ToxSci Advance Access published online on February 1, 2006
Toxicological Sciences, doi:10.1093/toxsci/kfj113
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1 Institute of Experimental Physiology, CONICET-National University of Rosario, ARGENTINA
* To whom correspondence should be addressed. We have shown that Ca2+-mediated PKC activation induces impairment of bile salt secretory function and F-actin redistribution in hepatocyte couplets. Since oxidative stress induces Ca2+ elevation, we tested here whether PKC inhibition or PKA activation, which often counteract 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 analogue, 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.
Received December 1, 2005
Accepted January 13, 2006
In Vitro Toxicology
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
Leonardo M. Pérez 1,
Piotr Milkiewicz 2,
Elwyn Elias 3,
Roger Coleman 4,
Enrique J. Sánchez Pozzi 1,
and
Marcelo G. Roma 1 *
2 Department of Gastroenterology, Pomeranian Medical School, Szczecin, POLAND; Liver and Hepatobiliary Unit, University of Birmingham, Birmingham, UK
3 Liver and Hepatobiliary Unit, University of Birmingham, Birmingham, UK
4 Department of Biosciences, University of Birmingham, Birmingham, UK
Marcelo G. Roma, E-mail: mroma{at}fbioyf.unr.edu.ar
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