Boyarsky G, Ganz M B, Sterzel R B, Boron W F
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510.
Am J Physiol. 1988 Dec;255(6 Pt 1):C857-69. doi: 10.1152/ajpcell.1988.255.6.C857.
We used the pH-sensitive dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) to further characterize the mechanisms of intracellular pH (pHi) regulation in renal mesangial cells. In the accompanying paper [Am. J. Physiol. 255 (Cell Physiol. 24): C844-C856, 1988], we showed that acid extrusion from mesangial cells is mediated by both an ethylisopropylamiloride (EIPA)-sensitive Na+-H+ exchanger and a 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS)-sensitive-HCO3(-)-dependent mechanism. In this study, we examined the ionic dependencies of pHi-regulatory mechanisms in the presence of CO2-HCO3-. We found that in CO2-HCO3-, approximately 90% of the net acid extrusion occurring during recovery from an acid load is blocked by removing external Na+. Short-term (less than 15 min) removal of external Cl- has little effect on the rate of recovery in CO2-HCO3-. In contrast longer periods of external Cl- removal (1-2 h) blocks 40-60% of the rate of recovery, which is consistent with the hypothesis that a large fraction of the SITS-sensitive-HCO3(-)-dependent recovery mechanism described in the preceding paper is also Na+- and Cl(-)-dependent. Therefore, this Cl(-)-dependent component is probably mediated by a Na+-dependent Cl(-)-HCO3- exchanger. As much as 16% of total acid extrusion is insensitive to EIPA and long-term Cl- removal but is blocked by SITS. Thus either 1-2 h of Cl- removal is insufficient to wash out all internal Cl-, or a small component of acid extrusion is mediated by a Cl(-)-independent mechanism, such as the electrogenic Na+/HCO3- cotransporter. We also studied the effect on pHi of the removal and readdition of external Cl-, observing pHi changes consistent with the existence of a Na+-independent Cl(-)-HCO3- exchanger, which would presumably function as an acid loader. In contrast to the Na+-H+ exchanger and Na+-dependent Cl(-)-HCO3- exchanger, which are stimulated at low pHi, the Cl(-)-HCO3- exchanger is stimulated at high pHi. Thus the acid-extruding and acid-loading mechanisms have opposite pHi dependencies.
我们使用对pH敏感的染料2,7-双（羧乙基）-5（6）-羧基荧光素（BCECF）来进一步阐明肾系膜细胞内pH（pHi）调节机制。在随附论文[《美国生理学杂志》255卷（细胞生理学24）：C844 - C856，1988年]中，我们表明系膜细胞的酸排出由对乙基异丙基amiloride（EIPA）敏感的Na⁺ - H⁺交换体和对4-乙酰氨基-4'-异硫氰基芪-2,2'-二磺酸（SITS）敏感的HCO₃⁻依赖性机制介导。在本研究中，我们在存在CO₂ - HCO₃⁻的情况下研究了pHi调节机制的离子依赖性。我们发现，在CO₂ - HCO₃⁻中，从酸负荷恢复过程中发生的约90%的净酸排出可通过去除细胞外Na⁺而被阻断。短期（少于15分钟）去除细胞外Cl⁻对CO₂ - HCO₃⁻中的恢复速率影响很小。相比之下，较长时间（1 - 2小时）去除细胞外Cl⁻会阻断40 - 60%的恢复速率，这与前一篇论文中描述的大部分对SITS敏感的HCO₃⁻依赖性恢复机制也是Na⁺和Cl⁻依赖性的假设一致。因此，这种Cl⁻依赖性成分可能由Na⁺依赖性Cl⁻ - HCO₃⁻交换体介导。高达16%的总酸排出对EIPA和长期Cl⁻去除不敏感，但被SITS阻断。因此，要么1 - 2小时的Cl⁻去除不足以洗去所有细胞内Cl⁻，要么一小部分酸排出由Cl⁻非依赖性机制介导，如电中性Na⁺/HCO₃⁻共转运体。我们还研究了去除和重新添加细胞外Cl⁻对pHi的影响，观察到与存在Na⁺非依赖性Cl⁻ - HCO₃⁻交换体一致的pHi变化，该交换体可能起酸装载器的作用。与在低pHi时被激活的Na⁺ - H⁺交换体和Na⁺依赖性Cl⁻ - HCO₃⁻交换体不同，Cl⁻ - HCO₃⁻交换体在高pHi时被激活。因此，酸排出和酸装载机制具有相反的pHi依赖性。
