Intramitochondrial Ascorbic Acid Enhances the Formation of Mitochondrial Superoxide Induced by Peroxynitrite via a Ca2+-Independent Mechanism
Abstract
:1. Introduction
2. Results
2.1. Relationships between Mitochondrial Superoxide Formation, with the Ensuing Downstream DNA Strand Scission, and Mitochondrial Ca2+ Accumulation in Cells Exposed to AA and Peroxynitrite
2.2. Mitochondrial Superoxide Formation and DNA Strand Scission in Human Monocytes and Macrophages Exposed to AA and Peroxynitrite
2.3. AA Promotes Peroxynitite-Dependent Mitochondrial Permeability Transition and Cytotoxicity via Ca2+-Independent Mechanisms
2.4. The Enhancing Effects of AA Are Mediated by an Increased Susceptibility of Complex III to Inhibition by Peroxynitrite
3. Discussion
- The enhancing effects of AA, detected in terms of mitochondrial superoxide formation and downstream DNA strand scission, were sensitive to rotenone, or myxothiazol, as in the case of CRDM and RDM, but not CDM.
- The enhancing effects of AA were insensitive to Ry, as for RDM, but not CRDM or CDM.
- There was no evidence of increased mitochondrial accumulation of Ca2+ after treatment with AA and/or peroxynitrite, as in the case of RDM. Increased mitochondrial accumulation of Ca2+ was instead associated with either CRDM or CDM.
- The DNA strand scission mediated by peroxynitrite in cells permeabilized after pre-exposure to AA, while suppressed by rotenone or myxothiazol, was insensitive to Ry, EGTA or inhibitors of mitochondrial Ca2+ uptake. All these treatments were instead effective in CRDM.
4. Materials and Methods
4.1. Chemicals
4.2. Cell Culture and Treatments
4.3. Measurement of AA Content
4.4. MitoSOX Red Oxidation
4.5. Aconitase Activity
4.6. Measurement of DNA Single-Strand Breakage by the Alkaline Halo Assay
4.7. Cytotoxicity Assay
4.8. Measurement of Mitochondrial Ca2+
4.9. MitoTracker Red CMXRos, Calcein Staining and Imaging
4.10. Measurement of Oxygen Consumption
4.11. Statistical Analysis
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Guidarelli, A.; Cerioni, L.; Fiorani, M.; Cantoni, O. Intramitochondrial Ascorbic Acid Enhances the Formation of Mitochondrial Superoxide Induced by Peroxynitrite via a Ca2+-Independent Mechanism. Int. J. Mol. Sci. 2017, 18, 1686. https://doi.org/10.3390/ijms18081686
Guidarelli A, Cerioni L, Fiorani M, Cantoni O. Intramitochondrial Ascorbic Acid Enhances the Formation of Mitochondrial Superoxide Induced by Peroxynitrite via a Ca2+-Independent Mechanism. International Journal of Molecular Sciences. 2017; 18(8):1686. https://doi.org/10.3390/ijms18081686
Chicago/Turabian StyleGuidarelli, Andrea, Liana Cerioni, Mara Fiorani, and Orazio Cantoni. 2017. "Intramitochondrial Ascorbic Acid Enhances the Formation of Mitochondrial Superoxide Induced by Peroxynitrite via a Ca2+-Independent Mechanism" International Journal of Molecular Sciences 18, no. 8: 1686. https://doi.org/10.3390/ijms18081686