Effect of extracellular Mg2+ on ROS and Ca2+ accumulation during reoxygenation of rat cardiomyocytes

The effects of Mg2+ on reactive oxygen species (ROS) and cell Ca2+ during r eoxygenation of hypoxic rat cardiomyocytes were studied. Oxidation of 2',7' -dichlorodihydrofluorescein (DCDHF) to dichlorofluorescein (DCF) and of dih ydroethidium (DHE) to ethidium (ETH) within cells were used as markers for intracellular ROS levels and were determined by flow cytometry. DCDHF/DCF i s sensitive to H2O2 and nitric oxide (NO), and DHE/ETH is sensitive to the superoxide anion (O-2(-).), respectively. Rapidly exchangeable cell Ca2+ wa s determined by Ca-45(2+) uptake. Cells were exposed to hypoxia for 1 h and reoxygenation for 2 h. ROS levels, determined as DCF fluorescence, were in creased 100-130% during reoxygenation alone and further increased 60% by in creasing extracellular Mg2+ concentration to 5 mM at reoxygenation. ROS lev els, measured as ETH fluorescence, were increased 16-24% during reoxygenati on but were not affected by Mg2+. Cell Ca2+ increased three- to fourfold du ring reoxygenation. This increase was reduced 40% by 5 mM Mg2+, 57% by 10 m uM 3,4-dichlorobenzamil (DCB) (inhibitor of Na+/Ca2+ exchange), and 75% by combining Mg2+ and DCB. H2O2 (25 and 500 muM) reduced Ca2+ accumulation by 38 and 43%, respectively, whereas the NO donor S-nitroso-N-acetyl-penicilla mine (1 mM) had no effect. Mg2+ reduced hypoxia/reoxygenation-induced lacta te dehydrogenase (LDH) release by 90%. In conclusion, elevation of extracel lular Mg2+ to 5 mM increased the fluorescence of the H2O2/NO-sensitive prob e DCF without increasing that of the O-2(-). -sensitive probe ETH, reduced Ca2+ accumulation, and decreased LDH release during reoxygenation of hypoxi c cardiomyocytes. The reduction in LDH release, reflecting the protective e ffect of Mg2+, may be linked to the effect of Mg2+ on Ca2+ accumulation and /or ROS levels.