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Peroxynitrite formation from activated human leukocytes
1Department of Physiology 2, Tokai University, Kanagawa, Japan.
Abstract:
We showed direct evidence of peroxynitrite formation from polymorphonuclear cells (PMN) with the nitration of 4-hydroxyphenylacetic acid (HPA) to 4-hydroxy-3-nitrophenylacetic acid (NO2HPA). Human PMN from healthy volunteers was stimulated with phorbol-12-myristate-13-acetate (PMA, 10 ng/ml) at 37 degrees C in 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid-buffered Hank's balanced salt solution (pH 7.4) with HPA (1 mM). NO2HPA was detected under PMA stimulation only in the presence of myeloperoxidase inhibitor. NO2HPA was eliminated by N-monomethyl-L-arginine (100 microM). The inhibition of myeloperoxidase appears to be essential to demonstrate the production of NO2HPA since myeloperoxidase itself or its product, hypochlorite, reacted with peroxynitrite and hampered the formation of NO2HPA.
Insights
Polymorphonuclear cells (PMN) generate peroxynitrite, evidenced by 4-hydroxyphenylacetic acid (HPA) nitration. This process requires myeloperoxidase inhibition for accurate detection of 4-hydroxy-3-nitrophenylacetic acid (NO2HPA).
Area of Science:
- Biochemistry
- Immunology
- Cell Biology
Background:
- Polymorphonuclear cells (PMN) are crucial immune cells involved in inflammatory responses.
- Peroxynitrite is a reactive nitrogen species implicated in cellular damage during inflammation.
- Understanding peroxynitrite formation mechanisms in PMNs is vital for inflammatory disease research.
Purpose of the Study:
- To provide direct evidence of peroxynitrite formation by human PMNs.
- To investigate the role of myeloperoxidase (MPO) in peroxynitrite production.
- To identify the nitration product of 4-hydroxyphenylacetic acid (HPA) formed by PMNs.
Main Methods:
- Human PMNs from healthy volunteers were stimulated with phorbol-12-myristate-13-acetate (PMA).
- The nitration of 4-hydroxyphenylacetic acid (HPA) to 4-hydroxy-3-nitrophenylacetic acid (NO2HPA) was measured.
- Experiments were conducted with and without myeloperoxidase inhibitors and N-monomethyl-L-arginine.
Main Results:
- Direct evidence of peroxynitrite formation was demonstrated by the nitration of HPA to NO2HPA.
- NO2HPA formation was observed only under PMA stimulation in the presence of an MPO inhibitor.
- N-monomethyl-L-arginine treatment eliminated NO2HPA, confirming its peroxynitrite-derived nature.
Conclusions:
- Human PMNs actively produce peroxynitrite, leading to the nitration of HPA.
- Myeloperoxidase inhibition is essential to detect PMN-derived peroxynitrite accurately.
- These findings elucidate a key mechanism in PMN-mediated inflammatory processes.