ATP in Lung Endothelial Barrier Enhancement

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）的一个突出特征，与细胞骨架重塑密切相关，后者导致细胞间接触破坏，包括由肌球蛋白轻链（MLC）磷酸化引发的收缩激活，随后是f -肌动蛋白应激纤维的形成和细胞旁间隙的形成。人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种反应中的关键作用。细胞外ATP是一种重要的血管介质，主要通过P2Y家族受体与特异性三聚体g蛋白偶联引起细胞对EC的影响。我们的新发现表明，生理上相关浓度的ATP会产生快速、持续和剂量依赖性的跨内皮电阻（TER）增加，这表明了由致水肿剂凝血酶引起的屏障增强和屏障功能障碍的强烈逆转。Gq和Gi2 α亚基的特异性缺失显著减弱了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升高与肌球蛋白相关磷酸酶（PPase） 1 （MLCP）活性升高密切相关。抑制PPase 1可消除ATP诱导的TER升高，并导致包括MLC、ezrin/radixin/moezin （ERM）和caldesmon在内的几个细胞骨架靶点磷酸化，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和血管扩张剂刺激蛋白（VASP）磷酸化的增加，VASP以磷酸化的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P2Y/G蛋白复合物的激活，随后是MLCP和PKA信号的协同激活。特异性Aim 1将定义特异性P2Y/ g蛋白复合物在MLCP-和pka依赖性信号通路激活中的作用。特异性目的2将确定MLCP及其细胞骨架靶点在atp诱导的EC屏障增强中的作用。特异性目的3将探讨PKA活性参与atp诱导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。特异性Aim 4将描述ATP在小鼠ALI模型中的潜在屏障保护作用。这些研究将提供atp诱导的肺EC屏障增强的新信号通路的理解，并为治疗肺部疾病提供新的方向和靶点。

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.