Garcia J G, Schaphorst K L, Verin A D, Vepa S, Patterson C E, Natarajan V
Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA.
J Appl Physiol (1985). 2000 Dec;89(6):2333-43. doi: 10.1152/jappl.2000.89.6.2333.
Diperoxovanadate (DPV), a potent tyrosine kinase activator and protein tyrosine phosphatase inhibitor, was utilized to explore bovine pulmonary artery endothelial cell barrier regulation. DPV produced dose-dependent decreases in transendothelial electrical resistance (TER) and increases in permeability to albumin, which were preceded by brief increases in TER (peak TER effect at 10-15 min). The significant and sustained DPV-mediated TER reductions were primarily the result of decreased intercellular resistance, rather than decreased resistance between the cell and the extracellular matrix, and were reduced by pretreatment with the tyrosine kinase inhibitor genistein but not by inhibition of p42/p44 mitogen-activating protein kinases. Immunofluorescent analysis after DPV challenge revealed dramatic F-actin polymerization and stress-fiber assembly and increased colocalization of tyrosine phosphoproteins with F-actin in a circumferential pattern at the cell periphery, changes that were abolished by genistein. The phosphorylation of focal adhesion and adherens junction proteins on tyrosine residues was confirmed in immunoprecipitates of focal adhesion kinase and cadherin-associated proteins in which dramatic dose-dependent tyrosine phosphorylation was observed after DPV stimulation. We speculate that DPV enhances endothelial cell monolayer integrity via focal adhesion plaque phosphorylation and produces subsequent monolayer destabilization of adherens junctions initiated by adherens junction protein tyrosine phosphorylation catalyzed by p60(src) or Src-related tyrosine kinases.
双氧钒酸酯（DPV）是一种有效的酪氨酸激酶激活剂和蛋白酪氨酸磷酸酶抑制剂，被用于研究牛肺动脉内皮细胞屏障调节。DPV导致跨内皮电阻（TER）呈剂量依赖性降低，白蛋白通透性增加，在这之前TER会有短暂升高（10 - 15分钟时TER达到峰值）。DPV介导的TER显著且持续降低主要是细胞间电阻降低的结果，而非细胞与细胞外基质之间电阻降低的结果，用酪氨酸激酶抑制剂染料木黄酮预处理可使其降低，但抑制p42/p44丝裂原活化蛋白激酶则不能。DPV刺激后进行免疫荧光分析，结果显示F - 肌动蛋白显著聚合和应力纤维组装，酪氨酸磷酸化蛋白与F - 肌动蛋白在细胞周边呈周向模式的共定位增加，这些变化被染料木黄酮消除。在粘着斑激酶和钙粘蛋白相关蛋白的免疫沉淀物中证实了粘着斑和粘附连接蛋白酪氨酸残基的磷酸化，在DPV刺激后观察到显著的剂量依赖性酪氨酸磷酸化。我们推测，DPV通过粘着斑磷酸化增强内皮细胞单层完整性，并随后由p60(src)或Src相关酪氨酸激酶催化的粘附连接蛋白酪氨酸磷酸化引发粘附连接的单层不稳定。
