MECHANISM FOR SIGNAL TRANSDUCTION OF SHEAR STRESS FORCES IN ENDOTHELIAL CELLS

血管壁切应力在血管病变发生发展中的作用 血管成形术后再狭窄，冠状动脉旁路移植闭塞和 动脉硬化。此外，这些过程也受到 血管细胞与内皮细胞的相互作用。这样做的目的是 项目是：1)表征在内皮细胞(EC)中， 剪切力对胞浆pH(PH)i和 [Ca~(2+)](CaI)。2)确定CAI的变化对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 细胞影响EC内CaI的动态平衡。层流剪应力的影响 通过考察EC在玻璃毛细管中的培养，研究了EC的PHI。 剪切应力使EC的PHI迅速降低 HCO3-。这种反应明显地被阴离子交换抑制剂钝化。 4-acetamido，4‘-isothio-cyanatostilbene-2，2’-disulfonic酸(SITS)，但 不受Na+去除的影响。在没有HCO3-的情况下，剪切力 导致PHI略有增加，但被取消 乙基异丙基阿米洛利(EIPA)，一种Na+/H+交换抑制剂。其效果 在没有明显剪切的情况下，考察了酸化对cai的影响。 应力要么通过去除NH4Cl，从碳酸氢盐- 自由到5%的CO2/HCO3--缓冲溶液，在恒定的缓冲pH下，或 从5%的CO2/HCO3-溶液改为20%的CO2/HCO3-溶液。不管 所采用的方法，细胞内酸化导致增加 在以荧光钙指示剂Indo-1为索引的CAI中。这一增长 5%CO_2/HCO_3~-改变时的Indo-1荧光比 20%的CO2/HCO3-溶液不会因为去除 缓激肽敏感性耗竭前后的缓冲钙离子 细胞内的钙储存。在其他实验中，我们检查了 外周血白细胞对EC细胞的黏附作用。当粒细胞或粒细胞接触时 单核细胞伴发EC时，EC的CAI迅速升高，表现为 朝着控制方向的部分恢复。这一回应在之后被废除 Thapsigarin耗竭EC内质网钙离子的同时 在无钙缓冲液中不受影响。也得到了类似的结果 当用黑色素瘤细胞代替白细胞时。EC与8的联系 FM惰性微珠未引起蔡氏染色的增加。因此，在血管中 内皮细胞，剪切力既激活了碱挤出机， 不依赖于Na+的Cl-/HCO3-交换，以及较小程度的酸 挤出机，Na+/H+交换；生理性碳酸氢盐的净效应 缓冲区是Phi中的减少量

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