MECHANICAL STRESSES ON BLOOD CELL ARACHIDONIC ACID METABOLISM

这项拟议研究的长期目标是了解其影响 流动、流体机械应力和机械应变 血管内皮细胞和白细胞的花生四烯酸代谢 血管内皮细胞基因表达和血小板的后续调节 白细胞与内皮细胞的相互作用。来自我们的初步研究 实验室已经显示出相当显著的压力-代谢耦合。 这些细胞类型中的每一个。拟议的工作将集中在了解 膜信号转导机制及其基因调控 独特的(和生理上相关的)刺激。潜在的调解人 包括由以下因素引起跨细胞膜的离子通量的变化 机械扰动(包括拉伸激活的离子通道)， 最有可能的是钙、钾和氢离子的调节。三 细胞内钙和pH分布的三维重建 将通过使用荧光视频重建获得 细胞内钙和pH的分布将利用 荧光视频显微镜与数字图像处理相结合。我们 假设应力(和应变)水平和应力(应变) 作用在细胞膜上的应力(和应变)的变化很重要 调制时间、应力(应变)大小和时间的因素 应力(应变)应用史。专门设计的 粘度计、流动室和单轴拉伸室 受雇的。 在基因调控水平上，聚合酶链式反应方法和 将使用诺斯分析来确定稳态的变化 信使RNA水平是由机械扰动引起的。改变的水平 代谢产物的分泌，如tPA，PAI-1和血管内皮细胞 膜相关黏附受体的表达增加 通过流体流动和应变将被量化。再一次，初步数据 从我们的实验室记录了许多强效物质分泌的变化 血管活性化合物。流动和应变对细胞表达的影响 黏附分子，如ICAM-I，VCAM-1和P-和E-选择素--也 AS对随后的中性粒细胞黏附的调节可能在 中风病理生理学。我们将扩大对人类脐带血的研究 静脉和牛肺和主动脉内皮细胞到一个新的 研制了牛脑内皮细胞制剂。关于流动和流动的研究 花生四烯酸代谢产物对内皮细胞通透性的控制 来自这些不同血管床的细胞单层将在 特别设计的带有核孔膜插件的流动室。 不同组织中代谢的流动和应变调节的比较 细胞类型对于体内卒中的应用将是重要的。 我们相信，我们的研究结果对于理解这一角色具有重要意义。 与缺血和再灌流相关的代谢事件中的血流。 产生的许多代谢物具有极高的生物活性，可能导致 血管通透性、细胞黏附和平滑肌张力的变化-ALL 其在卒中的发病机制中可能起重要作用。

The long term goal of the proposed research is to understand the effect of flow, fluid mechanical stresses and mechanical strain on the arachidonic acid metabolism of endothelial cells and leukocytes and subsequent modulation of endothelia cell gene expression and platelet and leukocyte interactions with endothelial cells. Initial studies from our laboratory have indicated quite striking stress-metabolism coupling in each of these cell types. Proposed work will center on understanding the mechanisms of membrane signal transduction and gene regulation for these unique (and physiologically relevant) stimuli. Potential mediators include alteration of ion fluxes across cell membranes caused by mechanical perturbations (including stretch activated ion channels), with calcium, potassium and hydrogen ion regulation being most likely. Three dimensional reconstructions of intracellular calcium and pH distribution will be obtained utilizing fluorescence video reconstructions of intracellular calcium and pH distribution will be obtained utilizing fluorescence video microscopy coupled with digital image processing. We hypothesize that both the level of stress (and strain) and the ate of change of stress (and strain) acting on the cell membrane are important factors in modulation of time, stress (strain) magnitude and the time history of stress (strain) applications. Specifically designed viscometers, flow chambers, and uniaxial stretching chambers will be employed. At the gene regulation level, polymerase chain reaction methods and northern analysis will be employed to ascertain changes in steady state messenger RNA levels caused by mechanical perturbations. Altered levels of secretion of metabolites, such as tPA, PAI-1 and endothelia, in addition to expression of membrane associated adhesion receptors caused by fluid flow and strain will be quantified. Again, preliminary data from our laboratory have documented changes in secretion of many potent vasoactive compounds. The roles of flow and strain on expression of cell adhesion molecules such as ICAM-I, VCAM-1 and P- and E-selectin-- as well as modulation of subsequent neutrophil adhesion may be very important in stroke pathophysiology. We will extend our studies of human umbilical vein and bovine pulmonary and aortic endothelial cells to a newly developed bovine brain endothelium preparation. Studies of flow and arachidonic acid metabolite control of the permeability of endothelial cell monolayers from these different vascular beds will be conducted in specially designed flow chambers with nuclepore membrane inserts. Comparison of flow and strain regulation of metabolism in these different cell types will be important with respect to in vivo stroke applications. We believe our results should be of importance in understanding the role of flow in metabolic events associated with ischemia and reperfusion. Many of the metabolites produced are extremely bioactive and can lead to changes in vessel permeability, cell adhesion and smooth muscle tone-all of which may be important in the pathogenesis of stroke.