The aim of this study was to investigate the role of secondary
free radicals and
calpain, a
calcium-activated
cysteine protease, in the development of
reperfusion injury in the heart. The time course of radical generation was assessed directly by Electron Paramagnetic Resonance (EPR) and spin trapping with N-ter butyl-alpha-phenylnitrone (PBN), in isolated perfused rat heart subjected to 30 minutes of global
ischemia and 30 minutes of reperfusion. The effect of
leupeptin, a
calpain inhibitor, was assessed on postischemic dysfunction. The
antioxidant properties of
leupeptin were also investigated by using
allophycocyanin, a fluorescent
protein sensitive to oxidative stress generated by the H2O2 + Cu++ system. Moreover, we measured the capacities of
leupeptin to scavenge
hydroxyl (.
OH) and
superoxide (O2-.) radicals using EPR technique. Our results show that myocardial reperfusion is associated with an increase of alkyl, alkoxyl
free radicals release; the administration of
catalase 5.10(5) UI/L significantly reduces this release, but didn't improve the postischemic contractile function of the heart. In our study
leupeptin 50 microM possess, in vitro,
antioxidant properties and scavenging abilities against .
OH and O2-., in return
leupeptin does not influence the cardiac functions during reperfusion period. In conclusion, our results confirm that myocardial reperfusion induces an important production of secondary
free radicals associated with contractile dysfunction. The role of
calpain in
myocardial ischemia-
reperfusion injury remains to be clarified 1) by assessing the activities of
calpain and calpastain, its main endogenous inhibitor, during these periods, 2) by measuring the ability of
leupeptin in inhibiting the
calpain dependent proteolysis.