Inhibition of skeletal sarcoplasmic reticulum Ca2+-ATPase activity by deferoxamine nitroxide free radical

Deferoxamine is an inhibitor of iron-dependent free radical reactions. Desp ite the antioxidant roles, prolonged clinical use of the chelator is far fr om benign, and paradoxically, deferoxamine has been shown to promote lipid peroxidation. The possible toxicity of the drug's metabolites, such as defe roxamine nitroxide free radical, deserves attention. We, therefore, tested the hypothesis that deferoxamine nitroxide radicals produced as a result of enzymatic one-electron oxidation of deferoxamine by horseradish peroxidase in the presence of H2O2 are capable of inactivating Ca2+-ATPase of skeleta l sarcoplasmic reticulum microsomes as a model system with which to explore the effect of the radical on a biological membrane. Ca2+-ATPase activity o f sarcoplasmic reticulum was depressed by exposure to Fenton's reagent (H2O 2/FeSO4); the observed effect was significantly enhanced by deferoxamine. W e found that the Fenton reaction produced hydroxyl radical, as determined b y electron spin resonance spectroscopy. The formation of hydroxyl radical w as completely inhibited by deferoxamine; instead, under the same experiment al conditions (in the presence of sarcoplasmic reticulum vesicles with or w ithout FeSO4 but without spin trap 5,5-dimethyl-1-pyrroline N-oxide), the s pectral shape and hyperfine coupling constants of electron spin resonance s ignals confirmed to be long-lived deferoxamine radical were obtained. Furth ermore, exposure of sarcoplasmic reticulum vesicles to deferoxamine radical formed by horseradish peroxidase via reaction with H2O2 caused an inhibiti on of the Ca2+-ATPase activity. The findings show that the sarcoplasmic ret iculum vesicles can act as peroxidases and suggest; that deferoxamine enhan ces the decreased Ca2+-ATPase activity afforded by H2O2/FeSO4 due to format ion of its metabolites, possibly deferoxamine nitroxide free radical.