cAMP in Enothelial Permeability

内皮细胞通透性中的 cAMP

• 批准号: 7217671
• 负责人: Troy Stevens
• 金额: $ 32.33万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7217671
• 关键词: adenylate cyclase; calcium flux; cell cell interaction; cyclic AMP; cytoplasm; electrophysiology; fluorescence microscopy; forskolin; intercellular connection; isozymes; laboratory rat; membrane; microcirculation; microtubules; molecular assembly /self assembly; protein localization; pulmonary artery; pulmonary circulation; spectrin; time resolved data; vascular endothelium; vascular endothelium permeability; video microscopy

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.

肺微血管内皮细胞（PMVEC）具有强粘附性的细胞-细胞连接 限制液体、溶质和大分子进入间质和肺泡的渗透性所必需的 室，这对于有效的气体交换很重要。 PMVEC 结强度是动态变化的 通过细胞内 cAMP 浓度进行调整。 6 型腺苷酸环化酶 (AC6) 在 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 (PDE4)，特别是 -D4 同工型，由血影蛋白定位在膜上。这 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 中，生成胞质 cAMP 池。 胞质内的 cAMP 合成会破坏而不是增强 PMVEC 屏障。确定 无论是膜还是胞质 AC 活性在内皮细胞屏障功能的控制中占主导地位，我们 利用可被毛喉素激活的嵌合哺乳动物可溶性 AC 酶。同时 毛喉素刺激膜和细胞质 AC 活性会破坏而不是增强 PMVEC 屏障，表明可溶性 AC 活性主要控制屏障强度。初步数据表明可溶 AC 与中心体及其相关微管相关，因此可能会重组 诱导 PMVEC 间隙所必需的微管结构。因此，该提案检验了总体假设 膜定位的 AC 产生增强的 cAMP 池，而胞质 AC 产生增强的 cAMP 池 破坏 PMVEC 屏障的 cAMP 池。具体目标测试相关假设：[1] AC6 生成由血影蛋白和 PDE4(D4) 相互作用维持的膜 cAMP 池； [2] 可溶 AC 产生控制微管组织的胞质 cAMP 池； [3] cAMP 访问 胞质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了解 cAMP 如何控制 PMVEC 屏障强度，并将 寻求解决铜绿假单胞菌等利用腺苷酸的细菌的致病机制 环化酶毒素破坏内皮细胞屏障并增加通透性。