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(申请人提供)：内皮细胞(EC)屏障功能障碍是急性肺损伤(ALI)的一个显著特征，与细胞骨架重塑密切相关，细胞重塑导致细胞-细胞接触中断，包括由肌球蛋白轻链(MLC)磷酸化启动的收缩激活，随后F-肌动蛋白应激纤维形成和细胞旁间隙形成。我们对决定屏障增强或保护的过程知之甚少；然而，我们发表的数据暗示了细胞骨架动力学在这一反应中的关键作用。细胞外三磷酸腺苷是一种重要的血管介质，它主要通过与特定的三聚体G蛋白偶联的P2Y家族受体对EC产生细胞效应。我们的新发现表明，生理相关浓度的ATP可迅速、持续且呈剂量依赖性地增加跨内皮细胞的电阻(TER)，这表明明显的屏障增强和有效地逆转了由水肿剂凝血酶引起的屏障功能障碍。Gq和GI2α亚基的特异性缺失显着减弱了ATP诱导的TER增加，表明这些G蛋白参与了ATP诱导的EC屏障的增强。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。抑制PPase 1可抑制ATP诱导的TER增加，并导致MLC、Ezrin/Radioxin/moezin(ERM)和caldesmon等细胞骨架靶蛋白的磷酸化，提示这些蛋白的去磷酸化可能参与了ATP的屏障增强作用。此外，蛋白激酶A(PKA)抑制可减弱ATP诱导的TER增加和血管扩张剂刺激蛋白(VASP)的磷酸化，VASP以磷酸化形式抑制应激纤维的形成，支持PKA/VASP途径参与ATP诱导的EC屏障增强。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依赖于特定的P2Y/G蛋白复合体的激活，随后MLCP和PKA信号的协调激活。具体目标1将确定特定的P2Y/G蛋白复合体在MLCP和PKA依赖的信号激活中的作用。特异靶2将确定MLCP及其细胞骨架靶点在三磷酸腺苷诱导的EC屏障增强中的作用。具体目标3将探讨PKA活性参与ATP诱导的EC屏障增强的分子机制，重点是Vasp和MLCP作为潜在的PKA靶点。具体目标4将表征三磷酸腺苷对小鼠ALI模型的潜在屏障保护作用。这些研究将有助于理解ATP诱导的肺内皮细胞屏障增强所涉及的新的信号通路，并有望为肺部疾病的治疗提供新的方向和靶点。