Urate and Tyrosine Nitration by Peroxynitrite in Ischemic Heart

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Abstract 1343 Poster Session IV, Tuesday, 5/4 (poster 221)

The covalent addition of nitro-(NO2) groups to tyrosine residues in proteins occurs rapidly in ischemic and septic hearts. Nitric oxide (NO) itself does not nitrate tyrosine directly, but peroxynitrite formed by the rapid reaction of NO with superoxide readily nitrates tyrosines in vitro. Recent controversies have arisen as to whether superoxide plus NO can nitrate free tyrosine at neutral pH and whether peroxynitrate infused into isolated hearts is protective. We found that the failure to observe tyrosine nitration at neutral pH was due to the generation of urate by xanthine oxidase used as a source of superoxide in previous studies. Using stopped flow spectroscopy, we found that peroxynitrite was formed in 100% yield by NO reacting with potassium superoxide and gave identical nitration yields with tyrosine as authentic peroxynitrite. Addition of physiological concentrations of urate substantially inhibited nitration. Proteins in heart homogenates are more resistant to nitration by peroxynitrite compared the brain or spinal cord due to low molecular weight antioxidants. Thiols and ascorbate account for only a small part of this protection. The formation of urate in hypoxic heart during sample preparation may account for the protection against peroxynitrite-mediated nitration. When peroxynitrite is infused into an isolated perfused heart, little tyrosine nitration is observed because the peroxynitrite decomposed rapidly in the buffer. Decomposition products formed by the reaction of peroxynitrite with urate and with glucose are known to vasodilate the coronary circulation and can account for the weak protection provided by peroxynitrite infusion. Our results indicate that the simultaneous production of superoxide and NO in the heart can produce nitrotyrosine, while the accumulation of urate from xanthine dehydrogenase/oxidase can provides some protection against peroxynitrite. Infusion of peroxynitrite into isolated heart does not mimic endogenous production of superoxide and NO within tissues because of rapid decomposition in perfusion buffers.

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(Spon by: Waldemar A. Carlo)

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