Measurement of nitric oxide and peroxynitrite generation in the postischemic heart: Evidence for peroxynitrite-mediated reperfusion injury

Altered nitric oxide (NO(·)) production is a critical factor in tissue reperfusion injury; however, controversy remains regarding these alterations and how they cause injury. Since superoxide (O ₂/ ^-·) generation is triggered during the early period of reperfusion the cytotoxic oxidant peroxynitrite (ONOO ^-) could be formed, but it is not known if this occurs. Therefore electron paramagnetic resonance and chemiluminescence studies were performed of the magnitude and time course of NO(·), O ₂/ ^-·, and ONOO ^- formation in the postischemic heart. Isolated rat hearts were subjected either to normal perfusion or to reperfusion after 30 min of ischemia in the presence of the NO(·) trap Fe ²⁺-N-methyl-D-glucamine dithiocarbamate with electron paramagnetic resonance measurements performed on the effluent. Although only trace signals were present prior to ischemia, prominent NO(·) adduct signals were seen during the first 2 min of reflow which were abolished by nitric oxide synthase (NOS) inhibition. Similar studies with the O ₂/ ^-· trap 5,5-dimethyl-1-pyrroline N-oxide demonstrated a burst of O ₂/ ^- · generation over the first 2 min of reflow. Chemiluminescence measurements using 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol) demonstrated a similar marked increase in ONOO ^- which was blocked by NOS inhibitors or superoxide dismutase. NOS inhibition or superoxide dismutase greatly enhanced the recovery of contractile function in postischemic hearts. Immunohistology demonstrated that the ONOO ^--mediated nitration product nitrotyrosine was formed in postischemic hearts but not in normally perfused controls. Thus, NO(·) formation is increased during the early period of reflow and reacts with O ₂/ ^-· to form ONOO ^-, which results in amino acid nitration and cellular injury.