Effect of calcium on reactive oxygen species in isolated rat cardiomyocytes during hypoxia and reoxygenation.
It has been suggested that calcium (Ca(2+)) overload and oxidative stress damage the myocardium during ischemia and reperfusion. We investigated the possible effect of varying extracellular Ca(2+)and total cell Ca(2+)on reactive oxygen species (ROS) levels in resting adult rat cardiomyocytes. Cardiomyocytes were isolated by trypsin/collagenase digestion and exposed to 1 h of hypoxia (H) (95% N(2)/5% CO(2), no glucose) and 2 h of reoxygenation (R) (95% air/5% CO(2), glucose 5.5 m M) in suspension. Cell Ca(2+)was measured by uptake of(45)Ca(2+). ROS was measured by flow cytometry of ethidium's red fluorescence formed by oxidation of dihydroethidium mostly by superoxide anion. Cell viability decreased during H and R, expressed as uptake of trypan blue, loss of rod shape morphology and release of lactate dehydrogenase. Rapidly exchangeable cell Ca(2+)was closely correlated with extracellular Ca(2+)concentration. Cell Ca(2+)was unchanged during H but increased three to four times after R. This increase was attenuated by adding 3,4-dichlorobenzamil, 10 microm at R, and amplified by adding ouabain 1 m M (from start), respectively. Levels of ROS in hypoxic cells were unchanged or slightly reduced at the end of H and increased significantly by 20% compared to control after R. Levels of ROS were significantly decreased by lowering total extracellular Ca(2+)from 1 m M to 0.1 m M or by decreasing free extracellular Ca(2+)with EGTA 0.9 m M at the onset of R. Keeping extracellular Ca(2+)constant, ROS levels were neither affected by attenuating the increase in cell Ca(2+)by DCB nor by amplifying the increase in cell Ca(2+)by ouabain. In conclusion, ROS (superoxide anion) levels increase rapidly after reoxygenation, are correlated with extracellular-free Ca(2+)and are reduced by lowering extracellular-free Ca(2+). Levels of ROS are apparently not consistently correlated with total cell Ca(2+).
Sharikabad
M N
MN
Division of Clinical Pharmacology and Toxicology, Ullevaal University Hospital, Oslo, Norway.
Hagelin
E M
EM
Hagberg
I A
IA
Lyberg
T
T
Brørs
O
O
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015687:Cell Hypoxia; D002469:Cell Separation; D048429:Cell Size; D002470:Cell Survival; D008297:Male; D009206:Myocardium / Q000166:cytology*; D010100:Oxygen / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*
D017382:Reactive Oxygen Species; D010100:Oxygen; D002118:Calcium