u-0126 and 2--7--dichlorofluorescein

u-0126 has been researched along with 2--7--dichlorofluorescein* in 1 studies

Other Studies

1 other study(ies) available for u-0126 and 2--7--dichlorofluorescein

Article	Year
Identification of the hydroxyl radical and other reactive oxygen species in human neutrophil granulocytes exposed to a fragment of the amyloid beta peptide. Free radical research, 2003, Volume: 37, Issue:3 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. Topics: Adult; Amyloid beta-Peptides; Blotting, Western; Butadienes; Chelating Agents; Ditiocarb; Dose-Response Relationship, Drug; Electron Spin Resonance Spectroscopy; Enzyme Inhibitors; Fluoresceins; Granulocytes; Humans; Hydroxyl Radical; Hypochlorous Acid; Luminol; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; NADPH Oxidases; Neutrophils; Nitriles; Oxygen; Parabens; Phospholipases A; Phospholipases A2; Phosphorylation; Reactive Oxygen Species; Salicylamides; Signal Transduction; Spectrometry, Fluorescence; Time Factors	2003

Article

Year

Identification of the hydroxyl radical and other reactive oxygen species in human neutrophil granulocytes exposed to a fragment of the amyloid beta peptide.

Free radical research, 2003, Volume: 37, Issue:3

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.

Topics: Adult; Amyloid beta-Peptides; Blotting, Western; Butadienes; Chelating Agents; Ditiocarb; Dose-Response Relationship, Drug; Electron Spin Resonance Spectroscopy; Enzyme Inhibitors; Fluoresceins; Granulocytes; Humans; Hydroxyl Radical; Hypochlorous Acid; Luminol; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; NADPH Oxidases; Neutrophils; Nitriles; Oxygen; Parabens; Phospholipases A; Phospholipases A2; Phosphorylation; Reactive Oxygen Species; Salicylamides; Signal Transduction; Spectrometry, Fluorescence; Time Factors

2003