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1 Division of Clinical Pharmacology and Toxicology, Clinical Chemistry Department, and 2 Research Forum, Ullevaal University Hospital, N-0407 Oslo, Norway
The effects of Mg2+ on reactive oxygen species
(ROS) and cell Ca2+ 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 H2O2 and
nitric oxide (NO), and DHE/ETH is sensitive to the superoxide anion
(O2
·), respectively. Rapidly exchangeable cell
Ca2+ was determined by 45Ca2+
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 Mg2+
concentration to 5 mM at reoxygenation. ROS levels, measured as ETH
fluorescence, were increased 16-24% during reoxygenation but were
not affected by Mg2+. Cell Ca2+ increased
three- to fourfold during reoxygenation. This increase was reduced 40%
by 5 mM Mg2+, 57% by 10 µM
3,4-dichlorobenzamil (DCB) (inhibitor of
Na+/Ca2+ exchange), and 75% by combining
Mg2+ and DCB. H2O2 (25 and 500 µM) reduced Ca2+ accumulation by 38 and 43%,
respectively, whereas the NO donor S-nitroso-N-acetyl-penicillamine (1 mM) had no
effect. Mg2+ reduced hypoxia/reoxygenation-induced lactate
dehydrogenase (LDH) release by 90%. In conclusion, elevation
of extracellular Mg2+ to 5 mM increased the fluorescence of
the H2O2/NO-sensitive probe DCF without
increasing that of the O2·-sensitive probe ETH,
reduced Ca2+ accumulation, and decreased LDH release during
reoxygenation of hypoxic cardiomyocytes. The reduction in LDH release,
reflecting the protective effect of Mg2+, may be linked to
the effect of Mg2+ on Ca2+ accumulation and/or
ROS levels.
hypoxia; magnesium; calcium; hydrogen peroxide; flow cytometry
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