RESUMEN
Endocytic internalization of the multidrug resistance-associated protein 2 (Mrp2) was previously suggested to be involved in estradiol-17beta-D-glucuronide (E217G)-induced cholestasis. Here we evaluated in the rat whether a similar phenomenon occurs with the bile salt export pump (Bsep) and the ability of DBcAMP to prevent it. E217G (15 micromol/kg i.v.) impaired bile salt (BS) output and induced Bsep internalization, as assessed by confocal microscopy and Western blotting. Neither cholestasis nor Bsep internalization occurred in TR- rats lacking Mrp2. DBcAMP (20 micromol/kg i.v.) partially prevented the decrease in bile flow and BS output and substantially prevented E217G-induced Bsep internalization. In hepatocyte couplets, E217G (50 microM) diminished canalicular accumulation of a fluorescent BS and decreased Bsep-associated fluorescence in the canalicular membrane; DBcAMP (10 microM) fully prevented both effects. In conclusion, our results suggest that changes in Bsep localization are involved in E217G-induced impairment of bile flow and BS transport and that DBcAMP prevents this effect by stimulating insertion of canalicular transporter-containing vesicles. Mrp2 is required for E217G to induce its harmful effect.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Endocitosis/efectos de los fármacos , Estradiol/análogos & derivados , Estradiol/farmacología , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/análisis , Actinas/análisis , Animales , Bilis/fisiología , Ácidos y Sales Biliares/metabolismo , Canalículos Biliares/química , Western Blotting , Bucladesina/farmacología , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Membrana Celular/química , Colestasis/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Hígado/química , Hígado/efectos de los fármacos , Hígado/metabolismo , Microscopía Confocal , Mutación , Ratas , Ratas Sprague-Dawley , Ratas WistarRESUMEN
BACKGROUND/AIMS: Bile salts (BS) are cytotoxic agents, but cell damage is not observed in the hepatobiliary system. We hypothesized that biliary lipid vesicles (unilamellae and multilamellae) could have a protective role against BS-induced cytotoxicity. METHODS: Biliary lipid lamellar secretion was induced by feeding rats with 0.5% diosgenin. Cytoprotection was assessed in bile duct-obstructed rats and by incubating human erythrocytes with sodium taurocholate. RESULTS: Biliary cholesterol concentration increased > 300% in diosgenin-fed rats; electron microscopic examination showed a great abundance of lipid lamellar vesicles in bile and within the canaliculi. After bile duct obstruction, serum hepatic enzyme activities were significantly lower in diosgenin-fed rats. Histologically severe and confluent hepatocellular necrosis was only observed in control rats. Biliary lamellar lipid material significantly reduced the BS-induced hemolytic effect in vitro in a concentration-dependent manner. This protective effect correlated to a progressive decrease in the intermicellar BS concentration. Phosphatidylcholine or cholesterol, alone or as lamellar structures, also showed cytoprotective effect in vitro but always less than native biliary lamellae. CONCLUSIONS: These results support the concept that native biliary cholesterol phospholipid lamellae represent an important cytoprotective factor for hepatocytes and biliary epithelial cells against BS-induced damage.