Vergun Olga, Votyakova Tatyana V, Reynolds Ian J
Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Biophys J. 2003 Nov;85(5):3358-66. doi: 10.1016/S0006-3495(03)74755-9.
In this study we measured DeltaPsim 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 approximately 70% of energized mitochondria we observed large amplitude spontaneous fluctuations in DeltaPsim with a time course comparable to that observed previously in mitochondria of intact cells. The other 30% of mitochondria maintained a stable DeltaPsim. 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 Ca(2+), 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 DeltaPsim were not connected to oxidant production since reducing illumination or the addition of antioxidants had no effect on DeltaPsim. Fluctuating mitochondria did not lose calcein, nor was there any effect of cyclosporin A on DeltaPsim, which ruled out a contribution of permeability transition. We conclude that the fluctuations in DeltaPsim reflect an intermediate, unstable state of mitochondria that may lead to or reflect mitochondrial dysfunction.
在本研究中，我们使用罗丹明123测量单个分离的脑线粒体中的ΔΨm。线粒体附着在盖玻片上，并用补充有苹果酸和谷氨酸的基于K⁺的HEPES缓冲培养基进行灌流。在大约70%的活跃线粒体中，我们观察到ΔΨm出现大幅度的自发波动，其时间进程与之前在完整细胞线粒体中观察到的相当。另外30%的线粒体保持稳定的ΔΨm。一些“稳定”的线粒体在记录期间开始自发波动。然而，最初波动的线粒体中没有一个变得稳定。从培养基中去除底物或施加少量Ca²⁺后，波动线粒体中的罗丹明123荧光迅速降至背景值，而非波动线粒体则延迟去极化，并且在完全去极化之前常常开始波动。ΔΨm的变化与氧化剂产生无关，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绿素，环孢素A对ΔΨm也没有任何影响，这排除了通透性转换的作用。我们得出结论，ΔΨm的波动反映了线粒体的一种中间不稳定状态，这种状态可能导致或反映线粒体功能障碍。
