Molecular and Cellular Biochemistry

, Volume 431, Issue 1–2, pp 197–210 | Cite as

Carnosine modulates nitric oxide in stimulated murine RAW 264.7 macrophages

  • Giuseppe Caruso
  • Claudia G. Fresta
  • Francisco Martinez-Becerra
  • Lopalco Antonio
  • Ryan T. Johnson
  • Richard P. S. de Campos
  • Joseph M. Siegel
  • Manjula B. Wijesinghe
  • Giuseppe Lazzarino
  • Susan M. Lunte
Article

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.

Keywords

Nitric oxide Nitrite Carnosine Microchip electrophoresis Macrophages Cytokines secretion 

Abbreviations

Arg

l-Arginine

β-Ala

β-Alanine

BSA

Bovine serum albumin

Car

Carnosine

Cit

l-Citrulline

CD86

Cluster of differentiation 86

CD206

Cluster of differentiation 206

CID

Collision-induced dissociation

DAF-FM DA

4-Amino-5-methylamino-2′,7′-difluorofluorescein diacetate

DEA/NO

Diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate

DMEM

Dulbecco’s modified Eagle’s medium

DMSO

Dimethyl sulfoxide

ESI-MS

Electrospray mass spectrometry

FBS

Fetal bovine serum

FMO

Fluorescence minus one

His

l-Histidine

IFN-γ

Interferon-γ

IL-1β

Interleukin-1β

IL-2

Interleukin-2

IL-4

Interleukin-4

IL-5

Interleukin-5

IL-6

Interleukin-6

IL-10

Interleukin-10

IL-12

Interleukin-12

iNOS

Inducible nitric oxide synthase

KC

Keratinocyte chemoattractant

LPS

Lipopolysaccharides

ME-LIF

Microchip electrophoresis with laser-induced fluorescence

MHCII

Major histocompatibility complex class II

NMR

Nuclear magnetic resonance

NO

Nitric oxide

NO2

Nitrite

PBS

Phosphate-buffered saline

PDMS

Polydimethylsiloxane

RNS

Reactive nitrogen species

ROS

Reactive oxygen species

SDS

Sodium dodecyl sulfate

TNF-α

Tumor necrosis factor α

Supplementary material

11010_2017_2991_MOESM1_ESM.pdf (324 kb)
Supplementary material 1 (PDF 324 KB)

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Giuseppe Caruso
    • 1
    • 2
  • Claudia G. Fresta
    • 1
    • 2
  • Francisco Martinez-Becerra
    • 2
    • 3
  • Lopalco Antonio
    • 2
  • Ryan T. Johnson
    • 4
  • Richard P. S. de Campos
    • 1
    • 2
    • 5
  • Joseph M. Siegel
    • 1
    • 2
  • Manjula B. Wijesinghe
    • 1
    • 2
  • Giuseppe Lazzarino
    • 6
  • Susan M. Lunte
    • 1
    • 2
    • 4
  1. 1.Ralph N. Adams Institute for Bioanalytical ChemistryUniversity of KansasLawrenceUSA
  2. 2.Department of Pharmaceutical ChemistryUniversity of KansasLawrenceUSA
  3. 3.Immunology Core Laboratory of the Kansas Vaccine InstituteUniversity of KansasLawrenceUSA
  4. 4.Department of ChemistryUniversity of KansasLawrenceUSA
  5. 5.Department of ChemistryState University of CampinasCampinasBrazil
  6. 6.Division of Medical Biochemistry, Department of Biomedical and Biotechnological SciencesUniversity of CataniaCataniaItaly

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