Identification of the Hydroxyl Radical and Other Reactive Oxygen Species in Human Neutrophil Granulocytes Exposed to a Fragment of the Amyloid Beta Peptide |
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Authors: | Jannike M. Andersen Oddvar Myhre Halvor Aarnes Tor Arne Vestad Frode Fonnum |
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Affiliation: | 1. Norwegian Defence Research Establishment, Division for Protection and Materiel, PO Box 25, N-2027 Kjeller, Norway;2. Department of Biology, University of Oslo, Oslo, Norway;3. Department of Physics, University of Oslo, Oslo, Norway |
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Abstract: | A fragment of the amyloid beta protein, g A(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 g A(25-35) stimulated formation of ROS in a concentration and time dependent manner. The inverted peptide, g A(35-25), gave no response. Also, luminol-amplified chemiluminescence was stimulated by g A(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 g A(25-35). The EPR spectra indicated a concentration dependent formation of superoxide ( O 2 m ) - 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 g A(25-35). The phospholipase A 2 (PLA 2 ) 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 g A(25-35) stimulates the NADPH oxidase by activating the ERK pathway and PLA 2 . Production of O 2 m can lead to HOCl and further formation of OH, which both have a cytotoxic potential. |
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Keywords: | Alzheimer's Disease Amyloid Beta Protein Human Neutrophil Granulocytes Reactive Oxygen Species Hydroxyl Radical Hypochlorous Acid |
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