Pérez Leonardo M, Milkiewicz Piotr, Ahmed-Choudhury Jalal, Elias Elwyn, Ochoa Justina E, Sánchez Pozzi Enrique J, Coleman Roger, Roma Marcelo G
Institute of Experimental Physiology, CONICET-University of Rosario, Suipacha 570, 2000 Rosario, Argentina.
Free Radic Biol Med. 2006 Jun 1;40(11):2005-17. doi: 10.1016/j.freeradbiomed.2006.01.034. Epub 2006 Feb 23.
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.
氧化应激会升高钙离子浓度，并且推测会激活依赖钙离子的蛋白激酶C（PKC）。我们分析了依赖钙离子的PKC在促氧化剂叔丁基过氧化氢（tBOOH）诱导的分离大鼠肝细胞膜泡形成和紧密连接损伤中的作用。tBOOH（100微摩尔）增加了活性氧（ROS），脂质过氧化增加（+217%，p<0.05）和2',7'-二氯荧光素的细胞内生成增加（+36%，p<0.05）表明了这一点。tBOOH还使胞质钙离子浓度和PKCα向膜的转位增加（分别增加+100%和+79%，p<0.05），PKCα转位是PKCα激活的一个指标。tBOOH增加了出现膜泡的肝细胞膜泡对数量（+278%，p<0.001）并导致F-肌动蛋白重新分布。tBOOH诱导紧密连接损伤，这可通过保留在胆小管泡中预先分泌的胆酰赖氨酰荧光素的肝细胞膜泡对百分比降低来表明（-54%，p<0.001）。tBOOH诱导紧密连接相关蛋白ZO-1重新分布。所有这些事件都被泛特异性PKC抑制剂H7和星形孢菌素、依赖钙离子的PKC抑制剂Gö6976、细胞内钙离子螯合剂BAPTA/AM以及PKA激活剂二丁酰环磷腺苷所阻止。此外，PKC抑制和PKA激活不仅阻止了tBOOH诱导的膜泡形成，而且使其完全逆转。相反，tBOOH诱导的ROS形成和钙离子升高保持不变。我们得出结论，ROS通过PKC介导的、依赖钙离子的机制诱导肝细胞肌动蛋白细胞骨架重排和紧密连接损伤，而PKA可抵消这种作用。
