Peroxynitrite, control

Not all oxidants formed biolc cally have the potential to promote lipid peroxidation. The free radicals superoxide and nitric oxide [or endothelium-derived relaxing aor (EDRF)] are known to be formed in ww but are not able to initiate the peroxidation of lipids (Moncada et tU., 1991). The protonated form of the superoxide radical, the hydroperoxy radical, is capable of initiating lipid peroxidation but its low pili of 4.5 effectively precludes a major contribution under most physiological conditions, although this has been suggested (Aikens and Dix, 1991). Interestingly, the reaction product between nitric oxide and superoxide forms the powerful oxidant peroxynitrite (Equation 2.6) at a rate that is essentially difiiision controlled (Beckman eta/., 1990 Huie and Padmaja, 1993). 

Reaction of nitric oxide with superoxide is undoubtedly the most important reaction of nitric oxide, resulting in the formation of peroxynitrite, one of the main reactive species in free radical-mediated damaging processes. This reaction is a diffusion-controlled one, with the rate constant (which has been measured by many workers, see, for example, Ref. [41]), of about 2 x 109 1 mol-1 s-1. Goldstein and Czapski [41] also measured the rate constant for Reaction (11) ... 

Simultaneous generation of nitric oxide and superoxide by NO synthases results in the formation of peroxynitrite. As the reaction between these free radicals proceeds with a diffusion-controlled rate (Chapter 21), it is surprising that it is possible to detect experimentally both superoxide and NO during NO synthase catalysis. However, Pou et al. [147] pointed out that the reason is the fact that superoxide and nitric oxide are generated consecutively at the same heme iron site. Therefore, after superoxide production NO synthase must cycle twice before NO production. Correspondingly, there is enough time for superoxide to diffuse from the enzyme and react with other biomolecules. 

FIGURE 7.9 (See color insert) Peroxynitrite production by activated macrophages. Cells isolated from control (CTL) and toxicant (TOX)-treated animals were cultured overnight in the presence of IFNa + LPS. Phorbol myristate acetate was then added. Thirty minutes later, the cells were loaded with DHR 123. After 5 minutes incubation, the cells were rinsed and analyzed for fluorescence associated with peroxynitrite production by confocal microscopy. 

It has now been more than a decade since Beckman and his collaborators first disclosed their observations that the combination of two relatively unreactive, yet biologically relevant free radicals, superoxide anion and nitric oxide, would produce a new highly reactive physiologically important reagent. The interaction of these two presumably innocuous species appears to be diffusion controlled and produces a thermally stable peroxy anion, peroxynitrite (equation 1). ... 

The iron responsive element, a critical factor in the control of proteins involved in iron utilization, has been identified as the cytoplasmic form of the iron-sulfur protein aconitase (Kennedy et al., 1992). Activated macrophages have been shown to activate this element, presumably by attack of the iron-sulfur cluster by NO (Drapier et al., 1993). It has been claimed that this attack is mediated by peroxynitrite (Castro et al., 1994 Hausladen and Fridovich, 1994, but this conclusion is not universally accepted. 

As noted earlier, peroxynitrite is formed with a diffusion-controlled rate from superoxide and nitric oxide (Reaction 10). As both these radicals are ubiquitous species, which present practically in all cells and tissues, peroxynitrite can be the most important species responsible for free radical-mediated damage in biological systems. Moreover, it is now known that NO synthases are capable of producing superoxide and nitric oxide simultaneously (see Chapter 22), greatly increasing the possible rate of peroxynitrite production. In addition, another enzyme xanthine dehydrogenase is also able to produce peroxynitrite in the presence of nitrite... 

The yellow ONOO- ion can be made in pure form by passing NO through a solution of tetramethylammonium superoxide in ammonia56 ONOO from other syntheses can be contaminated by reactants and nitrate. ONOOH is a weak acid with a pKa near 6.8 and isomerizes to N03 and H+ in seconds.57 At pH 12 the anion is stable for several hours at 0°C. It occurs solely as the cis isomer, due to a partial double bond between N and the first peroxide O.58 Peroxynitrite is toxic in vivo it is formed through the diffusion-controlled reaction of 02 with NO-.57 ONOOH oxidizes via direct and indirect pathways.59 Under physiological conditions the main reaction partners for ONOO" and ONOOH appear to be carbon dioxide60 and thiols,61 respectively. The ONOO—C02—adduct nitrates tyrosines. 

Relaxation of blood vessels appears to be at least partially under the control of endothelial cells and their secreted products, especially endothelium-derived relaxation factor (EDRF). Oxidized LDL directly inhibits the endothelial cell-associated vessel relaxation. The generation of increased reactive oxygen species in association with elevated levels of blood cholesterol has also been reported. One of these reactive oxygen species, superoxide (O2), may interact with vasoactive EDRF (nitric oxide) locally in the artery wall, preventing endothelial cell-dependent vasodilation. In addition, a product of the reaction of nitric oxide and superoxide, the reactive peroxynitrite, may act to stimulate lipoprotein oxidation, which, as noted above, is regarded as an early step in atherosclerotic plaque generation. 

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