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Updated: Sep 21, 2026
Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons
Published on: May 23, 2011
The role of reactive oxygen species in membrane potential changes in macrophages and astrocytes
I V Klyubin1, K M Kirpichnikova, A M Ischenko
1Institute of Cytology, Russian Academy of Sciences, St. Petersburg.
Abstract:
Involvement of reactive oxygen species (ROS) in changes of the plasma membrane potential of mouse peritoneal macrophages and astrocytes (U118 cell line) under the action of different agents has been studied. Membrane potential was measured using the voltage-dependent fluorescent oxonol dye DiBAC4(3). Agonists which stimulate macrophages to release ROS (the fMLP peptide and platelet activating factor) caused prolonged hyperpolarization. Experiments with the fluorescent probe 2',7'-dichlorofluorescein diacetate have shown that astrocytes release ROS upon the action of C5a complement anaphylatoxin (but not C3a). The effect of C5a was accompanied with hyperpolarization of the astrocyte plasma membrane. Treatment of the cells with agents which do not induce ROS generation (C3a, lipopolysaccharide, interferon-gamma) depolarized the plasma membrane. Hyperpolarization of both cell types was significantly decreased in the presence of superoxide dismutase (but not catalase). Moreover, the O2- -generating system caused a marked hyperpolarization of both cell types. The data obtained suggest that O2- is involved in the macrophage and astrocyte hyperpolarization response.
Insights
Reactive oxygen species (ROS), particularly superoxide (O2-), are involved in the plasma membrane potential changes in macrophages and astrocytes. Superoxide dismutase reduced hyperpolarization, suggesting its key role in these cellular responses.
Area of Science:
- Immunology
- Cell Biology
- Neuroscience
Background:
- Reactive oxygen species (ROS) play crucial roles in cellular signaling.
- Plasma membrane potential is critical for cell function, including immune cells and astrocytes.
- Understanding ROS involvement in membrane potential shifts is vital for cellular physiology.
Purpose of the Study:
- To investigate the role of ROS in modulating plasma membrane potential of mouse peritoneal macrophages and U118 astrocytes.
- To determine the specific ROS species involved in these membrane potential changes.
- To elucidate the signaling pathways linking ROS production to membrane potential alterations.
Main Methods:
- Measurement of plasma membrane potential using the voltage-dependent fluorescent dye DiBAC4(3).
- Assessment of ROS production using the fluorescent probe 2',7'-dichlorofluorescein diacetate.
- Application of various agonists (fMLP, PAF, C5a, C3a, LPS, IFN-γ) and ROS scavengers (superoxide dismutase, catalase).
Main Results:
- ROS-releasing agonists (fMLP, PAF, C5a) induced hyperpolarization in macrophages and astrocytes.
- Non-ROS-inducing agents (C3a, LPS, IFN-γ) caused depolarization.
- Superoxide dismutase significantly reduced hyperpolarization, while catalase had no effect.
- An O2- generating system induced hyperpolarization in both cell types.
Conclusions:
- Superoxide anion (O2-) is a key mediator of hyperpolarization in macrophages and astrocytes.
- ROS, specifically O2-, are directly involved in regulating plasma membrane potential in these cell types.
- Findings suggest a novel signaling mechanism involving ROS in immune and glial cell function.
