Life imaging of peroxynitrite in rat microglial and astroglial cells: Role of superoxide and antioxidants.

Free radicals, such as superoxide and nitric oxide, are known to play a role in a number of inflammatory and degenerative brain diseases, in which resident microglia upregulate the inducible nitric oxide synthase (iNOS) and thus produce large amounts of nitric oxide. Simultaneously, microglia generate superoxide mainly via NADPH-oxidase, which reacts at a diffusion-limited rate with nitric oxide to form the powerful oxidant peroxynitrite. We used mixed astroglial/microglial cultures to study the effects of iNOS induction by lipopolysaccharide and interferon-gamma on free radical formation. Using the fluorogenic compound 2,7-dihydrodichlorofluorescein diacetate, we monitored cellular peroxynitrite formation by confocal laser microscopy. Peroxynitrite formation in continuously nitric oxide-producing microglial cells was rather limited. However, activation of the superoxide-generating enzyme NADPH-oxidase dramatically increased DCF fluorescence within a few minutes. We conclude that superoxide is the limiting factor for peroxynitrite formation. Since the formation and oxidant activity of peroxynitrite depends strongly on the availability of cellular antioxidants, we investigated the capacity of several compounds to influence peroxynitrite formation. Among the substances under investigation in this study, glutathione and the synthetic compound ebselen had a major effect on preventing peroxynitrite formation, whereas ascorbate failed to decrease peroxynitrite levels.