ATP in Lung Endothelial Barrier Enhancement

罗维德。 内皮细胞屏障功能障碍是急性肺损伤(ALT)的一个显著特征，与 细胞骨架重塑，导致细胞-细胞接触中断，包括激活收缩 由肌球蛋白轻链(MLC)磷酸化引发，随后F-肌动蛋白应激纤维形成和形成 细胞旁间隙。对决定屏障增强或保护的过程知之甚少；然而，我们的 已发表的数据表明，细胞骨架动力学在这一反应中发挥了关键作用。胞外三磷酸腺苷是一种重要的 血管介体，主要通过与特异性三聚体G-受体偶联的P2Y家族受体对EC产生细胞效应。 蛋白质。我们的新发现表明，在生理上相关的浓度下，三磷酸腺苷能迅速、持续和 跨内皮细胞电阻(TER)呈剂量依赖性增加，表明屏障显著增强和 有效逆转水肿剂凝血酶引起的屏障功能障碍。Gq亚基的比耗竭 而GI2可显著抑制ATP诱导的TER升高，提示这些G蛋白参与了ATP诱导的TER升高。 诱导EC屏障增强。ATP诱导的TER增加与肌球蛋白相关的增加密切相关 磷酸酶(PPase)1(MLCP)活性。抑制PPase 1可阻断ATP诱导的TER升高并导致心肌梗死 MLC、Ezrin/Radioxin/moezin(ERM)和caldesmon等几个细胞骨架靶点的磷酸化 这些蛋白的去磷酸化可能参与了ATP的屏障增强作用。此外，蛋白质 抑制激酶A(PKA)可减弱ATP诱导的血管内皮生长因子受体(TER)和血管内皮生长因子磷酸化水平的升高 刺激蛋白(Vasp)，它以磷酸化的形式抑制支持参与的应力纤维的形成 PKA/VASP通路在三磷酸腺苷诱导的EC屏障增强中的作用。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的激活 蛋白质复合体随后协同激活MLCP和PKA信号。SA#1将定义特定的角色 P2Y/G蛋白复合体在激活MLCP和PKA依赖的信号转导中的作用SA#2将定义 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。SA#3将通过以下方式探索分子机制 哪个PKA活性参与了ATP诱导的EC屏障增强，Vasp和MLCP是潜在的PKA 目标。SA#4将在所有这些研究的小鼠模型中表征ATP的潜在屏障保护作用 将有助于理解参与三磷酸腺苷诱导的肺内皮细胞屏障增强的新的信号通路和 有望为肺部疾病的治疗提供新的方向和目标。

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.