Copyright © 2005, European Society of Cardiology
Does peroxynitrite sustain nuclear factor-
B?
Laboratorio de Fisiopatologia Renal, NMCE, Division of Nephrology, FCM, University of Campinas, Cidade Universitaria, Campinas 13083-970, Sao Paulo, Brazil
* Corresponding author. Tel.: +55 19 37887499; fax: +55 19 37887366. Email address: subrata{at}fcm.unicamp.br
Received 4 April 2005; accepted 15 June 2005
We read with interest the article by Hattori et al. [1] published in Cardiovascular Research in July, 2004. The authors showed almost complete suppression of lipopolysaccharide (LPS)-induced nuclear factor-
B (NF-B) activation by nitric oxide (NO) in vascular smooth muscle cells. They showed that this effect of NO was not mediated through the second messenger cGMP or through peroxynitrite, an NO-derived reactive nitrogen species (RNS). Instead, the authors demonstrated sustained activation of LPS-induced NF-B by peroxynitrite. Although the molecular mechanisms were not very clear, we considered this finding as a further step to explain the existing controversies regarding NO-mediated regulation of NF-B activity. Actually, we are trying to explain some of our in vivo findings in the light of this in vitro evidence. However, a detailed report by Park et al. [2] published in March, 2005, compelled us to reanalyze the article by Hattori et al. [1] and to mention some essential points.
Park et al. [2] also showed suppression of NF-B activity (both constitutively active and cytokine-activated) by NO. In striking contrast to the finding of Hattori et al. [1], however, Park et al. showed that NF-B could also be suppressed by peroxynitrite. Actually, Park et al. demonstrated that NO-mediated suppression of NF-B activation was mediated through peroxynitrite by using a peroxynitrite scavenger and also by performing a transfection study. By detailed investigation, Park et al. identified tyrosine nitration of the p65 subunit as the factor responsible for the NO-mediated inactivation of active NF-B. Since peroxynitrite and other RNS play a major role in tyrosine nitration [3,4], the finding of Park et al. seems scientifically conceivable. On the other hand, sustained activation of NF-B by peroxynitrite as shown by Hattori et al. could also be explained by its strong oxidant property [5]. However, this finding of Hattori et al. has become questionable for the following reasons.
The experiments from which Hattori et al. [1] have claimed that both pure peroxynitrite and peroxynitrite donor sustained, rather than suppressed, LPS-induced NF-B activation were not perfectly designed: no control or neutral substance was used in these experiments to show if LPS-induced NF-B activation is normally sustained in the absence of any potential interference. Thus, the sustained activation of NF-B observed with pure peroxynitrite and peroxynitrite donor might be a normal response to LPS and is, therefore, not necessarily an effect of peroxynitrite. As admitted by the authors [1] that high reactivity would consume all added peroxynitrite within seconds of addition to cells, we also are in doubt if the added peroxynitrite was active or effective under the conditions used in the study. This suspicion becomes stronger due to the fact that the yields of nitrotyrosine are very low in in vitro reactions when peroxynitrite is slowly infused or slowly generated from chemical precursors [6,7]. Whether added peroxynitrite, either in pure form or generated from a donor, was optimally effective or not, is, therefore, a pertinent point to consider regarding the study by Hattori et al.
Whether the suppression of NF-B by NO in the study by Hattori et al. [1] was mediated through peroxynitrite could not be confirmed, but the finding of Park et al. [2] leads us to think that peroxynitrite may be the candidate molecule. Since Hattori et al. did not use any peroxynitrite scavenger, it is difficult to ascertain if NO-mediated suppression of NF-B was independent of peroxynitrite in their study.
Hattori et al. [1] also observed suppression of LPS/interferon 
-induced NF-B activation by antioxidants, N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), in addition to NO. From this finding, the authors [1] postulated that the NF-B-suppressing effect of NO might be explained by its antioxidant property through scavenging of superoxide radical, which was also supported by Tritto and Ambrosio [8] in the editorial accompanying the article by Hattori et al. Although this explanation seems scientifically valid, it may not be the case in the study by Hattori et al. because superoxide dismutase, a specific scavenger of superoxide radical, was unable to suppress LPS-induced NF-B activity. However, it is very strange since NAC and PDTC markedly suppressed LPS-induced NF-B activity.
In conclusion, that NO may inactivate active NF-B by tyrosine nitration of p65 through peroxynitrite or other RNS, as shown by Park et al. [2], seems more convincing to us. The partially contradictory finding of Hattori et al. [1] might be explained by considering the suboptimum activity/effect of the peroxynitrite used in their study. This consideration suggests a unified, underlying molecular mechanism, i.e. tyrosine nitration of p65 by RNS, which will need further study to be proved. However, this discussion is important to avoid misleading readers by apparently contradictory findings.
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[Abstract/Free Full Text] - Park S.W., Huq M.D., Hu X., Wei L.N. Tyrosine nitration on p65: a novel mechanism to rapidly inactivate nuclear factor-B. Mol Cell Proteomics (2005) 4:300–309.
[Abstract/Free Full Text] - Beckman J.S. Oxidative damage and tyrosine nitration from peroxynitrite. Chem Res Toxicol (1996) 9:836–844.[CrossRef][Web of Science][Medline]
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[Abstract/Free Full Text] - Jourd'heuil D., Jourd'heuil F.L., Kutchukian P.S., Musah R.A., Wink D.A., Grisham M.B. Reaction of superoxide and nitric oxide with peroxynitrite. Implications for peroxynitrite-mediated oxidation reactions in vivo. J Biol Chem (2001) 276:28799–28805.
[Abstract/Free Full Text] - Hurst J.K. Whence nitrotyrosine? J Clin Invest (2002) 109:1287–1289.[CrossRef][Web of Science][Medline]
- Tritto I., Ambrosio G. The multi-faceted behavior of nitric oxide in vascular "inflammation": catchy terminology or true phenomenon? Cardiovasc Res (2004) 63:1–4.
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