Montrose M H, Murer H
J Membr Biol. 1986;93(1):33-42. doi: 10.1007/BF01871016.
Suspensions of LLC-PK1 cells (a continuous epitheliod cell line with renal characteristics) are examined for mechanisms of intracellular pH regulation using the fluorescent probe BCECF. Initial experiments determine suitable calibration procedures for use of the BCECF fluorescent signal. They also determine that the cell suspension contains cells which (after 4 hr in suspension) have Na+ and K+ gradients comparable to those of cells in monolayer culture. The steady-state intracellular pH (7.05 +/- 0.01, n = 5) of cells which have recovered in (pH 7.4) Na+-containing medium is not affected over several minutes by addition of 100 microM amiloride or removal of extracellular Na+ (Na+o less than 1 mM). In contrast, when the cells recover from an acid load (caused by NH4 preincubation and removal), the recovery is largely Na+ dependent and is sensitive to 100 microM amiloride. These results suggest that with resting pH near neutrality, both Na+o/H+i and Na+i/H+o exchange reactions are functionally inactive (compared to cellular buffering capacity). In contrast, Na+o/H+i exchange is activated by an increased cellular acid load. This activation may be observed directly either as a stimulation of net H+ efflux or net Na+ influx with decreasing intracellular pH. The extrapolation of this latter data suggests a "set point" of Na+/H+ exchange of approximately pH 7.0, consistent with the observed resting intracellular pH of approximately 7.05.
使用荧光探针BCECF检测LLC - PK1细胞（一种具有肾脏特性的连续上皮样细胞系）悬浮液的细胞内pH调节机制。初步实验确定了使用BCECF荧光信号的合适校准程序。他们还确定细胞悬浮液中的细胞（悬浮4小时后）具有与单层培养细胞相当的Na⁺和K⁺梯度。在含Na⁺（pH 7.4）的培养基中恢复的细胞的稳态细胞内pH（7.05±0.01，n = 5）在几分钟内不受添加100μM氨氯吡咪或去除细胞外Na⁺（Na⁺o小于1 mM）的影响。相反，当细胞从酸负荷（由NH₄预孵育和去除引起）中恢复时，恢复在很大程度上依赖于Na⁺，并且对100μM氨氯吡咪敏感。这些结果表明，在静息pH接近中性时，Na⁺o/H⁺i和Na⁺i/H⁺o交换反应在功能上均无活性（与细胞缓冲能力相比）。相反，Na⁺o/H⁺i交换通过细胞酸负荷增加而被激活。这种激活可以直接观察到，即随着细胞内pH降低，净H⁺外流或净Na⁺内流受到刺激。后一组数据的推断表明Na⁺/H⁺交换的“设定点”约为pH 7.0，与观察到的约7.05的静息细胞内pH一致。
