Calcium Inhibition of cAMP in Endothelial Cell Permeability

钙抑制 cAMP 在内皮细胞通透性中的作用

• 批准号: 6631289
• 负责人: Troy Stevens
• 金额: $ 25.75万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6631289
• 关键词: actins; adenylate cyclase; calcium channel blockers; calcium flux; cyclic AMP; inflammation; isozymes; laboratory rat; microcirculation; protein binding; pulmonary artery; pulmonary circulation; spectrin; stimulant /agonist; vascular endothelium; vascular endothelium permeability

Calcium agonists disrupt macrovascular and not microvascular cell barrier function, suggesting distinct responses to similar inflammatory mediators may represent a mechanism for targeting lung inflammation to specific vascular segments. Our prior work indicated that activation of store operated calcium entry by inflammatory calcium agonists inhibits adenylyl cyclase activity in pulmonary artery endothelial cells resulting in decreased cyclic AMP (cAMP) sufficient to increase permeability. Although microvascular endothelial cells (PMVECs) express a calcium inhibited isoform of adenylyl cyclase (AC6), calcium agonists neither decrease cAMP content nor disrupt the PMVEC barrier. Preliminary data indicate this calcium insensitivity is accompanied by a high rate of cAMP synthesis and turnover; when cAMP hydrolysis is disrupted in PMVECs calcium inhibits cAMP formation and inflammatory mediators induce intercellular gaps, demonstration AC6 critically regulates both PAEC and PMVEC barrier function. One cAMP-sensitive target that regulates endothelial cell permeability is membrane associated, on-erythroid spectrin. Disruption of spectrin binding to F-actin promotes intercellular gap formation in both PAECs spectrin. Disruption of spectrin binding to F-actin promotes intercellular gap formation in both PAECs and PMVECs, demonstrating spectrin's important role in regulating cell shape. Taken together our data support the idea that calcium inhibition of cAMP reduces the spectrin-F-actin association. In PAECs, whereas preservation of cAMP content promotes the spectrin-F-actin association in PMVECs. Thus, this proposal test the overall Hypothesis that calcium inhibition of cAMP formation decreases spectrin binding to F-actin important to increase lung endothelial cell permeability. Specific Aims test the related Hypotheses that: [1] cAMP accumulation regulates calcium inhibition of AC6 in PMVECs; [2] non-erythroid spectrin from F-actin increases macro- and microvascular endothelial cell permeability. Completion of this work will be important to further our understanding of the key signaling events that link calcium agonists to endothelial barrier disruption, a cardinal feature of multiple inflammatory lung diseases including acute respiratory distress syndrome, asthma, and reperfusion pulmonary edema.

钙激动剂破坏大血管而不是微血管细胞屏障功能，提示对类似炎症介质的不同反应可能代表了将肺部炎症靶向特定血管节段的机制。我们以前的工作表明，炎症性钙激动剂激活储存操作的钙内流抑制了肺动脉内皮细胞的腺酰环化酶活性，导致环磷酸腺苷(CAMP)减少，从而增加通透性。虽然微血管内皮细胞(PMVECs)表达一种钙抑制的腺酰环化酶(AC6)亚型，但钙激动剂既不能降低cAMP含量，也不能破坏PMVEC的屏障。初步数据表明，这种钙不敏感伴随着高cAMP合成和周转率；当PMVEC中cAMP的水解率被破坏时，钙抑制cAMP的形成，炎症介质诱导细胞间隙，证明AC6对PAEC和PMVEC的屏障功能都有关键的调节作用。调节内皮细胞通透性的一个cAMP敏感靶点是膜相关的红系血影蛋白。破坏血影蛋白与F-肌动蛋白的结合可促进两种血管内皮细胞血影蛋白细胞间隙的形成。破坏血影蛋白与F-肌动蛋白的结合可促进PAECs和PMVECs细胞间隙的形成，证明了血影蛋白在调节细胞形状中的重要作用。综上所述，我们的数据支持这一观点，即cAMP的钙抑制减少了血影蛋白-F-肌动蛋白的结合。在PAECs中，cAMP含量的保存促进了PMVECs中血影蛋白-F-肌动蛋白的结合。因此，这项建议验证了一个总体假设，即钙抑制cAMP的形成减少了血影蛋白与F-肌动蛋白的结合，F-肌动蛋白对增加肺内皮细胞的通透性至关重要。[1]cAMP积聚调节PMVECs中AC6的钙抑制作用；[2]来自F-肌动蛋白的非红系血影蛋白增加巨、微血管内皮细胞的通透性。这项工作的完成将对我们进一步了解将钙激动剂与内皮屏障破坏联系起来的关键信号事件具有重要意义，内皮屏障破坏是包括急性呼吸窘迫综合征、哮喘和再灌注性肺水肿在内的多种炎症性肺部疾病的基本特征。