cAMP in Enothelial Permeability

肺微血管内皮细胞（PMVEC）具有强粘附的细胞-细胞连接 这是限制液体、溶质和大分子渗透到间质和肺泡中所必需 隔室，这对于有效的气体交换是重要的。PMVEC结强度动态变化 通过细胞内cAMP浓度调节。6型腺苷酸环化酶（AC 6）在腺苷酸环化酶的下游合成cAMP。 细胞膜cAMP信号传导通过4型靶向生理相关效应分子 磷酸二酯酶（PDE 4），特别是-D4同种型，其通过血影蛋白定位于膜。的 膜定位的cAMP池增强PMVEC屏障功能。相比之下，铜绿假单胞菌 将可溶性腺苷酸环化酶毒素ExoY引入PMVEC，产生胞质cAMP池。 胞质溶胶内的cAMP合成破坏而不是加强PMVEC屏障。以确定 无论是膜还是胞浆AC活性在控制内皮细胞屏障功能中占主导地位，我们 利用嵌合哺乳动物可溶性AC酶，其可被毛喉素激活。同时 毛喉素刺激膜和胞质AC活性破坏，而不是加强，PMVEC 屏障，表明可溶性AC活性主要控制屏障强度。初步数据显示可溶性 AC与中心体及其相关的微管结合，因此可以重组 微管结构是诱导PMVEC间隙所必需的。因此，本提案检验了总体假设 膜定位的AC产生一个加强的cAMP池，而胞质AC产生一个加强的cAMP池。 破坏PMVEC屏障的cAMP池。具体目标测试相关假设：[1] AC 6 产生膜cAMP池，由血影蛋白和PDE 4（D4）相互作用维持; [2]可溶性 AC产生控制微管组织的胞质cAMP池;[3] 胞质区室分解微管并破坏内皮细胞屏障。完成 这项工作将有助于我们理解cAMP如何控制PMVEC屏障强度， 寻求解决细菌的致病机制，如铜绿假单胞菌，利用腺苷酸 环化酶毒素破坏内皮细胞屏障并增加渗透性。

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.