CA++ INHIBITION OF CAMP IN LUNG ENDOTHELIAL PERMEABILITY

CA 对 CAMP 对肺内皮通透性的抑制作用

• 批准号: 2839028
• 负责人: Troy Stevens
• 金额: $ 16.85万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-12-01 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2839028
• 关键词: adenylate cyclase; animal tissue; biological signal transduction; calcium flux; calcium ion; cyclic AMP; gap junctions; inflammation; intracellular transport; microcirculation; phosphorylation; pulmonary circulation; pulmonary edema; thapsigargin; thrombin; tissue /cell culture; vascular endothelium permeability; vasomotion

In diseases of non-cardiogenic pulmonary edema, endothelium becomes disrupted allowing passage of macromolecules into the extravascular space. Endothelial disruption in thee diseases is a regulated process that is likely initiated by release of toxic oxygen radicals and proteases by activated which cells, ischemia-reperfusion, and/or neurohumoral inflammatory and vasoactive factors. These stimuli elevate endothelial cell intracellular free Ca2+ ([Ca2=]I) which directly or indirectly reorganizes the cytoskeleton and promotes myosin light chain phosphorylation leading to actinomyosin interaction and contraction. Cytoskeletal reorganization and contraction together cause generation of intercellular gaps and permeability. While elevated [Ca2+]I promotes permeability, elevated cAMP opposes permeability. We therefore investigated the relationship between [Ca2=]I cAMP content in pulmonary microvascular endothelial cells. Expression of multiple isoforms of adenylyl cyclase were identified, including on Ca2+ inhibitable isoform; however, the activity of a Ca2+ inhibitable adenylyl cyclase predominated in intact cells and established an inverse relationship between [Ca2+]I and cAMP in permeability induced by [Ca2+]I agonists. This proposal tests the overall HYPOTHESIS that Ca2+ inhibition of cAMP formation, which is established by a Ca2+ inhibitable adenylyl cyclase, is an important amplification step in the development of endothelia permeability. The hypothesis will be tested using inflammatory mediators thrombin and thapsigargin, which elevate [Ca2+]I via different mechanisms. The SPECIFIC AIMS are to test the HYPOTHESES that: [1] ca2+ inhibitable adenylyl cyclase establishes an inverse relationship between [Ca2+]I and cAMP content in pulmonary microvascular endothelium; and, [2] Ca2+ inhibitable adenylyl cyclase promotes filamentous-actin reorganization, myosin light chain phosphorylation, intercellular gap formation, and permeability in pulmonary endothelium (macro and microvascular). It is hoped completion of these studies will improve our understanding of mechanisms which regulate endothelial barrier properties so that effective therapies can be developed for treatment of non-cardiogenic pulmonary edema.

在非心源性肺水肿疾病中，内皮细胞变得 破坏，允许大分子进入血管外 空间。 这些疾病中的内皮破坏是一个受调控的过程 这可能是由有毒氧自由基的释放引发的 通过激活细胞、缺血再灌注和/或 神经体液炎症和血管活性因子。 这些刺激会提升 内皮细胞胞内游离 Ca2+ ([Ca2=]I) 直接或 间接重组细胞骨架并促进肌球蛋白轻链 磷酸化导致放线肌球蛋白相互作用和收缩。 细胞骨架重组和收缩共同导致产生 细胞间隙和渗透性。 虽然 [Ca2+]I 升高会促进 通透性，升高的 cAMP 会对抗通透性。我们因此 探讨肺组织[Ca2=]I cAMP含量与肺组织中[Ca2=]I cAMP含量的关系 微血管内皮细胞。 多种异构体的表达 鉴定出腺苷酸环化酶，包括 Ca2+ 抑制亚型； 然而，Ca2+ 可抑制的腺苷酸环化酶的活性 在完整细胞中占主导地位并建立了反比关系 [Ca2+]I 和 cAMP 在 [Ca2+]I 激动剂诱导的通透性方面的差异。 该提案测试了 Ca2+ 抑制 cAMP 的总体假设 形成，这是由 Ca2+ 可抑制的腺苷酸环化酶建立的， 是内皮细胞发育中重要的放大步骤 渗透性。 该假设将使用炎症进行检验 介质凝血酶和毒胡萝卜素，通过不同的途径升高 [Ca2+]I 机制。 具体目标是检验以下假设： [1] ca2+ 可抑制的腺苷酸环化酶在两者之间建立了反比关系 肺微血管内皮中[Ca2+]I和cAMP含量；和， [2] Ca2+ 抑制腺苷酸环化酶促进丝状肌动蛋白 重组，肌球蛋白轻链磷酸化，细胞间隙 肺内皮细胞的形成和渗透性（宏观和 微血管）。 希望这些研究的完成能够改善 我们对调节内皮屏障机制的理解 特性，以便开发出有效的疗法来治疗 非心源性肺水肿。