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）屏障功能障碍是急性肺损伤（ALI）的一个显着特征，与细胞骨架重塑密切相关，细胞骨架重塑会导致细胞与细胞接触的破坏，并包括由肌球蛋白轻链 (MLC) 磷酸化，随后形成 F-肌动蛋白应力纤维和细胞旁间隙的形成。对于决定屏障增强或保护的过程知之甚少；然而，我们发表的数据表明细胞骨架动力学在这种反应中发挥着关键作用。细胞外 ATP 是一种重要的血管介质，主要通过与特定三聚体 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 会产生快速、持续和剂量依赖性的跨内皮电阻 (TER) 增加，表明屏障增强，并有效逆转由水肿剂凝血酶引起的屏障功能障碍。 Gq和Gi2的α亚基的特异性消耗显着减弱了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 可消除 ATP 诱导的 TER 增加，并导致多个细胞骨架靶标的磷酸化，包括 MLC、ezrin/radixin/moezin (ERM) 和 caldesmon，表明这些蛋白质的去磷酸化可能与 ATP 的屏障增强作用有关。此外，蛋白激酶 A (PKA) 抑制可减弱 ATP 诱导的 TER 增加和血管舒张刺激蛋白 (VASP) 的磷酸化，磷酸化形式可抑制应激纤维形成，支持 PKA/VASP 通路参与 ATP-诱导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 P2Y/G 蛋白复合物的激活，随后协调激活 MLCP 和 PKA 信号传导。具体目标 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。具体目标 2 将定义 MLCP 及其细胞骨架靶标在 ATP 诱导的 EC 屏障增强中的作用。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，重点关注 VASP 和 MLCP 作为潜在的 PKA 靶点。具体目标 4 将描述 ATP 在 ALI 小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 屏障增强所涉及的新信号通路的理解，并为治疗肺部疾病提供新的方向和目标。