M. Mohsen et al., DO NITROXIDES PROTECT CARDIOMYOCYTES FROM HYDROGEN-PEROXIDE OR SUPEROXIDE, Molecular and cellular biochemistry, 145(2), 1995, pp. 103-110
The aim of the research was to study the role played by extracellular
O-2(.-) radicals, which are implicated in cardiac cell damage and the
protective effect by cell-permeable, nitroxide, superoxide dismutase-m
imics. Cardiomyocytes cultures from 1-day-old rats served as the test-
system. Experiments were performed since 5th day in culture when >80%
of the cells were beating myocardial cells. Oxidative damage was induc
ed by 0.5 mM hypoxanthine and 0.06 U/ml xanthine oxidase or by 10 mM g
lucose and 0.15 U/ml glucose oxidase. The parameters used to evaluate
damages were spontaneous beating, lactate dehydrogenase release and AT
P level. The rhythmic pulsation was followed microscopically. To deter
mine the kinetics of cytosolic enzyme release from the cells, media sa
mples were collected at various points of time and assayed for enzyme
activity. To determine the cellular ATP, cells were washed with sodium
phosphate buffer, scraped off and boiled for 3 min with sodium phosph
ate buffer. Following centrifugation the supernatant was collected and
ATP was determined by the chemiluminogenic assay using firefly tails.
The present results indicate that nitroxide stable free radicals, in
the millimolar concentration range, provide full protection without to
xic side-effect. Unlike exogenously added SOD that failed to protect,
exogenous catalase provided almost full protection. In addition, the m
etal-chelating agent dipyridyl, but not diethylene-triamine-pentaaceta
te or desferrioxamine, protected the cultured cells. The present resul
ts suggest that H2O2 is the predominant toxic species mediating the ox
idative damage whereas extracellular superoxide radical does not contr
ibute to cultured cardiomyocyte damage. Since nitroxides do not remove
H2O2 they can protect the cells possibly by oxidizing the metal ions
and inhibiting the Fenton reaction. The superoxide dismutase-mimic act
ivity of nitroxides does not seem to underlie their protective effect,
however, the involvement of intracellular O-2(.-) cannot be excluded.