title: The NADPH oxidase inhibitor diphenyleneiodonium activates the human TRPA1 nociceptor.
abstract: Transient receptor potential ankyrin 1 (TRPA1) is a Ca(2+)-permeable nonselective cation channel expressed in neuronal and nonneuronal cells and plays an important role in acute and inflammatory pain. Here, we show that an NADPH oxidase (NOX) inhibitor, diphenyleneiodonium (DPI), functions as a TRPA1 activator in human embryonic kidney cells expressing human TRPA1 (HEK-TRPA1) and in human fibroblast-like synoviocytes. Application of DPI at 0.03-10 μM induced a Ca(2+) response in HEK-TRPA1 cells in a concentration-dependent manner. The Ca(2+) response was effectively blocked by a selective TRPA1 antagonist, HC-030031 (HC). In contrast, DPI had no effect on HEK cells expressing TRPV1-V4 or TRPM8. Four other NOX inhibitors, apocynin (APO), VAS2870 (VAS), plumbagin, and 2-acetylphenothiazine, also induced a Ca(2+) response in HEK-TRPA1 cells, which was inhibited by pretreatment with HC. In the presence of 5 mM glutathione, the Ca(2+) response to DPI was effectively reduced. Moreover, mutation of cysteine 621 in TRPA1 substantially inhibited the DPI-induced Ca(2+) response, while it did not inhibit the APO- and VAS-induced responses. The channel activity was induced by DPI in excised membrane patches with both outside-out and inside-out configurations. Internal application of neomycin significantly inhibited the DPI-induced inward currents. In inflammatory synoviocytes with TRPA1, DPI evoked a Ca(2+) response that was sensitive to HC. In mice, intraplantar injection of DPI caused a pain-related response which was inhibited by preadministration with HC. Taken together, our findings demonstrate that DPI and other NOX inhibitors activate human TRPA1 without mediating NOX.
authors.affiliation: Laboratory of Cellular Pharmacology, School of Pharmacy, Aichi-Gakuin University, Nagoya, Japan;
authors.forename: Hiroka
authors.initials: H
authors.lastname: Suzuki
authors.affiliation: Laboratory of Cellular Pharmacology, School of Pharmacy, Aichi-Gakuin University, Nagoya, Japan;
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Hatano
authors.affiliation: Laboratory of Cellular Pharmacology, School of Pharmacy, Aichi-Gakuin University, Nagoya, Japan;
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Muraki
authors.affiliation: Department of Drug Metabolism and Disposition, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan;
authors.forename: Yuka
authors.initials: Y
authors.lastname: Itoh
authors.affiliation: Laboratory of Neuropharmacology, School of Pharmacy, Aichi-Gakuin University, Nagoya, Japan; and.
authors.forename: Satoko
authors.initials: S
authors.lastname: Kimura
authors.affiliation: Department of Drug Metabolism and Disposition, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan;
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Hayashi
authors.affiliation: Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan.
authors.forename: Kikuo
authors.initials: K
authors.lastname: Onozaki
authors.affiliation: Laboratory of Neuropharmacology, School of Pharmacy, Aichi-Gakuin University, Nagoya, Japan; and.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Ohi
authors.affiliation: Laboratory of Neuropharmacology, School of Pharmacy, Aichi-Gakuin University, Nagoya, Japan; and.
authors.forename: Akira
authors.initials: A
authors.lastname: Haji
authors.affiliation: Laboratory of Cellular Pharmacology, School of Pharmacy, Aichi-Gakuin University, Nagoya, Japan; kmuraki@dpc.agu.ac.jp.
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Muraki
mesh_terms: D000818:Animals; D001522:Behavior, Animal / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology* / Q000633:toxicity; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007249:Inflammation / Q000378:metabolism; D008564:Membrane Potentials; D051379:Mice; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology* / Q000633:toxicity; D010146:Pain / Q000139:chemically induced / Q000503:physiopathology / Q000523:psychology; D017288:Pain Threshold / Q000187:drug effects; D013583:Synovial Membrane / Q000187:drug effects / Q000378:metabolism; D000074025:TRPA1 Cation Channel; D013997:Time Factors; D014162:Transfection; D050051:Transient Receptor Potential Channels / Q000819:agonists* / Q000235:genetics / Q000378:metabolism
chemical_list: D015220:Calcium Channels; D004791:Enzyme Inhibitors; D009419:Nerve Tissue Proteins; D009861:Onium Compounds; D000074025:TRPA1 Cation Channel; C473350:TRPA1 protein, human; D050051:Transient Receptor Potential Channels; C475685:Trpa1 protein, mouse; C007517:diphenyleneiodonium; D019255:NADPH Oxidases; D005978:Glutathione; D002118:Calcium
keywords: NADPH oxidase inhibitors; calcium channel; transient receptor potential