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Real‐Time Amperometric Analysis of Reactive Oxygen and Nitrogen Species Released by Single Immunostimulated Macrophages
Author(s) -
Amatore Christian,
Arbault Stéphane,
Bouton Cécile,
Drapier JeanClaude,
Ghandour Hala,
Koh Alaric C. W.
Publication year - 2008
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200700746
Subject(s) - peroxynitrite , reactive nitrogen species , reactive oxygen species , nitric oxide , amperometry , chemistry , macrophage , nitric oxide synthase , lipopolysaccharide , nitrite , peroxynitrous acid , superoxide , biochemistry , microbiology and biotechnology , in vitro , biology , immunology , enzyme , nitrate , electrochemistry , organic chemistry , electrode
Macrophages are key cells of the immune system. Immunologically activated macrophages are known to release a cocktail of reactive oxygen and nitrogen species. In this work, RAW 264.7 macrophages were activated by interferon‐γ and lipopolysaccharide, and the reactive mixture released by single cells was analyzed, in real time, by amperometry at platinized carbon microelectrodes. In comparison with untreated macrophages, significant increases in amperometric responses were observed for activated macrophages. Nitric oxide (NO . ), nitrite (NO 2 − ), and peroxynitrite (ONOO − ) were the main reactive species detected. The amounts of these reactive species were quantified, and their average fluxes released by a single, activated macrophage were evaluated. The detection of ONOO − is of particular interest, as its role and implications in various physiological conditions have been widely debated. Herein, direct evidence for the formation of ONOO − in stimulated macrophages is presented. Finally, the presence of 1400W, a selective inducible nitric oxide synthase (iNOS) inhibitor, led to an almost complete attenuation of the amperometric response of activated RAW 264.7 cells. The majority of the reactive species released by a macrophage are thus likely to be derived from NO . and superoxide (O 2 .− ) co‐produced by iNOS.