ToxSci Advance Access originally published online on February 1, 2006
Toxicological Sciences 2006 91(1):150-158; doi:10.1093/toxsci/kfj113
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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
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* Institute of Experimental Physiology, CONICET-National University of Rosario, Rosario, Argentina; Department of Gastroenterology, Pomeranian Medical School, Szczecin, Poland; Liver and Hepatobiliary Unit, University of Birmingham, Birmingham, United Kingdom; and Department of Biosciences, University of Birmingham, Birmingham, United Kingdom
Received September 27, 2005; accepted January 14, 2006
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 µM) and 2,3-dimethoxy-1,4-naphthoquinone (30 µM), 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 µM), the pan-specific PKC inhibitors H7 (100 µM) and staurosporine (1 µM), the inhibitor of Ca2+-dependent PKCs, Gö6976 (2 µM), and the PKA activator dibutyryl-cAMP (500 µM). 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.
Key Words: systems toxicology, oxidative injury; biotransformation and toxicokinetics; biliary excretion; xenobiotic transporters; in vitro alternatives; hepatocytes; systems toxicology; signal transduction.
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