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 (Deltapsi 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 (H(2)O(2)) was the main elevated species while superoxide anion (O(2).(-)) was nearly unchanged. The generation of H(2)O(2) 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 H(2)O(2) decreased greatly. The loss of the mitochondrial membrane potential (Deltapsi m) was also induced by manganese incubation. However, with the existence of extracellular GSH-EE the Deltapsi 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 H(2)O(2). GSH-EE could effectively clean the over-production of H(2)O(2), indicating that the low level of intracellular GSH was another main reason to the high accumulation of H(2)O(2). The mitochondrial membrane potential was also affected by manganese and rescued by GSH-EE, suggesting that H(2)O(2) was also involved in the manganese damages to the mitochondrial membrane potential.