Acid extrusion in S3 segment of rabbit proximal tubule. I. Effect of bilateral CO2/HCO3-.
Acid extrusion in S3 segment of rabbit proximal tubule. I. Effect of bilateral CO2/HCO3-.
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兔近曲小管 S3 段酸挤出。
DOI：
10.1152/ajprenal.1995.268.2.f179
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Boron,WF
中科院分区：
文献类型：
--
作者：
Chen,LK;Boron,WF
监测pH敏感染料二甲基羧基荧光素的吸收光谱，我们研究了细胞内pH值（pHi）的调节在离体灌流的兔近端小管S3段。为了解释先前的观察结果，存在CO2/HCO 3-（N. L.纳胡尔湖K. Chen和W. F.硼J.Gen.Physiol.102：1171-1205，1993），我们检查了双侧（即，管腔和基底外侧）CO2/HCO 3-对负责从酸负荷恢复pHi的酸挤出过程的影响。为了从pHi变化率计算通量，我们确定了内在细胞内缓冲能力的pHi依赖性，其在pHi 6.5时约为50 mM/pH，在pHi 7.4时线性下降至约20 mM。在一系列实验中，我们监测了从由NH 4 +/NH3预脉冲施加的酸负荷中恢复pHi的速率。在一个广泛的范围内的pH值，总净酸挤出约4倍，在双边存在的CO2/HCO 3-比在其缺席。在第二组实验中，其目的是确定CO2/HCO 3-对管腔Na+/H+交换的影响，我们监测的pH恢复率，通过添加Na+回仅管腔，在第一次去除Na+双边。在CO2/HCO 3-存在下，管腔Na（+）依赖性净酸挤出的初始速率约为229 μ M/s（pHi 6.92），比在不存在CO2/HCO 3-（pHi 6.66）下获得的约127 μ M/s的通量高约1.8倍（P < 0.005）。CO2/HCO 3-碱使通量与pHi的关系移动了0.3-0.4个pH单位。在最后一系列实验中，我们研究了CO2/HCO 3-对Na（+）-独立碱化的影响，这种碱化是在双边Na+去除引起的快速初始酸化之后进行的。在CO2/HCO 3-存在下，启动Na（+）非依赖性碱化的滞后时间仅为约36 s，而在没有CO2/HCO 3-存在下约为211 s（P < 0.002）。此外，Na（+）-独立的净酸挤出率是约2至3倍，在CO2/HCO 3-的存在下，在可比的pH值。这种Na（+）-非依赖性酸挤出对N-乙基马来酰亚胺（2 mM）不敏感，但通过消耗细胞内ATP的努力抑制了约94%（即，除去葡萄糖和氨基酸，加上加入2 mM氰化物和10 mM碘乙酸）。刺激管腔Na+/H+交换和Na（+）-非依赖性酸挤出似乎是主要的，如果不是全部，较高的稳态pHi所造成的双边添加CO2/HCO 3-的解释。
Monitoring the absorbance spectra of the pH-sensitive dye dimethylcarboxyfluorescein, we studied intracellular pH (pHi) regulation in the isolated perfused S3 segment of rabbit proximal tubule. To explain a previous observation, that steady-state pHi is higher in the presence than in the absence of CO2/HCO3- (N. L. Nakhoul, L. K. Chen, and W. F. Boron. J. Gen. Physiol. 102: 1171-1205, 1993), we examined the effect of bilateral (i.e., luminal and basolateral) CO2/HCO3- on the acid extrusion processes responsible for recovery of pHi from acid loads. To compute fluxes from rates of pHi change, we determined the pHi dependence of intrinsic intracellular buffering power, which was approximately 50 mM/pH at pHi 6.5 and fell linearly to approximately 20 mM at pHi 7.4. In one series of experiments, we monitored the rate of pHi recovery from an acid load imposed by an NH4+/NH3 prepulse. Over a broad range of pHi values, total net acid extrusion was approximately four times higher in bilateral presence of CO2/HCO3- than in its absence. In a second group of experiments, which were designed to determine the effect of CO2/HCO3- on luminal Na+/H+ exchange, we monitored the rate of pHi recovery elicited by adding Na+ back to only the lumen, after first removing Na+ bilaterally. Initial rate of luminal Na(+)-dependent net acid extrusion in presence of CO2/HCO3- was approximately 229 microM/s (pHi 6.92), approximately 1.8 times higher than the flux of approximately 127 microM/s (P < 0.005) obtained in absence of CO2/HCO3- (pHi 6.66). CO2/HCO3- alkali-shifted the flux vs. pHi relationship by 0.3-0.4 pH units. In a final series of experiments, we examined the effect of CO2/HCO3- on the Na(+)-independent alkalinization that follows the rapid, initial acidification elicited by bilateral Na+ removal. In the presence of CO2/HCO3-, lag time for initiation of the Na(+)-independent alkalinization was only approximately 36 vs. approximately 211 s (P < 0.002) in absence of CO2/HCO3-. Also, Na(+)-independent net acid extrusion rate was approximately two to three times higher in presence than in absence of CO2/HCO3- at comparable pHi. This Na(+)-independent acid extrusion was insensitive to N-ethylmaleimide (2 mM), but was inhibited approximately 94% by efforts to deplete intracellular ATP (i.e., removal of glucose and amino acids, plus addition of 2 mM cyanide and 10 mM iodoacetic acid). Stimulation of luminal Na+/H+ exchange and Na(+)-independent acid extrusion appears to be the major, if not the entire, explanation for the higher steady-state pHi caused by bilateral addition of CO2/HCO3-.