ENDOTHELIAL MODULATION OF LUNG VASCULAR PERMEABILITY

肺内皮细胞对蛋白质的通透性持续增加是一种 成人呼吸窘迫综合征的特征。此外， 炎症介质引起的面积/单位路径长度的增加 (ARHO/Ax)的内皮连接间隙是 微血管壁的溶质通透性升高。从长远来看 本研究的目的是为了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’环一磷酸(CAMP)，或蛋白激酶活性 C和A，以及细胞内游离钙浓度([Ca2+])将是 特别是在体外操作的。需要检验的假设有：1) 激动剂诱导的细胞内第二信使的变化 内皮细胞形态导致离散的溶质通透性 机制：受限扩散或对流；2)不同介质 离散时空诱导的不同细胞反应 细胞骨架蛋白[Ca~(2+)]的变化将在 具体目标1-3。《特定目标4》将检验克龙假说。这些 AIMS将使用两种不同来源的牛肺内皮细胞进行测试 地点：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 肺微血管对溶质的屏障模型。以下是 将使用以下分析方法：1)溶质渗透性机制 将分别测量内皮单分子层的阻隔性能 实验条件，2)内皮表面积将从 使用IMAGE-1的DIC数字图像 软件(通用成像公司)，3)激动者诱导的时空 用Fura-2测定细胞内钙离子浓度的变化 荧光数字图像。4)主体诱导的重排 内皮细胞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阴影处理，6) 细胞内[cAMP]和[cGMP]将用放射免疫法测定，7) 蛋白激酶活性将通过P标记的免疫亲和力来测量 蛋白SDS-聚丙烯酰胺凝胶电泳法。

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.