Abstract
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.
Highlights
Nitric oxide (NO) is an important gaseous molecule that transmits both intracellular and intercellular signals crucial for cell survival [1]
Effect of Car on NO and nitrite production by stimulated murine macrophage cells Fig. 1a shows the effect of LPS + IFN-γ stimulation on intracellular NO production in cultured macrophages
Results reported in the present study indicate that: i) Car (β-Ala-His), an endogenous dipeptide that is naturally present in several mammalian tissues [1], caused an apparent suppression of total NO production by cultured murine RAW 264.7 macrophages stimulated by LPS + IFN-γ; ii) this effect was specific to Car and was not observed in the presence of its constituent amino acids β-Ala and His; iii) the dose-dependent decrease in NO formation induced by Car in stimulated macrophages was accompanied by a drastic intracellular increase in its low toxic end product, nitrite; iiii) Car modulation of NO was accompanied by the modulation of macrophage activation markers and the secretion of IL-6 cytokine
Summary
Nitric oxide (NO) is an important gaseous molecule that transmits both intracellular and intercellular signals crucial for cell survival [1]. NO is generated in cells by a group of enzymes known as nitric oxide synthase (NOS), which generate NO through the conversion of L-arginine (Arg) to L-citrulline (Cit) [5]. It is well known that there are three major NOS isoforms: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). While nNOS and eNOS are constitutively expressed, iNOS, which is found primarily in glia and macrophages, is overexpressed under a variety of acute and chronic pathological conditions [6] and is a part of the immune response following tissue injury or inflammation [7]
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