Spontaneous changes in mitochondrial membrane potential in single isolated brain mitochondria.
Spontaneous changes in mitochondrial membrane potential in single isolated brain mitochondria.
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DOI：
10.1016/s0006-3495(03)74755-9
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发表时间：
2003-11
影响因子：
3.4
通讯作者：
O. Vergun;T. Votyakova;I. Reynolds
中科院分区：
文献类型：
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作者：
O. Vergun;T. Votyakova;I. Reynolds
在这项研究中，我们使用罗丹明123在单个分离的脑线粒体中测量ΔΨm。将线粒体附着在盖子上，并与添加苹果酸盐和谷氨酸盐的K+基hepes缓冲培养基混合。在约70%的激活线粒体中，我们观察到ΔΨm的大幅度自发波动，其时间过程与之前在完整细胞的线粒体中观察到的情况相当。另外30%的线粒体保持稳定ΔΨm。在记录期间，一些“稳定”的线粒体开始自发波动。然而，最初波动的线粒体没有一个变得稳定。在从培养基中去除底物或应用少量Ca2+后，罗丹明123荧光在波动线粒体中迅速下降到背景值，而非波动线粒体则延迟去极化，并且经常在完全去极化之前开始波动。ΔΨm的变化与氧化剂的产生无关，因为减少光照或添加抗氧化剂对ΔΨm没有影响。波动的线粒体没有丢失钙黄蛋白，环孢素A对ΔΨm也没有任何影响，这排除了通透性转变的贡献。我们得出结论，ΔΨm的波动反映了线粒体的中间不稳定状态，可能导致或反映线粒体功能障碍。
In this study we measured ΔΨm in single isolated brain mitochondria using rhodamine 123. Mitochondria were attached to coverslips and superfused with K+-based HEPES-buffer medium supplemented with malate and glutamate. In ∼70% of energized mitochondria we observed large amplitude spontaneous fluctuations in ΔΨm with a time course comparable to that observed previously in mitochondria of intact cells. The other 30% of mitochondria maintained a stable ΔΨm. Some of the "stable" mitochondria began to fluctuate spontaneously during the recording period. However, none of the initially fluctuating mitochondria became stable. Upon the removal of substrates from the medium or application of small amounts of Ca2+, rhodamine 123 fluorescence rapidly dropped to background values in fluctuating mitochondria, while nonfluctuating mitochondria depolarized with a delay and often began to fluctuate before complete depolarization. The changes in ΔΨm were not connected to oxidant production since reducing illumination or the addition of antioxidants had no effect on ΔΨm. Fluctuating mitochondria did not lose calcein, nor was there any effect of cyclosporin A on ΔΨm, which ruled out a contribution of permeability transition. We conclude that the fluctuations in ΔΨm reflect an intermediate, unstable state of mitochondria that may lead to or reflect mitochondrial dysfunction.