The regulation of cytosolic pH in isolated presynaptic nerve terminals from rat brain.
The regulation of cytosolic pH in isolated presynaptic nerve terminals from rat brain.
复制标题
大鼠脑中分离的突触前神经末梢细胞质 pH 值的调节。
DOI：
10.1085/jgp.91.2.289
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发表时间：
1988
期刊：
影响因子：
--
通讯作者：
Drapeau,P
中科院分区：
文献类型：
--
作者：
Nachshen,DA;Drapeau,P
使用荧光pH指示剂2 '，7'-双（羧乙基）-5，6-羧基荧光素（BCECF）测量从大鼠脑（突触体）分离的突触前神经末梢中的细胞溶质pH（pHi）。通过与BCECF的膜永久性乙酰氧基甲基酯衍生物一起孵育，使突触体负载BCECF，BCECF通过细胞内酯酶水解为母体化合物。通过两种不同的方法透化突触体膜后校准荧光信号来估计pHi。如果在用毛地黄皂苷透化后校准荧光信号，则估计负载有15-90 μ M BCECF的突触体具有6.94 +/- 0.02（平均值+/-标准误差; n = 54）的pHi;使用负载有10倍更少BCECF的突触体获得类似值（6.9 +/- 0.1; n = 5）。当通过用短杆菌肽和尼日利亚菌素将突触体膜透化至H+来校准荧光信号时，pHi估计为7.19 +/- 0.03（n = 12）。对于后一种方法，当突触体负载10倍以下的BCECF时，pHi = 6.95 +/- 0.09（n = 14）。因此，突触体中的pHi约为7.0，并且可以在较高的BCECF浓度下使用毛地黄皂苷校准方法更精确地监测。当突触体在含有20 mM NH 4Cl的培养基中孵育，然后稀释到不含NH 4Cl的培养基中时，pHi立即酸化至约6.6的水平。酸化后，pHi在几分钟内恢复。突触体的缓冲能力估计为约50 mM/pH单位。通过在无Na培养基中孵育、通过加入阿米洛利（KI = 3 μ M）和通过取消Nao/Nai梯度，回收率显著减慢。酸化后的pH值及其恢复不受孵育在含HCO 3的介质;二磺酸芪阴离子转运抑制剂（SITS和DIDS，1 mM）和甲基磺酸盐取代Cl不影响pH值的恢复率。看来，Na+/H+反向转运蛋白是哺乳动物脑神经末梢中pHi的主要调节剂。
Cytosolic pH (pHi) was measured in presynaptic nerve terminals isolated from rat brain (synaptosomes) using a fluorescent pH indicator, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The synaptosomes were loaded with BCECF by incubation with the membrane-permanent acetoxy-methyl ester derivative of BCECF, which is hydrolyzed by intracellular esterases to the parent compound. pHi was estimated by calibrating the fluorescence signal after permeabilizing the synaptosomal membrane by two different methods. Synaptosomes loaded with 15-90 microM BCECF were estimated to have a pHi of 6.94 +/- 0.02 (mean +/- standard error; n = 54) if the fluorescence signal was calibrated after permeabilizing with digitonin; a similar value was obtained using synaptosomes loaded with 10 times less BCECF (6.9 +/- 0.1; n = 5). When the fluorescence signal was calibrated by permeabilizing the synaptosomal membrane to H+ with gramicidin and nigericin, pHi was estimated to be 7.19 +/- 0.03 (n = 12). With the latter method, pHi = 6.95 +/- 0.09 (n = 14) when the synaptosomes were loaded with 10 times less BCECF. Thus, pHi in synaptosomes was approximately 7.0 and could be more precisely monitored using the digitonin calibration method at higher BCECF concentrations. When synaptosomes were incubated in medium containing 20 mM NH4Cl and then diluted into NH4Cl-free medium, pHi immediately acidified to a level of approximately 6.6. After the acidification, pHi recovered over a period of a few minutes. The buffering capacity of the synaptosomes was estimated to be approximately 50 mM/pH unit. Recovery was substantially slowed by incubation in an Na-free medium, by the addition of amiloride (KI = 3 microM), and by abolition of the Nao/Nai gradient. pHi and its recovery after acidification were not affected by incubation in an HCO3-containing medium; disulfonic stilbene anion transport inhibitors (SITS and DIDS, 1 mM) and replacement of Cl with methylsulfonate did not affect the rate of recovery of pHi. It appears that an Na+/H+ antiporter is the primary regulator of pHi in mammalian brain nerve terminals.