Peroxynitrite and
hydroxyl radical are reactive
oxidants produced during
myocardial reperfusion injury. In various cell types, including macrophages and smooth muscle cells,
peroxynitrite and
hydrogen peroxide cause
DNA single strand breakage, which triggers the activation of the nuclear
enzyme poly (ADP-ribose)
synthetase (PARS), resulting in cytotoxicity. Using
3-aminobenzamide and
nicotinamide, inhibitors of PARS, we investigated the role of PARS in the pathogenesis of myocardial
oxidant injury in H9c2 cardiac myoblasts in vitro.
Peroxynitrite (100-1000 microM),
hydrogen peroxide (0.3-10 microM) and the NO donor compounds S-nitroso-N-accetyl-DL-
penicillamine (SNAP) and diethyltriamine NONOate all caused a dose-dependent reduction of the mitochondrial respiration of the cells, as measured by the mitochondrial-dependent conversion of MTT to
formazan.
Peroxynitrite and
hydrogen peroxide, but not the NO donors caused activation of cellular PARS activity. The suppression of mitochondrial respiration by
peroxynitrite and
hydrogen peroxide, but not by the NO donors, was ameliorated by pharmacological inhibition of PARS. The protection by the PARS inhibitors diminished at extremely high concentrations of the
oxidants.
Hypoxia (1 h) followed by reoxygenation (1-24 h) also resulted in a significant activation of PARS, and caused a suppression of mitochondrial respiration, which was prevented by inhibition of PARS. Similar to the results obtained with the pharmacological inhibitors of PARS, a fibroblast cell line which derives from the PARS knockout mouse was protected against the suppression of mitochondrial respiration in response to
peroxynitrite and reoxygenation, but not to NO donors, when compared to the result of cells derived from wild-type animals. Based on our data, we suggest that activation of PARS plays a role in the myocardial
oxidant injury.