Nakhoul N L, Chen L K, Boron W F
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510-2989.
J Gen Physiol. 1993 Dec;102(6):1171-205. doi: 10.1085/jgp.102.6.1171.
We used the absorbance spectrum of the pH-sensitive dye dimethylcarboxyfluorescein to monitor intracellular pH (pHi) in the isolated perfused S3 segment of the rabbit proximal tubule, and examined the effect on pHi of switching from a HEPES to a CO2/HCO3- buffer in the lumen and/or the bath (i.e., basolateral solution). Solutions were titrated to pH 7.40 at 37 degrees C. With 10 mM acetate present bilaterally (lumen and bath), this causing steady-state pHi to be rather high (approximately 7.45), bilaterally switching the buffer from 32 mM HEPES to 5% CO2/25 mM HCO3- caused a sustained fall in pHi of approximately 0.26. However, with acetate absent bilaterally, this causing steady-state pHi to be substantially lower (approximately 6.9), bilaterally switching to CO2/HCO3- caused a transient pHi fall (due to the influx of CO2), followed by a sustained rise to a level approximately 0.18 higher than the initial one. The remainder of the experiments was devoted to examining this alkalinization in the absence of acetate. Switching to CO2/HCO3- only in the lumen caused a sustained pHi fall of approximately 0.15, whereas switching to CO2/HCO3- only in the bath caused a transient fall followed by a sustained pHi increase to approximately 0.26 above the initial value. This basolateral CO2/HCO3(-)-induced alkalinization was not inhibited by 50 microM DIDS applied shortly after CO2/HCO3- washout, but was slowed approximately 73% by DIDS applied more than 30 min after CO2/HCO3- washout. The rate was unaffected by 100 microM bilateral acetazolamide, although this drug greatly reduced CO2-induced pHi transients. The alkalinization was not blocked by bilateral removal of Na+ per se, but was abolished at pHi values below approximately 6.5. The alkalinization was also unaffected by short-term bilateral removal of Cl- or SO4=. Basolateral CO2/HCO3- elicited the usual pHi increase even when all solutes were replaced, short or long-term (> 45 min), by N-methyl-D-glucammonium/glucuronate (NMDG+/Glr-). Luminal CO2/HCO3- did not elicit a pHi increase in NMDG+/Glr-. Although the sustained pHi increase elicited by basolateral CO2/HCO3- could be due to a basolateral HCO3- uptake mechanism, net reabsorption of HCO3- by the S3 segment, as well as our ACZ data, suggest instead that basolateral CO2/HCO3- elicits the sustained pHi increase either by inhibiting an acid-loading process or stimulating acid extrusion across the luminal membrane (e.g., via an H+ pump).
我们使用对pH敏感的染料二甲基羧基荧光素的吸收光谱来监测兔近端小管分离灌注S3段中的细胞内pH（pHi），并研究了管腔和/或浴液（即基底外侧溶液）中从HEPES缓冲液切换到CO2/HCO3-缓冲液对pHi的影响。溶液在37℃下滴定至pH 7.40。双侧（管腔和浴液）存在10 mM乙酸盐时，这使得稳态pHi相当高（约7.45），双侧将缓冲液从32 mM HEPES切换到5% CO2/25 mM HCO3-导致pHi持续下降约0.26。然而，双侧不存在乙酸盐时，这使得稳态pHi显著更低（约6.9），双侧切换到CO2/HCO3-导致pHi短暂下降（由于CO2流入），随后持续上升至比初始值高约0.18的水平。其余实验致力于在不存在乙酸盐的情况下研究这种碱化现象。仅在管腔中切换到CO2/HCO3-导致pHi持续下降约0.15，而仅在浴液中切换到CO2/HCO3-导致短暂下降，随后pHi持续上升至比初始值高约0.26。这种基底外侧CO2/HCO3-诱导的碱化在CO2/HCO3-洗脱后不久应用50 μM DIDS时未被抑制，但在CO2/HCO3-洗脱后30分钟以上应用DIDS时减慢了约73%。尽管该药物极大地减少了CO2诱导的pHi瞬变，但100 μM双侧乙酰唑胺对速率没有影响。碱化本身不会因双侧去除Na+而被阻断，但在pHi值低于约6.5时被消除。短期双侧去除Cl-或SO4=对碱化也没有影响。即使所有溶质在短期或长期（> 45分钟）内被N-甲基-D-葡糖铵/葡糖醛酸盐（NMDG+/Glr-）替代，基底外侧CO2/HCO3-仍会引起通常的pHi升高。管腔CO2/HCO3-在NMDG+/Glr-中不会引起pHi升高。尽管基底外侧CO2/HCO3-引起的pHi持续升高可能是由于基底外侧HCO3-摄取机制，但S3段对HCO3-的净重吸收以及我们的乙酰唑胺数据表明，基底外侧CO2/HCO3-引起pHi持续升高要么是通过抑制酸加载过程，要么是通过刺激跨管腔膜的酸分泌（例如，通过H+泵）。
