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Related Experiment Videos

Distinction between nitrosating mechanisms within human cells and aqueous solution.

M G Espey1, K M Miranda, D D Thomas

  • 1Radiation Biology Branch, Division of Clinical Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA. sp@nih.gov

The Journal of Biological Chemistry
|June 19, 2001
PubMed
Summary

Nitric oxide (NO) autoxidation generates N(2)O(3), a key nitrosating species. Cellular environments and endogenous scavengers like ascorbate and glutathione critically influence NO reactivity and nitrosation, impacting cell function.

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Chemical Biology

Background:

  • Nitric oxide (NO) autoxidation produces dinitrogen trioxide (N(2)O(3)), a primary nitrosating agent.
  • Nitrosation of biomolecules can alter critical cellular functions.
  • Understanding NO reactivity in biological systems is crucial for cell signaling and pathology.

Purpose of the Study:

  • To investigate the biological mechanisms governing nitrogen oxide species reactivity during NO autoxidation.
  • To compare NO autoxidation mechanisms in aqueous buffers versus intact human tumor cells.
  • To elucidate the role of endogenous scavengers in modulating nitrosative processes.

Main Methods:

  • Utilized synthetic NO donor (DEA/NO) and a fluorescent probe (DAF) to monitor NO consumption and nitrosation.

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  • Employed 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (PTIO) to facilitate N(2)O(3) formation via NO(2) intermediacy.
  • Manipulated endogenous scavengers (ascorbate, glutathione) to assess their impact on nitrosative susceptibility.

    • Main Results:

    • NO consumption and nitrosation followed second-order kinetics in aqueous buffer but exhibited exponential kinetics within intact cells.
    • The N(2)O(3) formation pathway involving NO(2) intermediacy better reflected cellular nitrosation mechanisms compared to aqueous autoxidation.
    • Scavenger concentration, location, and affinity significantly determined the susceptibility of molecular targets to nitrosation.

    Conclusions:

    • Cellular nitrosation mechanisms differ from simple aqueous autoxidation, with NO(2) potentially playing a key role.
    • Endogenous scavengers compartmentalize and regulate nitrosation, protecting cellular targets.
    • Disruption of this protective architecture, often through scavenger depletion, can lead to nitrosative stress and compromise cellular function.