Oxidative stress induces actin-cytoskeletal and tight-junctional alterations in hepatocytes by a Ca2+-dependent, PKC-mediated mechanism: Protective effect of PKA

2006 
Abstract Oxidative stress elevates Ca 2+ and, presumably, activates Ca 2+ -dependent PKCs. We analyzed the participation of Ca 2+ -dependent PKCs in actin disorganization and tight-junctional impairment induced by the pro-oxidant tert -butylhydroperoxide ( t BOOH) in isolated rat hepatocyte couplets. t BOOH (100 μM) augmented radical oxygen species (ROS), as indicated by increased lipid peroxidation (+217%, p p 2+ and PKCα translocation to membrane, an indicator of PKCα activation, were also elevated by t BOOH (+100 and +79%, respectively, p t BOOH increased the number of couplets displaying membrane blebs (+278%, p t BOOH induced tight-junctional impairment, as indicated by a reduction in the percentage of couplets retaining presecreted cholyllysylfluorescein in their canalicular vacuoles (−54%, p t BOOH induced redistribution of the tight-junctional-associated protein ZO-1. All these events were prevented by the panspecific PKC inhibitors H7 and staurosporine, the Ca 2+ -dependent PKC inhibitor Go6976, the intracellular Ca 2+ chelator BAPTA/AM, and the PKA activator dibutyryl-cyclic AMP. Furthermore, PKC inhibition and PKA activation not only prevented but also fully reversed t BOOH-induced blebbing. Conversely, t BOOH-induced ROS formation and Ca 2+ elevation remained unchanged. We conclude that ROS induce hepatocellular actin-cytoskeleton rearrangement and tight-junctional impairment by a PKC-mediated, Ca 2+ -dependent mechanism, which is counteracted by PKA.
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