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 microM) 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 PKCalpha translocation to membrane, an indicator of PKCalpha 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.
Pérez
Leonardo M
LM
Institute of Experimental Physiology, CONICET-University of Rosario, Suipacha 570, 2000 Rosario, Argentina.
Milkiewicz
Piotr
P
Ahmed-Choudhury
Jalal
J
Elias
Elwyn
E
Ochoa
Justina E
JE
Sánchez Pozzi
Enrique J
EJ
Coleman
Roger
R
Roma
Marcelo G
MG
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism*; D004789:Enzyme Activation; D022781:Hepatocytes / Q000378:metabolism*; D008297:Male; D018384:Oxidative Stress; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D019108:Tight Junctions / Q000378:metabolism*; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
D000199:Actins; D020122:tert-Butylhydroperoxide; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D002118:Calcium