Related Experiment Video
Updated: Feb 8, 2026
Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo
Published on: November 5, 2013
Pyridine nucleotides regulate the superoxide anion flash upon permeabilization of mitochondrial membranes: An
Ekaterina S Kharechkina1, Anna B Nikiforova1, Alexey G Kruglov1
1Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia.
Abstract:
The permeabilization of mitochondrial membranes via permeability transition pore opening or by the pore-forming peptide alamethicin causes a flash of superoxide anion (SA) and hydrogen peroxide production and the inhibition of matrix aconitase. It was shown using the SA probe 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one (MCLA) that the substrates of NAD-dependent dehydrogenases, inhibitors of the respiratory chain, and NAD(P)H at millimolar concentrations suppressed or delayed SA flashes. In the presence of added NADH and NADPH, SA flashes were observed only after considerable oxidation of pyridine nucleotides. The production of SA was maximal at NADPH and NADH redox potentials from -315 to -295 mV and from -325 to -270 mV, respectively, depending on NAD(P)H concentration. SA generation supported by NADPH was severalfold greater than that supported by NADH. In intact mitochondria, NADPH- and NADH-dependent SA generation was negligible. Respiratory substrates at physiological or lower concentrations were incapable of suppressing the NADPH-supported SA flash. These data indicate that, in conditions close to pathophysiological, matrix NADPH oxidoreductase(s), presumably, an adrenodoxin reductase in complex with adrenodoxin, can essentially contribute to SA flashes associated with transient or irreversible permeability transition pore opening or membrane permeabilization by another mechanism.
Insights
Mitochondrial membrane permeabilization triggers superoxide anion (SA) flashes. Matrix NADPH oxidoreductases, particularly adrenodoxin reductase, significantly contribute to these SA flashes under pathophysiological conditions.
Area of Science:
- Mitochondrial biochemistry
- Oxidative stress
- Cellular signaling
Background:
- Mitochondrial membrane permeabilization, through mechanisms like permeability transition pore opening, leads to reactive oxygen species (ROS) production.
- Superoxide anion (SA) and hydrogen peroxide are key ROS implicated in cellular damage and signaling.
- Matrix aconitase inhibition is a known consequence of mitochondrial dysfunction.
Purpose of the Study:
- To investigate the role of NAD(P)H and specific oxidoreductases in mitochondrial superoxide anion (SA) flashes.
- To elucidate the contribution of matrix NADPH-dependent systems to SA generation during mitochondrial permeabilization.
- To understand the influence of redox potentials on SA production.
Main Methods:
- Utilized the SA probe 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one (MCLA) for SA detection.
- Assessed the effects of NAD-dependent dehydrogenase substrates, respiratory chain inhibitors, and NAD(P)H on SA flashes.
- Measured SA production under varying redox potentials of NADH and NADPH.
Main Results:
- SA flashes were suppressed or delayed by NAD(P)H at millimolar concentrations and required pyridine nucleotide oxidation.
- NADPH-supported SA generation was significantly higher than NADH-supported generation.
- NADPH- and NADH-dependent SA generation was minimal in intact mitochondria but substantial upon permeabilization.
- Respiratory substrates did not suppress NADPH-supported SA flashes at physiological concentrations.
Conclusions:
- Matrix NADPH oxidoreductases, likely involving adrenodoxin reductase and adrenodoxin, play a crucial role in SA flashes during mitochondrial permeability transition.
- These findings highlight the contribution of specific matrix redox systems to SA generation under near-pathophysiological conditions.
- The study implicates NADPH-dependent pathways in the ROS bursts associated with mitochondrial membrane permeabilization.
More Related Videos
06:35Author Spotlight: Innovative Techniques for ROS Detection and Implications for Platelet Research
Published on: March 29, 2024
06:57Author Spotlight: Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells
Published on: May 12, 2023
Related Concept Videos
01:28The Inner Mitochondrial Membrane
01:45Mitochondrial Membranes
01:34Protein Transport into the Inner Mitochondrial Membrane
Transport of mitochondrial precursors across the TIM23 channel is driven by...
01:08Nucleotide Excision Repair
01:12Porin Insertion in the Outer Mitochondrial Membrane
Three models describe the assembly of porins by the SAM complex and their insertion into the outer membrane. Model 1 suggests that porins are assembled outside the SAM channel as the...
01:19Membrane Asymmetry Regulating Transporters
Flippase
Eukaryotic flippases are type-IV P-type ATPases or P4-ATPases belonging to P-type ATPase family proteins that are membrane-bound pumps involved in the ATP-mediated transport of ions and molecules across the membrane. Flippases flip specific phospholipids from the outer to the inner leaflet of a membrane. All P4-ATPases have one...