Chen L K, Boron W F
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510.
Am J Physiol. 1995 Feb;268(2 Pt 2):F179-92. doi: 10.1152/ajprenal.1995.268.2.F179.
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-.
通过监测pH敏感染料二甲基羧基荧光素的吸收光谱，我们研究了兔近端小管分离灌注S3段中的细胞内pH（pHi）调节。为了解释先前的一项观察结果，即在有CO2/HCO3-存在时稳态pHi高于无CO2/HCO3-时（N. L. Nakhoul、L. K. Chen和W. F. Boron。《普通生理学杂志》102: 1171 - 1205, 1993），我们研究了双侧（即管腔侧和基底外侧）CO2/HCO3-对负责从酸负荷中恢复pHi的酸排出过程的影响。为了根据pHi变化率计算通量，我们测定了细胞内固有缓冲能力对pHi的依赖性，在pHi 6.5时约为50 mM/pH，并在pHi 7.4时线性下降至约20 mM。在一系列实验中，我们监测了由NH4+/NH3预脉冲施加酸负荷后pHi的恢复速率。在广泛的pHi值范围内，双侧存在CO2/HCO3-时的总净酸排出量比不存在时高约四倍。在第二组实验中，为了确定CO2/HCO3-对管腔Na+/H+交换的影响，我们在双侧首先去除Na+后，仅向管腔中添加Na+，监测由此引发的pHi恢复速率。在有CO2/HCO3-存在时，管腔依赖Na+的净酸排出初始速率约为229 μM/s（pHi 6.92），比在无CO2/HCO3-时（pHi 6.66）获得的约127 μM/s的通量高约1.8倍（P < 0.005）。CO2/HCO3-使通量与pHi的关系向碱侧移动0.3 - 0.4个pH单位。在最后一系列实验中，我们研究了CO2/HCO3-对双侧去除Na+引发的快速初始酸化后不依赖Na+的碱化作用的影响。在有CO2/HCO3-存在时，不依赖Na+的碱化作用起始的延迟时间仅约为36秒，而在无CO2/HCO3-时约为211秒（P < 0.002）。同样，在可比的pHi下，有CO2/HCO3-存在时不依赖Na+的净酸排出速率比不存在时高约两到三倍。这种不依赖Na+的酸排出对N-乙基马来酰亚胺（2 mM）不敏感，但通过耗尽细胞内ATP（即去除葡萄糖和氨基酸，加上添加2 mM氰化物和10 mM碘乙酸）可被抑制约94%。双侧添加CO2/HCO3-导致稳态pHi升高的主要（如果不是全部）原因似乎是管腔Na+/H+交换和不依赖Na+的酸排出的刺激。
