PI3K modulates the canalicular trasnporter internalization and the secretory failur induced by estradiol 17ß glucuronide: possible role for AKT

BOAGLIO, AC; ZUCCHETTI, AE; SANCHEZ POZZI, EJ; MOTTINO, AD; CROCENZI, FA; ROMA, MG

Copenhague

Congreso; International Liver Congress 2009 (EASL); 2009

EASL

E17G is an endogenous, cholestatic metabolite that induces endocytic internalization of the canalicular transporters relevant to bile secretion Bsep and Mrp2; this may justify its causal association with pregnancy-induced cholestasis. PI3K mediates cholestatic events, e.g. taurolithocholate- induced cholestasis (JBC 278: 17810, 2003). Since common pathomechanisms exist between both kinds of cholestasis, we assessed here whether PI3K is involved in E17G cholestatic effects.E17G (200 µM) activated PI3K from 20min onwards, as assessed by the phosphorylation of the final PI3K effector Akt in primary cultured hepatocytes. When preadministered to isolated rat hepatocyte couplets (IRHCs), the PI3K inhibitor wortmannin (WM; 100nM) prevented partially the reduction induced by E17G (12.5-800 µM) in the proportion of IRHCs accumulating apically the fluorescent Bsep and Mrp2 substrates cholyllysylfluorescein and glutathionylmethylfluorescein, respectively. The alternative PI3K inhibitor LY294002 (50 µM) and the "Akt inhibitor" (Calbiochem* #124005; 20 µM) showed similar protective effects. Immunostaining of Bsep and Mrp2 in IRHC followed by confocal microscopy and image analysis revealed that E17G induces endocytic internalization of the transporters. This redistribution was extensively prevented by WM. To evaluate this phenomenon in a more physiological model, we assessed the anticholestatic effect of WM in perfused rat livers. A bolus, intraportal injection of E17G (2 µmol) induced an acute decrease in bile flow within 10min, which did not recover during the remaining perfusion period (Figure 1). WM (100 nM) did not prevent this initial decay, but greatly accelerated the recovery to normality of bile flow. A similar behavior was observed for the biliary excretion of the Bsep and Mrp2 substrates [3H]taurocholate and glutathione, respectively.