title: Oxidative stress induces internalization of the bile salt export pump, Bsep, and bile salt secretory failure in isolated rat hepatocyte couplets: a role for protein kinase C and prevention by protein kinase A.
abstract: We have shown that Ca2+-mediated protein kinase C (PKC) activation induces impairment of bile salt secretory function and F-actin redistribution in hepatocyte couplets. Because oxidative stress induces Ca2+ elevation, we tested here whether PKC inhibition or protein kinase A (PKA) activation, which often counteracts PKC-dependent effects, can prevent and reverse these alterations. The pro-oxidant compounds tert-butylhydroperoxide (tBOOH, 100 microM) and 2,3-dimethoxy-1,4-naphthoquinone (30 microM), reduced by -41% and -29%, respectively, the percentage of couplets accumulating the fluorescent bile salt analog, cholyl-lysylfluorescein in their canalicular vacuoles (p < 0.01). tBOOH-induced bile salt secretory failure was accompanied by internalization of the canalicular bile salt export pump (Bsep), and disarrangement of cytoskeletal F-actin. All these deleterious effects were fully prevented by the intracellular Ca2+ chelator BAPTA/AM (20 microM), the pan-specific PKC inhibitors H7 (100 microM) and staurosporine (1 microM), the inhibitor of Ca2+-dependent PKCs, Gö6976 (2 microM), and the PKA activator dibutyryl-cAMP (500 microM). H7, Gö6976, and dibutyryl-cAMP not only prevented but also fully reversed the decrease in the cholyl-lysyl-fluorescein accumulation. In conclusion, these results suggest that low levels of oxidative stress impair bile salt secretion by internalizing Bsep through a Ca2+-dependent, PKC-mediated mechanism, and that inhibition of PKC, or activation of PKA, prevents and reverses these effects. Alterations in actin organization may be a causal factor.
authors.affiliation: Institute of Experimental Physiology, CONICET-National University of Rosario, Rosario, Argentina.
authors.forename: Leonardo M
authors.initials: LM
authors.lastname: Pérez
authors.forename: Piotr
authors.initials: P
authors.lastname: Milkiewicz
authors.forename: Elwyn
authors.initials: E
authors.lastname: Elias
authors.forename: Roger
authors.initials: R
authors.lastname: Coleman
authors.forename: Enrique J
authors.initials: EJ
authors.lastname: Sánchez Pozzi
authors.forename: Marcelo G
authors.initials: MG
authors.lastname: Roma
mesh_terms: D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism*; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D022781:Hepatocytes / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D009285:Naphthoquinones / Q000494:pharmacology; D018384:Oxidative Stress; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
chemical_list: D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters; C490548:Abcb11 protein, rat; D001647:Bile Acids and Salts; D009285:Naphthoquinones; D003994:Bucladesine; C063002:2,3-dimethoxy-1,4-naphthoquinone; D020122:tert-Butylhydroperoxide; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D002118:Calcium