Korbmacher C, Helbig H, Förster C, Wiederholt M
Institut für Klinische Physiologie, Freie Universität Berlin, Germany.
Biochim Biophys Acta. 1988 Sep 1;943(3):405-10. doi: 10.1016/0005-2736(88)90371-9.
Continuous intracellular pH (pHi) measurements were performed in SIRC rabbit corneal epithelial cells using the pH-sensitive absorbance of intracellularly trapped 5(and 6)-carboxy-4',5'-dimethylfluorescein. Steady-state pHi in nominally bicarbonate free Ringer's solution averaged 6.87 +/- 0.02 (mean +/- S.E., n = 53). After intracellular acidification induced by the NH4Cl-prepulse technique, there was a sodium-dependent pHi recovery towards the normal steady-state pHi. The initial pHi recovery rate was a saturable function of extracellular sodium concentration with an apparent Km for external sodium of about 25 mM and a Vmax of about 0.28 pH units/min. Virtually no pHi recovery was observed in the absence of extracellular sodium. Sodium removal during steady state acidified the cells by 0.36 +/- 0.05 pH units (mean +/- S.E., n = 13) within 5 min. There was a dose-dependent inhibition of pHi recovery after NH4Cl prepulse by amiloride with an IC50 of about 15 microM. Amiloride in a concentration of 1 mM almost completely abolished pHi recovery. Amiloride (1 mM) applied during steady state induced an intracellular acidification of 0.2 +/- 0.03 pH units (mean +/- S.E., n = 7) within 5 min. These findings suggest that a Na+/H+ exchange is present in SIRC rabbit corneal epithelial cells. Na+/H+ exchange seems to be the major process involved in pHi recovery in SIRC cells after an intracellular acid load. Na+/H+ exchange also plays a role in the maintenance of steady-state pHi.
利用细胞内捕获的5（和6）-羧基-4'，5'-二甲基荧光素的pH敏感吸光度，在SIRC兔角膜上皮细胞中进行了连续的细胞内pH（pHi）测量。在名义上不含碳酸氢盐的林格氏溶液中，稳态pHi平均为6.87±0.02（平均值±标准误，n = 53）。在通过NH4Cl预脉冲技术诱导细胞内酸化后，存在钠依赖性的pHi恢复至正常稳态pHi。初始pHi恢复率是细胞外钠浓度的饱和函数，细胞外钠的表观Km约为25 mM，Vmax约为0.28 pH单位/分钟。在没有细胞外钠的情况下，几乎没有观察到pHi恢复。稳态期间去除钠在5分钟内使细胞酸化0.36±0.05 pH单位（平均值±标准误，n = 13）。氨氯地平对NH4Cl预脉冲后的pHi恢复有剂量依赖性抑制作用，IC50约为15 microM。1 mM浓度的氨氯地平几乎完全消除了pHi恢复。在稳态期间应用氨氯地平（1 mM）在5分钟内诱导细胞内酸化0.2±0.03 pH单位（平均值±标准误，n = 7）。这些发现表明SIRC兔角膜上皮细胞中存在Na+/H+交换。Na+/H+交换似乎是SIRC细胞在细胞内酸负荷后pHi恢复中涉及的主要过程。Na+/H+交换在稳态pHi的维持中也起作用。
