Montrose M H, Murer H
Institute of Physiology, University of Zurich, Switzerland.
Am J Physiol. 1990 Jul;259(1 Pt 1):C110-20. doi: 10.1152/ajpcell.1990.259.1.C110.
Opossum kidney (OK) cells (an epithelial cell line) were examined by flame photometry of cellular Na+ and K+ and by microfluorometric measurements of the intracellular pH (pHi) of single cells loaded with 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). The work concentrates on defining resting pHi values under different experimental conditions and examines factors that contribute to the maintenance of resting pHi. To use nigericin to calibrate the intracellular response of BCECF, cellular K+ levels were measured by a null point analysis, and the stability and magnitude of cellular Na+ and K+ levels were determined vs. time. Resting pHi in medium without added CO2 was high when measured by null point analysis of the population (pHi 7.6) and from measurements of single cells that have recovered from an acid load caused by NH4 prepulse (pHi 7.76 +/- 0.03, n = 20 cells). In single-cell measurements, addition of CO2-HCO3- to the medium results in cellular acidification of the steady-state pHi by 0.35 +/- 0.04 pH units. In medium equilibrated with room air, the resting pHi is shown to be a dynamic steady state composed of net flux due to apical Na(+)-dependent transport (Na(+)H+ exchange) plus acidifying processes. It is concluded that although 5-[N-ethyl-N-isopropyl]amiloride (EIPA) inhibits the forward reaction of Na(+)-H+ exchange, EIPA is either ineffective as an inhibitor of the reverse reaction of Na(+)-H+ exchange or Na(+)-H+ exchange does not reverse measurably in the OK cells.
通过火焰光度法检测负鼠肾（OK）细胞（一种上皮细胞系）中的细胞内钠离子（Na⁺）和钾离子（K⁺），并通过微量荧光法测量负载2',7'-双（2-羧乙基）-5,6-羧基荧光素（BCECF）的单细胞的细胞内pH值（pHi）。这项工作着重于确定不同实验条件下的静息pHi值，并研究有助于维持静息pHi的因素。为了使用尼日利亚菌素校准BCECF的细胞内响应，通过零点分析测量细胞内钾离子水平，并确定细胞内钠离子和钾离子水平随时间的稳定性和大小。在没有添加二氧化碳的培养基中，通过群体零点分析测量时静息pHi较高（pHi 7.6），并且从由铵预脉冲引起的酸负荷中恢复的单细胞测量结果显示（pHi 7.76±0.03，n = 20个细胞）。在单细胞测量中，向培养基中添加二氧化碳-碳酸氢根会使稳态pHi发生细胞酸化，降低0.35±0.04个pH单位。在与室内空气平衡的培养基中，静息pHi显示为由顶端钠离子依赖性转运（钠氢交换）引起的净通量加上酸化过程组成的动态稳态。得出的结论是，尽管5-[N-乙基-N-异丙基]氨氯地平（EIPA）抑制钠氢交换的正向反应，但EIPA作为钠氢交换逆向反应的抑制剂无效，或者钠氢交换在OK细胞中不会发生可测量的逆向反应。
