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Microchip electrophoresis with laser-induced fluorescence detection for the determination of the ratio of nitric oxide to superoxide production in macrophages during inflammation.

ABSTRACT: It is well known that excessive production of reactive oxygen and nitrogen species is linked to the development of oxidative stress-driven disorders. In particular, nitric oxide (NO) and superoxide (O2•-) play critical roles in many physiological and pathological processes. This article reports the use of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate and MitoSOX Red in conjunction with microchip electrophoresis and laser-induced fluorescence detection for the simultaneous detection of NO and O2•- in RAW 264.7 macrophage cell lysates following different stimulation procedures. Cell stimulations were performed in the presence and absence of cytosolic (diethyldithiocarbamate) and mitochondrial (2-methoxyestradiol) superoxide dismutase (SOD) inhibitors. The NO/O2•- ratios in macrophage cell lysates under physiological and proinflammatory conditions were determined. The NO/O2•- ratios were 0.60?±?0.07 for unstimulated cells pretreated with SOD inhibitors, 1.08?±?0.06 for unstimulated cells in the absence of SOD inhibitors, and 3.14?±?0.13 for stimulated cells. The effect of carnosine (antioxidant) or Ca2+ (intracellular messenger) on the NO/O2•- ratio was also investigated. Graphical Abstract Simultaneous detection of nitric oxide and superoxide in macrophage cell lysates.

SUBMITTER: Caruso G

PROVIDER: S-EPMC5525329 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Microchip electrophoresis with laser-induced fluorescence detection for the determination of the ratio of nitric oxide to superoxide production in macrophages during inflammation.

Caruso Giuseppe G Fresta Claudia G CG Siegel Joseph M JM Wijesinghe Manjula B MB Lunte Susan M SM

Analytical and bioanalytical chemistry 20170529 19

It is well known that excessive production of reactive oxygen and nitrogen species is linked to the development of oxidative stress-driven disorders. In particular, nitric oxide (NO) and superoxide (O<sub>2</sub><sup>•-</sup>) play critical roles in many physiological and pathological processes. This article reports the use of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate and MitoSOX Red in conjunction with microchip electrophoresis and laser-induced fluorescence detection for the si ...[more]

PMID: 28555342

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Project description:Initiation of nitric oxide (NO.)-mediated apoptotic cell death in RAW 264.7 macrophages is associated with up-regulation of mitochondrial manganese superoxide dismutase (MnSOD; SOD2) and down-regulation of cytosolic copper zinc superoxide dismutase (CuZnSOD; SOD1) at their individual mRNA and protein levels. To evaluate the decreased CuZnSOD expression and the initiation of apoptosis we stably transfected macrophages to overexpress human CuZnSOD. Individual clones revealed a 2-fold increase in CuZnSOD activity. Expression of a functional and thus protective CuZnSOD was verified by attenuated superoxide (O2(.)-)-mediated apoptotic as well as necrotic cell death. In this study we showed that SOD-overexpressing macrophages (R-SOD1-12) were also protected against NO.-initiated programmed cell death. Protection was substantial towards NO. derived from exogenously added NO donors or when NO. was generated by inducible NO synthase activation, and was evident at the level of p53 accumulation, caspase activation and DNA fragmentation. Stimulation of parent and SOD-overexpressing cells with a combination of lipopolysaccharide and murine interferon gamma produced equivalent amounts of nitrite/nitrate, which ruled out attenuated inducible NO. synthase activity during protection. Because protection by a O2(.)--scavenging system during NO. -intoxication implies a role of NO. and O2(.)- in the progression of cell damage, we used uric acid to delineate the role of peroxynitrite during NO.-elicited apoptosis. The peroxynitrite scavenger uric acid left S-nitrosoglutathione or spermine-NO-elicited apoptosis unaltered, blocking only 3-morpholinosydnonimine-mediated cell death. As a result we exclude peroxynitrite from contributing, to any major extent, to NO. -mediated apoptosis. Therefore protection observed with CuZnSOD overexpression is unlikely to stem from interference with peroxynitrite formation and/or action. Unequivocally, the down-regulation of CuZnSOD is associated with NO. cytotoxicity, whereas CuZnSOD overexpression protects macrophages from apoptosis.

Dataset Information

Microchip electrophoresis with laser-induced fluorescence detection for the determination of the ratio of nitric oxide to superoxide production in macrophages during inflammation.

Publications

Microchip electrophoresis with laser-induced fluorescence detection for the determination of the ratio of nitric oxide to superoxide production in macrophages during inflammation.

Similar Datasets

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