Contribution of nitric oxide synthase to human neutrophil chemiluminescence.
A chemiluminescence (CL) assay has been used to measure the reactive oxygen species (ROS)-generating capacity of phagocytes. Primed neutrophils produce ROS and nitric oxide (NO) upon induction of nitric oxide synthase (NOS) activity. NO and superoxide (O(2)(-)) form peroxynitrite (ONOO(-)), and emit CL. We examined the involvement of NOS in the CL response of neutrophils using a method based on the modulation of enzyme activity of NOS by the substrate L-arginine and an inhibitor; L-NAME. We used lipopolysaccharide (LPS) as the neutrophil-priming agent. Addition of sodium azide (NaN(3)) with horseradish peroxidase (HRP) to luminol-dependent CL, gave a CL response that was significantly enhanced when 10 mmol/L L-arginine was present (p <0.05), suggesting that NOS activity contributed to the CL response of human neutrophils. LPS-primed luminol-dependent CL was significantly inhibited by L-NAME compared with D-NAME. The proportion of the difference between the two inhibitors in luminol-dependent CL was 12.3 +/- 15.0%. Therefore, approximately 12% of the LPS-primed luminol-dependent CL decrease induced by L-NAME indicated the contribution of NOS activity to the CL response.
Kudoh
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S
Department of Hygiene, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori, 036-8562, Japan. satoru@cc.hirosaki-u.ac.jp
Suzuki
K
K
Yamada
M
M
Liu
Q
Q
Nakaji
S
S
Sugawara
K
K
D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008163:Luminescent Measurements; D008165:Luminol; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism
D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D017382:Reactive Oxygen Species; D008165:Luminol; D019001:Nitric Oxide Synthase; D019331:NG-Nitroarginine Methyl Ester