ENDOTHELIAL MODULATION OF LUNG VASCULAR PERMEABILITY

肺内皮细胞对蛋白质渗透性的持续增加是一种 成人呼吸窘迫综合征的标志 此外，委员会认为， 炎症介质引发的面积/单位路径长度增加 (Arho/Ax）是内皮细胞连接间隙的基础。 微血管壁的溶质渗透性升高。 长期 这项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'环磷酸（cAMP）或蛋白激酶活性 C和A，以及细胞内游离Ca 2+浓度（[Ca 2 +]）将是 专门在体外进行操作。 待检验的假设为：1） 激动剂诱导的细胞内第二信使的变化调节 导致离散溶质渗透性的内皮形态 机制：受限扩散或对流和2）不同介质 通过离散时空诱导不同的细胞反应， 细胞骨架蛋白[Ca 2 +]的改变将在 具体目标1-3. 具体目标4将检验老妪假说。 这些 aims将使用来自两种不同的牛肺内皮细胞进行测试。 位置：大血管（肺动脉内皮细胞，AEC）和 大血管（MEC）。 AEC和MEC的单层将用作细胞膜。 肺微血管对溶质屏障的模型。 以下 将使用：1）定义溶质渗透性机制的溶质渗透性机制 将测量内皮单层的屏障性质 2）内皮表面积将从以下测量： 使用Image-1的微分干涉衬度（DIC）数字图像 软件（通用成像公司），3）激动剂诱导的时空 细胞内[Ca 2 +]的改变将通过fura-2测定。 荧光数字图像。4)药物诱导重排 内皮F-肌动蛋白/肌球蛋白II将通过荧光数字 活细胞中的图像，5）通过旋转电子显微镜分析 将进行内皮细胞骨架的阴影，6） 将通过放射免疫测定法测量细胞内[cAMP]和[cGMP]，7） 蛋白激酶活性将通过P-标记的免疫亲和法测定。 蛋白质SDS聚丙烯酰胺凝胶电泳。

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.