Adaptive regulation of bile salt transporters in kidney and liver in obstructive cholestasis in the rat.

BACKGROUND & AIMS: Cholestasis results in adaptive regulation of bile salt transport proteins in hepatocytes that may limit liver injury. However, it is not known if changes also occur in the expression of bile salt transporters that reside in extrahepatic tissues, particularly the kidney, which might facilitate bile salt excretion during obstructive cholestasis. METHODS: RNA and protein were isolated from liver and kidney 14 days after common bile duct ligation in rats and assessed by RNA protection assays, Western analysis, and tissue immunofluorescence. Sodium-dependent bile salt transport was also measured in brush border membrane vesicles from the kidney. RESULTS: After common bile duct ligation, serum bile salts initially rose and then declined to lower levels after 3 days. In contrast, urinary bile salt excretion rose progressively over the 2-week period. By that time, the ileal sodium-dependent bile salt transporter messenger RNA and protein expression in total liver had increased to 300% and 200% of controls, respectively, while falling to 46% and 37% of controls, respectively, in the kidney. Sodium-dependent uptake of (3)H-taurocholate in renal brush border membrane vesicles was decreased. In contrast, the multidrug resistance-associated protein 2 expression in the kidney was increased 2-fold, even 1 day after ligation. Immunofluorescent studies confirmed the changes in the expression of these transporters in liver and kidney. CONCLUSIONS: These studies show that the molecular expression of bile salt transporters in the kidney and cholangiocytes undergo adaptive regulation after common bile duct obstruction in the rat. These responses may facilitate extrahepatic pathways for bile salt excretion during cholestasis.

Cl(-)-dependent secretory mechanisms in isolated rat bile duct epithelial units.

Cholangiocytes absorb and secrete fluid, modifying primary canalicular bile. In several Cl(-)-secreting epithelia, Na(+)-K(+)-2Cl(-) cotransport is a basolateral Cl(-) uptake pathway facilitating apical Cl(-) secretion. To determine if cholangiocytes possess similar mechanisms independent of CO2/HCO, we assessed Cl(-)-dependent secretion in rat liver isolated polarized bile duct units (IBDUs) by using videomicroscopy. Without CO2/HCO, forskolin (FSK) stimulated secretion entirely dependent on Na(+) and Cl(-) and inhibited by Na(+)-K(+)-2Cl(-) inhibitor bumetanide. Carbonic anhydrase inhibitor ethoxyzolamide had no effect on FSK-stimulated secretion, indicating negligible endogenous CO2/HCO transport. In contrast, FSK-stimulated secretion was inhibited approximately 85% by K(+) channel inhibitor Ba(2+) and blocked completely by bumetanide plus Ba(2+). IBDU Na(+)-K(+)-2Cl(-) cotransport activity was assessed by recording intracellular pH during NH4Cl exposure. Bumetanide inhibited initial acidification rates due to NH entry in the presence and absence of CO2/HCO. In contrast, when stimulated by FSK, a 35% increase in Na(+)-K(+)-2Cl(-) cotransport activity occurred without CO2/HCO. These data suggest a cellular model of HCO-independent secretion in which Na(+)-K(+)-2Cl(-) cotransport maintains high intracellular Cl(-) concentration. Intracellular cAMP concentration increases activate basolateral K(+) conductance, raises apical Cl(-) permeability, and causes transcellular Cl(-) movement into the lumen. Polarized IBDU cholangiocytes are capable of vectorial Cl(-)-dependent fluid secretion independent of HCO. Bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransport, Cl(-)/HCO exchange, and Ba(2+)-sensitive K(+) channels are important components of stimulated fluid secretion in intrahepatic bile duct epithelium.

Molecular identification and functional characterization of Mdr1a in rat cholangiocytes.

BACKGROUND & AIMS: The multidrug resistance P-glycoprotein 170 gene products (mdr1a and 1b) are glycosylated plasma membrane proteins that function as adenosine triphosphate-dependent transmembrane export pumps for lipophilic xenobiotics of widely different structure. We assessed whether these P-glycoproteins are functionally expressed in cholangiocytes. METHODS: A reverse-transcription polymerase chain reaction was performed on RNA from a normal rat cholangiocyte cell line using mdr1-specific primers. Northern and Western blot analyses were performed on cholangiocytes immunoisolated from 2-week bile duct-ligated rats and cholangiocytes and isolated cholangiocyte membrane subfractions, respectively. Functional assays were performed in isolated bile duct units from bile duct-ligated rats and incubated with rhodamine 123, a P-glycoprotein substrate, with or without the P-glycoprotein inhibitors verapamil or GF120918. RESULTS: A 400-base pair fragment with 99% homology to the cytosolic domain of rat intestinal mdr1a (5' 1953-2350 3') was identified that hybridized to a 5.2-kilobase RNA transcript in a normal rat cholangiocyte cell line, isolated rat cholangiocytes, and ileum. Western analysis localized mdr1 to the apical membrane of cholangiocytes. Confocal microscopy showed active secretion of rhodamine 123 into the lumen of isolated bile duct units that was abolished by vanadate and P-glycoprotein competitive antagonists, verapamil and GF120918, in a dose-dependent manner. CONCLUSIONS: These findings provide the first molecular and functional evidence for the expression of mdr1a on the luminal membrane of cholangiocytes, where it may have a protective role.

Effect of pharmacological modulation of liver P-glycoproteins on cyclosporin A biliary excretion and cholestasis: a study in isolated perfused rat liver.

In different cell types P-glycoproteins (P-gp) are involved in the transport of cyclosporin A (CyA). The aim of this study was to evaluate the effect of the pharmacological modulation of the hepatic P-gp on biliary secretion of CyA and on cholestasis induced by acute administration of CyA in the isolated perfused rat liver (IPRL). Verapamil was used as a P-gp specific inhibitor and acetylaminofluorene (AAF) as a P-gp inducer. CyA biliary excretion was determined by administering in the IPRL a tracer dose of [3H]CyA with or without verapamil or AAF. The effect on bile flow was evaluated by administering increasing doses of CyA (2.8, 8, and 20 mg/kg body wt) in the IPRL. Morphological evidence of damage was evaluated by optical and electron microscopy in the liver as well as in primary culture of rat hepatocytes exposed to CyA +/- verapamil. Verapamil significantly inhibited the biliary excretion of a tracer dose of [3H]CyA (0.15+/-0.04 vs 0.33+/-0.07%; P < 0.05). In contrast, pretreatment with AAF significantly increased the biliary excretion of [3H]CyA, (0.61+/-0.10 vs 0.33+/-0.07%; P < 0.05). CyA induced a dose-dependent inhibition of bile flow with a maximal effect at 20 mg/kg CyA (-49.3+/-4.5% decrease of basal bile flow). CyA cholestasis was significantly worsened by the P-gp inhibitor, verapamil (-75.5+/-7.5%; P < 0.05), but it was unaffected by induction of P-gp via AAF pretreatment (-44.9+/-1.7%). During CyA cholestasis, the cumulative biliary excretion of [3H]CyA was lower than in the absence of cholestasis (0.22+/-0.05 vs 0.33+/-0.07%; P < 0.05), was inhibited by verapamil (0.08+/-0.01%; P < 0.05), but was unaffected by AAF (0.23+/-0.05%). No morphological evidence of damage was observed in the liver, and no evidence of cytoskeleton derangement was seen in primary cultures of rat hepatocytes exposed to CyA +/- verapamil. We demonstrated that pharmacological modulation of P-gp may influence the biliary excretion of CyA. The acute cholestatic effect of CyA is worsened by P-gp inhibitors, while it is unaffected by P-gp inducers. This indicates CyA should not be given with other P-gp substrates or inhibitors.

Endoscopic palliative therapy in neoplastic diseases of the esophagus.

Over the past 20 years, fiberoptic endoscopy, followed by video-endoscopy, has taken on an increasingly important role in the diagnostics and therapy for digestive tract diseases. Especially in the field of oncology, endoscopy is fundamental not only for the diagnosis and staging of diseases of the gastroenteric tract but also as definitive and/or palliative therapy for tumors that do not respond to radical treatment. Endoscopic techniques are widely employed in diseases of the esophagus and the head and neck district. In fact, it is generally accepted that only 35% of patients with cancer of the esophagus or of the cardias may benefit from surgery with 5-year survival rates of about 5% in Western countries (1, 2).

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.

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.

Regulation of endocytic-transcytotic pathways and bile secretion by phosphatidylinositol 3-kinase in rats.

BACKGROUND & AIMS: Phosphatidylinositol 3-kinases (PI3-K) are a family of enzymes that play key roles in control of cell growth, membrane recycling, and vesicular endoexocytotic processes. The aim of this study was to investigate the effect of a specific PI3-K inhibitor, wortmannin, on bile secretion, cytoskeleton organization, and endotranscytotic pathways in rats. METHODS: Isolated perfused rat liver (IPRL) and isolated rat hepatocyte couplets (IRHCs) were used. RESULTS: Wortmannin induced a 25% inhibition of basal bile flow in IPRL (P < 0.01). Horseradish peroxidase biliary excretion in the IPRL was markedly decreased by wortmanin. In IRHC incubated with 25 nmol/L wortmannin for 10 minutes at 37 degrees C, morphological studies showed early significant dilatation of bite canalicular lumen (P < 0.001). At short intervals (3 minutes), uptake of the fluid-phase marker, Lucifer yellow, was markedly decreased by exposure to wortmannin (P < 0.001). At longer times (20 minutes), Lucifer yellow was retained in basolateral area of IRHC as compared with control cells, where the marker was rapidly transported to the pericanalicular area. In IRHC, wortmannin induced a marked disorganization of microfilaments. CONCLUSIONS: Wortmannin inhibits basal bile flow, endocytosis, and transcytotic transport of fluid-phase markers in the liver, and causes an early dilatation of the canalicular lumen and disorganization of microfilaments. These findings suggest that PI3-K is involved in the regulation of vesicle trafficking, cytoskeleton organization, and the process of bile formation.

Cytotoxicity of bile salts against biliary epithelium: a study in isolated bile ductule fragments and isolated perfused rat liver.

We evaluated cytotoxic effects of different unconjugated and glycine- and taurine-conjugated bile salts (BS) against bile duct epithelial cells in isolated bile ductule fragments and isolated perfused rat liver. Ultrastructural morphometric studies were performed in polarized rat bile ductule fragments exposed in vitro to increasing concentrations (10-100 micromol/L) of lithocholate (LCA), deoxycholate (DCA), chenodeoxycholate (CDCA), cholate (CA), ursodeoxycholate (UDCA), their taurine-conjugates, and glycoconjugates of cholic (GCA) or chenodeoxycholic acid (GCDCA) for 20, 30, or 75 minutes. To evaluate the cytotoxicity of unconjugated hydrophobic bile salts against biliary epithelium (BDE) in the whole liver, livers were isolated from rats with impaired taurine-conjugation capacity (beta-alanine treatment) and perfused for 70 minutes with 2 micromol/min LCA (n = 6), CDCA (n = 6), CA (n = 6), or 0.5 micromol/min tauro-LCA (n = 4). In isolated bile ductule fragments, hydrophobic unconjugated bile salts (LCA, CDCA, DCA) induced a marked damage of intracellular organelles, mainly mitochondria. The damage started at a concentration of 10 micromol/L and became prominent at concentrations higher than 50 micromol/L. No damage of the apical and basolateral membrane was seen and tight junctions appeared intact. UDCA, taurine and glycoconjugated bile salts failed to induce any evident ultrastructural alteration. In taurine-depleted isolated livers, perfused with LCA, CDCA, or CA, bile duct epithelial cells showed no evidence of intracellular damage, despite the increased biliary excretion of unconjugated BS. Marked alterations of the apical cell membrane were seen only in livers perfused with LCA and in isolated segments of the biliary epithelium. In contrast with biliary epithelium, hepatocytes showed prominent subcellular damage with CA and CDCA, and profound alterations of the canalicular membrane with LCA and tauro-LCA. We have shown that, in vitro, BDE cells are not damaged by taurine- or glycine-conjugated BS, but they are very sensitive to cytotoxicity of hydrophobic unconjugated BS. Such sensitivity is not present in the whole liver, probably because of the specificity of BS transport processes, the microvascular architecture of the bile ductal system, and the presence in bile of a physiological surfactant, such as phospholipids.

Hormonal regulation of bicarbonate secretion in the biliary epithelium.

Bicarbonate excretion in bile is a major function of the biliary epithelium. It is driven by the apically located Cl-/HCO3- exchanger which is functionally coupled with a cAMP-dependent Cl- channel (CFTR). A number of hormones and/or neuropeptides with different mechanisms and at different intracellular levels regulate, in concert, the processes underlying bicarbonate excretion in the biliary epithelium. Secretin induces a bicarbonate rich choleresis by stimulating the activity of the Cl-/HCO3- exchanger by cAMP and protein kinase A mediated phosphorylation of CFTR regulatory domain. Protein phosphatase 1/2A are involved in the run-down of secretory stimulus after secretin removal. Acetylcholine potentiates secretin-choleresis by inducing a Ca(++)-calcineurin mediated "sensitization" of adenyl cyclase to secretin. Bombesin and vasoactive intestinal peptide also enhance the Cl-/HCO3- exchanger activity, but the intracellular signal transduction pathway has not yet been defined. Somatostatin and gastrin inhibit basal and/or secretin-stimulated bicarbonate excretion by down-regulating the secretin receptor and decreasing cAMP intracellular levels induced by secretin.

Inhibition of biliary bicarbonate secretion in ethinyl estradiol-induced cholestasis is not associated with impaired activity of the Cl-/HCO-3 exchanger in the rat.

BACKGROUND/AIMS: Bicarbonate is a major component of bile salt independent bile flow, which is impaired in ethinyl estradiol (EE)-cholestasis. To examine this subject in EE-cholestasis, we studied: 1) basal and glucagon-stimulated biliary bicarbonate secretion both in vivo and in the isolated perfused rat liver (IPRL); 2) H+/HCO-3 transport processes in isolated rat hepatocyte couplets. METHODS: Rats received EE (5 mg.kg b.w.-1) for 5 days. Intracellular pH (pHi) was measured (BCECF-AM) using a single-cell microfluorimetric setup. RESULTS: Bile flow was markedly (p < 0.01) decreased in EE-treated rats. Bicarbonate concentration in bile was decreased (p < 0.01) and bicarbonate secretion was 2.5-fold lower in EE-treated animals than in controls, both in bile-fistula rats [19.5 +/- 5.1 (n = 23) vs 54.2 +/- 5.7 (n = 20) nmol.min-1g liver-1; p < 0.01] and in the IPRL [11 +/- 2 (n = 8) vs 24 +/- 3 (n = 8) nmol.min-1.g liver-1; p < 0.01]. In control IPRL, a bile/perfusate gradient for bicarbonate is maintained, while it is lost in EE-treated IPRL because of the lower bicarbonate concentration in bile. Glucagon stimulated bile flow and bicarbonate secretion to a similar extent in EE-treated and control IPRL (+25% vs +23%). Resting pHi of EE-treated hepatocyte couplets was higher in comparison with controls in KRB [7.25 +/- 0.07 (n = 35) vs 7.20 +/- 0.05 (n = 33); p < 0.02] but similar in Hepes [7.08 +/- 0.07 (n = 24) vs 7.05 +/- 0.06 (n = 26)]. Basal activity of the Cl-/HCO-3 exchanger was similar in EE-treated and control hepatocyte couplets [H+ flux = 2.87 +/- 1.12 (n = 18) vs 3.01 +/- 1.23 mM/min (n = 15)] and was stimulated to a similar extent by glucagon. Na+/HCO3-symport activity was increased in EE-treated hepatocyte couplets (p < 0.05) while the Na+/H+ exchanger was unchanged. CONCLUSIONS: Bicarbonate biliary secretion is markedly impaired during EE-cholestasis in association with a marked decrease of bile salt independent bile flow. However, the Cl-/HCO-3 exchanger and its hormonal regulation are normal, indicating that the lower bicarbonate excretion in EE-cholestasis is not due to a compromised activity of this anion exchanger. Since the bile/perfusate gradient for bicarbonate is dissipated in EE-treated IPRL, the impaired bicarbonate excretion could be caused by a reflux of biliary bicarbonate via leaky tight junctions.

Effect of glucagon on intracellular pH regulation in isolated rat hepatocyte couplets.

To elucidate mechanisms of glucagon-induced bicarbonate-rich choleresis, we investigated the effect of glucagon on ion transport processes involved in the regulation of intracellular pH (pHi) in isolated rat hepatocyte couplets. It was found that glucagon (200 nM), without influencing resting pHi, significantly stimulates the Cl-/HCO3- exchange activity. The effect of glucagon was associated with a sevenfold increase in cAMP levels in rat hepatocytes. The activity of the Cl-/HCO3- exchanger was also stimulated by DBcAMP + forskolin. The effect of glucagon on the Cl-/HCO3- exchange was individually blocked by two specific and selective inhibitors of protein kinase A, Rp-cAMPs (10 microM) and H-89 (30 microM), the latter having no influence on the glucagon-induced cAMP accumulation in isolated rat hepatocytes. The Cl- channel blocker, NPPB (10 microM), showed no effect on either the basal or the glucagon-stimulated Cl-/HCO3 exchange. In contrast, the protein kinase C agonist, PMA (10 microM), completely blocked the glucagon stimulation of the Cl-/HCO3- exchange; however, this effect was achieved through a significant inhibition of the glucagon-stimulated cAMP accumulation in rat hepatocytes. Colchicine pretreatment inhibited the basal as well as the glucagon-stimulated Cl-/HCO3- exchange activity. The Na+/H+ exchanger was unaffected by glucagon either at basal pHi or at acid pHi values. In contrast, glucagon, at basal pHi, stimulated the Na(+)-HCO3- symport. The main findings of this study indicate that glucagon, through the cAMP-dependent protein kinase A pathway, stimulates the activity of the Cl-/HCO3- exchanger in isolated rat hepatocyte couplets, a mechanism which could account for the in vivo induced bicarbonate-rich choleresis.

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.

Effect of S-adenosyl-L-methionine on ethanol cholestasis and hepatotoxicity in isolated perfused rat liver.

We investigated whether S-adenosyl-L-methionine (SAMe) influences the inhibitory effect of ethanol on bile secretion and ethanol hepatotoxicity in the isolated perfused rat liver. SAMe (25 mg/kg intramuscularly three times a day) was administered for three days consecutively. Liver was then isolated and perfused with taurocholate to stabilize bile secretion and exposed to 1% ethanol for 70 min. The effect of ethanol on bile flow, bile salt biliary secretion, oxygen liver consumption, AST and LDH release in the perfusate, and hepatic concentration of glutathione, malondialdehyde, and diene conjugates was compared between SAMe-treated livers (N = 11) and paired controls (N = 11). Control experiments without ethanol were also performed (N = 6). Exposure to 1% ethanol induced a significantly (P < 0.03) higher inhibition of bile flow (-35% vs 17%) and bile salt secretion (-28% vs 16%) in untreated compared with SAMe-treated livers. During 1% ethanol exposure, the release of LDH and AST in the perfusate was significantly lower (P < 0.02) in SAMe-treated livers. Oxygen liver consumption was markedly inhibited by 1% ethanol administration (P < 0.02 vs controls without ethanol), an effect almost totally prevented by SAMe treatment (P < 0.02 vs ethanol controls). The hepatic concentration of total glutathione was significantly (P < 0.02) decreased by 1% ethanol exposure, but this effect was less pronounced in SAMe-treated than in untreated controls (P < 0.02). The hepatic levels of malondialdehyde and diene conjugates were not significantly changed by ethanol exposure in either SAMe-treated or control livers in comparison to ethanol-free controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional and ultrastructural features of ethanol/bile salts interaction in the isolated perfused rat liver.

We investigated whether bile salts (BS) with different hydrophobic-hydrophilic properties interact with ethanol on bile secretion, enzyme (aspartate transaminase [AST], lactate dehydrogenase [LDH]) release in the perfusate, liver ultrastructure, and vesicular exocytosis in the isolated perfused rat liver. Ethanol (0.1 or 1%) promoted a rapid decrease of bile flow and BS secretion in livers perfused with taurocholate (TCA), the physiologic BS in the rat (-28% decrease of baseline values with 0.1% and -34% with 1% ethanol). The inhibitory effect of ethanol on bile flow and BS secretion was significantly (P < .02) attenuated by perfusing liver with the hydrophilic BS, tauroursodeoxycholate (TUDCA), and it was exacerbated (P < .02) by perfusion with the hydrophobic BS, taurodeoxycholate (TDCA). The release of AST and LDH in the perfusate was unaffected by 0.1% ethanol, but increased threefold to fivefold by 1% ethanol in TCA-perfused livers. This cytolitic effect of ethanol was not observed in TUDCA-perfused livers, but it was enhanced (P < .03) by perfusion with TDCA. No ultrastructural abnormalities were found in either TCA- or TUDCA-perfused livers, with or without 1% ethanol. Only minimal changes were found in livers perfused with TDCA alone, but, in the presence of TDCA, 1% ethanol induces marked mitochondrial damage. The biliary excretion of the fluid phase marker horseradish peroxidase was inhibited by ethanol, an effect reversed by TUDCA (P < .02) and exacerbated by TDCA (P < .04). In conclusion, this study demonstrates that hydrophilic BS such as TUDCA counteract the inhibitory effect of ethanol on bile secretion and vesicular exocytosis as well as the ethanol-induced cytolitic effect in the isolated perfused rat liver. In the presence of hydrophobic BS such as TDCA, the exposure to ethanol promotes a marked inhibition of bile secretion and vesicular exocytosis as well as prominent mitochondrial damage.

Effect of Brefeldin A on transcytotic vesicular pathway and bile secretion: a study on the isolated perfused rat liver and isolated rat hepatocyte couplets.

This study investigated the effect of Brefeldin A (BFA) on the transcytotic vesicular pathway labeled with horseradish peroxidase (HRP) in both isolated rat hepatocyte couplets (IRHC) and the isolated perfused rat liver (IPRL). To evaluate the role of the transcytotic vesicular pathway on bile secretion, the effect of BFA on bile secretion in the IPRL was then investigated. In the basolateral area of IRHC, BFA showed no effect on the density and percentage of area of HRP-labeled vesicles. However, HRP-labeled vesicles tended to accumulate in the juxtanuclear area of BFA-treated hepatocytes (P < .001 vs. controls). In the pericanalicular area, on the other hand, HRP-labeled vesicles were depleted compared with controls (P < .001). In keeping with these findings, although the early peak remained unchanged, BFA inhibited as much as 50% of the late peak of HRP excretion in bile, after a pulse load of HRP in the IPRL. Bile flow and the biliary secretion of bile salts (BS) and phospholipids were not modified by BFA in isolated livers perfused without BS in the perfusate or with 1 mumol/min taurocholate (TCA). In BFA-treated livers, peak bile flow and BS output decreased by 20% (P < .05 vs. controls) only when a 5 mumol TCA bolus was administered. In conclusion, this study demonstrates that BFA inhibits the transcytotic vesicular pathway in the liver. However, BFA has no significant effect on bile secretion either in basal conditions or during perfusion with physiological amounts of BS. BFA slightly decreases bile flow and BS output only after an overload of BS, providing evidence against the physiological relevance of the transcytotic vesicular pathway in the process of bile formation.