Premium
Ca 2+ ‐dependent protein kinase C isoforms are critical to estradiol 17β‐ D ‐glucuronide–induced cholestasis in the rat
Author(s) -
Crocenzi Fernando A.,
Sánchez Pozzi Enrique J.,
Ruiz María Laura,
Zucchetti Andrés E.,
Roma Marcelo G.,
Mottino Aldo D.,
Vore Mary
Publication year - 2008
Publication title -
hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.488
H-Index - 361
eISSN - 1527-3350
pISSN - 0270-9139
DOI - 10.1002/hep.22532
Subject(s) - multidrug resistance associated protein 2 , protein kinase c , cholestasis , bone canaliculus , medicine , endocrinology , biology , glutathione , hepatocyte , metabolite , glucuronide , kinase , biochemistry , transporter , enzyme , atp binding cassette transporter , in vitro , gene
The endogenous estradiol metabolite estradiol 17β‐ 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 Ca 2+ ‐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 μmol/liver; intraportal, single injection) maximally decreased bile flow, total glutathione, and [ 3 H] taurocholate excretion by 61%, 62%, and 79%, respectively; incorporation of the specific cPKC inhibitor Gö6976 (500 nM) in the perfusate almost totally prevented these decreases. In dose‐response studies using IRHC, E 2 17G (3.75–800 μM) decreased the canalicular vacuolar accumulation of the Bsep substrate cholyl‐lysylfluorescein with an IC50 of 54.9 ± 7.9 μM. Gö6976 (1 μM) increased the IC50 to 178.4 ± 23.1 μM, and similarly prevented the decrease in the canalicular vacuolar accumulation of the Mrp2 substrate, glutathione methylfluorescein. Prevention of these changes by Gö6976 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 Gö6976. The cPKC isoform PKCα, but not the Ca 2+ ‐independent PKC isoform, PKCϵ, translocated to the plasma membrane after E 2 17G administration in primary cultured rat hepatocytes; Gö6976 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. (H EPATOLOGY 2008;48:1885‐1895.)