Harris R C, Seifter J L, Lechene C
Am J Physiol. 1986 Nov;251(5 Pt 1):C815-24. doi: 10.1152/ajpcell.1986.251.5.C815.
Na-H exchange was studied using electron probe analysis and microfluorescent pH analysis of individual cells, in 3-day primary cultures of rat proximal tubule cells (RPTC) obtained from 40- to 50-day-old Sprague-Dawley rats. After Na-K pump inhibition, the initial rate of net Na influx was inhibited 87% by 1 mM amiloride. K influx, an estimate of Na-K pump activity, was increased approximately three times in cells containing high Na (0.114 mM K X mM P-1 X min-1) compared with control cells containing low Na (0.038 mM K X mM P-1 X min-1). Single cell measurements of RPTC loaded with the cytoplasmic pH indicator 5- (and -6) carboxy-4',5'-dimethylfluorescein indicated that there was reversible intracellular acidification in the absence of external Na or in the presence of amiloride. When intracellular acidification was induced by the addition and subsequent removal of NH4Cl, recovery of intracellular pH was inhibited in the absence of external Na or in the presence of amiloride. Using a similar protocol, it was found that after intracellular acidification, the rate of Na influx increased at least 5.9 times, and intracellular Na content was increased 3.15 +/- 0.64 times at 60 s. There was an initial 50% inhibition of Na-K pump activity within the first 60 s compared with control (nonacidified) RPTC, secondarily followed by an increase in Na-K pump activity. Amiloride (0.5 mM) inhibited the acidification-induced increase in Na influx, and persistent acidification led to a persistent inhibition of Na-K pump activity compared with control. In summary, these results demonstrate that Na-H exchange mediates the majority of net Na influx into RPTC under our basal conditions and is necessary for maintenance of intracellular pH homeostasis. In RPTC, Na-H exchange is further activated by intracellular acidification, leading to a net increase in intracellular Na content, which secondarily stimulates Na-K pump activity. The initial inhibition of Na-K pump activity may be due to a direct effect of intracellular acidification.
采用电子探针分析和单个细胞微荧光pH分析方法，对从40至50日龄斯普拉格-道利大鼠获取的近端肾小管细胞（RPTC）进行3天原代培养，研究钠-氢交换。在抑制钠钾泵后，1 mM氨氯吡脒使净钠内流的初始速率受到87%的抑制。钾内流是钠钾泵活性的一个指标，与含低钠（0.038 mM钾×毫摩尔磷-1×分钟-1）的对照细胞相比，含高钠（0.114 mM钾×毫摩尔磷-1×分钟-1）的细胞中钾内流增加约三倍。用细胞质pH指示剂5-（及-6）羧基-4′,5′-二甲基荧光素加载的RPTC单细胞测量结果表明，在无细胞外钠或存在氨氯吡脒的情况下，细胞内发生可逆性酸化。当通过添加并随后去除氯化铵诱导细胞内酸化时，在无细胞外钠或存在氨氯吡脒的情况下，细胞内pH的恢复受到抑制。采用类似方案发现，细胞内酸化后，钠内流速率至少增加5.9倍，且在60秒时细胞内钠含量增加3.15±0.64倍。与对照（未酸化）RPTC相比，最初60秒内钠钾泵活性有50%的抑制，随后钠钾泵活性增加。氨氯吡脒（0.5 mM）抑制酸化诱导的钠内流增加，与对照相比，持续酸化导致钠钾泵活性持续受到抑制。总之，这些结果表明，在我们的基础条件下，钠-氢交换介导了大部分净钠流入RPTC，并且是维持细胞内pH稳态所必需的。在RPTC中，钠-氢交换通过细胞内酸化进一步激活，导致细胞内钠含量净增加，进而刺激钠钾泵活性。钠钾泵活性的最初抑制可能是由于细胞内酸化的直接作用。
