title: Ca2+-dependent protein kinase C isoforms induce cholestasis in rat liver.
abstract: Bile secretion is regulated by different signaling transduction pathways including protein kinase C (PKC). However, the role of different PKC isoforms for bile formation is still controversial. This study investigates the effects of PKC isoform selective activators and inhibitors on PKC translocation, bile secretion, bile acid uptake, and subcellular transporter localization in rat liver, isolated rat hepatocytes and in HepG2 cells. In rat liver activation of Ca(2+)-dependent cPKCalpha and Ca(2+)-independent PKCepsilon by phorbol 12-myristate 13-acetate (PMA, 10nmol/liter) is associated with their translocation to the plasma membrane. PMA also induced translocation of the cloned rat PKCepsilon fused to a yellow fluorescent protein (YFP), which was transfected into HepG2 cells. In the perfused liver, PMA induced marked cholestasis. The PKC inhibitors Gö6850 (1 micromol/liter) and Gö6976 (0.2 micromol/liter), a selective inhibitor of Ca(2+)-dependent PKC isoforms, diminished the PMA effect by 50 and 60%, respectively. Thymeleatoxin (Ttx,) a selective activator of Ca(2+)-dependent cPKCs, did not translocate rat PKCepsilon-YFP transfected in HepG2 cells. However, Ttx (0.5-10 nmol/liter) induced cholestasis similar to PMA and led to a retrieval of Bsep from the canalicular membrane in rat liver while taurocholate-uptake in isolated hepatocytes was not affected. Gö6976 completely blocked the cholestatic effect of Ttx but had no effect on tauroursodeoxycholate-induced choleresis. The data identify Ca(2+)-dependent PKC isoforms as inducers of cholestasis. This is mainly due to inhibition of taurocholate excretion involving transporter retrieval from the canalicular membrane.
authors.affiliation: Department of Gastroenterology, Hepatology, and Infectiology, Heinrich-Heine University, D-40225 Düsseldorf, Germany. Kubitz@med.uni-duesseldorf.de
authors.forename: Ralf
authors.initials: R
authors.lastname: Kubitz
authors.forename: Nirmalendu
authors.initials: N
authors.lastname: Saha
authors.forename: Thomas
authors.initials: T
authors.lastname: Kühlkamp
authors.forename: Supiya
authors.initials: S
authors.lastname: Dutta
authors.forename: Stephan
authors.initials: S
authors.lastname: vom Dahl
authors.forename: Matthias
authors.initials: M
authors.lastname: Wettstein
authors.forename: Dieter
authors.initials: D
authors.lastname: Häussinger
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D001646:Bile / Q000378:metabolism; D001692:Biological Transport; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002227:Carbazoles / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002779:Cholestasis / Q000209:etiology*; D003001:Cloning, Molecular; D003720:Densitometry; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008099:Liver / Q000378:metabolism / Q000473:pathology*; D008164:Luminescent Proteins / Q000737:chemistry; D008297:Male; D008301:Maleimides / Q000494:pharmacology; D018613:Microscopy, Confocal; D008954:Models, Biological; D010477:Perfusion; D010703:Phorbol Esters / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D021381:Protein Transport; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction; D013656:Taurocholic Acid / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate; D013997:Time Factors; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D002227:Carbazoles; D003902:Detergents; D007211:Indoles; D008164:Luminescent Proteins; D008301:Maleimides; D010703:Phorbol Esters; D020033:Protein Isoforms; C054562:yellow fluorescent protein, Bacteria; C081021:Go 6976; D013656:Taurocholic Acid; C080287:thymeleatoxin; C497257:Prkce protein, rat; C496628:PRKCA protein, human; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium