Stimulation of dichlorofluorescin oxidation by capsaicin and analogues in RAW 264 monocyte/macrophages: Lack of involvement of the vanilloid receptor

In studies into the oxidative burst in RAW 264 monocyte/macrophages, it was observed that caysaicin, a vanilloid receptor agonist, stimulated dichloro fluorescin (DCFH) oxidation in a concentration-dependent manner, which coul d be blocked by capsazepine, a vanilloid receptor antagonist. However, by u se of a number of vanilloid agonists (including N-octyl-3-chloro-4-hydroxyp henylacetamide, 4m), we demonstrated that there was no relationship between vanilloid agonist potency and the capacity to stimulate DCFH oxidation. Th e oxidative burst stimulators Tween 20 and phorbol myristyl acetate (PMA) a lso stimulated reactive oxygen species generation which again was inhibited by capsazepine. Use of the selective inhibitor diphenyliodonium iodide rul ed our a role for plasma membrane NAD(P)H oxidase as the site of capsaicin- and 4m-stimulated DCFH oxidation. However, this DCFH oxidation was modulat ed by a number of inhibitors of mitochondrial respiration. Rotenone enhance d DCFH oxidation induced by capsaicin and 4m, whilst malonic acid and potas sium cyanide inhibited this response. 2,4-Dinitrophenol, an inhibitor of ox idative phosphorylation, was without effect. The antioxidant trolox c inhib ited DCFH oxidation stimulated by capsaicin, 4m, and PMA, whereas N-acetylc ysteine, a precursor of glutathione, was without effect. Capsazepine inhibi ted DCFH oxidation in unstimulated cells and in cells treated with menadion e, a redox-cycling quinone. Capsazepine was also a potent antioxidant when measured in a Fe3 + reduction assay. We concluded that DCFH oxidation stimu lated by vanilloid analogues was nor mediated via a vanilloid receptor, but rather by impairment of mitochondrial electron transport. (C) 2000 Elsevie r Science Inc.