Ou-yang Y, Mellergård P, Siesjö B K
Laboratory of Experimental Brain Research, Lund University Hospital, Sweden.
J Cereb Blood Flow Metab. 1993 Sep;13(5):827-40. doi: 10.1038/jcbfm.1993.105.
Intracellular pH (pHi) and the mechanisms of pHi regulation in cultured rat cortical neurons were studied with microspectrofluorometry and the pH-sensitive fluorophore 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. Steady-state pHi was 7.00 +/- 0.17 (mean +/- SD) and 7.09 +/- 0.14 in nominally HCO3(-)-free and HCO3(-)-containing solutions, respectively, and was dependent on extracellular Na+ and Cl-. Following an acid transient, induced by an NH1 prepulse or an increase in CO2 tension, pHi decreased and then rapidly returned to baseline, with an average net acid extrusion rate of 2.6 and 2.8 mmol/L/min, in nominally HCO3(-)-free and HCO3(-)-containing solutions, respectively. The recovery was completely blocked by removal of extracellular Na+ and was partially inhibited by amiloride or 5-N-methyl-N-isobutylamiloride. In most cells pHi recovery was completely blocked in the presence of harmaline. The recovery of pHi was not influenced by addition of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or removal of Cl-. The rapid regulation of pHi seen following a transient alkalinization was not inhibited by amiloride or by removal of extracellular Na+, but was partially inhibited by DIDS and by removal of extracellular Cl-. The results are compatible with the presence of at least two different pHi-regulating mechanisms: an acid-extruding Na+/H+ antiporter, possibly consisting of different subtypes, and a passive Cl-/HCO3- exchanger, mediating loss of HCO3- from the cell.
利用显微分光荧光测定法和pH敏感荧光团2',7'-双（羧乙基）-5,6-羧基荧光素，研究了培养的大鼠皮层神经元的细胞内pH值（pHi）及其调节机制。在名义上不含HCO3(-)和含HCO3(-)的溶液中，稳态pHi分别为7.00±0.17（平均值±标准差）和7.09±0.14，且依赖于细胞外Na+和Cl-。在由NH4预脉冲或CO2张力增加诱导的酸性瞬变后，pHi下降，然后迅速恢复到基线，在名义上不含HCO3(-)和含HCO3(-)的溶液中，平均净酸外排速率分别为2.6和2.8 mmol/L/分钟。去除细胞外Na+可完全阻断恢复过程，而氨氯吡咪或5-N-甲基-N-异丁基氨氯吡咪可部分抑制该过程。在大多数细胞中，在harmaline存在的情况下，pHi恢复被完全阻断。添加4,4'-二异硫氰基芪-2,2'-二磺酸（DIDS）或去除Cl-对pHi恢复没有影响。短暂碱化后出现的pHi快速调节不受氨氯吡咪或去除细胞外Na+的抑制，但部分受DIDS和去除细胞外Cl-的抑制。这些结果与至少两种不同的pHi调节机制的存在相一致：一种酸外排Na+/H+反向转运体，可能由不同亚型组成，以及一种被动Cl-/HCO3-交换体，介导细胞内HCO3-的丢失。
