Enhanced hydrogen peroxide generation accompanies the beneficial bioenergetic effects of methylene blue in isolated brain mitochondria

• Published in: Free radical biology & medicine Vol. 77; pp. 317 - 330
• Main Authors: Tretter, L., Horvath, G., Hölgyesi, A., Essek, F., Adam-Vizi, V.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2014
• Subjects: Adenosine Triphosphate - biosynthesis; Animals; Brain mitochondria; Calcium - metabolism; Drug Evaluation, Preclinical; Energy Metabolism - drug effects; Free radicals; Guinea Pigs; H2O2 generation; Hydrogen Peroxide - metabolism; Membrane Potential, Mitochondrial - drug effects; Methylene blue; Methylene Blue - pharmacology; Mitochondria - drug effects; Mitochondria - metabolism; Mitochondrial membrane potential; Neuroprotective Agents - pharmacology; Oxidation-Reduction; Oxygen Consumption - drug effects; Reactive oxygen species; Respiration; RET; Reactive oxygen species; Free radicals; ΔΨμ; α-GPDH; α-GP; PTP; Methylene blue; Brain mitochondria; MB; ROS; H2O2 generation; Respiration; Mitochondrial membrane potential; HO generation
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2014.09.024

The redox dye methylene blue (MB) is proven to have beneficial effects in various models of neurodegenerative diseases. Here we investigated the effects of MB (100nM, 300nM, and 1μM) on key bioenergetic parameters and on H2O2 production/elimination in isolated guinea pig brain mitochondria under normal as well as respiration-impaired conditions. As measured by high-resolution Oxygraph the rate of resting oxygen consumption was increased, but the ADP-stimulated respiration was unaffected by MB with any of the substrates (glutamate malate, succinate, or α-glycerophosphate) used for supporting mitochondrial respiration. In mitochondria treated with inhibitors of complex I or complex III MB moderately but significantly increased the rate of ATP production, restored ΔΨm, and increased the rate of Ca2+ uptake. The effects of MB are consistent with transferring electrons from upstream components of the electron transport chain to cytochrome c, which is energetically favorable when the flow of electrons in the respiratory chain is compromised. On the other hand, MB significantly increased the production of H2O2 measured by Amplex UltraRed fluorimetry under all conditions, in resting, ATP-synthesizing, and respiration-impaired mitochondria, with each substrate combination supporting respiration. Furthermore, it also decreased the elimination of H2O2. Generation of H2O2 without superoxide formation, observed in the presence of MB, is interpreted as a result of reduction of molecular oxygen to H2O2 by the reduced MB. The elevated generation and impaired elimination of H2O2 should be considered for the overall oxidative state of mitochondria treated with MB. [Display omitted] •Methylene blue (MB) stimulates ATP synthesis in respiration-impaired mitochondria.•MB rescues ΔΨm and improves Ca2+ uptake in respiration-impaired mitochondria.•MB stimulates generation and decreases elimination of H2O2 in brain mitochondria.•Increased H2O2 generation indicates an oxidative burden in MB-treated mitochondria.•Stimulated H2O2 generation could be a signal for upregulation of antioxidant mechanisms.