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Estradiol 17b-D-glucuronide (E 2 17G) is an endogenous, cholestatic metabolite that induces endocytic internalization of the canalicular transporters relevant to bile secretion: bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2). We assessed whether phosphoinositide 3-kinase (PI3K) is involved in E 2 17G-induced cholestasis. E 2 17G activated PI3K according to an assessment of the phosphorylation of the final PI3K effector, protein kinase B (Akt). When the PI3K inhibitor wortmannin (WM) was preadministered to isolated rat hepatocyte couplets (IRHCs), it partially prevented the reduction induced by E 2 17G in the proportion of IRHCs secreting fluorescent Bsep and Mrp2 substrates (cholyl lysyl fluorescein and glutathione methylfluorescein, respectively). 2-Morpholin-4-yl-8-phenylchromen-4-one, another PI3K inhibitor, and an Akt inhibitor (Calbiochem 124005) showed similar protective effects. IRHC immunostaining and confocal microscopy analysis revealed that endocytic internalization of Bsep and Mrp2 induced by E 2 17G was extensively prevented by WM; this effect was fully blocked by the microtubule-disrupting agent colchicine. The protection of WM was additive to that afforded by the classical protein kinase C (cPKC) inhibitor 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile (Gö6976); this suggested differential and complementary involvement of the PI3K and cPKC signaling pathways in E 2 17G-induced cholestasis. In isolated perfused rat liver, an intraportal injection of E 2 17G triggered endocytosis of Bsep and Mrp2, and this was accompanied by a sustained decrease in the bile flow and the biliary excretion of the Bsep and Mrp2 substrates [ 3 H]taurocholate and glutathione until the end of the perfusion period. Unlike Gö6976, WM did not prevent the initial decay, but it greatly accelerated the recovery to normality of these parameters and the reinsertion of Bsep and Mrp2 into the canalicular membrane in a microtubule-dependent manner. Conclusion: The PI3K/Akt signaling pathway is involved in the biliary secretory failure induced by E 2 17G through sustained internalization of canalicular transporters endocytosed via cPKC. (HEPATOLOGY 2010;52:1465-1476 B ile formation is a highly regulated process. It depends on the coordinated action of a number of transporters in the sinusoidal and canalicular domains of hepatocytes. The transfer of solutes across the canalicular membrane is the rate-limiting step in bile formation; therefore, functional alterations in canalicular transporters are more likely to impair bile flow generation. Bile salt export pump (Bsep; adenosine triphosphate-binding cassette b11) and multidrug resistance-associated protein 2 (Mrp2; adenosineAbbreviations: Akt, protein kinase B; BSA, bovine serum albumin; Bsep, bile salt export pump; CLF, cholyl lysyl fluorescein; CMFDA, 5-chloromethylfluorescein diacetate; cPKC, classical protein kinase C; cVA, canalicular vacuolar accumulation; DMSO, dimethyl sulfoxide; E 2 17G, estrad...

The occurrence of all-trans (-)-14,15-epoxygeranyl-geraniol in Pterodon pubescens Benth. is established, and its prophylactic activity against infection by Schitsosma mansoni demonstrated. Two other diterpenes present in the oil are inactive.