期刊
FREE RADICAL BIOLOGY AND MEDICINE
卷 40, 期 11, 页码 2005-2017出版社
ELSEVIER SCIENCE INC
DOI: 10.1016/j.freeradbiomed.2006.01.034
关键词
adenosine 3 ': 5 '-cyclic monophosphate; F-actin; fluorescent bile-salt analogue; Ca2+-dependent PKCs; PKA; tert-butylhydroperoxide; signaling; tight-junctional permeability; free radicals
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 mu M) 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 PKC alpha translocation to membrane, an indicator of PKCa. 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 G66976, 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, tBOOHinduced 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. (c) 2006 Elsevier Inc. All rights reserved.
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