ATP in Lung Endothelial Barrier Enhancement

肺内皮屏障增强中的 ATP

• 批准号: 7026844
• 负责人: ALEXANDER D VERIN
• 金额: $ 38.23万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7026844
• 关键词: G protein; actin binding protein; acute disease /disorder; adenosine triphosphate; biological signal transduction; cell surface receptors; cytoprotection; cytoskeleton; disease /disorder model; enzyme activity; gene expression; laboratory mouse; lung injury; myosin light chain kinase; protein kinase A; protein protein interaction; protein structure function; respiratory epithelium; respiratory function; vascular endothelium permeability; vasodilators

DESCRIPTION (provided by applicant): Endothelial cell (EC) barrier dysfunction, a prominent feature of acute lung injury (ALI), is tightly linked to cytoskeletal remodeling, which leads to the disruption of cell-cell contacts and includes activation of contraction initiated by myosin light chain (MLC) phosphorylation followed by F-actin stress fiber formation and formation of paracellular gaps. Little is known about processes which determine barrier enhancement or protection; however, our published data implicate a critical role for cytoskeletal dynamics in this response. Extracellular ATP is an important vascular mediator, which elicits cellular effects on EC mainly through P2Y family receptors coupled to specific trimeric G-proteins. Our novel findings indicate that ATP at physiologically relevant concentrations produces rapid, sustained and dose-dependent increases in transendothelial electrical resistance (TER), indicating profound barrier enhancement and potently reversed barrier dysfunction elicited by the edemagenic agent, thrombin. Specific depletion of alpha subunits of Gq and Gi2 significantly attenuated ATP-induced increase in TER indicating the involvement of these G-proteins in ATP- induced EC barrier enhancement. The ATP-induced increase in TER is tightly linked to an increase in myosin-associated phosphatase (PPase) 1 (MLCP) activity. Inhibition of PPase 1 abolished the ATP-induced increase in TER and lead to phosphorylation of several cytoskeletal targets including MLC, ezrin/radixin/moezin (ERM) and caldesmon suggesting that dephosphorylation of these proteins may be involved in the barrier-enhancing effect of ATP. In addition, protein kinase A (PKA) inhibition attenuates both ATP-induced increases in TER and phosphorylation of vasodilator-stimulated protein (VASP), which in the phosphorylated form inhibits stress fiber formation supporting the involvement of the PKA/VASP pathway in ATP-induced EC barrier enhancement. Our working hypothesis is that ATP-induced EC barrier enhancement and cytoskeletal remodeling is dependent, at least in part, upon activation of specific P2Y/G protein complexes followed by coordinated activation of MLCP and PKA signaling. Specific Aim 1 will define the role of specific P2Y/G-protein complexes in the activation of MLCP- and PKA-dependent signaling. Specific Aim 2 will define the involvement of MLCP and its cytoskeletal targets in ATP-induced EC barrier enhancement. Specific Aim 3 will explore the molecular mechanisms by which PKA activity is involved in ATP-induced EC barrier enhancement focusing on VASP and MLCP as potential PKA targets. Specific Aim 4 will characterize the potential barrier-protective effects of ATP in murine models of ALI. These studies will provide an understanding of the novel signaling pathways involved in ATP-induced lung EC barrier enhancement and promise new directions and targets for treatment of lung disorders.

描述（由申请人提供）：内皮细胞（EC）屏障功能障碍，急性肺损伤（ALI）的突出特征，与细胞骨架重塑密切相关，这会导致细胞细胞接触的破坏，并包括由肌蛋白蛋白轻链（MLC）F-aptration r-aptration r-aptration fr-aptration fr-aptration tak r-aptration the phoptration tak r-aptration tak r-aptration tak r-appactriartion遵循磷酸化的形式。关于确定障碍增强或保护的过程知之甚少；但是，我们已发布的数据暗示了在此响应中细胞骨架动力学的关键作用。细胞外ATP是一种重要的血管介质，它主要通过与特定三聚体G蛋白结合的P2Y家族受体对EC产生影响。我们的新发现表明，ATP在生理相关的浓度中会产生跨内皮电阻（TER）的快速，持续和剂量依赖性增加，表明屏障的增强深度增强，并有效地逆转了由肿瘤药物引起的屏障功能障碍。 GQ和GI2的α亚基的特异性耗竭显着减弱了ATP诱导的TER增加，表明这些G蛋白参与ATP诱导的EC屏障增强。 ATP诱导的TER增加与肌球蛋白相关磷酸酶（PPase）1（MLCP）活性的增加密切相关。 PPase 1的抑制作用消除了ATP诱导的TER的增加，并导致几种细胞骨架靶标的磷酸化，包括MLC，Ezrin/radixin/Moezin（ERM）和Caldesmon和Caldesmon表明这些蛋白质的去磷酸化可能涉及这些蛋白质的障碍物。此外，蛋白激酶A（PKA）抑制作用减弱了ATP诱导的血管舒张剂刺激蛋白（VASP）的TER和磷酸化的增加，这种蛋白质（VASP）以磷酸化形式抑制了应激纤维形成，从而支持PKA/VASP途径在PKA/VASP中的参与。我们的工作假设是，ATP诱导的EC屏障增强和细胞骨架重塑至少部分取决于激活特定的P2Y/G蛋白复合物，然后协调MLCP和PKA信号传导。特定的目标1将定义特定P2Y/G蛋白复合物在MLCP和PKA依赖性信号传导激活中的作用。特定的目标2将定义MLCP及其细胞骨架靶标参与ATP诱导的EC屏障增强。具体目标3将探索PKA活性参与ATP诱导的EC屏障增强的分子机制，该增强的重点是VASP和MLCP作为潜在的PKA靶标。特定的目标4将表征ATP在ALI鼠模型中的潜在屏障保护作用。这些研究将提供对ATP诱导的肺EC屏障增强的新型信号通路的理解，并保证了治疗肺部疾病的新方向和目标。