Breyer M D, Jacobson H R
Department of Internal Medicine, Nashville Veterans Administration Hospital, Tennessee.
J Clin Invest. 1989 Sep;84(3):996-1004. doi: 10.1172/JCI114264.
The collecting duct of the inner stripe outer medulla (OMCDi) is a major site of distal nephron acidification. Using the pH sensitive fluorescent dye 2'-7'-bis(carboxyethyl)-5,6,-carboxyfluorescein (BCECF) and quantitative spectrofluorometry to measure intracellular pH in isolated perfused OMCDi, we have characterized basolateral transport processes responsible for regulation of intracellular pH. Experiments suggesting the existence of basolateral Cl-/base exchange were performed. In HCO3- containing buffers, bath Cl- replacement resulted in reversible alkalinization of the OMCDi from 7.22 +/- 0.05 to 7.57 +/- 0.12. Similarly 0.1 mM bath 4',4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) alkalinized the OMCDi from 7.14 +/- 0.09 to 7.34 +/- 0.09 and blocked further alkalinization by bath Cl- removal (delta = + 0.02 pH units). The concentration dependence kinetics of Cl-/base exchange revealed a K1/2 of 10 mM for external Cl- with a Vmax of 0.50 pH U/min. Experiments suggesting the existence of basolateral Na+/H+ exchange were also performed. Replacement of bath Na+ by tetramethylammonium resulted in reversible cell acidification (7.14 +/- 0.09 to 6.85 +/- 0.1). Tubules that were acidified by a brief exposure to NH4Cl displayed recovery of cell pH back to baseline at a rate that was highly dependent on bath Na+ concentration. Half maximal recovery rate was achieved at 7 mM bath Na+ and Vmax was 0.605 pH U/min. The Na+-dependent rate of cell pH recovery after acidification was blocked by 0.2 mM bath amiloride. These results suggest that intracellular pH in the OMCDi is regulated by parallel basolateral Na+/H+ exchange and Cl-/base exchange.
外髓质内带集合管（OMCDi）是远端肾单位酸化的主要部位。我们使用pH敏感荧光染料2'-7'-双（羧乙基）-5,6-羧基荧光素（BCECF）和定量荧光光谱法来测量分离灌注的OMCDi中的细胞内pH，从而对负责调节细胞内pH的基底外侧转运过程进行了表征。进行了提示基底外侧Cl⁻/碱基交换存在的实验。在含HCO₃⁻的缓冲液中，浴液中Cl⁻的置换导致OMCDi从7.22±0.05可逆地碱化至7.57±0.12。同样，0.1 mM浴液中的4',4'-二异硫氰基芪-2,2'-二磺酸（DIDS）使OMCDi从7.14±0.09碱化至7.34±0.09，并阻止了因浴液中Cl⁻去除而导致的进一步碱化（δ = + 0.02 pH单位）。Cl⁻/碱基交换的浓度依赖性动力学显示，外部Cl⁻的K1/2为10 mM，Vmax为0.50 pH U/分钟。还进行了提示基底外侧Na⁺/H⁺交换存在的实验。用四甲基铵替代浴液中的Na⁺导致细胞可逆性酸化（从7.14±0.09降至6.85±0.1）。短暂暴露于NH₄Cl而酸化的肾小管显示细胞pH恢复到基线的速率高度依赖于浴液中Na⁺的浓度。在浴液中Na⁺浓度为7 mM时达到最大恢复速率的一半，Vmax为0.605 pH U/分钟。酸化后细胞pH恢复的Na⁺依赖性速率被0.2 mM浴液中的氨氯吡咪阻断。这些结果表明，OMCDi中的细胞内pH由平行的基底外侧Na⁺/H⁺交换和Cl⁻/碱基交换调节。
