ROCK mediates thrombin's endothelial barrier dysfunction.

Thrombin-induced endothelial monolayer hyperpermeability is thought to result from increased F-actin stress fiber-related contractile tension, a process regulated by the small GTP-binding protein Rho. We tested whether this process was dependent on the Rho-associated protein kinase, ROCK, using a specific ROCK inhibitor, Y-27632. The effects of Y-27632 on thrombin-induced myosin light chain phosphorylation (MLCP) and tyrosine phosphorylation of p125 focal adhesion kinase (p125(FAK)) and paxillin were measured by Western blotting. F-actin organization and content were analyzed by digital imaging, and endothelial monolayer permeability was measured in bovine pulmonary artery endothelial cell (EC) monolayers using a size-selective permeability assay. Y-27632 enhanced EC monolayer barrier function due to a decline in small-pore number that was associated with increased EC surface area, reduced F-actin content, and reorganization of F-actin to beta-catenin-containing cell-cell adherens junctions. Although Y-27632 prevented thrombin-induced MLCP, stress fiber formation, and the increased phosphotyrosine content of paxillin and p125(FAK), it attenuated but did not prevent the thrombin-induced formation of large paracellular holes. These data indicate that thrombin-induced stress fiber formation is ROCK dependent. In contrast, thrombin-induced paracellular hole formation occurs in a ROCK-independent manner, whereas thrombin-induced monolayer hyperpermeability appears to be partially ROCK dependent.

凝血酶诱导的内皮单层高通透性被认为是由F - 肌动蛋白应力纤维相关收缩张力增加所致，这一过程受小GTP结合蛋白Rho调节。我们使用一种特异性的Rho相关蛋白激酶（ROCK）抑制剂Y - 27632来检测这一过程是否依赖于ROCK。通过蛋白质印迹法测定Y - 27632对凝血酶诱导的肌球蛋白轻链磷酸化（MLCP）以及p125焦点黏附激酶（p125(FAK)）和桩蛋白的酪氨酸磷酸化的影响。通过数字成像分析F - 肌动蛋白的组织和含量，并使用尺寸选择性通透性测定法在牛肺动脉内皮细胞（EC）单层中测量内皮单层通透性。Y - 27632增强了内皮细胞单层的屏障功能，这是由于小孔数量减少，同时伴有内皮细胞表面积增加、F - 肌动蛋白含量降低以及F - 肌动蛋白重新组织到含β - 连环蛋白的细胞 - 细胞黏附连接。尽管Y - 27632阻止了凝血酶诱导的MLCP、应力纤维形成以及桩蛋白和p125(FAK)磷酸酪氨酸含量的增加，但它减弱了但并未阻止凝血酶诱导的大的细胞旁孔的形成。这些数据表明凝血酶诱导的应力纤维形成依赖于ROCK。相反，凝血酶诱导的细胞旁孔形成以不依赖于ROCK的方式发生，而凝血酶诱导的单层高通透性似乎部分依赖于ROCK。