PH REGULATION IN SINGLE GLOMERULAR MESANGIAL CELLS .1. ACID EXTRUSION IN ABSENCE AND PRESENCE OF HCO-3-
PH REGULATION IN SINGLE GLOMERULAR MESANGIAL CELLS .1. ACID EXTRUSION IN ABSENCE AND PRESENCE OF HCO-3-
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DOI：
10.1152/ajpcell.1988.255.6.c844
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发表时间：
1988-12-01
影响因子：
--
通讯作者：
BORON, WF
中科院分区：
文献类型：
--
作者：
BOYARSKY, G;GANZ, MB;BORON, WF
我们已经开发了一种技术，以测量在单个肾小球系膜细胞培养的pH敏感染料（2，7-biscarboxyethyl-5（6）-carboxyfluorescein）的荧光。使用尼日利亚菌素高K+方法校准染料的细胞内荧光激发比。在没有CO2-HCO 3-的情况下，由NH 4+前脉冲加载酸的系膜细胞表现出自发的细胞内pH（pHi）恢复，其被乙基异丙基氨氯吡咪（EIPA）或去除外部Na+阻断。该pHi恢复最可能反映Na+-H+交换剂的活性。当细胞从N-2-羟乙基哌嗪-N“-2-乙磺酸（HEPES）缓冲溶液转换为含有CO2-HCO 3-的缓冲溶液时，由于CO2进入而突然酸化，随后pHi自发恢复至高于HEPES中普遍存在的稳态值。回收率和较高的稳态pHi都意味着CO2-HCO 3-的应用引入了从细胞的净酸挤出的增加。CO2-HCO 3-中总净酸挤出的三分之一是EIPA敏感的，并且最有可能由Na+-H+交换剂介导。其余三分之二的酸挤出可能是由于背景酸负荷率降低和/或引入新的HCO 3依赖性酸挤出机制所致。HCO 3诱导的碱化不能用HCO 3诱导的酸负荷率降低来解释。后者可以通过在不存在HCO 3-的情况下应用EIPA并观察pHi下降速率来估计。我们发现，这种酸负载率只有约五分之一的总净酸挤出率在HCO 3-的存在下。事实上，HCO 3-中三分之二的净酸挤出被HCO 3-依赖性转运的抑制剂4-乙酰氨基-4 ''-异硫氰基芪-2，2 ''-二磺酸（SITS）阻断。此外，EIPA和SIT的效果是相加的。因此，在存在CO2-HCO 3-的情况下，SITS-敏感性-HCO 3-依赖性转运蛋白是酸排出的主要机制。该机制也解释了添加CO2-HCO 3-时稳态pHi的增加。
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 NH4+ 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-.