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Studies on nitric oxide free radicals generated from polymorphonuclear leukocytes (PMN) stimulated by phorbol
Abstract:
The interaction of NO and O2- free radicals generated from PMA (phorbol myristate acetate)-stimulated PMN (polymorphonuclear leukocytes) was studied by a nitroxide spin trap, DMPO (5,5-dimethyl-1-pyrroline-1-oxide). It was found that addition of L-arginine to the system would significantly decrease the trapped O2- by DMPO and addition of NG-monomethyl-arginine (NGMA) would significantly increase the trapped O2- by DMPO. It was proved that the formation of ONOO- by the reaction of NO and O2- was the main reason for the decrease of trapped O2- in the experiment with xanthine/xanthine oxidase and irradiation of riboflavin systems. The yield of NO during this process was calculated. The generation dynamic of NO was studied by a luminol-dependent chemiluminescence technique and it was found that after stimulation of PMN by PMA, there would be an immediate, significant chemiluminescence, which came mainly from the active oxygen free radicals generated by PMN. If L-arginine was added to this system, the chemiluminescence would increase about 100-fold, but NGMA inhibited the increase of the chemiluminescence. Ten minutes after addition of L-arginine, this increase did not change, the chemiluminescence peak decreased gradually, but the half life increased. The ESR and chemiluminescence properties of NO and ONOO- synthesized were also studied in model systems.
Insights
L-arginine affects nitric oxide (NO) and superoxide (O2-) radical interactions in neutrophils. L-arginine addition decreases O2- trapping, while NG-monomethyl-arginine (NGMA) increases it, indicating peroxynitrite formation.
Area of Science:
- Biochemistry
- Cellular Biology
- Free Radical Chemistry
Background:
- Polymorphonuclear leukocytes (PMNs) generate reactive oxygen and nitrogen species upon stimulation.
- The interaction between nitric oxide (NO) and superoxide (O2-) is crucial in cellular signaling and oxidative stress.
- Understanding these interactions is key to elucidating inflammatory processes.
Purpose of the Study:
- To investigate the interaction dynamics between NO and O2- free radicals generated by phorbol myristate acetate (PMA)-stimulated PMNs.
- To quantify the role of L-arginine and NG-monomethyl-arginine (NGMA) in modulating these radical interactions.
- To elucidate the formation and impact of peroxynitrite (ONOO-) in this system.
Main Methods:
- Electron Spin Resonance (ESR) spectroscopy using DMPO (5,5-dimethyl-1-pyrroline-1-oxide) spin trap.
- Luminol-dependent chemiluminescence assay to study NO generation dynamics.
- Utilizing xanthine/xanthine oxidase and riboflavin irradiation systems for controlled radical generation.
- Synthesis and characterization of NO and ONOO- in model systems.
Main Results:
- L-arginine significantly decreased O2- trapping by DMPO, while NGMA increased it.
- Peroxynitrite (ONOO-) formation was identified as the primary cause for decreased O2- trapping.
- PMN stimulation with PMA induced immediate chemiluminescence, enhanced ~100-fold by L-arginine and inhibited by NGMA.
- Chemiluminescence kinetics showed altered peak and half-life upon L-arginine addition over time.
Conclusions:
- L-arginine availability influences the balance between NO and O2- in stimulated PMNs.
- ONOO- formation is a significant consequence of NO and O2- interaction in this biological context.
- Chemiluminescence measurements provide a dynamic readout of NO generation and subsequent radical reactions.