Ca(2+)-dependent protein kinase C isoforms are critical to estradiol 17beta-D-glucuronide-induced cholestasis in the rat.
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.
Crocenzi
Fernando A
FA
Institute of Experimental Physiology, National University of Rosario, Rosario, Argentina.
Sánchez Pozzi
Enrique J
EJ
Ruiz
María Laura
ML
Zucchetti
Andrés E
AE
Roma
Marcelo G
MG
Mottino
Aldo D
AD
Vore
Mary
M
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
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