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(The FASEB Journal. 2002;16:1854.)
© 2002 FASEB

Protein tyrosine nitration and peroxynitrite: Reply

Silvia Pfeiffer and Bernd Mayer

Department of Pharmacology and Toxicology arl-Franzens-University Graz A-8010 Graz Austria

IN A RECENT comment in this journal, Deen et al. (1) criticized our conclusions suggesting that peroxynitrite plays no essential role in tyrosine nitration in activated macrophages (2) . The authors argue that the mismatch in the time course of NO and superoxide formation we observed may have been due to fast reaction of superoxide with iNOS-derived NO to form peroxynitrite, such that superoxide was "invisible" in the cytochrome c assay.

Deen et al. apparently have overlooked all other evidence supporting our conclusion, in particular, the insensitivity of in vivo nitration to superoxide scavenging. Even so, their argument would hold only if the sharp decrease in superoxide formation were paralleled by iNOS expression. However, superoxide had already ceased several hours before the expression of iNOS became detectable (measured as release of NO, nitrite accumulation in the cell culture medium, and appearance of iNOS protein in Western blots). This markedly delayed expression of iNOS leaves little chance for the peroxynitrite reaction to occur. This conclusion is further supported by our observation that DHR oxidation, a widely accepted measure for peroxynitrite formation (3) , was not increased over basal at any time of cell activation (up to 48 h). Moreover, albeit not shown for peritoneal macrophages, the presence of the NOS inhibitor L-NAME did not affect superoxide release from cytokine-activated RAW 264.7 macrophages, which exhibited a similar mismatch in the time course of NO and superoxide formation (4) .

Work from at least five independent laboratories demonstrates that peroxynitrite generated at relatively low steady-state concentrations from continuous fluxes of NO/superoxide does not cause significant nitration of free or protein-bound tyrosine (see refs 5 , 6 ). Our paper extends those studies to the in vivo situation and adds another piece to the overwhelming evidence against peroxynitrite as a mediator of tyrosine nitration in inflammatory diseases.

REFERENCES

  1. Deen, W. M., Tannenbaum, S. R., Beckman, J. S. (2002) Protein tyrosine nitration and peroxynitrite: comment. FASEB J 16,1144[Free Full Text]
  2. Pfeiffer, S., Lass, A., Schmidt, K., Mayer, B. (2001) Protein tyrosine nitration in mouse peritoneal macrophages activated in vitro and in vivo: evidence against an essential role of peroxynitrite. FASEB J 15,2355-2364[Abstract/Free Full Text]
  3. Kooy, N. W., Royall, J. A., Ischiropoulos, H., Beckman, J. S. (1994) Peroxynitrite-mediated oxidation of dihydrorhodamine 123. Free Rad. Biol. Med. 16,149-156[CrossRef][Medline]
  4. Pfeiffer, S., Lass, A., Schmidt, K., Mayer, B. (2001) Protein tyrosine nitration in cytokine-activated murine macrophages—involvement of a peroxidase/nitrite pathway rather than peroxynitrite. J. Biol. Chem. 276,34051-34058[Abstract/Free Full Text]
  5. Pfeiffer, S., Schmidt, K., Mayer, B. (2000) Dityrosine formation outcompetes tyrosine nitration at low steady-state concentrations of peroxynitrite—implications for tyrosine modification by nitric oxide/superoxide in vivo. J. Biol. Chem. 275,6346-6352[Abstract/Free Full Text]
  6. Espey, M. G., Xavier, S., Thomas, D. D., Miranda, K. M., Wink, D. A. (2002) Direct real-time evaluation of nitration with green fluorescent protein in solution and within human cells reveals the impact of nitrogen dioxide vs. peroxynitrite mechanisms. Proc. Natl. Acad. Sci. USA 99,3481-3486[Abstract/Free Full Text]




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