ROLE OF CALCIUM-CALMODULIN PATHWAY IN ESTRADIOL-17ß- GLUCURONIDE INDUCED IMPAIRMENT OF CANALICULAR SECRETION IN ISOLATED RAT HEPATOCYTE COUPLETS

ZUCCHETTI, ANDRÉS ERNESTO; TOLEDO, FLAVIA D.; BAROSSO, ISMAEL R.; OCHOA, J. ELENA; CROCENZI, FERNANDO A.; SÁNCHEZ POZZI, ENRIQUE J.

Viena

Congreso; 45th annual meeting of the European Association for the Study of the Liver; 2010

European Associaton for the Study of the Liver

The endogenous estradiol metabolite, estradiol 17ß-D-glucuronide (E17G), induces an acute cholestasis in rat liver due in part to retrieval of canalicular transporters such as the bile salt export pump (Bsep, Abcc11) and the multidrug resistance associated protein 2 (Mrp2, Abcc2), in a process that involves estrogen receptor (J. Hepatol 50: s103, 2009). Calmodulin interacts with estrogen receptor and potentiates estrogen activation of the receptor (Cell Signal 19: 439-443, 2007). The aim of this study was to evaluate the involvement of calcium-calmodulin pathway in E17G-induced changes in canalicular excretion.Methods: Canalicular transport activities: Isolated rat hepatocytes couplets were cultured for 5 h, exposed to verapamyl (V, calcium channel blocker, 10 µM), trifluoroperazin (T, Calcium-calmodulin inhibitor, 10 µM) or W7 (W, Calcium-calmodulin kinase II [CaMKII] inhibitor, 100 µM) for 15 min and then incubated with E17G (100 µM) for 20 min. Finally, all preparations were incubated with cholyl-lysylfluorescein (CLF, fluorescent bile salt substrate of Bsep) or CMFDA (intracellularly converted in gluthation-methylfluorescein [GMF], fluorescent substrate of Mrp2). Couplets accumulating CLF or GMF in their vacuole were counted in a fluorescent microscope and informed as a percentage (%AC).CaMKII activation: Isolated hepatocyte were cultivated in collagen sandwich for 5 days, exposed to E17G (200 µM) for 15 and 30 min and then lysed. Western blot of the samples were performed using antibodies against total and phosphorylated CaMKII. The ratio of the densitometry of phosphorylated/total CaMKII bands was used as estimation of kinase activation. Results were expressed as mean±SD and compared by ANOVA followed by Student-Newman-Keuls test.Results (n = 3): Canalicular transport activities of CLF and GMF, respectively (as % of Control):E17G: 66±4a, 65±4a; E17G+V: 84±4a,b, 81±11a,b; E17G+T: 90±11b, 95±6b; E17G+W:    97±13b, 94±1b.a, significantly different from Control (p < 0.01); b, significantly different from E17G (p < 0.05). V, T and W did not affect %AC of the substrates.CaMKII activation [as phosphorylated/total CaMKII (arbitrary units)]Control:  100±0,  E17G 15 min: 126±25, E17G 30 min: 172±34aa, significantly different from Control (p < 0.05).Conclusions: E17G activates calcium-calmodulin pathway as demonstrated by CaMKII activation, and this pathway participates in estrogen-induced alteration in canalicular secretion.