I250 1 I 251 I TYROSINE NITRATION IS NEGLIGIBLE AT LOW PEROXYNITRITE STEADY-STATE LEVELS

r, Kurt Schmidt and Bemd Mayer

Institut fiir Pharmakologie und Toxikologie, Karl-Franzens- Universitxt Graz, Universititsplatz 2, A-8010 Graz, Austria

It is generally assumed that cellular tyrosine nitration is mediated by peroxynitrite, which is formed in a very rapid reac- tion from NO and 02’-. However, we have recently shown that NO and 0~‘~ generated simultaneously at equal initial rates from spermine NONOate and hypoxanthine/xanthine oxidase, respec- tively, exhibit much lower nitrating efficiency than authentic peroxynitrite’. The present study extends those earlier findings to several other NO/w--generating systems and provides evidence that the apparent lack of tyrosine nitration by NO/Oz’- is due to a pronounced decrease of nitration yields at low steady-state con- centrations of authentic peroxynitritc. The results demonstrate that very high fluxes of NO/O2’- (> 2 @l/s) are required to render peroxynitrite an efficient trigger of tyrosine nitration. Although such high fluxes of NO/O2’- are unlikely to occur normally in cells, local bursts of NO/O2’- could give rise to sufficiently high steady-state concentrations of peroxynitrite in certain cellular compartments, making the NO/Oi- pathway biologically relevant for protein nitration in certain pathophysiological conditions.

‘Pfeiffer, S. & Mayer, B., J. Biol. Chem. 273, 27280-5, 1998

EPR SPECTROSCOPIC EVIDENCE OF ALTERED MYOGLOBIN STATES IN CARDIAC ALLOGRAFI REJECTION. Cnle?l M Pieper. AIlan M. Roza, Christopher C. Felix, Mark B. Adnms,Ching-Snn La, Medical College of Wisconsin, Milwaukee, WI &-r Medmm, Inc., Snn Dqo, CA

Oxidation of ferrous myoglobin (Mb) to ferric Mb (met-Mb) or nitrosylation to NO-Mb results in the inability to bind oxygen. Using EPR spectroscopy, we evaluated potential molecular targets of nitric oxide (NO) in a rat model of cardiac rejection. We confirmed iNOS gene expression by immunostaining. Only background EI’R signals were observed at g=2.02 and g=1.94 (reduced Fe-S clusters) and g=2.004 (semiquinone) in normal, isografts or native hearts of allograft recipients. In contrast for allografts at postoperative day (POD) #4 to 6, there was a progressive increase in an EPR signal at g=2.08 (NO-Mb) and a triplet at g=2.014 with 17.5 G hyperfine splitting (nitrosoheme). These signals occurred prior to rejection at POD #7 and were never seen in isografts or native recipient hearts. An additional signal at g=6.0 consistent with high spin met-Mb was revealed in 4000 Gauss scans. The met-Mb:NO-Mb signal ratio was greater at POD #4 vs. #6 suggesting an early phase of oxidative stress. Chronic treatment with NOX-101, an iron-thiol-based NO chelator, decreased plasma nitrate + nitrite indicating in vivo NO scavenging and decreased met-Mb and NO-Mb formation. A single NOX-101 injection failed to decrease both Mb forms indicating no direct action. NO-Mb and met-Mb were eliminated by low-dose cyclosporine (CsA) treatment or by a short-term combination of CsA + NOX-101. This combination therapy resulted in a profound synergistic increase in graft survival vs. either agent alone. Long-term combination therapy (POD #l-100) followed by discontinuing drugs resulted in permanent graft acceptance with no evidence for either met-Mb or NO-Mb at POD #450. Our findings suggest that both oxidative and nitrosative stress targeting Mb are implicated in cardiac allograft rejection.

PREVENTION BY HEMOGLOBIN OF PEROXYNITRITE-MEDIATED TYROSYNE NITRATIONS

iuseppe Scorzn and Maurizio Minetti. Istituto Superiorr

Hemoglobin in erythrocytes is an important target of peroxynitrite generated in blood. Using direct ESR at 37-C and spin trapping techniques, we showed that peroxynitrite reacts with oxyhemoglobin (oxyHb) in a direct bimolecular reaction oxidizing the heme to a ferry1 species and forming long-lived globin tyrosyl radicals. Owing to the high concentration of Hb, this new pathway may be of dominating importance in vivo and may represent the major pathway of peroxynitrite detoxification. To test this hypothesis, we measured the Ht-dependent inhibition of nitrotyrosine formation. Our results showed that oxyHb inhibits in a dose-dependent manner the peroxynitrite-dependent nitration of the dipeptide Ala-Tyr. Notably, a complete inhibition of nitrotyrosine formation was obtained at 1:l herne/peroxynitrite ratio or at higher ratios. Moreover, oxyHb entrapped inside resealed ghosts afforded a strong inhibition of peroxynitrite-mediated tyrosine nitration of membrane proteins. This effect was coupled with Hb nitration and a week formation of non -SH dependent Hb dimers. We conclude that the oxyHb reaction may be an important pathway to remove peroxynitrite from the vasculature and to prevent tyrosine nitration of erythrocyte proteins.

MECHANISMS OF NITRIC OXIDE’S ANTIOXIDANT AND PROOXIDANT EFFECTS ON OXIDATIVE KILLING OF CELLS 1~1,~ L Pm* Makesk S. Joskz and lack R. Lancaster, \r. LSUMC, New Orlean’s, LA.

The effect of nitric oxide (NO) on oxidant-mediated toxicity was investigated in a rat hepatoma cell line to study the ‘Janus-faced’actions of NO as both an oxidant and an antioxidant. Oxidant-mediated cell injury and death has been shown to involve various mechanisms of insult including lipid peroxidation, mitochondrial dysfunction, glutathione depletion, and DNA damage. Cells treated with tert-butyl hydroperoxide (tBH), hydrogen peroxide (H,O,) or glucose oxidase (GO) in combination with a range of NO concentrations results in a biphasic killing pattern as measured by lactate dehydrogenase release. Lower NO levels protect against cell killing and higher NO levels augment killing without causing toxicity alone. The mechanisms of killing by the oxidant alone and in the presence of high NO appear to involve different death pathways. Killing by the oxidant alone is prevented by a lipid radical scavenger (N,N’-diphenyl-p-phenylenediamine, DPPD), an iron chelator, and peroxynitrite scavengers. Protection by the putative peroxynitrite inhibitors suggests an antioxidant role for these compounds since NO is not involved. Therefore killing by either tBH, H,O, or GO is iron and lipid radical-mediated. However, the mechanism of killing by the oxidant with high NO involves different pathways as only the proposed poly (ADP-ribose) synthetase (PARS) inhibitor, 3-aminobenzamide, protects from killing.

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