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Updated: Jun 15, 2026
Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons
Published on: May 23, 2011
Membrane potential-related effect of calcium on reactive oxygen species generation in isolated brain mitochondria
Zsofia Komary1, Laszlo Tretter, Vera Adam-Vizi
1Department of Medical Biochemistry, Semmelweis University, Neurobiochemical Group, Hungarian Academy of Sciences, Hungary.
Abstract:
The effect of Ca2+ applied in high concentrations (50 and 300 microM) was addressed on the generation of reactive oxygen species in isolated mitochondria from guinea-pig brain. The experiments were performed in the presence of ADP, a very effective inhibitor of mitochondrial permeability transition. Moderate increase in H2O2 release from mitochondria was induced by Ca2+ applied in 50 microM, but not in 300 microM concentration as measured with Amplex red fluorescent assay starting with a delay of 100-150 sec after exposure to Ca2+. Parallel measurements of membrane potential (DeltaPsim) by safranine fluorescence showed a transient depolarization by Ca2+ followed by the recovery of DeltaPsim to a value, which was more negative than that observed before addition of Ca2+ indicating a relative hyperpolarization. NAD(P)H fluorescence was also increased by Ca2+ given in 50 microM concentration. In mitochondria having high DeltaPsim in the presence of oligomycin or ATP, the basal rate of release of H2O2 was significantly higher than that observed in a medium containing ADP and Ca2+ no longer increased but rather decreased the rate of H2O2 release. With 300 microM Ca2+ only a loss but no tendency of a recovery of DeltaPsim was detected and H2O2 release was unchanged. It is suggested that in the presence of nucleotides the effect of Ca2+ on mitochondrial ROS release is related to changes in DeltaPsim; in depolarized mitochondria, in the presence of ADP, moderate increase in H2O2 release is induced by calcium, but only in
Insights
Calcium ions (Ca2+) at 50 microM moderately increase hydrogen peroxide (H2O2) release from guinea-pig brain mitochondria, but higher concentrations do not. This effect depends on mitochondrial membrane potential and nucleotide presence, not permeability transition.
Area of Science:
- Mitochondrial biochemistry
- Cellular redox signaling
Background:
- Mitochondria play a crucial role in cellular energy production and signaling.
- Calcium ions (Ca2+) are important cellular messengers that can influence mitochondrial function.
- Reactive oxygen species (ROS) generation by mitochondria is linked to various physiological and pathological processes.
Purpose of the Study:
- To investigate the effect of different concentrations of Ca2+ on reactive oxygen species (ROS) generation in isolated guinea-pig brain mitochondria.
- To explore the relationship between Ca2+ concentration, mitochondrial membrane potential (DeltaPsim), and ROS production.
- To determine if these effects are mediated by the mitochondrial permeability transition.
Main Methods:
- Isolated guinea-pig brain mitochondria were used.
- Hydrogen peroxide (H2O2) release was measured using the Amplex red fluorescent assay.
- Mitochondrial membrane potential (DeltaPsim) was assessed via safranine fluorescence.
- NAD(P)H fluorescence was also monitored.
- Experiments were conducted in the presence of ADP or ATP/oligomycin.
Main Results:
- A moderate increase in H2O2 release was observed with 50 microM Ca2+ in the presence of ADP, accompanied by transient depolarization and subsequent hyperpolarization of DeltaPsim.
- Higher Ca2+ concentration (300 microM) did not increase H2O2 release and caused irreversible DeltaPsim loss.
- In highly polarized mitochondria (with ATP/oligomycin), Ca2+ did not stimulate H2O2 release.
- The observed effects were independent of mitochondrial permeability transition.
Conclusions:
- Ca2+ can modulate mitochondrial ROS generation in a concentration-dependent manner.
- The effect of Ca2+ on ROS production is closely linked to alterations in mitochondrial membrane potential and the presence of nucleotides.
- Low to moderate Ca2+ concentrations can stimulate ROS release under specific conditions (e.g., presence of ADP, subsequent hyperpolarization), while high concentrations or different energetic states do not.
- These findings highlight the complex interplay between Ca2+, mitochondrial bioenergetics, and redox signaling.
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