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):C844-56. doi: 10.1152/ajpcell.1988.255.6.C844.
We have developed a technique to measure the fluorescence of a pH-sensitive dye (2,7-biscarboxyethyl-5(6)-carboxyfluorescein) in single glomerular mesangial cells in culture. The intracellular fluorescence excitation ratio of the dye was calibrated using the nigericin-high-K+ approach. In the absence of CO2-HCO3-, mesangial cells that are acid loaded by an NH+4 prepulse exhibit a spontaneous intracellular pH (pHi) recovery that is blocked either by ethylisopropylamiloride (EIPA) or removal of external Na+. This pHi recovery most probably reflects the activity of a Na+-H+ exchanger. When the cells are switched from a N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution to one containing CO2-HCO3-, there is an abrupt acidification due to CO2 entry, which is followed by a spontaneous recovery of pHi to a steady-state value higher than that prevailing in HEPES. Both the rate of recovery and the higher steady-state pHi imply that the application of CO2-HCO3- introduces an increase in net acid extrusion from the cell. One third of total net acid extrusion in CO2-HCO3- is EIPA sensitive and most likely is mediated by the Na+-H+ exchanger. The remaining two thirds of acid extrusion could be caused by a decrease in the background acid-loading rate and/or the introduction of a new, HCO3- -dependent acid-extrusion mechanism. The HCO3- -induced alkalinization cannot be accounted for by a HCO3- -induced reduction in the acid-loading rate. The latter can be estimated by applying EIPA in the absence of HCO3- and observing the rate of pHi decline. We found that this acid-loading rate is only about one fifth as great as the total net acid extrusion rate in the presence of HCO3-. Indeed, two thirds of net acid extrusion in HCO3- is blocked by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), an inhibitor of HCO3- -dependent transport. Furthermore, the effects of EIPA and SITS were additive. Thus, in the presence of CO2-HCO3-, a SITS-sensitive-HCO3- -dependent transporter is the dominant mechanism of acid extrusion. This mechanism also accounts for the increase in steady-state pHi on addition of CO2-HCO3-.
我们开发了一种技术，用于测量培养的单个肾小球系膜细胞中pH敏感染料（2,7-双羧乙基-5(6)-羧基荧光素）的荧光。使用尼日利亚菌素-高钾方法校准该染料的细胞内荧光激发比。在没有CO2-HCO3-的情况下，经NH4+预脉冲酸化的系膜细胞表现出自发性细胞内pH（pHi）恢复，该恢复被乙基异丙基氨氯吡咪（EIPA）或去除细胞外Na+所阻断。这种pHi恢复很可能反映了Na+-H+交换器的活性。当细胞从N-2-羟乙基哌嗪-N'-2-乙磺酸（HEPES）缓冲溶液切换到含有CO2-HCO3-的溶液时，由于CO2进入会突然酸化，随后pHi会自发恢复到高于HEPES中pHi的稳态值。恢复速率和更高的稳态pHi都表明，应用CO2-HCO3-会使细胞的净酸排泄增加。在CO2-HCO3-存在下，总净酸排泄的三分之一对EIPA敏感，很可能由Na+-H+交换器介导。其余三分之二的酸排泄可能是由于背景酸加载速率降低和/或引入了一种新的、依赖HCO3-的酸排泄机制。HCO3-诱导的碱化不能用HCO3-诱导的酸加载速率降低来解释。后者可以通过在没有HCO3-的情况下应用EIPA并观察pHi下降速率来估计。我们发现，这种酸加载速率仅约为存在HCO3-时总净酸排泄速率的五分之一。实际上，HCO3-存在时三分之二的净酸排泄被4-乙酰氨基-4'-异硫氰基芪-2,2'-二磺酸（SITS）阻断，SITS是一种依赖HCO3-转运的抑制剂。此外，EIPA和SITS的作用是相加的。因此，在存在CO2-HCO3-的情况下，一种对SITS敏感的、依赖HCO3-的转运体是酸排泄的主要机制。这种机制也解释了添加CO2-HCO3-后稳态pHi的升高。
