Abstract
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 Fe2+-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.
Highlights
Altered nitric oxide (NO1⁄7) production is a critical factor in tissue reperfusion injury; controversy remains regarding these alterations and how they cause injury
NO1⁄7 formation is increased during the early period of reflow and reacts with O2. to form ONOO؊, which results in amino acid nitration and cellular injury
Since it was demonstrated previously that there is a burst of O2. generation in the postischemic heart, it was hypothesized that if NO1⁄7 is increased upon reperfusion it might react with this O2., resulting in the formation of the potent cytotoxic oxidant ONOOϪ [6]
Summary
NO1⁄7, nitric oxide; O2., superoxide; ONOOϪ, peroxynitrite; NOS, nitric oxide synthase; MGD, N-methyl-Dglucamine dithiocarbamate; Fe-MGD, ferrous iron complex of MGD; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; SOD, superoxide dismutase; TBS, Tris-buffered saline; L-NAME, N-nitro-L-arginine methyl ester; L-NMMA, NG-methyl-L-arginine. Generation in the postischemic heart, it was hypothesized that if NO1⁄7 is increased upon reperfusion it might react with this O2., resulting in the formation of the potent cytotoxic oxidant ONOOϪ [6]. Increased NO1⁄7 and ONOOϪ has been hypothesized to be a critical biochemical mechanism of postischemic injury in the heart and in other tissues [9, 12, 13], there is only indirect evidence to support this since no direct measurements of NO1⁄7 and ONOOϪ formation during reperfusion have been performed. To assess the functional importance of this process in the pathogenesis of myocardial injury, hemodynamic studies of the recovery of contractile function were performed, in the presence and absence of NOS inhibition or superoxide dismutase (SOD), along with histochemical measurements of the formation of the ONOOϪ-mediated nitration product nitrotyrosine
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