cAMP in Enothelial Permeability

肺微血管内皮细胞(PMVECs)具有强烈的细胞-细胞连接 以限制液体、溶质和大分子对间质和肺泡的渗透性 隔室，这对于高效的气体交换非常重要。PMVEC结强度是动态的 由细胞内cAMP浓度调节。6型腺酰环化酶(AC6)在 细胞膜。CAMP信号通过4型作用于与生理相关的效应分子 磷酸二酯酶(PDE4)，特别是-D4亚型，它是由血影蛋白膜定位的。这个 膜定位cAMP池增强PMVEC屏障功能。相比之下，铜绿假单胞菌 将一种可溶性的腺酰环化酶毒素ExoY引入PMVECs，产生胞质cAMP池。 胞浆内cAMP的合成破坏而不是加强PMVEC屏障。要确定 无论是膜或胞浆AC活性在控制内皮细胞屏障功能中起主导作用，我们 利用了一种嵌合的哺乳动物可溶性AC酶，该酶可以被Forsklin激活。同时进行 Forskolin对膜和胞浆AC活性的刺激作用破坏而不是增强PMVEC 屏障，表明可溶性AC活性主要控制屏障强度。初步数据显示可溶 急性冠脉综合征与中心体及其相关的微管联系在一起，因此可以重组 诱导PMVEC间隙所必需的微管结构。因此，这一提议检验了总体假设 膜定位的ACS产生一个加强的cAMP池，而胞质的ACS产生一个 破坏PMVEC屏障的营地泳池。具体目标检验相关假设：[1]AC6 产生由血影蛋白和PDE4(D4)相互作用维持的膜cAMP池；[2]可溶 ACS生成控制微管组织的胞质cAMP池；以及，[3]访问 胞浆室分解微管，破坏内皮细胞屏障。完成 这项工作将有助于我们理解cAMP如何作用于控制PMVEC屏障强度，并将 寻求解决像铜绿假单胞菌这样的细菌的致病机制，这些细菌利用腺苷 环化毒素，破坏内皮细胞屏障，增加通透性。

Pulmonary microvascular endothelial cells (PMVECs) possess strongly adherent cell-cell junctions that are necessary to limit fluid, solute and macromolecule permeability into interstitial and alveolar compartments, which is important for efficient gas exchange. PMVEC junction strength is dynamically adjusted by intracellular cAMP concentrations. The type 6 adenylyl cyclase (AC6) synthesizes cAMP at the cell membrane. cAMP signaling is targeted to physiologically relevant effector molecules by type 4 phosphodiesterases (PDE4), specifically the -D4 isoform, which is membrane-localized by spectrin. The membrane-localized cAMP pool strengthens PMVEC barrier function. In contrast, Pseudomonas aeruginosa introduces a soluble adenylyl cyclase toxin, ExoY, into PMVECs that generates a cytosolic cAMP pool. cAMP synthesis within the cytosol disrupts, rather than strengthens, the PMVEC barrier. To determine whether membrane or cytosolic AC activity dominates in control of endothelial cell barrier function, we utilized a chimeric mammalian soluble AC enzyme that could be activated by forskolin. Simultaneous stimulation of membrane and cytosolic AC activity by forskolin disrupts, rather than strengthens, the PMVEC barrier, indicating soluble AC activity dominantly controls barrier strength. Preliminary data suggest soluble ACs associate with the centrosome and its associated microtubules, and may therefore reorganize microtubule architecture necessary to induce PMVEC gaps. Thus, this proposal tests the overall hypothesis that membrane-localized ACs produce a cAMP pool that strengthens, whereas cytosolic ACs produce a cAMP pool that disrupts, the PMVEC barrier. Specific Aims test the related Hypotheses that: [1] AC6 generates a membrane cAMP pool that is maintained by a spectrin and PDE4(D4) interaction; [2] Soluble ACs generate a cytosolic cAMP pool that controls microtubule organization; and, [3] cAMP that accesses the cytosolic compartment disassembles microtubules and disrupts the endothelial cell barrier. Completion of this work will contribute to our understanding of how cAMP acts to control PMVEC barrier strength, and will seek to resolve pathogenic mechanisms of bacteria like Pseudomonas aeruginosa, which utilize adenylyl cyclase toxins to disrupt the endothelial cell barrier and increase permeability.