tetramethylrhodamine and Hypoxia

Melatonin, a hormonal product of the pineal gland, is now known to be a multi-faceted free radical scavenger and anti-oxidant. Since little information is available regarding the action of melatonin on the heart, we studied the effects of melatonin on adult ventricular myocytes subjected to chemical hypoxia and reoxygenation.. Adult rat ventricular myocytes were preloaded with tetramethylrhodamine (TMRM) in combination with one of the following fluorophores: dichlorodihydrofluorescein diacetate (DCDHF), dihydrorhodamine 123 (DHR) or fluo 3 (Fluo) and then investigated with confocal laser scanning microscopy. Chemical hypoxia was induced by addition of 1.5 mM KCN and 20 mM deoxyglucose to the superfusion buffer. Melatonin (50-100 microM) was added at intervals during the protocol.. Cells subjected to 12.5 min chemical hypoxia showed marked morphological changes, increased fluorescence intensity of DCDHF, DHR and Fluo, suggesting Ca2+ accumulation and generation of H2O2 and reactive oxygen species. The number of cells showing increased fluorescence also increased significantly. Melatonin (50 and 100 microM) caused a significant reduction in morphological changes, number of cells with increased fluorescence and fluorescence intensity of DHR and Fluo, (but not DCDHF).. Melatonin effectively reduced damage induced by chemical hypoxia in adult cardiomyocytes, probably by virtue of its effects on reactive oxygen species generation and intracellular Ca2+ accumulation.

Topics: Aniline Compounds; Animals; Calcium; Cells, Cultured; Fluoresceins; Fluorescent Dyes; Heart Ventricles; Hypoxia; Image Processing, Computer-Assisted; Male; Melatonin; Microscopy, Confocal; Myocardium; Perfusion; Rats; Reperfusion Injury; Rhodamines; Time Factors; Xanthenes