Reusch H P, Lowe J, Ives H E
Division of Nephrology, University of California, San Francisco 94143.
Am J Physiol. 1995 Jan;268(1 Pt 1):C147-53. doi: 10.1152/ajpcell.1995.268.1.C147.
In many systems, osmotically induced cell shrinkage activates the Na+/H+ exchanger. To assess the role of H(+)-extruding transporters in the response to osmotic shrinkage in vascular smooth muscle (VSM) and Chinese hamster ovary (CHO) cells, intracellular pH (pHi) was measured with 2',7'-bis(carboxy-ethyl)-5(6)- carboxyfluorescein-acetoxymethyl ester (BCECF-AM) after exposing cells to hypertonic medium. In nominally HCO(3-)-free medium, addition of 200 mM sucrose caused pHi to increase 0.33 pH unit on average in VSM cells but only 0.13 pH unit in CHO cells. Permeant solutes failed to increase pHi significantly. Cytochalasin B (1-20 microM), colchicine (1-10 microM), Ca2+ removal, and downregulation of protein kinase C activity did not affect osmotic activation of H+ extrusion in either cell type. Additional work was carried out to determine why osmotic activation of H+ extrusion was less in CHO than in VSM cells. In CHO cells, the osmotically induced delta pHi was only weakly sensitive to amiloride, suggesting that osmotic forces may activate an H+ transport system other than Na+/H+ exchange. In the presence of 10 mM HCO3-, osmotically induced delta pHi decreased by 60% in VSM cells but increased by 50% in CHO cells compared with the delta pHi in HCO(3-)-free medium. Lastly, removal of extracellular Cl- did not affect osmotically induced delta pHi in VSM cells but completely abolished the response in CHO cells. We conclude that in VSM cells osmotically induced changes in pHi are mediated by Na+/H+ exchange, whereas in CHO cells they are most likely mediated by a Na(+)-dependent Cl-/HCO3- exchanger.
在许多系统中，渗透压诱导的细胞收缩会激活钠氢交换体。为了评估氢离子排出转运体在血管平滑肌（VSM）和中国仓鼠卵巢（CHO）细胞对渗透压收缩反应中的作用，在将细胞暴露于高渗培养基后，用2',7'-双（羧基乙基）-5（6）-羧基荧光素乙酰氧基甲酯（BCECF-AM）测量细胞内pH（pHi）。在名义上无HCO₃⁻的培养基中，添加200 mM蔗糖使VSM细胞中的pHi平均增加0.33个pH单位，但在CHO细胞中仅增加0.13个pH单位。渗透性溶质未能显著增加pHi。细胞松弛素B（1 - 20 μM）、秋水仙碱（1 - 10 μM）、去除Ca²⁺以及下调蛋白激酶C活性均不影响这两种细胞类型中氢离子排出的渗透压激活。还进行了额外的研究以确定为何CHO细胞中氢离子排出的渗透压激活比VSM细胞中少。在CHO细胞中，渗透压诱导的ΔpHi对氨氯吡脒仅具有微弱的敏感性，这表明渗透压可能激活了钠氢交换以外的氢离子转运系统。在存在10 mM HCO₃⁻的情况下，与无HCO₃⁻培养基中的ΔpHi相比，渗透压诱导的ΔpHi在VSM细胞中降低了60%，但在CHO细胞中增加了50%。最后，去除细胞外Cl⁻不影响VSM细胞中渗透压诱导的ΔpHi，但完全消除了CHO细胞中的反应。我们得出结论，在VSM细胞中，渗透压诱导的pHi变化由钠氢交换介导，而在CHO细胞中，它们最有可能由钠依赖性氯/碳酸氢根交换体介导。
