Diperoxovanadate alters endothelial cell focal contacts and barrier function: role of tyrosine phosphorylation
Diperoxovanadate alters endothelial cell focal contacts and barrier function: role of tyrosine phosphorylation
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DOI：
10.1152/jappl.2000.89.6.2333
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发表时间：
2000-12-01
影响因子：
3.3
通讯作者：
Natarajan, V
中科院分区：
文献类型：
--
作者：
Garcia, JGN;Schaphorst, KL;Natarajan, V
二过钒酸盐 (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，用于探索牛肺动脉内皮细胞屏障的调节。 DPV 产生剂量依赖性的跨内皮电阻 (TER) 降低和白蛋白渗透性增加，随后 TER 短暂增加（TER 效应在 10-15 分钟达到峰值）。 DPV 介导的 TER 显着且持续的减少主要是细胞间阻力降低的结果，而不是细胞与细胞外基质之间阻力降低的结果，并且通过用酪氨酸激酶抑制剂金雀异黄素预处理来减少，但不通过抑制 p42/p44 丝裂原激活蛋白激酶来减少。 DPV 攻击后的免疫荧光分析显示，F-肌动蛋白聚合和应力纤维组装显着，酪氨酸磷蛋白与 F-肌动蛋白在细胞外周以圆周模式共定位增加，但金雀异黄素消除了这些变化。粘着斑和粘附连接蛋白在酪氨酸残基上的磷酸化在粘着斑激酶和钙粘蛋白相关蛋白的免疫沉淀中得到证实，其中在 DPV 刺激后观察到显着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，并通过 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随后的粘附连接单层不稳定。
Diperoxavanadate (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.