Biliary secretory function in rats chronically intoxicated with aluminum.
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The effects of a chronic aluminum (Al) exposure on biliary secretory function, with special emphasis on hepatic handling of non-bile salt organic anions, was investigated. Male Wistar rats received, intraperitoneally, either 27 mg/kg body weight of Al, as Al hydroxide [Al (+) rats], or the vehicle saline [Al (-) rats] three times a week for 3 months. Serum and hepatic Al levels were increased by the treatment (approximately 9- and 4-fold, respectively). This was associated with enhanced malondialdehyde formation (+110%) and a reduction in GSH content (-17%) and in the activity of the antioxidant enzymes catalase (-84%) and GSH peroxidase (-46%). Bile flow (-23%) and the biliary output of bile salts (-39%), cholesterol (-43%), and proteins (-38%) also decreased. Compartmental analysis of the plasma decay of the model organic anion bromosulphophthalein revealed that sinusoidal uptake and canalicular excretion of the dye were significantly decreased in Al (+) rats (-53 and -43%, respectively). Expression of multidrug resistance-associated protein 2 (Mrp2), the main, multispecific transporter involved in the canalicular excretion of organic anions, was also decreased (-40%), which was associated with a significant decrease in the cumulative biliary excretion of the Mrp2 substrate, dinitrophenyl-S-glutathione (-50%). These results show that chronic Al exposure leads to oxidative stress, cholestasis, and impairment of the hepatic handling of organic anions by decreasing both sinusoidal uptake and canalicular excretion. The alteration of the latter process seems to be causally related to impairment of Mrp2 expression. We have addressed some possible mechanisms involved in these deleterious effects. (+info)
Intracellular trafficking of bile salt export pump (ABCB11) in polarized hepatic cells: constitutive cycling between the canalicular membrane and rab11-positive endosomes.
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The bile salt export pump (BSEP, ABCB11) couples ATP hydrolysis with transport of bile acids into the bile canaliculus of hepatocytes. Its localization in the apical canalicular membrane is physiologically regulated by the demand to secrete biliary components. To gain insight into how such localization is regulated, we studied the intracellular trafficking of BSEP tagged with yellow fluorescent protein (YFP) in polarized WIF-B9 cells. Confocal imaging revealed that BSEP-YFP was localized at the canalicular membrane and in tubulo-vesicular structures either adjacent to the microtubule-organizing center or widely distributed in the cytoplasm. In the latter two locations, BSEP-YFP colocalized with rab11, an endosomal marker. Selective photobleaching experiments revealed that single BSEP-YFP molecules resided in canalicular membranes only transiently before exchanging with intracellular BSEP-YFP pools. Such exchange was inhibited by microtubule and actin inhibitors and was unaffected by brefeldin A, dibutyryl cyclic AMP, taurocholate, or PI 3-kinase inhibitors. Intracellular carriers enriched in BSEP-YFP elongated and dissociated as tubular elements from a globular structure adjacent to the microtubule-organizing center. They displayed oscillatory movement toward either canalicular or basolateral membranes, but only fused with the canalicular membrane. The pathway between canalicular and intracellular membranes that BSEP constitutively cycles within could serve to regulate apical pools of BSEP as well as other apical membrane transporters. (+info)
Bezafibrate stimulates canalicular localization of NBD-labeled PC in HepG2 cells by PPARalpha-mediated redistribution of ABCB4.
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Fibrates, including bezafibrate (BF), upregulate the expression of ATP binding cassette protein B4 (ABCB4) through gene transcription in mice. To determine the effects of BF on the expression levels of ABCB4 and on the stimulation of biliary phosphatidylcholine (PC) transport in human HepG2 hepatoblastoma cells, mRNA and protein levels as well as subcellular localization were investigated in the cells treated with BF. The canalicular accumulation of a fluorescent PC was assessed by confocal laser scanning microscopy. Treatment with 300 micromol/l BF for 24 h increased levels of ABCB4 mRNA but not protein by up to 151%. BF caused redistribution of ABCB4 into pseudocanaliculi formed between cells. In association with this redistribution, BF accelerated the accumulation of fluorescent PC in bile canaliculi (up to 163% of that in nontreated cells). Suppression of peroxisome proliferator-activated receptor alpha (PPARalpha) expression by either a small interfering RNA duplex or morpholino antisense oligonucleotide attenuated the BF-induced redistribution of ABCB4. These findings suggest that BF may enhance the capacity of human hepatocytes to direct PC into bile canaliculi via PPARalpha-mediated redistribution of ABCB4 to the canalicular membrane. This provides a rationale for the use of BF to improve cholestasis and/or cholangitis that is attributable to hypofunction of ABCB4. (+info)
Role of microtubules in estradiol-17beta-D-glucuronide-induced alteration of canalicular Mrp2 localization and activity.
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Estradiol-17beta-D-glucuronide (E2-17G) induces a marked but reversible inhibition of bile flow in the rat together with endocytic retrieval of multidrug resistance-associated protein 2 (Mrp2) from the canalicular membrane to intracellular structures. We analyzed the effect of pretreatment (100 min) with the microtubule inhibitor colchicine or lumicholchicine, its inactive isomer (1 micromol/kg iv), on changes in bile flow and localization and function of Mrp2 induced by E2-17G (15 micromol/kg iv). Bile flow and biliary excretion of bilirubin, an endogenous Mrp2 substrate, were measured throughout, whereas Mrp2 localization was examined at 20 and 120 min after E2-17G by confocal immunofluorescence microscopy and Western analysis. Colchicine pretreatment alone did not affect bile flow or Mrp2 localization and activity over the short time scale examined (3-4 h). Administration of E2-17G to colchicine-pretreated rats induced a marked decrease (85%) in bile flow and biliary excretion of bilirubin as well as internalization of Mrp2 at 20 min. These alterations were of a similar magnitude as in rats pretreated with lumicolchicine followed by E2-17G. Bile flow and Mrp2 localization and activity were restored to control levels within 120 min of E2-17G in animals pretreated with lumicolchicine. In contrast, in colchicine-pretreated rats followed by E2-17G, bile flow and Mrp2 activity remained significantly inhibited by 60%, and confocal and Western studies revealed sustained internalization of Mrp2 120 min after E2-17G. We conclude that recovery from E2-17G cholestasis, associated with exocytic insertion of Mrp2 in the canalicular membrane, but not its initial E2-17G-induced endocytosis, is a microtubule-dependent process. (+info)
Long-term effect of treating pregnant rats with ursodeoxycholic acid on the congenital impairment of bile secretion induced in the pups by maternal cholestasis.
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Transient latent cholestasis in young rats born from mothers with obstructive cholestasis during pregnancy (OCP) has been reported. The cause of this congenital impairment and the long-term effect on the pups of treating their mothers with ursodeoxycholic acid (UDCA) during pregnancy were investigated. Complete biliary obstruction was imposed on day 14 of pregnancy and UDCA treatment was begun on day 15. Serum bile acids (BAs) concentrations were elevated in 4-week-old pups born from OCP, but not OCP + UDCA, mothers. However, gas chromatographic/mass spectrometric analysis of BA species in basal bile indicated the presence of significant differences among all experimental groups (control, OCP, and OCP + UDCA). Canalicular plasma membrane fluidity was reduced in OCP, but not in OCP + UDCA, pups. Screening by reverse transcription followed by real-time quantitative polymerase chain reaction of the steady-state levels of mRNA of genes related to hepatobiliary function revealed changes (upregulation of Cyp7a1 and Mrp1 and down-regulation of Abcg5 and Abcg8) in OCP group, which were prevented by UDCA treatment. Electron microscopy examination showed multilamellar bodies occupying part of the canalicular lumen in OCP pups. Their number and size were reduced in animals born from OCP + UDCA mothers. In OCP, but not OCP + UDCA, the stimulation of bile flow and BA output induced by taurocholate administration were reduced and cholesterol/BA output ratio was increased, whereas phospholipid/BA output ratio was enhanced in both groups (OCP > OCP + UDCA). In conclusion, UDCA treatment of rats with cholestasis during pregnancy has long-term beneficial effects on their offspring by preventing in part the congenital impairment in hepatobiliary function of the pups that affects their biliary lipid secretion. (+info)
Role of farnesoid X receptor in the enhancement of canalicular bile acid output and excretion of unconjugated bile acids: a mechanism for protection against cholic acid-induced liver toxicity.
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Mice lacking the farnesoid X receptor (FXR) involved in the maintenance of hepatic bile acid levels are highly sensitive to cholic acid-induced liver toxicity. Serum aspartate aminotransferase (AST) activity was elevated 15.7-fold after feeding a 0.25% cholic acid diet, whereas only slight increases in serum AST (1.7- and 2.5-fold) were observed in wild-type mice fed 0.25 and 1% cholic acid diet, respectively. Bile salt export pump mRNA and protein levels were increased in wild-type mice fed 1% cholic acid diet (2.1- and 3.0-fold) but were decreased in FXR-null mice fed 0.25% cholic acid diet. The bile acid output rate was 2.0- and 3.7-fold higher after feeding of 0.25 and 1.0% cholic acid diet in wild-type mice, respectively. On the other hand, no significant increase in bile acid output rate was observed in FXR-null mice fed 0.25% cholic acid diet in contrast to a significant decrease observed in mice fed a 1.0% cholic acid diet in spite of the markedly higher levels of hepatic tauro-conjugated bile acids. Unconjugated cholic acid was not detected in the bile of wild-type mice fed a control diet, but it was readily detected in wild-type mice fed 1% cholic acid diet. The ratio of biliary unconjugated cholic acid to total cholic acid (unconjugated cholic acid and tauro-conjugated cholic acid) reached 30% under conditions of hepatic taurine depletion. These results suggest that the cholic acid-induced enhancement of canalicular bile acid output rates and excretion of unconjugated bile acids are involved in adaptive responses for prevention of cholic acid-induced toxicity. (+info)
Xenobiotics inhibit hepatic uptake and biliary excretion of taurocholate in rat hepatocytes.
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Reports suggest that troglitazone, and to a lesser extent bosentan, may alter bile acid homeostasis by inhibiting the bile salt export pump. The present studies examined the hypothesis that these xenobiotics may modulate multiple hepatic bile acid transport mechanisms. In suspended rat hepatocytes, troglitazone (10 microM) decreased the initial rate of taurocholate uptake approximately 3-fold; the initial uptake rate of estradiol-17beta-D-glucuronide, a substrate of the organic anion transporting polypeptides, also was decreased approximately 4-fold. Bosentan (100 microM) decreased the initial uptake rate of taurocholate and estradiol-17beta-D-glucuronide by approximately 12- and approximately 7-fold, respectively. In sandwich-cultured rat hepatocytes, 10-min accumulation of taurocholate in cells + bile canaliculi (408 +/- 57 pmol/mg protein) was decreased significantly by troglitazone (157 +/- 17 pmol/mg protein, respectively) only in the presence of Na+, the driving force for the sodium taurocholate cotransporting polypeptide. A similar decrease with 10-fold higher concentrations of bosentan was noted. The biliary excretion index of taurocholate (55 +/- 8%) was decreased in the presence of 10 microM troglitazone (27 +/- 2%) and 100 microM bosentan (10 +/- 6%). In conclusion, xenobiotics may alter hepatic bile acid transport by inhibiting both hepatic uptake and biliary excretion. (+info)
Bile secretory function in the obese Zucker rat: evidence of cholestasis and altered canalicular transport function.
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BACKGROUND: Obese Zucker rats (ZR) have been used as an experimental model for non-alcoholic fatty liver disease and are particularly susceptible to various types of liver injury. Bile secretory function has not been assessed in ZR. AIM: To study bile secretion and expression of the main hepatobiliary transporters in ZR. METHODS: Bile flow and biliary secretion of lipids and glutathione were determined in eight and 14 week old obese ZR and their lean controls. Protein mass and mRNA of the Na(+)/taurocholate cotransporting polypeptide (Ntcp), the bile salt export pump (Bsep), and the multidrug resistant associated protein 2 (Mrp2) were assessed by western and northern blot, respectively. The effects of administration of a tumour necrosis factor alpha inactivator (etanercept) and an insulin sensitiser (rosiglitazone) were assessed in obese ZR while leptin was given to non-obese rats to study its effect on Mrp2 expression. RESULTS: ZR exhibited increased body weight and hyperlipidaemia. Only 14 week old obese ZR has fatty liver. Decreased bile flow and biliary lipid and glutathione secretion as well as reduced hepatic transport of both taurocholate and bromosulphthalein were found in obese ZR. Hepatic Mrp2 protein mass was markedly reduced (-70%) in obese rats while Ntcp and Bsep protein levels were similar to lean rats. Downregulation of Mrp2 seems to involve both transcriptional and post-transcriptional mechanisms probably related to insulin and leptin resistance. CONCLUSIONS: Obese ZR exhibit an impaired bile secretory function with significant functional and molecular alterations consistent with mild cholestasis. A defective hepatobiliary transport capacity may be a contributory factor in rendering the obese ZR more susceptible to liver injury. (+info)