title: Stimulation of dichlorofluorescin oxidation by capsaicin and analogues in RAW 264 monocyte/macrophages: lack of involvement of the vanilloid receptor.
abstract: In studies into the oxidative burst in RAW 264 monocyte/macrophages, it was observed that capsaicin, a vanilloid receptor agonist, stimulated dichlorofluorescin (DCFH) oxidation in a concentration-dependent manner, which could be blocked by capsazepine, a vanilloid receptor antagonist. However, by use of a number of vanilloid agonists (including N-octyl-3-chloro-4-hydroxyphenylacetamide, 4m), we demonstrated that there was no relationship between vanilloid agonist potency and the capacity to stimulate DCFH oxidation. The oxidative burst stimulators Tween 20 and phorbol myristyl acetate (PMA) also stimulated reactive oxygen species generation, which again was inhibited by capsazepine. Use of the selective inhibitor diphenyliodonium iodide ruled out a role for plasma membrane NAD(P)H oxidase as the site of capsaicin- and 4m-stimulated DCFH oxidation. However, this DCFH oxidation was modulated by a number of inhibitors of mitochondrial respiration. Rotenone enhanced DCFH oxidation induced by capsaicin and 4m, whilst malonic acid and potassium cyanide inhibited this response. 2,4-Dinitrophenol, an inhibitor of oxidative phosphorylation, was without effect. The antioxidant trolox c inhibited DCFH oxidation stimulated by capsaicin, 4m, and PMA, whereas N-acetylcysteine, a precursor of glutathione, was without effect. Capsazepine inhibited DCFH oxidation in unstimulated cells and in cells treated with menadione, a redox-cycling quinone. Capsazepine was also a potent antioxidant when measured in a Fe3+ reduction assay. We concluded that DCFH oxidation stimulated by vanilloid analogues was not mediated via a vanilloid receptor, but rather by impairment of mitochondrial electron transport.
authors.affiliation: School of Medical Biosciences, Medical School, Queens Medical Centre, University of Nottingham, UK.
authors.forename: M J
authors.initials: MJ
authors.lastname: Garle
authors.forename: A
authors.initials: A
authors.lastname: Knight
authors.forename: A T
authors.initials: AT
authors.lastname: Downing
authors.forename: K L
authors.initials: KL
authors.lastname: Jassi
authors.forename: R H
authors.initials: RH
authors.lastname: Clothier
authors.forename: J R
authors.initials: JR
authors.lastname: Fry
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002211:Capsaicin / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology*; D002273:Carcinogens / Q000494:pharmacology; D002839:Chromans / Q000494:pharmacology; D005452:Fluoresceins / Q000378:metabolism*; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D009000:Monocytes / Q000187:drug effects* / Q000378:metabolism; D010084:Oxidation-Reduction; D011136:Polysorbates / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D011955:Receptors, Drug / Q000378:metabolism*; D013501:Surface-Active Agents / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014812:Vitamin K / Q000494:pharmacology
chemical_list: D000975:Antioxidants; D002273:Carcinogens; D002839:Chromans; D005452:Fluoresceins; D011136:Polysorbates; D017382:Reactive Oxygen Species; D011955:Receptors, Drug; D013501:Surface-Active Agents; D014812:Vitamin K; C037631:2',7'-dichlorofluorescein; C071423:capsazepine; D013755:Tetradecanoylphorbol Acetate; D002211:Capsaicin; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; D000111:Acetylcysteine