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.

PÉREZ L.M.; MILKIEWICZ P.; ELIAS E.; COLEMAN R.; SÁNCHEZ POZZI E.J.; ROMA M.G.

Toxicological Sciences

Oxford University Press

Año: 2006 vol. 91 p. 150 - 158

1096-6080

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, Go6976 (2 microM), and the PKA activator dibutyryl-cAMP (500 microM). H7, Go6976, 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.