MECHANISM FOR SIGNAL TRANSDUCTION OF SHEAR STRESS FORCES IN ENDOTHELIAL CELLS

血管壁切应力在血管内皮细胞的形成中起着重要作用。 血管成形术后再狭窄、冠状动脉旁路移植物闭塞和 动脉粥样硬化 此外，这些过程也受到 血管细胞与内皮的相互作用。 这样做的目的 项目是：1）表征，在内皮细胞（EC）中， 对胞质pH（pH）i的剪切应力的急剧变化， [Ca2+]（Cai）。 2)确定Cai变化对EC pHi的影响 以及3）确定内皮细胞和其他细胞之间的相互作用是如何发生的。 细胞影响EC中的Cai稳态。 层流剪应力效应 通过检查在玻璃毛细管中培养的EC来研究对EC pHi的影响。 剪切应力力导致EC的pHi在以下存在下快速降低： HCO3-。 这种反应被阴离子交换抑制剂明显减弱 4-乙酰氨基，4 '-异硫氰基芪-2，2'-二磺酸（SITS） 不受Na+去除的影响。 在不存在HCO 3-的情况下，剪切应力力 引起pHi的小幅增加， 乙基异丙基氨氯吡咪（EIPA），一种Na+/H+交换抑制剂。 效果 在没有显著剪切的情况下， 通过去除NH 4Cl，从碳酸氢盐- 在恒定缓冲液pH下游离于5% CO2/HCO 3缓冲溶液中，或 从5%CO2/HCO 3-溶液变为20%CO2/HCO 3-溶液。 无论 所采用的方法，细胞内酸化导致增加 用荧光Ca 2+指示剂indo-1指示的Cai。 的增加 通过从5%CO2/HCO 3-改变到5%CO2/HCO 3-诱导的indo-1荧光比率， 20%CO2/HCO 3-溶液中， 缓冲液Ca 2+之前或之后的缓激肽敏感性耗竭 细胞内钙库。 在其他实验中，我们研究了 白细胞粘附于EC的Cai上。 当粒细胞或 单核细胞与EC有一个快速增加EC的蔡，表现出 部分恢复到控制状态 这一回应在 用毒胡萝卜素耗竭EC内质网Ca 2+， 在无钙缓冲液中不受影响。 获得了类似的结果 当黑色素瘤细胞被用来代替白细胞时。 欧盟委员会与8 fm惰性珠没有引起Cai的增加。 因此，在血管 内皮细胞，剪切应力力激活碱挤出器， Na+-独立的Cl-/HCO 3-交换，以及在较小程度上的酸 挤出机，Na+/H+交换;生理碳酸氢盐中的净效应 缓冲液是pHi降低

Wall shear stress in blood vessels plays a role in the development of restenosis after angioplasty, coronary artery bypass graft occlusion and atherosclerosis. In addition these processes are also modulated by the interaction of vascular cells with the endothelium. The purpose of this project is: 1) To characterize, in endothelial cells (EC), the effect of acute changes in shear stress forces on cytosolic pH (pH)i and [Ca2+](Cai). 2) To determine the effects of changes in Cai on EC's pHi and 3) to determine how the interaction between the endothelium and other cells affects Cai homeostasis in EC. The effect of laminar shear stress on EC's pHi was studied by examining EC cultured in glass capillary tubes. Shear stress forces led to a rapid decrease in EC's pHi in the presence of HCO3-. This response was markedly blunted by the anion exchange inhibitor 4-acetamido,4'-isothio-cyanatostilbene-2,2'-disulfonic acid (SITS) but unaffected by Na+ removal. In the absence of HCO3-, shear stress forces caused a small increase in pHi which was abolished by ethylisopropylamiloride (EIPA), a Na+/H+ exchange inhibitor. The effect of acidification on Cai was examined in the absence of significant shear stress forces either by removal of NH4Cl, changing from a bicarbonate- free to a 5% CO2/HCO3--buffered solution at constant buffer pH, or changing from a 5% CO2/HCO3- to a 20% CO2/HCO3- solution. Regardless of the method employed, intracellular acidification resulted in an increase in Cai indexed by the fluorescent Ca2+ indicator indo-1. The increase in the indo-1 fluorescence ratio induced by changing from a 5% CO2/HCO3- to a 20% CO2/HCO3- solution was not significantly altered by removal of buffer Ca2+ either before or after depletion of bradykinin-sensitive intracellular Ca2+ stores. In other experiments we examined the effect of leukocyte adhesion on EC's Cai. Upon contact between granulocytes or monocytes with EC there was a rapid increase in EC's Cai which exhibited a partial recovery toward control. This response was abolished after depletion of EC's endoplasmic reticulum Ca2+ with thapsigargin while it was not affected in a Ca2+-free buffer. Similar results were obtained when melanoma cells were used in place of leukocytes. EC contact with 8 fm inert beads did not elicit an increase in Cai. Thus, in vascular endothelial cells, shear stress forces activate both an alkali extruder, Na+-independent Cl-/HCO3- exchange, and to a smaller extent an acid extruder, Na+/H+ exchange; the net effect in a physiologic bicarbonate buffer is a decrease in pHi