Excitotoxic mitochondrial depolarisation requires both calcium and nitric oxide in rat hippocampal neurons
Excitotoxic mitochondrial depolarisation requires both calcium and nitric oxide in rat hippocampal neurons
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DOI：
10.1111/j.1469-7793.1999.00797.x
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发表时间：
1999-11-01
影响因子：
5.5
通讯作者：
Duchen, MR
中科院分区：
文献类型：
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作者：
Keelan, J;Vergun, O;Duchen, MR
1. 谷氨酸神经毒性归因于细胞Ca2+超载。由于线粒体去极化可能是细胞死亡过程中的关键一步，我们已经使用数字成像技术来检查谷氨酸中毒期间细胞质Ca2+浓度（[Ca2+](c)）和线粒体电位（δ Psi(m)）之间的关系，并确定线粒体功能障碍的机制。在离体11天的>细胞（DIV）中，暴露于50 mM钾或100 μ m谷氨酸对δ Psi(m)有不同的影响。KCl引起小的瞬时δ Psi(m)损失，但在谷氨酸反应中，δ Psi(m)损失严重。在7-10 DIV的细胞中，谷氨酸只引起适度的可逆的δ Psi(m)下降。使用fura-2测量[Ca2+](c)，对KCl和谷氨酸的反应没有显着差异。然而，使用低亲和力指示剂fura-2FF发现[Ca2+](c)对KCl和谷氨酸的反应存在差异，这与δ Psi(m)的损失明显相关。线粒体深度去极化的神经元也显示出fura-2FF比率的大量继发性增加。谷氨酸诱导的δ Psi(m)损失依赖于Ca2+内流。然而，L-NAME对一氧化氮合酶（NOX）的抑制显著减弱了δ Psi(m)的损失。此外，在7-10 div的培养液中，无论是对KCI还是对谷氨酸的反应，当与高[Ca2+]结合时，单独没有影响的笼中NO的光解作用促进了线粒体的深度去极化(c)。在对谷氨酸仅表现出适度线粒体反应的细胞中，通过添加NO诱导线粒体去极化，随后是[Ca2+]的二次升高(c)。这些数据表明，在谷氨酸中毒期间，[Ca2+](c)和一氧化氮协同作用导致线粒体功能障碍和[Ca2+](c)稳态受损。
1. Glutamate neurotoxicity has been attributed to cellular Ca2+ overload. As mitochondrial depolarisation may represent a pivotal step in the progression to cell death, we have used digital imaging techniques to examine the relationship between cytosolic Ca2+ concentration ([Ca2+](c)) and mitochondrial potential (Delta Psi(m)) during glutamate toxicity, and to define the mechanisms underlying mitochondrial dysfunction.2. In cells of > 11 days in vitro (DIV), exposure to 50 mM potassium or 100 mu m glutamate had different consequences for Delta Psi(m). KCl caused a small transient loss of Delta Psi(m) but in response to glutamate there was a profound loss of Delta Psi(m). In cells of 7-10 DIV, glutamate caused only a modest and reversible drop in Delta Psi(m).3. Using fura-2 to measure [Ca2+](c), responses to KCl and glutamate did not appear significantly different. However, use of the low affinity indicator fura-2FF revealed a difference in the [Ca2+](c) responses to KCl and glutamate, which clearly correlated with the loss of Delta Psi(m). Neurons exhibiting a profound mitochondrial depolarisation also showed a large secondary increase in the fura-2FF ratio.4. The glutamate-induced loss of Delta Psi(m) was dependent on Ca2+ influx. However, inhibition of nitric oxide synthase (NOX) by L-NAME significantly attenuated the loss of Delta Psi(m). Furthermore, photolysis of caged NO at levels that had no effect alone promoted a profound mitochondrial depolarisation when combined with high [Ca2+](c), either in response to KCI or to glutamate in cultures at 7-10 DIV.5. In cells that showed only modest mitochondrial responses to glutamate, induction of a mitochondrial depolarisation by the addition of NO was followed by a secondary rise in [Ca2+](c). These data suggest that [Ca2+](c) and nitric oxide act synergistically to cause mitochondrial dysfunction and impaired [Ca2+](c) homeostasis during glutamate toxicity.