title: Ca(2+)-dependent protein kinase C isoforms are critical to estradiol 17beta-D-glucuronide-induced cholestasis in the rat.
abstract: The endogenous estradiol metabolite estradiol 17beta-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 mumol/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 muM) decreased the canalicular vacuolar accumulation of the Bsep substrate cholyl-lysylfluorescein with an IC50 of 54.9 +/- 7.9 muM. Gö6976 (1 muM) increased the IC50 to 178.4 +/- 23.1 muM, 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 PKCalpha, but not the Ca(2+)-independent PKC isoform, PKCepsilon, 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute of Experimental Physiology, National University of Rosario, Rosario, Argentina.
authors.forename: Fernando A
authors.initials: FA
authors.lastname: Crocenzi
authors.forename: Enrique J
authors.initials: EJ
authors.lastname: Sánchez Pozzi
authors.forename: María Laura
authors.initials: ML
authors.lastname: Ruiz
authors.forename: Andrés E
authors.initials: AE
authors.lastname: Zucchetti
authors.forename: Marcelo G
authors.initials: MG
authors.lastname: Roma
authors.forename: Aldo D
authors.initials: AD
authors.lastname: Mottino
authors.forename: Mary
authors.initials: M
authors.lastname: Vore
mesh_terms: D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002227:Carbazoles / Q000494:pharmacology; D002478:Cells, Cultured; D002779:Cholestasis / Q000139:chemically induced* / Q000378:metabolism*; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004958:Estradiol / Q000009:adverse effects / Q000031:analogs & derivatives* / Q000494:pharmacology; D005260:Female; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051744:Protein Kinase C-epsilon / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters; C490548:Abcb11 protein, rat; C423013:Abcc2 protein, rat; D002227:Carbazoles; D004791:Enzyme Inhibitors; C081021:Go 6976; C025483:estradiol-17 beta-glucuronide; D004958:Estradiol; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D002118:Calcium