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Updated: May 1, 2026
Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells
Published on: March 16, 2017
Inducible nitric oxide synthase is key to peroxynitrite-mediated, LPS-induced protein radical formation in murine
Ashutosh Kumar1, Shih-Heng Chen2, Maria B Kadiiska1
1Free Radical Metabolism Group, Laboratory of Toxicology & Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Abstract:
Microglia are the resident immune cells in the brain. Microglial activation is characteristic of several inflammatory and neurodegenerative diseases including Alzheimer's disease, multiple sclerosis, and Parkinson's disease. Though lipopolysaccharide (LPS)-induced microglial activation in models of Parkinson's disease is well documented, the free radical-mediated protein radical formation and its underlying mechanism during LPS-induced microglial activation are not known. Here we have used immuno-spin trapping and RNA interference to investigate the role of inducible nitric oxide synthase (iNOS) in peroxynitrite-mediated protein radical formation in murine microglial BV2 cells treated with LPS. Treatment of BV2 cells with LPS resulted in morphological changes, induction of iNOS, and increased protein radical formation. Pretreatments with FeTPPS (a peroxynitrite decomposition catalyst), L-NAME (total NOS inhibitor), 1400W (iNOS inhibitor), and apocynin significantly attenuated LPS-induced protein radical formation and tyrosine nitration. Results obtained with coumarin-7-boronic acid, a highly specific probe for peroxynitrite detection, correlated with LPS-induced tyrosine nitration, which demonstrated involvement of peroxynitrite in protein radical formation. A similar degree of protection conferred by 1400W and L-NAME led us to conclude that only iNOS, and no other forms of NOS, is involved in LPS-induced peroxynitrite formation. Subsequently, siRNA for iNOS, the iNOS-specific inhibitor 1400W, the NF-κB inhibitor PDTC, and the p38 MAPK inhibitor SB202190 was used to inhibit iNOS directly or indirectly. Inhibition of iNOS precisely correlated with decreased protein radical formation in LPS-treated BV2 cells. The time course of protein radical formation also matched the time course of iNOS expression. Taken together, these results prove the role of iNOS in peroxynitrite-mediated protein radical formation in LPS-treated microglial BV2 cells.
Insights
Lipopolysaccharide (LPS) triggers protein radical formation in brain immune cells via inducible nitric oxide synthase (iNOS). Inhibiting iNOS reduces this process, revealing a key mechanism in neuroinflammation.
Area of Science:
- Neuroscience
- Immunology
- Biochemistry
Background:
- Microglia are key brain immune cells involved in neuroinflammation.
- Microglial activation is implicated in neurodegenerative diseases like Parkinson's.
- The mechanism of free radical formation during microglial activation is not fully understood.
Purpose of the Study:
- To investigate the role of inducible nitric oxide synthase (iNOS) in peroxynitrite-mediated protein radical formation.
- To elucidate the mechanism underlying lipopolysaccharide (LPS)-induced microglial activation.
Main Methods:
- Used immuno-spin trapping and RNA interference in BV2 microglial cells.
- Treated cells with LPS and various inhibitors (FeTPPS, L-NAME, 1400W, apocynin, PDTC, SB202190).
- Employed coumarin-7-boronic acid for peroxynitrite detection.
Main Results:
- LPS treatment induced microglial morphological changes, iNOS expression, and protein radical formation.
- Inhibitors of iNOS and peroxynitrite significantly reduced protein radical formation and tyrosine nitration.
- iNOS was identified as the primary source of peroxynitrite in LPS-activated microglia.
Conclusions:
- Inducible nitric oxide synthase (iNOS) plays a critical role in peroxynitrite-mediated protein radical formation in LPS-activated microglia.
- This finding provides insight into the molecular mechanisms of neuroinflammation.
- Targeting iNOS may offer therapeutic potential for neurodegenerative diseases.
