Involvement of Hydrogen Peroxide in the Manganese-Induced Myocytes Mitochondrial Membrane Potential Loss

Isolated rat ventricular myocytes were incubated in different manganese concentrations and after that the reactive oxygen species (ROS) generation, the level of reduced glutathione (GSH), and the changes of mitochondrial membrane potential ( m) were investigated by a flow cytometer (FACScan) as well as laser scanning confocal microscopy (LSCM). Results showed that, although the total ROS in the cell were increased after manganese treatment, hydrogen peroxide (H2O2) was the main elevated species while superoxide anion (O2-) was nearly unchanged. The generation of H2O2 became obvious when the myocytes were incubated in the higher concentration of manganese. The content of GSH in myocytes also decreased after manganese exposure. When the myocytes were incubated with both manganese and GSH-ethylester (GSH-EE), which is permeable to the cell membrane, the generation of H2O2 decreased greatly. The loss of the mitochondrial membrane potential ( m) was also induced by manganese incubation. However, with the existence of extracellular GSH-EE the m were rescued. The results suggested that manganese action may lead to the ROS stress upon myocytes which most probably rise from high generation of intracellular H2O2. GSH-EE could effectively clean the over-production of H2O2, indicating that the low level of intracellular GSH was another main reason to the high accumulation of H2O2. The mitochondrial membrane potential was also affected by manganese and rescued by GSH-EE, suggesting that H2O2 was also involved in the manganese damages to the mitochondrial membrane potential.