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Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons
Published on: May 23, 2011
Ca2+-induced permeabilization promotes free radical release from rat brain mitochondria with partially inhibited
Tatyana V Votyakova1, Ian J Reynolds
1Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Abstract:
Mitochondrial complex I dysfunction has been implicated in a number of brain pathologies, putatively owing to an increased rate of reactive oxygen species (ROS) release. However, the mechanisms regulating the ROS burden are poorly understood. In this study we investigated the effect of Ca2+ loads on ROS release from rat brain mitochondria with complex I partially inhibited by rotenone. The addition of 20 nm rotenone to brain mitochondria increased ROS release. Ca2+ (100 microm) alone had no effect on ROS release, but greatly potentiated the effects of rotenone. The effect of Ca2+ was decreased by ruthenium red. Ca2+-challenged mitochondria lose about 88% of their glutathione and 46% of their cytochrome c under these conditions, although this depends only on Ca2+ loading and not complex I inhibition. ADP in combination with oligomycin decreased the loss of glutathione and cytochrome c and free radical generation. Cyclosporin A alone was ineffective in preventing these effects, but augmented the protection provided by ADP and oligomycin. Non-specific permeabilization of mitochondria with alamethicin also increased the ROS signal, but only when combined with partial inhibition of complex I. These results demonstrate that Ca2+ can greatly increase ROS release by brain mitochondria when complex I is impaired.
Insights
Calcium overload significantly increases reactive oxygen species (ROS) release from brain mitochondria with impaired Complex I. This finding sheds light on mitochondrial dysfunction in brain pathologies.
Area of Science:
- Biochemistry
- Neuroscience
- Cell Biology
Background:
- Mitochondrial complex I dysfunction is linked to brain diseases, potentially due to increased reactive oxygen species (ROS).
- The precise mechanisms controlling ROS production under these conditions remain unclear.
Purpose of the Study:
- To investigate how calcium (Ca2+) loads influence ROS release from rat brain mitochondria with partially inhibited Complex I.
Main Methods:
- Utilized rat brain mitochondria with Complex I partially inhibited by rotenone.
- Assessed ROS release, glutathione and cytochrome c levels, and the effects of Ca2+, ruthenium red, ADP, oligomycin, cyclosporin A, and alamethicin.
Main Results:
- Rotenone-induced Complex I inhibition increased ROS release.
- Ca2+ potentiated rotenone's effect on ROS release, an effect reduced by ruthenium red.
- Ca2+ challenge led to significant glutathione and cytochrome c loss, dependent on Ca2+ loading.
- ADP and oligomycin mitigated ROS generation and loss of glutathione/cytochrome c.
- Alamethicin-induced permeabilization increased ROS only when Complex I was inhibited.
Conclusions:
- Ca2+ significantly exacerbates ROS release in brain mitochondria when Complex I function is compromised.
- Mitochondrial Ca2+ overload contributes to oxidative stress and potential cell damage in neurodegenerative conditions.
