MECHANISM FOR SIGNAL TRANSDUCTION OF SHEAR STRESS FORCES IN ENDOTHELIAL CELLS

血管内皮细胞，位于流动的血液和 血管壁具有独特的血液动力学剪切力。 切应力对血管内皮细胞膜厚度影响的研究 和胞浆[Ca~(2+)]i，细胞培养在1mm2的玻璃中 毛细管，装有荧光指示剂羧基- Seminphtharhodafluor-1(Phi的snarf-1)或Indo-1([Ca~(2+)]i)和 对改进型倒置荧光显微镜工作台进行了研究。 这些毛细管有助于测量PHI或[钙]i 使用CO2/HCO3时不允许气体扩散的封闭系统-- 缓冲溶液；流量的微小变化会导致相对 剪应力变化较大。我们最近报道说， 内皮细胞发生依赖于流动的细胞内酸化 在短暂暴露于连续层流剪应力期间 因NaO-平行激活而与重碳酸盐形成生理缓冲 独立的Cl-/HCO3-交换和Na/H交换(Science 258：656- 659,1992)。细胞内pH(Phi)的这种变化在 30分钟暴露在13.4达因厘米-2的剪应力下，尽管 酸化的部分恢复发生在30分钟内 暴露在2.7达因厘米-2或更小的剪应力下。至 确定PHI在30分钟内部分恢复的机制 在2.7达因厘米~(-2)或更小的剪应力作用下的微小接触，细胞 暴露于乙基异丙基阿米洛利(EIPA)，一种Na/H交换 抑制剂或无钠缓冲液，以抑制钠依赖的交换 机械装置。当EIPA没有影响时，缓冲液Na的去除 显著抑制PHI的恢复。这些结果表明， 而依赖于Na O的Cl-/HCO3-交换也被激活 血流动力学切应力暴露。还进行了研究，以 切变作用30分钟后PHI反应的特征 压力力。在回到控制条件后，一个缓慢发展的 内皮PHI增加了大约0.20个pH单位 已在返回控制状态时注明。在这种碱化之后， PHI在15-20分钟内恢复到控制值。因此，Phi出现了 在血管内皮细胞的反应中发挥重要作用 剪切力。

The vascular endothelium, positioned between the flowing blood and the vessel wall, is uniquely exposed to hemodynamic shear stress forces. To study the effect of shear stress forces on vascular endothelial pHi and cytosolic [Ca2+] ([Ca2+]i), cells were cultured in 1 mm2 glass capillary tubes, loaded with the fluorescent indicator carboxy- seminaphtharhodafluor-1 (SNARF-1 for pHi) or indo-1 ([Ca2+]i) and studied on the stage of a modified inverted fluorescence microscope. These capillary tubes facilitate pHi or [Ca2+]i measurements in a closed system which does not allow gas diffusion when using CO2/HCO3-- buffered solutions; small changes in flow rate result in relatively large changes in shear stress forces. We have recently reported that flow-dependent intracellular acidification occurs in endothelial cells during brief exposures to continuous laminar shear stress forces in a physiologic buffer with bicarbonate due to parallel activation of Na+o- independent Cl-/HCO3- exchange and Na+/H+ exchange (Science 258: 656- 659, 1992). This change in intracellular pH (pHi) is sustained during a 30 minute exposure to shear stress forces of 13.4 dyne cm-2, although partial recovery from the acidification occurs during a 30 minute exposure to shear stress forces of 2.7 dyne cm-2 or less. To determine the mechanism of the partial recovery of pHi during a 30 minute exposure to shear stress forces of 2.7 dyne cm-2 or less, cells were exposed to ethylisopropylamiloride (EIPA), a Na+/H+ exchange inhibitor or to Na+-free buffer to inhibit Na+-dependent exchange mechanisms. While EIPA had no effect, removal of buffer Na+ significantly inhibited the pHi recovery. These results suggest that while Na+o-dependent Cl-/HCO3- exchange is also activated by hemodynamic shear stress exposure. Studies were also performed to characterize the pHi response following a 30 minute exposure to shear stress forces. After return to control conditions, a slowly-developing increase in endothelial pHi of approximately 0.20 pH units has been noted on return to control conditions. Following this alkalinization, pHi recovers to control values over 15-20 minutes. Thus, pHi appears to play a significant role in the response of the vascular endothelium to shear stress forces.