INVESTIGADORES
CROCENZI Fernando Ariel
congresos y reuniones científicas
Título:
Role of GPR30-adenylyl cyclase (AC)-PKA pathway in estradiol 17ß-glucuronide (E17G)-induced cholestasis in isolated rat hepatocytes couplets (IRHC) and isolated perfused rat liver (IPRL).
Autor/es:
ZUCCHETTI, ANDRÉS ERNESTO; BAROSSO, ISMAEL R.; BOAGLIO, ANDREA C; OCHOA, J. ELENA; DAVIO, CARLOS; ROMA, MARCELO G; CROCENZI, FERNANDO A.; SÁNCHEZ POZZI, ENRIQUE J
Lugar:
Barcelona
Reunión:
Congreso; 47th Annual Meeting of the European Association for the Study of the Liver (EASL); 2012
Institución organizadora:
EASL
Resumen:
Background and Aims: 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-ACPKA 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 (10 nM), AC inhibitors SQ22,536 (SQ, 10 mM), MDL12,330
(MDL, 20 mM),
2-3_dideoxyadenosine (DDA,
1 mM)
and PKA inhibitors H89 (1 mM),
KT5720 (KT, 0.25 mM)
and RP-cAMPS (RP, 10 mM)
and then incubated with E17G (100
mM)
20min. For transport studies, we assessed the canalicular vacuolar accumulation
(cVA) of cholyl-lysylfluorescein (CLF, ABCB11 substrate) or
chloromethylfluorescein diacetate (intracellularly converted in
glutathionmethylfluorescein, 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 (3 mmol/liver). Inhibitors G15,
DDA and H89 were administered 10min before E17G. Finally, biliary flow, and
bile salt and dinitrophenyl-glutathion excretion were measured for 1 h in
5minperiods.
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. Function
studies 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 dinitrophenylglutathion
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.