ATP in Lung Endothelial Barrier Enhancement

已送达。 内皮细胞（EC）屏障功能障碍是急性肺损伤（ALT）的一个突出特征，与以下因素密切相关： 细胞骨架重塑，导致细胞-细胞接触的破坏，包括收缩的激活 由肌球蛋白轻链（MLC）磷酸化引发，随后是F-肌动蛋白应激纤维形成和 细胞间隙对决定屏障增强或保护的过程知之甚少;然而，我们的 已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是重要的 血管介质，其主要通过与特异性三聚体G- proteins.我们的新发现表明，ATP在生理相关浓度下产生快速，持续， 跨内皮电阻（TER）呈剂量依赖性增加，表明屏障显著增强， 有效逆转由水肿剂凝血酶引起的屏障功能障碍。Gq α亚基的特异性缺失 和Gi 2显着减弱ATP诱导的TER增加，表明这些G蛋白参与ATP诱导的TER增加。 诱导EC屏障增强。ATP诱导的TER增加与肌球蛋白相关的 磷酸酶（PPase）1（MLCP）活性。PPase 1的抑制消除了ATP诱导的TER增加，并导致 几种细胞骨架靶点的磷酸化，包括MLC、ezrin/radixin/moezin（ERM）和钙调蛋白， 这些蛋白质的去磷酸化可能参与ATP的屏障增强作用。此外，蛋白质 激酶A（PKA）抑制减弱ATP诱导的TER增加和血管舒张剂磷酸化， 刺激蛋白（VASP），其磷酸化形式抑制应力纤维形成，支持参与 PKA/VASP途径在ATP诱导的EC屏障增强中的作用。我们的假设是ATP诱导的EC 屏障增强和细胞骨架重塑至少部分依赖于特异性P2 Y/G的激活， 蛋白质复合物，然后协调激活MLCP和PKA信号传导。SA#1将定义特定 P2 Y/G-蛋白复合物在MLCP和PKA依赖性信号传导的激活中的作用SA#2将定义参与 MLCP及其在ATP诱导的EC屏障增强中的细胞骨架靶点。SA #3将探索分子机制， 其中PKA活性参与ATP诱导的EC屏障增强，重点关注VASP和MLCP作为潜在的PKA 目标的SA#4将描述ATP在ALL小鼠模型中的潜在屏障保护作用。 将提供一个新的信号通路参与ATP诱导的肺EC屏障增强的理解， 为肺部疾病的治疗提供了新的方向和靶点。

ROVIDED. Endothelial cell (EC) barrier dysfunction, a prominent feature of acute lung injury (ALT), 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 a subunits of Gq and Gi2 significantly attenuated ATP-induced increase in TER indicating the involvement of these G-proteins inATP- 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 includingMLC, 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 vasolidator- 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. SA#1will define the role of specific P2Y/G-protein complexes in the activation of MLCP- and PKA-dependent signaling. SA#2 will define the involvement of MLCP and its cytoskeletal targets in ATP-induced EC barrier enhancement. SA #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. SA#4 will characterize the potential barrier-protective effects of ATP in murine models of ALL 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.