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Fig. 5. EPR spectra of NO trapping in hearts labeled with Fe—MGD. (A) Frozen tissue from a normally perfused heart; (B) frozen tissue from a heart subjected to 30 min of ischemia.

limited solubility in water, therefore, the ferrous iron complex of N-methyl-D-glucamine-dithiocarbamate (MGD), Fe2+—MGD2 (Fe—MGD), was also developed and has been applied for measuring NO in living tissues [66]. Alterations in NO generation had been hypothesized to be a critical cause of injury in the ischemic heart; however, the alterations in NO which occur were unknown. Therefore, we performed EPR studies measuring NO in isolated rat hearts subjected to global ischemia, using Fe—MGD, which binds NO giving rise to a characteristic triplet EPR spectrum with g = 2.04, aN = 13.2 G. While only a small triplet signal was observed in normally perfused, control heart (Fig. 5A), a tenfold increase in this signal occurred after 30 min ischemia indicating an NO formation (Fig. 5B). NO formation increased as a function of the duration of the ischemia [3,65]. With short ischemic durations of 30 min or less, NO generation was decreased by the nitric oxide synthase (NOS) blocker l-NAME [6,7]. Blockade of NO generation with l-NAME also resulted in increased recovery of contractile function after reperfusion [3]. At 1 mM concentration, l-NAME, totally inhibits the enzyme. Infusion of 1 mM l-NAME into normally perfused hearts also caused maximum depression of the coronary flow. Even though l-NAME totally blocked NOS-mediated vasorelaxation, formation of the NO triplet signal seen in ischemic hearts after 30 min or longer was only partially inhibited, with a 60—80% decrease. With higher l-NAME concentrations or with l-NMMA, no further inhibition occurred. This lack of total inhibition of NO formation with blockade of NOS suggested the existence of a NOS independent pathway of NO formation [1,40,65]. To confirm this, direct measurements of NO formation via its binding to intrinsic met allo-heme centers within the tissue were also performed. The binding of NO to heme proteins gives rise to a unique EPR spectrum with axial symmetry and with a 5-coordinate complex, a characteristic inverted triplet is seen due to the hyperfine coupling of the nitrogen nucleus of bound NO. These complexes are inherently O2 labile and are best observed at low O2 tensions as occur with prolonged ischemia. In control hearts, no NO-heme signal was seen; however, in ischemic tissue after 2—12 h, increasingly prominent NO-heme signal was seen, confirming that increased NO formation occurs during myocardial ischemia. Pre-treatment of hearts with l-NAME partially decreased these NO signals with only a 30-50% decrease after 4-8 h of ischemia, further supporting the existence of a NOS-independent pathway of NO generation. Myocardial ischemia results in intracellular acidosis and severe hypoxia leading to a highly reduced state that could cause nitrite reduction to NO. To determine if nitrite, NO-, was reduced during ischemia to form NO, experiments were performed measuring NO with Fe-MGD in hearts subjected to ischemia in the presence of isotopically labeled 15NO- [7]. Since 15N has a nuclear spin of 1/2, doublet hyperfine splitting will be observed in the EPR spectra of NO complexes instead of the triplet splitting observed for the natural abundance 14N that has a nuclear spin of 1. In the normally perfused control hearts, 15NO- did not result in significant NO formation, however in labeled hearts which were subjected to 30 min of ischemia, marked 15NO formation was seen. In matched experiments with natural abundance 14NO-, large 14NO triplet signals were seen. Thus, NO- is reduced to NO in the ischemic heart. Nitrosyl-heme formation was also measured in ischemic hearts labeled with 1 mM 15NO-. A prominent doublet nitrosyl-heme signal was seen due to the formation and binding of 15NO to these proteins, further confirming that NO is generated from NO- (Fig. 6B). With 14NO- , a similar magnitude triplet 14NO signal was observed. Further experiments performed measuring the concentration of nitrite within the heart prior

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Fig. 6. EPR spectra of the nitroso-heme complexes formed in heart tissue after 8 h of ischemia in the presence of 1 mM 14NO- (A) or 15NO- (B). In the presence of 14NO-, a prominent triplet nitroso-heme signal is seen (A), while in the presence of 15NO- , a doublet signal is observed (B), indicating that the NO formation was directly derived from nitrite.

to ischemia, using an NO analyzer [7,35], showed that relatively large nitrite concentrations of 12 ^M, were present.

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