MECHANICAL STRESSES ON BLOOD CELL ARACHIDONIC ACID METABOLISM

拟议研究的长期目标是了解 流动，流体机械应力和机械应变 内皮细胞和白细胞的花生四烯酸代谢， 随后调节内皮细胞基因表达和血小板， 白细胞与内皮细胞的相互作用。 我们的初步研究 实验室已经表明，在植物中存在相当显著胁迫-代谢耦合， 每一种细胞类型。 拟议的工作将集中在了解 细胞膜信号转导和基因调控机制 独特的（和生理相关的）刺激。 潜在调解人 包括由以下因素引起穿过细胞膜的离子通量的改变 机械扰动（包括拉伸激活离子通道）， 钙、钾和氢离子调节是最有可能的。 三 细胞内钙和pH分布的三维重建 将利用荧光视频重建获得 细胞内钙和pH分布将利用 荧光视频显微术结合数字图像处理。 我们 假设压力（和应变）水平和 作用在细胞膜上的应力（和应变）的变化是重要的 时间、应力（应变）大小和时间的调制因素 应力（应变）应用历史。 专门设计 粘度计、流动室和单轴拉伸室将被 就业。 在基因调控水平，聚合酶链反应方法和 将采用北方分析确定稳态变化 机械扰动引起的信使RNA水平。 改变的水平 代谢产物如tPA、派-1和内皮细胞的分泌， 除了引起膜相关粘附受体的表达外， 流体流动和应变将被量化。 同样，初步数据 我们的实验室已经记录了许多强效的 血管活性化合物 流动和应变对细胞表达的影响 粘附分子，如ICAM-1，VCAM-1和P-和E-选择素-以及 因为调节随后的中性粒细胞粘附可能是非常重要的 中风病理生理学 我们将扩大我们对人类脐带的研究， 静脉和牛肺和主动脉内皮细胞对新的 研制了牛脑内皮细胞制剂。 研究流动和 花生四烯酸代谢产物对内皮细胞通透性的调控 来自这些不同血管床的细胞单层将在 专门设计的带有核孔膜插入物的流动室。 比较这些不同的代谢流和应变调节 细胞类型对于体内中风应用是重要的。 我们相信我们的研究结果对于理解 与缺血和再灌注相关的代谢事件中的血流。 产生的许多代谢产物具有极强的生物活性，可导致 血管通透性、细胞粘附和平滑肌张力的变化-所有 其中可能在中风的发病机制中起重要作用。

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.