MECHANISM FOR SIGNAL TRANSDUCTION OF SHEAR STRESS FORCES IN ENDOTHELIAL CELLS

内皮细胞剪切应力信号传导机制

• 批准号: 3767792
• 负责人: M C CAPOGROSSI
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3767792
• 关键词: acidity /alkalinity; amiloride; antiport; bicarbonates; biological signal transduction; cell adhesion; cell cell interaction; cytoplasm; fluorescent dye /probe; hemodynamics; hydrogen; mechanical stress; membrane transport proteins; neoplastic cell; sodium; tissue /cell culture; vascular endothelium

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.

血管内皮位于流动的血液和血管之间， 血管壁唯一地暴露于血液动力学剪切应力力。 研究剪切力对血管内皮细胞pHi的影响 和胞浆[Ca 2 +]（[Ca 2 +]i），将细胞在1 mm 2玻璃中培养 毛细管，装有荧光指示剂羧基- 萘并[a]芘或-1（SNARF-1代表pHi）或indo-1（[Ca 2 +]i），以及 在改进的倒置荧光显微镜的载物台上进行研究。 这些毛细管有助于在一个特定的环境中进行pHi或[Ca 2 +]i测量。 使用CO2/HCO 3时不允许气体扩散的封闭系统-- 缓冲溶液;流速的微小变化导致相对 剪切应力的大变化。 我们最近报道说， 在内皮细胞中发生流动依赖性细胞内酸化 在短暂暴露于连续层流剪切应力力的过程中， 生理缓冲液与碳酸氢盐由于平行激活Na+o- 独立的Cl-/HCO 3-交换和Na+/H+交换（Science 258：656- 659，1992）。 细胞内pH（pHi）的这种变化在细胞生长期间持续。 暴露于13.4达因cm-2的剪切应力30分钟，尽管 在30分钟内从酸化中部分回收 暴露于2.7达因cm-2或更小的剪切应力力。 到 确定在30分钟内pHi部分恢复的机制， 细胞暴露于2.7 dyne cm-2或更小的剪切应力的分钟 暴露于乙基异丙基氨氯吡咪（EIPA），Na+/H+交换 抑制剂或无Na+缓冲液以抑制Na+依赖性交换 机制等 虽然EIPA没有影响，但去除缓冲液Na+ 显著抑制pHi恢复。 这些结果表明 而Na+ o依赖的Cl-/HCO 3-交换也被激活， 血流动力学剪切应力暴露。 还进行了研究， 表征暴露于剪切30分钟后的pHi响应 应力 回到控制条件后， 内皮pHi增加约0.20 pH单位， 在返回到控制条件时记录。 在碱化之后， pHi在15-20分钟内恢复到对照值。 因此，pHi似乎 在血管内皮的反应中起重要作用 剪切应力。