Role of GPR30-adenylyl cyclase-PKA pathway in estradiol 17ß-glucuronide induced cholestasis in isolated rat hepatocytes couplets and isolated perfused rat liver.

ZUCCHETTI, AE; BAROSSO, IR; BOAGLIO, AC; OCHOA, JE; DAVIO, C; ROMA, MG; CROCENZI, FA; SÁNCHEZ POZZI, EJ

Barcelona

Congreso; International Liver Congress 2012; 2012

EASL

E17G internalizes canalicular transporters, considered as model of intrahepatic cholestasis of pregnancy. Signaling pathways participating in this alteration represent a topic of recent development. The majority of the pathways activated by E17G are similar to those activated by non-conjugated estradiol. GPR30-AC-PKA pathway is activated by estradiol in liver. Hence, we aimed to evaluate the role of this pathway in E17G-induced alteration of two canalicular transporters: ABCB11 (bile salt export pump) and ABCC2 (multidrug resistance-associated protein 2). Methods: IRHC were exposed 15min to either: GPR30 inhibitor G15 (10nM), AC inhibitors SQ22,536 (SQ, 10µM), MDL12,330 (MDL, 20µM), 2'-3'dideoxyadenosine (DDA, 1µM) and PKA inhibitors H89 (1µM), KT5720 (KT, 0.25µM) and RP-cAMPS (RP, 10µM) and then incubated with E17G (100µM) 20min. For transport studies, we assessed the canalicular vacuolar accumulation (cVA) of cholyl-lysylfluorescein (CLF, ABCB11 substrate) or chloro-methylfluorescein diacetate (intracellularly converted in glutathion-methylfluorescein, GS-MF, ABCC2 substrate). Transporters localization was studied by immunofluorescence, followed by confocal microscopy. cAMP was determined by PKA competition assay, after treatment with E17G for 10min. PKA activation was determined by western blot using an antibody against phospho-PKA substrate. In IPRL, cholestasis was induced by intraportal injection of E17G (3µmol/liver). Inhibitors G15, DDA and H89 were administered 10min before E17G. Finally, biliary flow, and bile salt and dinitrophenyl-glutathion excretion were measured for 1h in 5min-periods. Results (n=3): E17G increased cAMP levels (2.2±0.2pmol/100000 couplets vs control: 0.8±0.3pmol/100000 couplets, p< 0.05). PKA was activated by E17G (density of the 25-kDa phospho-PKA substrate increased by 107±5% against control, p< 0.05). All pathways inhibitors prevented PKA activation.   Control E17G E17G+G15 E17G+SQ E17G+MDL E17G+DDA E17G+H89 E17G+KT E17G+RP cVACLF 100±0 56±3a 91±6b 95±3b 92±4b 94±2b 88±3b 85±2b 90±2b cVAGS-MF 100±0 59±4a 93±5b 94±2b 90±6b 93±5b 87±5b 89±3b 89±3b[Function Studies] a different from Control (p< 0.05); b different from E17G (p< 0.05) Localization studies: E17G induced retrieval of both transporters. Pretreatment with all inhibitors totally prevents transporter delocalization. In IPRL, E17G diminished bile flow, bile salt and dinitrophenyl-glutathion excretion (-69±14%, p< 0.001). All pathway inhibitors significantly prevented this drop. Conclusions: Activation of GPR30-AC-PKA pathway is a key factor in the alteration of canalicular transporters induced by E17G.