Peroxynitrite: just an oxidative/nitrosative stressor or a physiological regulator as well?

It is widely accepted now that enhanced peroxynitrite (ONOO⁻) formation contributes to oxidative and nitrosative stress in a variety of cardiovascular and other pathologies (see for reviews: Ferdinandy & Schulz (2001, 2003), Denicola & Radi (2005)). Therefore, targeting ONOO⁻ directly by ONOO⁻ decomposition catalysts and ONOO⁻ scavengers or indirectly by inhibitors of downstream targets of peroxynitrite such as poli(ADP-ribose)-polimerase or matrix metalloproteinases are exciting new strategies for cytoprotection (Salvemini et al., 1998; Ferdinandy et al., 2000; Virag et al., 2003; Giricz et al., 2006). In contrast, increasing evidence suggests that physiological levels of ONOO⁻ may act as a regulator of several physiological functions (Ferdinandy & Schulz, 2001; 2003; Herold & Fago, 2005; Ji et al., 2006). However, still very little is known about the physiological roles of endogenous peroxynitrite formation, possibly due to the number of technical limitations of detecting low, physiological levels of ONOO⁻ in biological systems (Tarpey & Fridovich, 2001; Daiber et al., 2003).

ONOO⁻ is a powerful oxidant species, which can be formed in vivo by the nonenzymatic reaction of nitric oxide (NO) and superoxide anion at an extremely rapid rate limited only by diffusion (Figure 1). At physiological pH, ONOO⁻ is protonated to form peroxynitrous acid which rapidly decomposes forming highly reactive oxidant species especially in the presence of CO₂ (see for review Szabó, 1996). Unfortunately, due to its very short half life at physiological pH, endogenous formation of ONOO⁻ cannot be directly detected in biological systems (Tarpey & Fridovich, 2001; Alvarez & Radi, 2003; Daiber et al., 2003). Although nitration of tyrosine residues is being recognized as a marker for ONOO⁻ formation, the specificity and sensitivity of nitrotyrosine formation, especially in case of physiological rate of ONOO⁻ production, is not sufficient (van der Vliet et al., 1995; Ferdinandy & Schulz, 2001; 2003; Tarpey & Fridovich, 2001). Nitrotyrosine can be formed by ONOO⁻-independent pathways as well, for example, via the actions of peroxidases in the presence of nitrite (Eiserich et al., 1998). Moreover, the exogenous administration of ONOO⁻ in experimental settings (e.g. via the blood) does not accurately reflect the effects of endogenous generation of ONOO⁻ within the cells. Exogenous ONOO⁻ rapidly reacts with plasma proteins and thiols to form the NO donor S-nitrosothiols (see for review Ferdinandy & Schulz (2001)). Thus, ONOO⁻ is likely to be detoxified before it has a chance to reach tissues downstream of the injection site, let alone the intracellular compartment (Ishida et al., 1999). As NO itself is a cardioprotective and antioxidant molecule (Wink et al., 1993; Rubbo et al., 1996; Ferdinandy & Schulz, 2003) tissue protection may be seen when exogenous ONOO⁻ is administered intravenously (Lefer et al., 1997; Nossuli et al., 1997; 1998). Exogenously applied ONOO⁻, however, may show toxic effects when it does not have the opportunity to combine with sulphydryl groups or other antioxidant defenses before reaching its cellular targets. This is dependent upon the concentration of ONOO⁻ and the antioxidant capacity of the cell or tissue of interest. Indeed, ONOO⁻ has been shown to be detrimental to cellular functions when it was applied for example, in crystalloid buffer systems, in which the concentrations of extracellular antioxidants and both free and protein-bound thiols are limited (Schulz et al., 1997; Digerness et al., 1999; Ferdinandy et al., 2000).

Figure 1.

Cellular mechanisms of the actions of NO, superoxide (O₂^−•), and ONOO⁻. NO is an important cardioprotective molecule via its vasodilator, antioxidant, antiplatelet, and antineutrophil actions and it is essential for normal cellular function. However, excess NO could be detrimental if it combines with O₂^−• to form ONOO⁻ which rapidly decomposes to highly reactive oxidant species leading to tissue injury. There is a critical balance between cellular concentrations of NO, O₂^−•, and superoxide dismutase (SOD) which physiologically favor NO production but in pathological conditions such as, for example, ischemia and reperfusion result in ONOO⁻ formation. ONOO⁻ might be converted to NO donors if it combines with SH-group containing molecules (X-SH) to form S-nitroso compounds (X-SNO) including S-nitrosoglutathione. S-nitrosylation and S-glutathiolation are proposed mechanisms by which ONOO⁻ regulates protein functions. Increasing evidence suggests that physiological levels of ONOO⁻ act as regulator of several physiological functions. MMP, matrix metalloproteinase; NOS, NO synthase; PARP, poly-ADP ribose polymerase; XOR, xanthine oxidoreductase; SERCA, sarcoplasmic reticulum Ca²⁺-ATPase; K_ATP, ATP sensitive potassium channel.

In this issue of British Journal of Pharmacology, Graves et al. (2006) show that L-β,β-dimethylcysteine (L-penicillamine), a potential ONOO⁻ scavenger, inhibits the dose-dependent vasodilator responses to moderate doses of peroxynitrite administered repeatedly in vivo. This group has also shown recently that the vasodilator response elicited by exogenous ONOO⁻ involves activation of ATP-sensitive potassium channels (K_ATP) (Graves et al., 2005b). As the glibenclamide-sensitive vasodilator response was still seen after repeated injections of increasing doses of ONOO⁻, when depletion of antioxidants is suspected, ONOO⁻ may open K_ATP independently from generation of S-nitrosothiols (Graves et al., 1998). However, when 10 repeated injections of a high dose of ONOO⁻ (10 μmol kg⁻¹) were administered, a loss of K_ATP function has been observed (Graves et al., 2005a). Vasodilation and opening of K_ATP is not the only potential regulator function of ONOO⁻, where ONOO⁻ is not definitely detrimental to the tissues.

Increasing evidence suggests that ONOO⁻ may act as a regulator of various physiologic cellular functions. Endogenous ONOO⁻ has been shown to trigger ischemic stress adaptation of the rat myocardium (Altug et al., 2000; Csonka et al., 2001; see for review: Ferdinandy & Schulz, 2003), and to activate stress response pathways such as the tyrosine kinase-dependent MAP-kinase and ERK pathways (see for review Klotz et al. (2002)). Cerioni et al. (2006) has recently demonstrated that nontoxic concentrations of peroxynitrite induced mitochondrial translocation of PKC-alpha and activated cell survival pathways in U937 cells. It has been recently shown that activation of microsomal glutathione-S-transferase-1 by peroxynitrite is mediated by nitration of tyrosine residue 92, and represents one of the few examples in which a gain in function has been associated with nitration of a specific tyrosine residue by ONOO⁻ (Ji et al., 2006). Reactions of ONOO⁻ with globins are suspected to play crucial roles in regulating normal physiological responses (see for review Herold & Fago, 2005). Moreover, ONOO⁻ appears to be essential to the reversible S-glutathiolation of sarcoplasmic reticulum Ca²⁺-ATPase, thereby regulating muscle relaxation (Viner et al., 1999; Adachi et al., 2004). ONOO⁻ and NO donors can stimulate myocardial contractility independently of guanylyl cyclase activation, suggesting a role for S-nitrosylation reactions in the positive inotropic effects of NO/peroxynitrite in intact hearts (Paolocci et al., 2000). S-nitrosylation and S-glutathiolation are proposed mechanisms by which ONOO⁻ regulates protein functions, although it should be noted that the role of NO and ONOO⁻ in these reactions is still not clear and little is known about the oxidative actions ONOO⁻ which seems to be more important than the nitrosative effect of ONOO⁻ (Ji et al., 1999; Viner et al., 1999; Okamoto et al., 2001; Steffen et al., 2001). Nevertheless, it is plausible to speculate that ONOO⁻ via its oxidative and nitrosative actions plays an important role in several physiological regulatory mechanisms that is becoming increasingly clear.

In summary, although it is widely accepted that enhanced ONOO⁻ formation is cytotoxic, increasing evidence suggests that physiologic levels of ONOO⁻ contribute to regulation of normal cellular functions. However, due to the numerous limitations of ONOO⁻ detection using the currently available techniques, the conclusions should be drawn cautiously from studies based on ONOO⁻ measurements. The development of more sensitive techniques to detect ONOO⁻ and/or the discovery of specific and sensitive markers for endogenous ONOO⁻ formation at a physiological rate will definitely enhance the exploration of the physiological roles of ONOO⁻.