0.03). In conclusion, we showed that TUDCA almost completely counteracts the cholestatic and cytolitic effects promoted by ethanol in the isolated perfused rat liver.     

Current concepts of hepatic uptake, intracellular transport and biliary secretion of bile acids: physiological basis and pathophysiological changes in cholestatic liver dysfunction

Hepatic sinusoidal uptake of bile acids is mediated by defined carrier proteins against unfavourable concentration and electrical gradients. Putative carrier proteins have been identified using bile acid photoaffinity labels and more recently using immunological probes, such as monoclonal antibodies. At the sinusoidal domain, proteins with molecular weights of 49 and 54 kDa have been shown to be carriers for bile acid transport. The 49 kDa protein has been associated with the Na(+)-dependent uptake of conjugated bile acids, while the 54 kDa carrier has been involved in the Na(+)-independent bile acid uptake process. Within the hepatocyte, cytosolic proteins, such as the glutathione S-transferase (also designated the Y protein), the Y binders and the fatty acid binding proteins, are able to bind bile acids and possibly facilitate their movement to the canalicular domain. At the canalicular domain a 100 kDa carrier protein has been isolated and it has been shown by several laboratories that this particular protein is concerned with canalicular bile acid transport. The system is ATP-dependent and follows Michaelis-Menten kinetics. Interference with bile acid transport has been demonstrated by several chemicals. The mechanisms by which these chemicals inhibit bile acid transport may explain the apparent cholestatic properties observed in patients and experimental animals treated with these agents. Several studies have shown that Na+/K(+)-ATPase activity is markedly decreased in cholestasis induced by ethinyloestradiol, taurolithocholate and chlorpromazine. However, other types of interference have been described and the cholestatic effects may be the result of several mechanisms. Cholestasis is associated with several adaptive changes that may be responsible for the accumulation of bile acids and other cholephilic compounds in the blood of these patients. It may be speculated that the nature of these changes is to protect liver parenchymal cells from an accumulation of bile acids to toxic levels. However, more detailed quantitative experiments are necessary to answer questions with regard to the significance of these changes and the effect of various hepatobiliary disorders in modifying these mechanisms. It is expected that the mechanisms by which bile acid transport is regulated and efforts to understand the molecular basis for these processes will be among the areas of future research.     

Ursodeoxycholic acid in the treatment of liver diseases

Ursodeoxycholic acid is a dihydroxy bile acid with a rapidly expanding spectrum of usage in acute and chronic liver diseases. The various mechanisms of action of this hydrophilic bile acid include direct cytoprotection, detergent action on dysfunctional microtubules, immunomodulation and induction of hypercholeresis. Its efficacy in primary biliary cirrhosis and primary sclerosing cholangitis as an adjunct to medical therapy has been well established. Newer indications include its use in the management of chronic hepatitis, cirrhosis, post liver transplant rejection, graft-versus-host disease and acute viral hepatitis, where it not only relieves symptoms of cholestasis but also arrests ongoing hepatocyte necrosis.     

Molecular regulation of sinusoidal liver bile acid transporters during cholestasis

Impairment of the hepatic transport of bile acids and other organic anions will result in the clinically important syndrome of cholestasis. Cloning of a number of specific hepatic organic anion transporters has enabled studies of their molecular regulation during cholestasis. The best characterized transport system is a 50-51 kDa sodium-dependent taurocholate cotransporting polypeptide (ntcp), which mediates the sodium-dependent uptake of conjugated bile acids at the sinusoidal plasma membrane of hepatocytes. Under physiologic conditions and after depletion of biliary constituents, ntcp remains constitutively expressed throughout the liver acinus. However, both function and expression of ntcp are rapidly down-regulated in rat liver in various models of experimental cholestasis, such as cholestasis induced by common bile duct ligation, estrogen, endotoxin or cytokine treatment. In addition to ntcp, the sinusoidal organic anion transporting polypeptide oatp-1 is also down-regulated at the protein and steady-state mRNA levels in estrogen-cholestasis, but does not affect sodium-independent uptake of taurocholate. The regulation of a recently cloned member of the organic anion transporter family (oatp-2), which is highly expressed in liver, remains to be studied under cholestatic conditions.     

The clinical significance of type 1 fiber predominance

Having recently demonstrated that taurine supplementation prevents total parenteral nutrition (TPN)-induced cholestasis, we chose to use this model to examine plasma membrane composition in relation to bile formation. Male guinea pigs received daily a mixture of glucose and of the amino acid solution Travasol with or without added taurine (1.2 mM). After 3 days, bile was collected and liver plasma membrane fractions enriched in sinusoidal lateral membrane and bile canalicular membrane domains were isolated. In animals receiving TPN alone, bile flow and biliary secretory rate of bile acid and bicarbonate decreased significantly compared with controls. Although membrane ATPases (Na+K+ and Mg+) were unchanged, TPN induced an increase in the lipid to protein ratio and a decrease of polyunsaturated fatty acids, in conjunction with a higher content of diene conjugates in sinusoidal lateral membrane fractions. Taurine corrected these changes and, in addition, reduced significantly the cholesterol to phospholipid ratio in both membrane fractions. The data show that changes in liver cell membranes occur in TPN-induced cholestasis and suggest that free radical injury may play a role. As taurine prevented cholestasis as well as membrane changes, it is suggested that taurine should be added to amino acid solutions used for parenteral nutrition.     

Postcholestatic alkaline phosphatase activity after relief of bile duct obstruction in the rat

The effects of obstructive cholestasis on the activity of alkaline phosphatase have been extensively studied in serum and liver tissue. However, very little is known about the activity of this enzyme in the postcholestatic condition after relief of the biliary obstruction. The purpose of this study has been to characterize alkaline phosphatase activity in serum, liver and bile in the postcholestatic period and to relate it to changes in bile acid secretory rate. Serum activity and biliary secretory rates of alkaline phosphatase were markedly increased in rats subjected to a reversible obstructive cholestasis for 24 hr or 48 hr and progressively declined along the postcholestatic period to values not significantly different from those of control rats within 48 hr. A significant direct linear relationship between the biliary secretory rates of enzyme activity and bile salts was apparent both in cholestatic groups and in the control groups. The slope of the regression line (units of alkaline phosphatase secreted per micromole of bile salts) was 1.5-fold to 3-fold higher in cholestatic animals. Remarkably, a positive y-intercept of regression lines suggested that a significant fraction of the enzyme was secreted independently of bile salts; this fraction was 18-fold and 34-fold greater in 24-hr and 48 hr cholestatic rats, respectively, compared with that in controls. Sodium taurocholate administered intravenously, either as a bolus or as an infusion at increasing submaximal rates, resulted in parallel increases of bile salt and alkaline phosphatase secretory rates into bile. The enzyme activity secreted per micromole of taurocholate was significantly greater in cholestatic than in control rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Liver tissue schistosomiasis mansoni vaccine, as a third generation livestock immunogenes

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

Nitric oxide and guanosine 3',5'-cyclic monophosphate stimulate bile secretion in isolated rat hepatocyte couplets, but not in isolated bile duct units

Nitric oxide (NO) and guanosine 3',5'-cyclic monophosphate (cGMP) have recently been shown to stimulate bile acid-independent bile flow in the isolated perfused rat liver (IPRL). However, the cellular origin and mechanisms of this choleresis have not yet been determined. To address these questions, we examined the effects of NO and cGMP on bile secretion in isolated rat hepatocyte couplets (IRHC) and in isolated bile duct units (IBDU), both of which are isolated cell systems in which cell polarity is maintained and secretion can be measured directly. Changes in the area of the canalicular and ductular lumens were determined in IRHC and IBDU, respectively, as indicators of the rate of fluid secretion using video microscopy. In addition, Cl-/HCO3- exchanger activity in IBDU was evaluated by measuring changes in intracellular pH (pHi) after Cl- removal/readmission by microfluorometric methods. In the presence of HCO3-, both the NO donor, S-nitroso-acetyl-penicillamine (SNAP), and the cell-permeant cGMP analogue, dibutyryl cGMP (DBcGMP), stimulated canalicular bile secretion (P <.05), as did the cell-permeant cAMP analogue, dibutyryl cAMP (DBcAMP) (P <.05). Removal of HCO3- from the buffer completely abolished the choleretic effects of DBcGMP, but had no effect on NO-induced choleresis. In contrast, secretion in IBDU was not stimulated following incubations with SNAP or DBcGMP over 30 minutes, whereas DBcAMP and secretin, a cholangiocyte secretagogue and cAMP agonist, both had a marked effect on ductular secretion over this same time interval (P <.05). SNAP also had no effect on Cl-/HCO3- exchanger activity in IBDU, and inhibition of endogenous NO synthesis by NG-monomethyl-L-arginine (L-NMMA) did not alter secretin-induced stimulation of ductular bile secretion and Cl-/HCO3- exchanger activity. In summary, NO and cGMP stimulate bile secretion exclusively at the the level of hepatocytes, whereas cAMP mediates choleresis at both hepatocyte and bile duct levels. These findings may have important implications for the regulation of ductular bile secretion by hormones and neuropeptides, as well as under pathological conditions with increased hepatic NO synthesis.     

Terbinafine-induced prolonged cholestasis with reduction of interlobular bile ducts

The antifungal drug terbinafine has infrequently been incriminated in the occurrence of acute liver injury. We report a case of prolonged cholestasis that occurred in a 75-year-old woman, following terbinafine administration. Jaundice followed by pruritus appeared after four weeks of therapy and was associated with mixed hepatocellular and cholestatic liver tests abnormalities. Following drug withdrawal, serum bilirubin returned to normal values within three months, but anicteric cholestasis persisted for over six months. A liver biopsy performed after six months showed centrilobular cholestasis, discrete portal fibrosis, and a reduction in the number of interlobular biliary ducts. Terbinafine should be added to the list of drugs that can cause reduction in interlobular bile ducts.     

Maitotoxin, a cationic channel activator

ICAM-1 mediates the recruitment of neutrophils through the endothelium to the site of inflammation by the ICAM-1/Mac-1 and ICAM-1/LFA-1 adhesion pathways. In extrahepatic cholestasis, recruitment of neutrophils is a main feature of the inflammatory infiltrate in areas of parenchymal damage. The aim of the present study was to describe the light and electron microscopical localization of ICAM-1 expression in the liver of cholestatic patients. The peroxidase-antiperoxidase technique was used. Increased ICAM-1 expression was detected on sinusoidal endothelial and Kupffer cells. A de novo ICAM-1 expression was described on some Ito cells and the sinusoidal hepatocyte membrane in areas of parenchymal injury. In the portal areas of livers of cholestatic patients, ICAM-1 was observed on the endothelial surface of portal veins and on hepatic arteries. Occasionally, ICAM-1 was found on the surface of bile duct epithelia. It is suggested that ICAM-1 expression is up-regulated by cytokines like TNF-alpha, IL-1 and interferons released from activated Kupffer cells. The mechanisms of ICAM-1 upregulation and neutrophil recruitment in the liver during extrahepatic cholestasis are discussed.     

Endothelin-1 induces cholestasis which is mediated by an increase in portal pressure

Endothelin-1 (ET-1) is a potent vasoactive peptide which generally exerts its effect on target cells by increasing [Ca++]i. Both vasoconstriction (resulting in an increase in perfusion pressure) and increased [Ca++]i are actions of ET-1 that may result in cholestasis. Single-pass isolated perfused rat liver (IPRL) were used, and [Ca++]i was measured in both populations of hepatocytes and single cells. ET-1 (0.1-100 nM) induced a dose-dependent increase in perfusion pressure and decrease in bile flow. Perfusion pressure increased by 112% (p < 0.001) and bile flow decreased by 17% (p < 0.008) in response to 2 nM ET-1. At this concentration of ET-1, but not at higher concentrations, the cholestasis was abolished (p > 0.18 vs basal) and the rise in perfusion pressure was decreased (by 62%; p < 0.002) by the vasodilator papaverine. This ET-1 concentration also had no measurable effect on [Ca++]i in isolated hepatocytes. Taken together these findings indicate that ET-1 inhibits bile flow in IPRL and suggests that this effect is mediated by vasoconstriction and not by changes in hepatocyte cytosolic Ca++.     

The Toll pathway is required in the epidermis for muscle development in the Drosophila embryo

BACKGROUND & AIMS: Hydrophobic bile acids have been implicated in the pathogenesis of cholestatic liver injury. The hypothesis that hydrophobic bile acid toxicity is mediated by oxidant stress in an in vivo rat model was tested in this study. METHODS: A dose-response study of bolus intravenous (i.v.) taurochenodeoxycholic acid (TCDC) in rats was conducted. Rats were then pretreated with parenteral alpha-tocopherol, and its effect on i.v. TCDC toxicity was evaluated by liver blood tests and by assessing mitochondrial lipid peroxidation. RESULTS: Four hours after an i.v. bolus of TCDC (10 mumol/100 g weight), serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels peaked, hepatic mitochondria showed evidence of increased lipid peroxidation, and serum bile acid analysis was consistent with a cholestatic injury. Liver histology at 4 hours showed hepatocellular necrosis and swelling and mild portal tract inflammation. Treatment with parenteral alpha-tocopherol was associated with a 60%-70% reduction in AST and ALT levels, improved histology, and a 60% reduction in mitochondrial lipid peroxidation in rats receiving TCDC. CONCLUSIONS: These data show that hepatocyte injury and oxidant damage to mitochondria caused by i.v. TCDC can be significantly reduced by pretreatment with the antioxidant vitamin E. These in vivo findings support the role for oxidant stress in the pathogenesis of bile acid hepatic toxicity.     

Hormone-induced bile flow and hepatobiliary calcium fluxes are attenuated in the perfused liver of rats made cholestatic with ethynylestradiol in vivo and with phalloidin in vitro

The actions of vasopressin and glucagon, administered alone or together, were assessed on bile flow in perfused livers from rats made cholestatic by the injection of ethynylestradiol and from those allowed to recover from such treatment. Concomitant measurements were made of biliary calcium output as well as changes in the perfusate Ca2+ concentration, glucose output, and oxygen uptake. Experiments were also conducted where cholestasis was induced in vitro in the perfused liver by the infusion of phalloidin. In each case cholestasis was demonstrated to have occurred by a reduction in bile flow by approximately 50%. The data show that the transient increase in bile flow and bile calcium seen in control rat liver soon after the administration of vasopressin, particularly when coadministered with glucagon, is largely absent in cholestasis induced by ethynylestradiol and attenuated in cholestasis induced by phalloidin. At the same time the pattern of perfusate Ca2+ fluxes in ethynylestradiol-induced cholestasis shifts to one reflecting net efflux of the ion from the liver. The responses to glucagon administration alone contrast with those of vasopressin in that in the perfused liver of ethynylestradiol-treated rats, glucagon induces a pronounced and sustained increase in bile flow. In cholestasis induced by both ethynylestradiol and phalloidin, glucagon fails to induce an initial transient decrease in bile flow. The effects of glucagon, including enhancement of vasopressin-stimulated bile flow in control and in ethynylestradiol-treated rats, can be mimicked by dibutyryl cyclic adenosine monophosphate (cAMP). Changes in glucose output and oxygen uptake induced by both hormones are only slightly attenuated. The data show that the modulation of bile flow that occurs rapidly after the administration of vasopressin and glucagon to control perfused rat liver is altered in conditions of cholestasis induced by either ethynylestradiol or phalloidin.     

The effect of idebenone, a coenzyme Q analogue, on hydrophobic bile acid toxicity to isolated rat hepatocytes and hepatic mitochondria

Oxidant stress induced by hydrophobic bile acids has been implicated in the pathogenesis of liver injury in cholestatic liver disorders. We evaluated the effect of idebenone, a coenzyme Q analogue, on taurochenodeoxycholic acid (TCDC)-induced cell injury and oxidant stress in isolated rat hepatocytes and on glycochenodeoxycholic acid (GCDC)-induced generation of hydroperoxides in fresh hepatic mitochondria. Isolated rat hepatocytes in suspension under 9% oxygen atmosphere were preincubated with 0, 50, and 100 micromol/l idebenone for 30 min and then exposed to 1000 micromol/l TCDC for 4 h. LDH release (cell injury) and thiobarbituric acid reactive substances (measure of lipid peroxidation) increased after TCDC exposure but were markedly suppressed by idebenone pretreatment. In a second set of experiments, the addition of 100 micromol/l idebenone up to 3 h after hepatocytes were exposed to 1000 micromol/l TCDC resulted in abrogation of subsequent cell injury and markedly reduced oxidant damage to hepatocytes. Chenodeoxycholic acid concentrations increased to 5.15 nmol/10(6) cells after 2 h and to 7.05 after 4 h of incubation of hepatocytes with 1000 micromol/l TCDC, and did not differ in the presence of idebenone. In freshly isolated rat hepatic mitochondria, when respiration was stimulated by succinate, 10 micromol/l idebenone abrogated the generation of hydroperoxides during a 90-minute exposure to 400 micromol/l GCDC. These data demonstrate that idebenone functions as a potent protective hepatocyte antioxidant during hydrophobic bile acid toxicity, perhaps by reducing generation of oxygen free radicals in mitochondria.     

Basal total opioid peptide release in the striatum of rats with cholestasis from bile duct resection: a study by the use of in vivo microdialysis

The opiate withdrawal-like reaction experienced by patients with cholestatic liver disease after the ingestion of the opiate antagonist nalmefene led to the hypothesis that increased opioidergic neurotransmission/neuromodulation in the central nervous system (CNS) contributes to the pathophysiology of cholestasis. The state of antinociception, which is stereospecifically reversed by naloxone, documented in rats with cholestasis from bile duct resection supports this hypothesis. To further study the opioid system in this animal model of cholestasis, we studied the release of endogenous opioid peptides into the extracellular fluid of the dorso-lateral striatum by the technique of in-vivo microdialysis. Total opioid peptide concentration in the dialysate was measured by a solid phase radioimmunoassay with an antibody directed against the N-terminus of the Tyr-Gly-Gly-Phe-X amino acid sequence after acetylation. Basal total opioid peptide release was significantly higher after surgery in both sham resected and bile duct resected animals. However, basal (unstimulated) total opioid peptide release in the striatum of rats was not altered by cholestasis. It is inferred that the opioidergic abnormalities of cholestasis are not associated with an appreciable increase in the release of endogenous opioids into the extracellular fluid of the striatum. Abnormal processing of specific opioid peptides in cholestasis however, cannot be excluded.