Saito Y, Ozawa T, Nishiyama A
Department of Physiology, Tohoku University School of Medicine, Sendai, Japan.
Pflugers Arch. 1990 Dec;417(4):382-90. doi: 10.1007/BF00370657.
Kinetic properties of the Na(+)-H+ antiport in the acinar cells of the isolated, superfused mouse lacrimal gland were studied by measuring intracellular pH (pHi) and Na+ activity (aNai) with the aid of double-barreled H(+)- and Na(+)-selective microelectrodes, respectively. Bicarbonate-free solutions were used throughout. Under untreated control conditions, pHi was 7.12 +/- 0.01 and aNai was 6.7 +/- 0.6 mmol/l. The cells were acid-loaded by exposure to an NH4+ solution followed by an Na(+)-free N-methyl-D-glucamine (NMDG+) solution. Intracellular Na+ and H+ concentrations were manipulated by changing the duration of exposure to the above solutions. Subsequent addition of the standard Na+ solution rapidly increased pHi. This Na(+)-induced increase in pHi was almost completely inhibited by 0.5 mmol/l amiloride and was associated with a rapid, amiloride-sensitive increase in aNai. The rate of pHi recovery induced by the standard Na+ solution increased in a saturable manner as pHi decreased, and was negligible at pHi 7.2-7.3, indicating an inactivation of the Na(+)-H+ antiport. The apparent Km for intracellular H+ concentration was 105 nmol/l (pH 6.98). The rate of acid extrusion from the acid-loaded cells increased proportionally to the increase in extracellular pH. Depletion of aNai to less than 1 mmol/l by prolonged exposure to NMDG+ solution significantly increased the rate of Na(+)-dependent acid extrusion. The rate of acid extrusion increased as the extracellular Na+ concentration increased following Michaelis-Menten kinetics (Vmax was 0.55 pH/min and the apparent Km was 75 mmol/l at pHi 6.88).(ABSTRACT TRUNCATED AT 250 WORDS)
通过分别借助双管H⁺和Na⁺选择性微电极测量细胞内pH（pHi）和Na⁺活性（aNai），研究了分离的、经超灌注的小鼠泪腺腺泡细胞中Na⁺-H⁺反向转运体的动力学特性。实验全程使用无碳酸氢盐溶液。在未处理的对照条件下，pHi为7.12±0.01，aNai为6.7±0.6 mmol/L。细胞通过暴露于NH₄⁺溶液，随后再暴露于无Na⁺的N-甲基-D-葡糖胺（NMDG⁺）溶液来进行酸负载。通过改变暴露于上述溶液的持续时间来控制细胞内Na⁺和H⁺浓度。随后加入标准Na⁺溶液会使pHi迅速升高。这种由Na⁺诱导的pHi升高几乎完全被0.5 mmol/L的氨氯吡咪抑制，并且与aNai的快速、氨氯吡咪敏感的升高相关。标准Na⁺溶液诱导的pHi恢复速率随着pHi降低以饱和方式增加，在pHi 7.2 - 7.3时可忽略不计，表明Na⁺-H⁺反向转运体失活。细胞内H⁺浓度的表观Km为105 nmol/L（pH 6.98）。从酸负载细胞中挤出酸的速率与细胞外pH的升高成比例增加。通过长时间暴露于NMDG⁺溶液使aNai降至低于1 mmol/L会显著增加Na⁺依赖性酸挤出的速率。随着细胞外Na⁺浓度按照米氏动力学增加，酸挤出速率增加（在pHi 6.88时，Vmax为0.55 pH/分钟，表观Km为75 mmol/L）。（摘要截短于250字）
