Identification of the hydroxyl radical and other reactive oxygen species in human neutrophil granulocytes exposed to a fragment of the amyloid beta peptide.
A fragment of the amyloid beta protein, betaA(25-35), was investigated for its effect on production of reactive oxygen species (ROS) in human neutrophil granulocytes. The formation and identification of ROS were examined by using a 2',7'-dichlorofluorescin (DCF) fluorescence assay, a luminol chemiluminescence assay, electron paramagnetic resonance (EPR) spectroscopy with DEPMPO as a spin trap, and hydroxylation of 4-hydroxybenzoate (4-HBA). The DCF assay showed that betaA(25-35) stimulated formation of ROS in concentration and time dependent manner. The inverted peptide, betaA(35-25), gave no response. Also, luminol-amplified chemiluminescence was stimulated by betaA(25-35). Incubation with diethyldithiocarbamate (a superoxide dimustase inhibitor) and salicylhydroxamate (SHA; a myeloperoxidase inhibitor) reduced the chemiluminescence. This indicates that hypochlorous acid (HOCl) is formed after exposure to betaA(25-35). The EPR spectra indicated a concentration dependent formation of superoxide (O2*-)- and hydroxyl (*OH)-radicals. Hydroxylation of 4-HBA to 3,4,-dihydroxybenzoate confirmed production of *OH. This response was attenuated by SHA, indicating involvement of HOCl in formation of *OH. The DCF fluorescence was inhibited with U0126 (an extracellular signal regulated protein kinase (ERK) inhibitor). Further analysis with western blot confirmed phosphorylation of ERK1/2 after exposure to betaA(25-35). The phospholipase A2 (PLA2) inhibitor 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid, and diphenyleneiodonium, which inhibits the NADPH oxidase, also led to a reduction of the DCF fluorescence. The present findings indicate that betaA(25-35) stimulates the NADPH oxidase by activating the ERK pathway and PLA2. Production of O2*- can lead to HOCl and further formation of *OH, which both have a cytotxic potential.
Andersen
Jannike M
JM
Norwegian Defence Research Establishment, Division for Protection and Materiel, PO Box 25, N-2027 Kjeller, Norway. jannike.andersen@ffi.no
Myhre
Oddvar
O
Aarnes
Halvor
H
Vestad
Tor Arne
TA
Fonnum
Frode
F
D000328:Adult; D016229:Amyloid beta-Peptides / Q000737:chemistry*; D015153:Blotting, Western; D002070:Butadienes / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D004050:Ditiocarb / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005452:Fluoresceins / Q000494:pharmacology; D006098:Granulocytes / Q000378:metabolism*; D006801:Humans; D017665:Hydroxyl Radical; D006997:Hypochlorous Acid / Q000378:metabolism; D008165:Luminol / Q000378:metabolism; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D010226:Parabens / Q000494:pharmacology; D010741:Phospholipases A / Q000037:antagonists & inhibitors; D054467:Phospholipases A2; D010766:Phosphorylation; D017382:Reactive Oxygen Species; D012457:Salicylamides / Q000494:pharmacology; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D016229:Amyloid beta-Peptides; D002070:Butadienes; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D009570:Nitriles; D010226:Parabens; D017382:Reactive Oxygen Species; D012457:Salicylamides; C113580:U 0126; D017665:Hydroxyl Radical; C037631:2',7'-dichlorofluorescein; D008165:Luminol; D006997:Hypochlorous Acid; C005703:salicylhydroxamic acid; D004050:Ditiocarb; D019255:NADPH Oxidases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010741:Phospholipases A; D054467:Phospholipases A2; C038193:4-hydroxybenzoic acid; D010100:Oxygen