Oxygen radical-mediated lipid peroxidation and inhibition of Ca 2+-ATPase activity of cardiac sarcoplasmic reticulum

Abstract

Oxygen radicals have been implicated as important mediators of myocardial ischemic and reperfusion injury. A major product of oxygen radical formation is the highly reactive hydroxyl radical via a biological Fenton reaction. The sarcoplasmic reticulum is one of the major target organelles injured by this process. Using a oxygen radical generating system consisting of dihydroxyfumarate and Fe 3+-ADP, we studied lipid peroxidation and Ca 2+-ATPase of cardiac sarcoplasmic reticulum. Incubation of sarcoplasmic reticulum with dihydroxyfumarate plus Fe 3+-ADP significantly inhibited enzyme activity. Addition of superoxide dismutase, superoxide dismutase plus catalase (15μg/ml) or iron chelator, deferoxamine (1.25–1000 μ m) protected Ca 2+-ATPase activity. Time course studies showed that this system inhibited enzyme activity in 7.5 to 10 min. Similar exposure of sarcoplasmic reticulum to dihydroxyfumarate plus Fe 3+-ADP stimulated malondialdehyde formation. This effect was inhibited by superoxide dismutase, catalase, singlet oxygen, and hydroxyl radical scavengers. EPR spin-trapping with 5,5-dimethyl-1-pyroline- N-oxide verified production of the hydroxyl radical. The combination of dihydroxyfumarate and Fe 3+-ADP resulted in a spectrum of hydroxyl radical spin trap adduct, which was abolished by ethanol, catalase, mannitol, and superoxide dismutase. The results demonstrate the role of oxygen radicals in causing inactivation of Ca 2+-ATPase and inhibition of lipid peroxidation of the sarcoplasmic reticulum which could possibly be one of the important mechanisms of oxygen radical-mediated myocardial injury.