To elucidate mechanisms of glucagon-induced bicarbonaterich choleresis, we investigated the effect of glucagon on ion transport processes involved in the regulation of intracellular pH (pH1) in isolated rat hepatocyte couplets. It was found that glucagon (200 nM), without influencing resting pH1, 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, uM) and H-89 (30 ,uM), the latter having no influence on the glucagon-induced cAMP accumulation in isolated rat hepatocytes. The Cl -channel blocker, NPPB (10 pmM), showed no effect on either the basal or the glucagon-stimulated Cl-/ HCO3 exchange. In contrast, the protein kinase C agonist, PMA (10 jpM), completely blocked the glucagon stimulation of the C1-/HCO3 exchange; however, this effect was achieved through a significant inhibition of the glucagonstimulated 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 pH1 or at acid pH, values. In contrast, glucagon, at basal pH1, stimulated the Na+-HCO-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. (J. Clin. Invest. 1995.

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.

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.

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)

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.