Pedersen S F, Jørgensen N K, Damgaard I, Schousboe A, Hoffmann E K
Department of Biochemistry, August Krough Institute, Copenhagen, Denmark.
J Neurosci Res. 1998 Feb 15;51(4):431-41. doi: 10.1002/(SICI)1097-4547(19980215)51:4<431::AID-JNR3>3.0.CO;2-D.
Maintenance and regulation of intracellular pH (pHi) was studied in single cultured mouse neocortical neurons using the fluorescent probe 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). Reversal of the Na+ gradient by reduction of the extracellular Na+ concentration ([Na+]o) resulted in rapid intracellular acidification, inhibited by 5'-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of Na+/H+ exchange. In the presence of EIPA and/or 4',4'-diisothiocyano-stilbene-2',2'-sulfonic acid (DIDS), an inhibitor of Na+-coupled anion exchangers and Na+-HCO3- cotransport, a slow decline of pHi was seen. Following intracellular acidification imposed by an NH4Cl prepulse, pHi recovered at a rapid rate, which was reduced by reduction of [Na+]o and was virtually abolished by EIPA and DIDS in combination. Creating an outward Cl- gradient by removal of extracellular Cl- significantly increased the rate of pHi recovery. In HCO3(-)-free media, the pHi recovery rate was reduced in control cells and was abolished at zero [Na+]o and by EIPA. After intracellular alkalinization imposed by an acetate prepulse, pHi recovery was unaffected by DIDS but was significantly reduced in the absence of extracellular Cl-, as well as in the presence of Zn2+, which is a blocker of proton channels. Together, this points toward a combined role of DIDS-insensitive Cl-/HCO3- and passive H+ influx in the recovery of pHi after alkalinization.
利用荧光探针2',7'-双-(2-羧乙基)-5,6-羧基荧光素（BCECF），对培养的单个小鼠新皮质神经元中的细胞内pH（pHi）的维持和调节进行了研究。通过降低细胞外Na⁺浓度（[Na⁺]o）使Na⁺梯度反转，导致细胞内迅速酸化，5'-(N-乙基-N-异丙基)amiloride（EIPA）可抑制这种酸化，EIPA是一种Na⁺/H⁺交换抑制剂。在存在EIPA和/或4',4'-二异硫氰酸根合芪-2',2'-磺酸（DIDS）（一种Na⁺偶联阴离子交换器和Na⁺-HCO₃⁻共转运抑制剂）的情况下，可观察到pHi缓慢下降。在NH₄Cl预脉冲引起细胞内酸化后，pHi迅速恢复，[Na⁺]o降低会使其恢复速率降低，而EIPA和DIDS联合使用则几乎完全消除了这种恢复。通过去除细胞外Cl⁻建立外向Cl⁻梯度，可显著提高pHi的恢复速率。在无HCO₃⁻培养基中，对照细胞的pHi恢复速率降低，在[Na⁺]o为零时以及使用EIPA时恢复被消除。在乙酸盐预脉冲引起细胞内碱化后，pHi的恢复不受DIDS影响，但在没有细胞外Cl⁻以及存在质子通道阻滞剂Zn²⁺的情况下，恢复显著降低。总之，这表明DIDS不敏感的Cl⁻/HCO₃⁻和被动H⁺内流在碱化后pHi的恢复中起联合作用。
