White R J, Reynolds I J
Center for Neuroscience, University of Pittsburgh School of Medicine, Pennsylvania 15261.
J Neurosci. 1995 Feb;15(2):1318-28. doi: 10.1523/JNEUROSCI.15-02-01318.1995.
Utilizing Indo-1 microfluorimetry, we have investigated the role of mitochondria and Na+/Ca2+ exchange in buffering calcium loads induced by glutamate stimulation or depolarization of cultured rat forebrain neurons. A 15 sec pulse of 3 microM glutamate or 50 mM potassium with veratridine was followed by a 2 min wash with a solution containing either Na(+)-free buffer or the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), or both. For glutamate-induced Ca2+ loads, a Na(+)-free wash delayed recovery to baseline by twofold, mitochondrial uncoupling delayed recovery by greater than fourfold, and the combined treatment essentially prevented recovery of [Ca2+]i for the duration of the wash. Although the depolarization stimulus was able to elicit a larger peak [Ca2+]i, the neurons required significantly less time to recover from depolarization-induced Ca2+ loads after identical wash manipulations, indicating a fundamental difference between calcium loads induced by glutamate as opposed to those induced by depolarization. We show evidence that the delayed recovery is not primarily the result of perturbations in intracellular pH regulation and have also demonstrated that a substantial portion of the delayed recovery is independent of Ca2+ entry during the washout phase. We conclude that glutamate and depolarization both induce Ca2+ loads whose buffering is critically dependent on functional mitochondria and secondarily reliant on Na+/Ca2+ exchange. The two systems overlap and seem to be responsible for buffering most of the glutamate-induced Ca2+ load, because manipulations that compromised both systems completely disabled the neurons' ability to recover [Ca2+]i to baseline.
利用Indo-1显微荧光测定法，我们研究了线粒体和Na⁺/Ca²⁺交换在缓冲培养的大鼠前脑神经元谷氨酸刺激或去极化诱导的钙负荷中的作用。用含有3 microM谷氨酸或50 mM钾与藜芦碱的溶液进行15秒的脉冲处理，随后用含有无钠缓冲液或线粒体解偶联剂羰基氰化物对三氟甲氧基苯腙（FCCP）或两者的溶液洗涤2分钟。对于谷氨酸诱导的Ca²⁺负荷，无钠洗涤将恢复到基线的时间延迟了两倍，线粒体解偶联将恢复延迟了四倍以上，并且联合处理在洗涤期间基本上阻止了[Ca²⁺]i的恢复。尽管去极化刺激能够引发更大的[Ca²⁺]i峰值，但在相同的洗涤操作后，神经元从去极化诱导的Ca²⁺负荷中恢复所需的时间明显更少，这表明谷氨酸诱导的钙负荷与去极化诱导的钙负荷之间存在根本差异。我们提供的证据表明，延迟恢复并非主要是细胞内pH调节紊乱的结果，并且还证明了延迟恢复的很大一部分与洗脱阶段的Ca²⁺内流无关。我们得出结论，谷氨酸和去极化均诱导Ca²⁺负荷，其缓冲关键依赖于功能性线粒体，其次依赖于Na⁺/Ca²⁺交换。这两个系统重叠，似乎负责缓冲大部分谷氨酸诱导的Ca²⁺负荷，因为损害这两个系统的操作完全破坏了神经元将[Ca²⁺]i恢复到基线的能力。
