Direct measurement of nitric oxide generation in the ischemic heart using electron paramagnetic resonance spectroscopy

Nitric oxide, NO^., exerts numerous important regulatory functions in biological tissues and has been hypothesized to have a role in the pathogenesis of cellular injury in a number of diseases. It has been suggested that alterations in NO^. generation are a critical cause of injury in the ischemic heart. However, the precise alterations in NO^. generation which occur are not known, and there is considerable controversy regarding whether myocardial ischemia results in increased or decreased NO^. formation. Therefore, electron paramagnetic resonance studies were performed to directly measure NO^. in isolated rat hearts subjected to global ischemia, using the direct NO^. trap Fe²⁺-N-methyl-D-glucamine dithiocarbamate, which specifically binds NO^. giving rise to a characteristic triplet EPR spectrum with g = 2.04 and a(N) = 13.2 G. While only a small triplet signal was observed in normally perfused hearts, a 10-fold increased in this triple EPR spectrum was observed after 30 min of ischemia indicating a marked increase in NO^. formation and trapping. Measurements were performed as a function of the duration of ischemia, and it was determined that with increased duration of ischemia NO^. formation and trapping was also increased. NO^. generation was inhibited by the nitric oxide synthase blocker, N-nitro-L-arginine methyl ester (L-NAME), suggesting that NO^. was generated via nitric oxide synthase. Blockade of NO^. generation with L-NAME resulted in more than a 2-fold increase in the recovery of contractile function in hearts reperfused after 30 min of global ischemia. Thus, ischemia causes a marked duration-dependent increase of NO^. in the heart which may in turn mediate postischemic injury.