title: Carnosine modulates nitric oxide in stimulated murine RAW 264.7 macrophages.
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.
authors.affiliation: Ralph N. Adams Institute for Bioanalytical Chemistry, University of Kansas, Lawrence, KS, USA.|Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Caruso
authors.affiliation: Ralph N. Adams Institute for Bioanalytical Chemistry, University of Kansas, Lawrence, KS, USA.|Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA.
authors.forename: Claudia G
authors.initials: CG
authors.lastname: Fresta
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA.|Immunology Core Laboratory of the Kansas Vaccine Institute, University of Kansas, Lawrence, KS, USA.
authors.forename: Francisco
authors.initials: F
authors.lastname: Martinez-Becerra
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA.
authors.forename: Lopalco
authors.initials: L
authors.lastname: Antonio
authors.affiliation: Department of Chemistry, University of Kansas, Lawrence, KS, USA.
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Johnson
authors.affiliation: Ralph N. Adams Institute for Bioanalytical Chemistry, University of Kansas, Lawrence, KS, USA.|Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA.|Department of Chemistry, State University of Campinas, Campinas, Brazil.
authors.forename: Richard P S
authors.initials: RPS
authors.lastname: de Campos
authors.affiliation: Ralph N. Adams Institute for Bioanalytical Chemistry, University of Kansas, Lawrence, KS, USA.|Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Siegel
authors.affiliation: Ralph N. Adams Institute for Bioanalytical Chemistry, University of Kansas, Lawrence, KS, USA.|Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA.
authors.forename: Manjula B
authors.initials: MB
authors.lastname: Wijesinghe
authors.affiliation: Division of Medical Biochemistry, Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy. lazzarig@unict.it.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Lazzarino
authors.affiliation: Ralph N. Adams Institute for Bioanalytical Chemistry, University of Kansas, Lawrence, KS, USA. slunte@ku.edu.|Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA. slunte@ku.edu.|Department of Chemistry, University of Kansas, Lawrence, KS, USA. slunte@ku.edu.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Lunte
mesh_terms: D000818:Animals; D002336:Carnosine / Q000494:pharmacology*; D007371:Interferon-gamma / Q000494:pharmacology; D015850:Interleukin-6 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D000067996:RAW 264.7 Cells
chemical_list: C000611649:IFNG protein, mouse; D015850:Interleukin-6; D008070:Lipopolysaccharides; C577686:interleukin-6, mouse; D009569:Nitric Oxide; D007371:Interferon-gamma; D002336:Carnosine; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse
keywords: Carnosine; Cytokines secretion; Macrophages; Microchip electrophoresis; Nitric oxide; Nitrite