Nitric oxide synthase independent nitric oxide formation occurs in the ischemic heart and causes postischemic injury

We have recently shown that NO generation is greatly increased in ischémie tissues and results in cellular injury. While it has been generally assumed that the formation of NO in biological tissues is solely due to direct synthesis by specific nitric oxide synthase enzymes (NOS), we observe that in ischémie tissues such as the heart, large quantities of NO can be formed by a mechanism which is not enzyme dependent. Electron paramagnetic resonance, EPR, speclroscopy was applied to directly measure the mechanisms of NO generation in the ischémie heart. NO' bound to either intrinsic heme proteins or to the spin trap Fe-MGD gives rise to characteristic triplet signals which enable definitive measurement of NO formed within the heart. While only trace NO triplet signals were seen prior to ischemia, during ischemia these signals greatly increased indicating that marked NO generation occurred. While with short ischémie durations of 30 min or less, inhibition of NOS decreased NO formation, inhibition was incomplete and with increasing durations of ischemia less that 50% inhibition was seen. When hearts were labeled with ¹⁵N nitrite and then subjected to ischemia prominent doublet signals were observed arising from the ¹⁵N coupling to the unpaired electron, demonstrating that during ischemia nitrite was directly reduced giving rise to large amounts of NO. With long periods of ischemia progressing to necrosis this mechanism of NO formation predominated and it was further observed that this NO generation resulted in myocardial injury with a loss of contractile function. The existence of this enzyme independent mechanism of NO formation has important implications in our understanding of the pathogenesis and treatment of tissue injury.