ENDOTHELIAL MODULATION OF LUNG VASCULAR PERMEABILITY

肺血管通透性的内皮调节

• 批准号: 2224903
• 负责人: RICHARD C SCHAEFFER
• 金额: $ 18.86万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-12-15 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2224903
• 关键词: SDS polyacrylamide gel electrophoresis; calcium; charge coupled device camera; cyclic AMP; cytoskeletal proteins; cytoskeleton; digital imaging; electron microscopy; fluorescent dye /probe; intracellular; microcirculation; protein kinase A; protein kinase C; pulmonary artery; pulmonary circulation; radioimmunoassay; second messengers; solute; thrombin; vascular endothelium permeability

A persistent increase in lung endothelial permeability to protein is a hallmark of the Adult Respiratory Distress Syndrome. Moreover, inflammatory mediator-initiated increase in the Area/Unit pathlength (Arho/Ax) of the Endothelial Junctional Space is the foundation of elevated solute permeability of the microvascular wall. The long term objectives of this research are to better understand the endothelial mechanisms that modulate pulmonary microvascular permeability to macromolecules. Intracellular level of cyclic nucleotide, adenosine 3',5' cyclic monophosphate (cAMP), or the activities of protein kinase C and A, and intracellular free Ca2+ concentration ([Ca2+]) will be specifically manipulated in vitro. The hypotheses to be tested are: 1) Agonist-induced changes of intracellular second messengers modulate endothelial morphology leading to the discrete Solute Permeability Mechanism: Restricted Diffusion or Convection and 2) Different mediator induced distinct cellular responses by discrete spatiotemporal alterations in [Ca2+] of cytoskeleton protein will be assessed in specific aims 1-3. Specific aim 4 will test the Crone Hypothesis. These aims will be tested using bovine lung endothelial cells from two distinct locations: large vessel (pulmonary artery endothelial cells, AEC) and macrovessel (MEC). Monolayers of AEC and MEC will be used as cellular models of the lung microvascular barrier to solutes. The following assays will be used: 1) The solute permeability mechanisms that define the barrier properties of endothelial monolayer will be measured for each experimental condition, 2) Endothelial surface area will be measured from differential interference contrast (DIC) digital images using Image-1 software (universal Imaging Corp), 3) Agonist-induced spatiotemporal alterations of intracellular [Ca2+] will be measured by fura-2 fluorescence digital images. 4) Agent-induced rearrangement of endothelial F-actin/myosin II will be measure by fluorescence digital images in living cells, 5) Electron microscopic analysis by rotary shadowing of the endothelial cytoskeleton will be performed, 6) Intracellular [cAMP] and [cGMP] will be measured by radioimmunoassay, 7) Protein kinase activities will be measured by P-labelled immunoaffinity protein SDS polyacrylamide gel electrophoresis.

肺内皮细胞对蛋白质的通透性持续增加是 成人呼吸窘迫综合征的标志。 而且， 炎症介质引发的面积/单位路径长度的增加 内皮连接空间 (Arho/Axe) 是 微血管壁溶质通透性升高。 长期来看 本研究的目的是更好地了解内皮细胞 调节肺微血管通透性的机制 大分子。 细胞内环核苷酸、腺苷水平 3',5'环单磷酸（cAMP），或蛋白激酶的活性 C 和 A，以及细胞内游离 Ca2+ 浓度 ([Ca2+]) 为 在体外进行专门操作。 要检验的假设是：1） 激动剂诱导的细胞内第二信使变化调节 内皮形态导致离散的溶质渗透性 机制：受限扩散或对流以及 2) 不同介体 通过离散时空诱导不同的细胞反应 细胞骨架蛋白 [Ca2+] 的改变将在 具体目标1-3。 具体目标 4 将检验老婆假设。 这些 将使用来自两种不同的牛肺内皮细胞来测试目标 位置：大血管（肺动脉内皮细胞，AEC）和 大血管（MEC）。 AEC 和 MEC 的单层将用作细胞 肺微血管溶质屏障模型。 下列 将使用以下分析： 1) 定义溶质渗透性机制 将测量每个内皮单层的屏障特性 实验条件，2) 内皮表面积将从 使用 Image-1 的微分干涉衬度 (DIC) 数字图像 软件（Universal Imaging Corp），3) 激动剂诱导的时空 细胞内[Ca2+]的变化将通过fura-2测量 荧光数字图像。 4）代理诱导的重排 内皮F-肌动蛋白/肌球蛋白II将通过荧光数字测量 活细胞中的图像，5) 旋转电子显微镜分析 将进行内皮细胞骨架的遮蔽，6) 细胞内[cAMP]和[cGMP]将通过放射免疫测定法测量，7) 蛋白激酶活性将通过 P 标记的免疫亲和力来测量 蛋白质SDS聚丙烯酰胺凝胶电泳。