Nakhoul N L, Lopes A G, Chaillet J R, Boron W F
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510.
J Gen Physiol. 1988 Sep;92(3):369-93. doi: 10.1085/jgp.92.3.369.
We used the absorbance spectrum of 4',5'-dimethyl-5-(and 6) carboxyfluorescein to measure intracellular pH (pHi) in the isolated, perfused S3 segment of the rabbit proximal tubule. Experiments were conducted in HCO3- -free solutions. pHi recovered from an acid load imposed by an NH4+ prepulse, indicating the presence of one or more active acid-extrusion mechanisms. Removal of Na+ from bath and lumen caused pHi to decrease by approximately 0.6, whereas Na+ readdition caused complete pHi recovery. Removal of Na+ from the bath caused only a slow pHi decrease that was enhanced about fourfold when Na+ was subsequently removed from the lumen also. Similarly, the pHi recovery produced by the readdition of Na+ to the bath and lumen was about ninefold faster than when Na+ was returned to the bath only. Amiloride (1-2 mM) inhibited the pHi recovery that was elicited by returning 15 or 29 mM Na+ to lumen by only approximately 30%. However, in the absence of external acetate (Ac-), 1 mM amiloride inhibited approximately 66% of the pHi recovery induced by the readdition of 29 mM Na+ to the lumen only. The removal of external Ac- reduced the pHi recovery rate from an NH4+-induced acid load by approximately 47%, and that elicited by Na+ readdition, by approximately 67%. Finally, when bilateral removal of Na+ was maintained for several minutes, pHi recovered from the initial acidification, slowly at first, and then more rapidly, eventually reaching a pHi approximately 0.1 higher than the initial one. This Na+-independent pHi recovery was not significantly affected by lowering [HEPES]o from 32 to 3 mM or by adding N'N'-dicyclohexylcarbodiimide (10(-4) M) to the lumen, but it was reduced approximately 57% by iodoacetate (0.5 mM) plus cyanide (1 mM). We conclude that in the nominal absence of HCO3-, three transport systems contribute to acid extrusion by S3 cells: (a) a Na+-independent mechanism, possibly an H+ pump; (b) a Na-H exchanger, confined primarily to the luminal membrane; and (c) an Ac- and luminal Na+-dependent mechanism. The contribution of these three mechanisms to total acid extrusion, assessed by the rapid readdition of Na+, was approximately 13, approximately 30, and approximately 57%, respectively.
我们利用4',5'-二甲基-5-(及6)-羧基荧光素的吸收光谱来测量兔近端小管分离灌注S3段的细胞内pH值（pHi）。实验在无HCO3-的溶液中进行。pHi从由NH4+预脉冲施加的酸负荷中恢复，表明存在一种或多种活性酸排出机制。从浴液和管腔中去除Na+导致pHi下降约0.6，而重新添加Na+导致pHi完全恢复。从浴液中去除Na+仅导致pHi缓慢下降，而随后从管腔中也去除Na+时，下降幅度增强约四倍。同样，向浴液和管腔中重新添加Na+所产生的pHi恢复速度比仅向浴液中返回Na+时快约九倍。氨氯吡脒（1 - 2 mM）仅抑制通过向管腔中返回15或29 mM Na+所引发的pHi恢复约30%。然而，在无外部乙酸盐（Ac-）的情况下，1 mM氨氯吡脒仅抑制通过向管腔中重新添加29 mM Na+所诱导的pHi恢复的约66%。去除外部Ac-使从NH4+诱导的酸负荷中恢复的pHi速率降低约47%，而由重新添加Na+所引发的恢复降低约67%。最后，当双侧去除Na+持续几分钟时，pHi从初始酸化中恢复，起初缓慢，然后更快，最终达到比初始pHi高约0.1的水平。这种不依赖Na+的pHi恢复不受将[HEPES]o从32 mM降至3 mM或向管腔中添加N'N'-二环己基碳二亚胺（10(-4) M）的显著影响，但被碘乙酸盐（0.5 mM）加氰化物（1 mM）降低约57%。我们得出结论，在名义上无HCO3-的情况下，三种转运系统有助于S3细胞排出酸：（a）一种不依赖Na+的机制，可能是H+泵；（b）一种Na-H交换体，主要局限于管腔膜；（c）一种依赖Ac-和管腔Na+的机制。通过快速重新添加Na+评估，这三种机制对总酸排出的贡献分别约为13%、约30%和约57%。
