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Do nitroxides protect cardiomyocytes from hydrogen peroxide or superoxide?

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Abstract

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-mimics. 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 induced by 0.5 mM hypoxanthine and 0.06 U/ml xanthine oxidase or by 10 mM glucose and 0.15 U/ml glucose oxidase. The parameters used to evaluate damages were spontaneous beating, lactate dehydrogenase release and ATP level. The rhythmic pulsation was followed microscopically. To determine the kinetics of cytosolic enzyme release from the cells, media samples 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 phosphate 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 toxic side-effect. Unlike exogenously added SOD that failed to protect, exogenous catalase provided almost full protection. In addition, the metal-chelating agent dipyridyl, but not diethylene-triamine-pentaacetate or desferrioxamine, protected the cultured cells. The present results suggest that H2O2 is the predominant toxic species mediating the oxidative damage whereas extracellular superoxide radical does not contribute 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 activity of nitroxides does not seem to underlie their protective effect, however, the involvement of intracellular O .-2 cannot be excluded.

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Abbreviations

CHDO:

2-spirocyclohexane doxyl (2-cyclohexane-5,5-demethyl-3-oxazolidinoxyl)

DF:

desferrioxamine

DTPA:

diethylene-triamine-pentaacetate

EPR:

electron paramagnetic resonance

HX:

hypoxanthine

LDH:

lactate dehydrogenase

SOD:

superoxide dismutase

SEM:

standard error of mean: TEMPOL, 4-hydroxy-2,2,6,6-tetramethyl-piperidinoxyl

TEMPAMINE:

4-amino-2,2,6,6-tetramethyl-piperidinoxyl

XO:

xanthine oxidase

CAT:

catalase

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Authors and Affiliations

  1. Department of Molecular Biology, Hebrew University-Hadassah Medical School, 91120, Jerusalem, Israel

    Mohammad Mohsen, Renliang Zhang & Amram Samuni

  2. Laboratory for Myocardial Research, Hebrew University-Hadassah Medical School, 91120, Jerusalem, Israel

    Arié Pinson

Authors
  1. Mohammad Mohsen
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  2. Arié Pinson
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  3. Renliang Zhang
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  4. Amram Samuni
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Mohsen, M., Pinson, A., Zhang, R. et al. Do nitroxides protect cardiomyocytes from hydrogen peroxide or superoxide?. Mol Cell Biochem 145, 103–110 (1995). https://doi.org/10.1007/BF00935482

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  • DOI: https://doi.org/10.1007/BF00935482

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