Effect of extracellular Mg(2+) on ROS and Ca(2+) accumulation during reoxygenation of rat cardiomyocytes.
The effects of Mg(2+) on reactive oxygen species (ROS) and cell Ca(2+) during reoxygenation of hypoxic rat cardiomyocytes were studied. Oxidation of 2',7'-dichlorodihydrofluorescein (DCDHF) to dichlorofluorescein (DCF) and of dihydroethidium (DHE) to ethidium (ETH) within cells were used as markers for intracellular ROS levels and were determined by flow cytometry. DCDHF/DCF is sensitive to H(2)O(2) and nitric oxide (NO), and DHE/ETH is sensitive to the superoxide anion (O(2)(-).), respectively. Rapidly exchangeable cell Ca(2+) was determined by (45)Ca(2+) uptake. Cells were exposed to hypoxia for 1 h and reoxygenation for 2 h. ROS levels, determined as DCF fluorescence, were increased 100-130% during reoxygenation alone and further increased 60% by increasing extracellular Mg(2+) concentration to 5 mM at reoxygenation. ROS levels, measured as ETH fluorescence, were increased 16-24% during reoxygenation but were not affected by Mg(2+). Cell Ca(2+) increased three- to fourfold during reoxygenation. This increase was reduced 40% by 5 mM Mg(2+), 57% by 10 microM 3,4-dichlorobenzamil (DCB) (inhibitor of Na(+)/Ca(2+) exchange), and 75% by combining Mg(2+) and DCB. H(2)O(2) (25 and 500 microM) reduced Ca(2+) accumulation by 38 and 43%, respectively, whereas the NO donor S-nitroso-N-acetyl-penicillamine (1 mM) had no effect. Mg(2+) reduced hypoxia/reoxygenation-induced lactate dehydrogenase (LDH) release by 90%. In conclusion, elevation of extracellular Mg(2+) to 5 mM increased the fluorescence of the H(2)O(2)/NO-sensitive probe DCF without increasing that of the O(2)(-).-sensitive probe ETH, reduced Ca(2+) accumulation, and decreased LDH release during reoxygenation of hypoxic cardiomyocytes. The reduction in LDH release, reflecting the protective effect of Mg(2+), may be linked to the effect of Mg(2+) on Ca(2+) accumulation and/or ROS levels.
Sharikabad
M N
MN
Division of Clinical Pharmacology and Toxicology, Clinical Chemistry Department, Ullevaal University Hospital, N-0407 Oslo, Norway. m.n.sharikabad@ioks.uio.no
Ostbye
K M
KM
Lyberg
T
T
Brørs
O
O
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D015687:Cell Hypoxia; D002614:Chelating Agents / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D005434:Flow Cytometry; D006321:Heart / Q000187:drug effects*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D066298:In Vitro Techniques; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008027:Light; D008274:Magnesium / Q000494:pharmacology*; D008297:Male; D009206:Myocardium / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D026423:S-Nitroso-N-Acetylpenicillamine; D012542:Scattering, Radiation
D002021:Buffers; D002614:Chelating Agents; D016877:Oxidants; D017382:Reactive Oxygen Species; C041470:3',4'-dichlorobenzamil; D026423:S-Nitroso-N-Acetylpenicillamine; D000584:Amiloride; D006861:Hydrogen Peroxide; D007770:L-Lactate Dehydrogenase; D010396:Penicillamine; D008274:Magnesium; D002118:Calcium