Role of Ca2+-Dependent protein kinase C isoforms in estradiol 17beta-D-glucuronide-induced cholestasis in the rat

CROCENZI FA; SÁNCHEZ POZZI EJ; RUIZ ML; ZUCHETTI AE; ROMA MG; MOTTINO AD; VORE M

Boston, EEUU

Congreso; Annual Meeting of the American Association for the Study of Liver Diseases (AASLD); 2007

The endogenous estradiol metabolite, estradiol 17β-D-glucuronide (E17G), induces an acute cholestasis in rat liver due in part to retrieval of the canalicular bile salt export pump (Bsep, Abcc11) and the multidrug resistance protein 2 (Mrp2, Abcc2), and the consequent loss of their function. Activation of the classical, Ca2+-dependent isoforms of protein kinase C (cPKC) has been shown to affect bile formation and normal transporter localization (J Biol Chem 279:10323). We evaluated the involvement of cPKC isoforms in E17G-induced changes in canalicular transporter function and localization. Methods: The non-recirculating isolated perfused liver (IPL) from female rats was used to assess the effect of cPKC inhibition by Gö6976 (500 nM in perfusate) on changes in bile flow, total glutathione (GSH) and 3H-taurocholate (3H-TC) excretion induced by E17G. Bile was collected before and every 5 min after E17G (2 ìmol, intraportal bolus dose) for 60 min. Liver samples were taken 20 min after E17G for localization of Bsep and Mrp2 by confocal immunofluorescence laser microscopy. Isolated hepatocytes were cultured for 5 h and exposed to E17G (50 ìM) for 5-20 min. After subcellular fractionation, activation of the cPKC isoform, PKCα and the novel Ca2+-independent PKC isoform, PKCε , were evaluated by western blotting of cytosolic and total membrane fractions. Results: E17G induced an acute, significant (p<0.05 vs control) decrease in bile flow (61%) and biliary excretion of total GSH (62%) and [3H]TC (79%). The selective cPKC-isoform inhibitor Gö6976 partially protected against the decreases in bile flow (15%), total GSH (23%) and [3H]TC (20%; p <0.05 vs E17G alone). In E17G-treated livers, both Bsep and Mrp2 demonstrated endocytic retrieval from the canalicular membrane; Gö6976 completely protected against E17G-induced retrieval measured by Image-J (NIH) software analysis. In hepatocytes, E17G induced a 60% increase (p<0.05) in membrane-bound PKCα within 5 min, an indicator of PKC activation. This increase persisted for 15 min and returned to control values by 20 min. The proportion of PKCε associated with the membrane fraction was not modified by E17G. Conclusions: E17G selectively activates cPKC isoforms within minutes. Selective blockage of cPKC activation with Gö6976 completely prevented retrieval of Mrp2 and Bsep, and markedly protected against their loss of function. These findings support a central role for cPKC isoforms in E17G-induced cholestasis by a mechanism involving mainly transporter retrieval from the canalicular membrane.