Calcium Inhibition of cAMP in Endothelial Cell Permeability

钙激动剂破坏大血管而非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段的机制。我们之前的工作表明，炎症性钙激动剂激活储存操作钙进入抑制肺动脉内皮细胞腺苷酸环化酶活性，导致环AMP （cAMP）降低，足以增加通透性。尽管微血管内皮细胞（PMVECs）表达一种钙抑制腺苷酸环化酶（AC6），但钙激动剂既不会降低cAMP含量，也不会破坏PMVEC屏障。初步数据表明，这种钙不敏感伴随着高速率的cAMP合成和转换；当PMVEC中cAMP水解被破坏时，钙抑制cAMP的形成，炎症介质诱导细胞间隙，证明AC6对PAEC和PMVEC屏障功能都有关键调节作用。一个camp敏感的调控内皮细胞通透性的靶标是膜相关的红系谱蛋白。谱蛋白与f -肌动蛋白结合的破坏促进两种PAECs谱蛋白的细胞间隙形成。spectrin与F-actin结合的破坏促进PAECs和PMVECs细胞间隙的形成，证明了spectrin在调节细胞形状方面的重要作用。综上所述，我们的数据支持钙抑制cAMP减少谱蛋白- f -肌动蛋白关联的观点。在paec中，而在pmvec中，cAMP含量的保存促进了谱蛋白- f -肌动蛋白的关联。因此，这一提议验证了钙抑制cAMP形成的总体假设，即降低了谱蛋白与f -肌动蛋白的结合，这对增加肺内皮细胞的通透性很重要。具体目的验证了相关假设：[1]cAMP积累调节PMVECs中AC6的钙抑制；来自f -肌动蛋白的非红系谱蛋白增加大血管内皮细胞和微血管内皮细胞的通透性。这项工作的完成将有助于我们进一步了解钙激动剂与内皮屏障破坏之间的关键信号事件，内皮屏障破坏是多种炎症性肺部疾病（包括急性呼吸窘迫综合征、哮喘和再灌注肺水肿）的主要特征。

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.