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

Oxidative stress elevates Ca²⁺ and, presumably, activates Ca²⁺-dependent PKCs. We analyzed the participation of Ca²⁺-dependent PKCs in actin disorganization and tight-junctional impairment induced by the pro-oxidant tert-butylhydroperoxide (tBOOH) in isolated rat hepatocyte couplets. tBOOH (100 μM) augmented radical oxygen species (ROS), as indicated by increased lipid peroxidation (+217%, p < 0.05) and intracellular production of 2′,7′-dichlorofluorescein (+36%, p < 0.05). Cytosolic Ca²⁺ and PKCα translocation to membrane, an indicator of PKCα activation, were also elevated by tBOOH (+100 and +79%, respectively, p < 0.05). tBOOH increased the number of couplets displaying membrane blebs (+278%, p < 0.001) and caused redistribution of F-actin. tBOOH induced tight-junctional impairment, as indicated by a reduction in the percentage of couplets retaining presecreted cholyllysylfluorescein in their canalicular vacuoles (-54%, p < 0.001). tBOOH induced redistribution of the tight-junctional-associated protein ZO-1. All these events were prevented by the panspecific PKC inhibitors H7 and staurosporine, the Ca²⁺-dependent PKC inhibitor Gö6976, the intracellular Ca²⁺ chelator BAPTA/AM, and the PKA activator dibutyryl-cyclic AMP. Furthermore, PKC inhibition and PKA activation not only prevented but also fully reversed tBOOH-induced blebbing. Conversely, tBOOH-induced ROS formation and Ca²⁺ elevation remained unchanged. We conclude that ROS induce hepatocellular actin-cytoskeleton rearrangement and tight-junctional impairment by a PKC-mediated, Ca²⁺-dependent mechanism, which is counteracted by PKA.