ATP in Lung Endothelial Barrier Enhancement

肺内皮屏障增强中的 ATP

• 批准号: 7435762
• 负责人: ALEXANDER D VERIN
• 金额: $ 35.68万
• 依托单位: AUGUSTA UNIVERSITY
• 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/7435762
• 关键词: Acute Lung Injury; Adhesions; Adhesives; Agonist; Apoptosis; Attenuated; Biochemical; Blood Platelets; Blood Vessels; Cell Wall; Cells; Complex; Condition; Cyclic AMP-Dependent Protein Kinases; Cytoskeletal Proteins; Data; Disruption; Dose; Electrical Resistance; Endothelial Cells; Endothelium; Enzymes; Extracellular Fluid; F-Actin; Family; Figs - dietary; Functional disorder; GTP-Binding Proteins; Homeostasis; Hour; Inflammatory; Investigation; Lead; Leukocytes; Link; Liquid substance; Lung; Lung diseases; Mediating; Mediator of activation protein; Mitogen-Activated Protein Kinases; Modeling; Molecular; Mus; Myosin ATPase; Myosin Light Chains; Pathway interactions; Permeability; Phosphoric Monoester Hydrolases; Phosphorylation; Physiological; Principal Investigator; Process; Protein Dephosphorylation; Proteins; Publishing; Pulmonary artery structure; Regulation; Role; Signal Pathway; Signal Transduction; Smooth Muscle; Source; Stress Fibers; Thrombin; Tissues; Vascular Permeabilities; Work; caldesmon; cell growth; clinically relevant; extracellular; ezrin; injury prevention; lung injury; migration; novel; programs; protective effect; protein activation; radixin protein; receptor coupling; response

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.

骑着。 内皮细胞（EC）屏障功能障碍是急性肺损伤（ALT）的突出特征，与 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）磷酸化发起，然后是F-肌动蛋白应力纤维形成和形成 细胞间隙。关于确定障碍增强或保护的过程知之甚少；但是，我们的 已发布的数据暗示了在此反应中细胞骨架动力学的关键作用。细胞外ATP是重要的 血管介质，它主要通过P2Y家族受体对EC的影响引起EC的影响 蛋白质。我们的新发现表明，ATP在生理上相关的浓度会产生快速，持续和 剂量依赖性增加了跨内皮电阻（TER），表明屏障的增强和 e虫剂，凝血酶引起的有效逆转障碍功能障碍。 GQ亚基的特异性耗竭 GI2显着减弱了ATP诱导的TER的增加，表明这些G蛋白INATP- 诱导EC屏障增强。 ATP引起的TER的增加与肌球蛋白相关的增加紧密相关 磷酸酶（PPase）1（MLCP）活性。抑制PPase 1消除了ATP诱导的TER的增加，并导致 包括MLC，Ezrin/radixin/Moezin（ERM）和Caldesmon在内的几个细胞骨架靶标的磷酸化 这些蛋白质的去磷酸化可能与ATP的屏障增强作用有关。另外，蛋白质 激酶A（PKA）抑制作用会减弱ATP诱导的血管脂剂的TER和磷酸化的增加和磷酸化 - 刺激的蛋白质（VASP）以磷酸化形式抑制了应力纤维的形成，支持参与 ATP诱导的EC屏障增强中的PKA/VASP途径。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取决于特定P2Y/G的激活 蛋白质复合物随后是MLCP和PKA信号的协调激活。 sa＃1will定义特定的作用 MLCP和PKA依赖性信号传导激活中的P2Y/G蛋白复合物。 SA＃2将定义 MLCP及其在ATP诱导的EC屏障增强中的细胞骨架靶标。 SA＃3将通过 哪些PKA活性参与ATP诱导的EC屏障增强，重点是VASP和MLCP作为潜在的PKA 目标。 SA＃4将表征ATP在所有这些研究的鼠模型中的潜在屏障保护作用 将提供对ATP诱导的肺EC屏障增强的新型信号通路的理解 承诺新的方向和目标治疗肺部疾病。