Ca2+-Dependent Protein Kinase C lsoforms Are Critical to Estradiol 17β-D-Glucuronide-induced Cholestasis in the Rat

The endogenous estradiol metabolite estradiol 17 beta-D-glucuronide (E(2)17G) induces an acute cholestasis in rat liver coincident with retrieval of the canalicular transporters bile salt export pump (Bsep, Abcc11) and multidrug resistance-associated protein 2 (Mrp2, Abcc2) and their associated loss of function. We assessed the participation of Ca2+-dependent protein kinase C isoforms (cPKC) in the cholestatic manifestations of E(2)17G in perfused rat liver (PRL) and in isolated rat hepatocyte couplets (IRHCs). In PRL, E(2)17G (2 mu mol/liver; intraportal, single injection) maximally decreased bile flow, total glutathione, and [H-3] taurocholate excretion by 61%, 62%, and 79%, respectively; incorporation of the specific cPKC inhibitor Go6976 (500 nM) in the perfusate almost totally prevented these decreases. In dose-response studies using IRHC, E(2)17G (3.75-800 mu M) decreased the canalicular vacuolar accumulation of the Bsep substrate cholyl-lysylfluorescein with an IC50 of 54.9 +/- 7.9 mu M. Go6976 (1 mu M) increased the IC50 to 178.4 +/- 23.1 mu M, and similarly prevented the decrease in the canalicular vacuolar accumulation of the Mrp2 substrate, glutathione methylfluorescein. Prevention of these changes by Go6976 coincided with complete protection against E(2)17G-induced retrieval of Bsep and Mrp2 from the canalicular membrane, as detected both in the PRL and IRHC. E(2)17G also increased paracellular permeability in IRHC, which was only partially prevented by Go6976. The cPKC isoform PKC alpha, but not the Ca2+-independent PKC isoform, PKC epsilon, translocated to the plasma membrane after E(2)17G administration in primary cultured rat hepatocytes; Go6976 completely prevented this translocation, thus indicating specific activation of cPKC. This is consistent with increased autophosphorylation of cPKC by E(2)17G, as detected via western blotting. Conclusion: Our findings support a central role for cPKC isoforms in E(2)17G-induced cholestasis, by inducing both transporter retrieval from the canalicular membrane and opening of the paracellular route. (HEPATOLOGY 2008;48:1885-1895.)