Excess
nitric oxide (NO) production occurs in several pathological states, including neurodegeneration,
ischemia, and
inflammation, and is generally accompanied by increased oxidative/nitrosative stress.
Carnosine [β-
alanine-
histidine (β-
Ala-His)] has been reported to decrease oxidative/nitrosative stress-associated cell damage by reducing the amount of NO produced. In this study, we evaluated the effect of
carnosine on NO production by murine RAW 264.7 macrophages stimulated with lipopolysaccharides + interferon-γ. Intracellular NO and intracellular and extracellular
nitrite were measured by microchip electrophoresis with
laser-induced fluorescence and by the Griess assay, respectively. Results showed that
carnosine causes an apparent suppression of total NO production by stimulated macrophages accompanied by an unexpected simultaneous drastic increase in its intracellular low toxicity endproduct,
nitrite, with no inhibition of
inducible nitric oxide synthase (iNOS). ESI-MS and NMR spectroscopy in a cell-free system showed the formation of multiple adducts (at different ratios) of
carnosine-NO and
carnosine-
nitrite, involving both constituent
amino acids (β-Ala and His) of
carnosine, thus providing a possible mechanism for the changes in free NO and
nitrite in the presence of
carnosine. In stimulated macrophages, the addition of
carnosine was also characterized by changes in the expression of macrophage activation markers and a decrease in the release of
IL-6, suggesting that
carnosine might alter M1/M2 macrophage ratio. These results provide evidence for previously unknown properties of
carnosine that modulate the NO/
nitrite ratio of stimulated macrophages. This modulation is also accompanied by changes in the release of pro-inflammatory molecules, and does not involve the inhibition of iNOS activity.