title: Inhibitory effects of AG490 on H2O2-induced TRPM2-mediated Ca(2+) entry.
abstract: Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca(2+)-permeable channel that controls Ca(2+) signalling. The activation of Janus kinase 2 (Jak2) by oxidative stress is implicated in the production of inflammatory mediators. We found that AG490, a Jak2 inhibitor, had an inhibitory effect on H2O2-induced TRPM2 activation. The purpose of this study was to examine the underlying mechanisms of the inhibitory effects of AG490. Activation of TRPM2 in TRPM2-expressing human embryonic kidney 293 (TRPM2/HEK) cells or the human monocytic cell line U937 was monitored by fluorescence-based Ca(2+) imaging and patch-clamp techniques. Treatment with AG490 almost completely blocked H2O2-induced increase in intracellular Ca(2+) in TRPM2/HEK and U937 cells. In the patch-clamp study, AG490 inhibited the H2O2-evoked inward current but not the ADP-ribose-induced inward current in TRPM2/HEK cells. In contrast, Jak inhibitor 1 (pyridone 6) and staurosporine, both of which inhibit Jak2, had no effect on H2O2-induced increase in intracellular Ca(2+). Moreover, AG490 decreased intracellular reactive oxygen species level, which was measured by using a hydroperoxide-sensitive fluorescent dye, on incubation with H2O2. In the cell-free assay system, AG490 scavenged hydroxyl radicals but not H2O2. These findings indicate that AG490 significantly reduces H2O2-induced TRPM2 activation, presumably by scavenging hydroxyl radicals rather than Jak2-dependent mechanisms. Although transient receptor potential ankyrin 1 (TRPA1) channel is also activated by H2O2, the H2O2-induced Ca(2+) entry through TRPA1 was only slightly delayed by AG490. This validates the potential use of AG490, as one of the materials for characterizing the role of TRPM2 channels in pathological models.
authors.affiliation: Laboratory of Pharmacology, Department of Clinical Pharmacy, Yokohama College of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama 245-0066, Japan; Division of Physiology and Pathology, Department of Pharmacology, Toxicology and Therapeutics, Showa University School of Pharmacy, Tokyo 142-8555, Japan. Electronic address: shun@hamayaku.ac.jp.
authors.forename: Shunichi
authors.initials: S
authors.lastname: Shimizu
authors.affiliation: Division of Physiology and Pathology, Department of Pharmacology, Toxicology and Therapeutics, Showa University School of Pharmacy, Tokyo 142-8555, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Yonezawa
authors.affiliation: Department of Food and Nutrition Science, Sagami Women׳s Junior College, Kanagawa 252-0838, Japan.
authors.forename: Tamio
authors.initials: T
authors.lastname: Hagiwara
authors.affiliation: Department of Oral Biology, Graduate School of Dentistry, Tohoku University, Sendai 980-8575, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yoshida
authors.affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Takahashi
authors.affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Hamano
authors.affiliation: Department of Pharmacogenomics, Showa University School of Pharmacy, Tokyo 142-8555, Japan.
authors.forename: Takaharu
authors.initials: T
authors.lastname: Negoro
authors.affiliation: Laboratory of Pharmacology, Department of Clinical Pharmacy, Yokohama College of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama 245-0066, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Toda
authors.affiliation: Department of Oral Biology, Graduate School of Dentistry, Tohoku University, Sendai 980-8575, Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Wakamori
authors.affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Mori
authors.affiliation: Division of Physiology and Pathology, Department of Pharmacology, Toxicology and Therapeutics, Showa University School of Pharmacy, Tokyo 142-8555, Japan.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Ishii
mesh_terms: D001562:Benzimidazoles / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity*; D053613:Janus Kinase 1 / Q000037:antagonists & inhibitors; D053614:Janus Kinase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D018408:Patch-Clamp Techniques; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011728:Pyridones / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D000074025:TRPA1 Cation Channel; D050053:TRPM Cation Channels / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D050051:Transient Receptor Potential Channels / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology*; D020298:U937 Cells
chemical_list: C524063:2-tert-butyl-9-fluoro-3,6-dihydro-7H-benz(h)imidazo(4,5-f)isoquinoline-7-one; D001562:Benzimidazoles; D015220:Calcium Channels; D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D009419:Nerve Tissue Proteins; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011728:Pyridones; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D000074025:TRPA1 Cation Channel; C473350:TRPA1 protein, human; D050053:TRPM Cation Channels; C121934:TRPM2 protein, human; D050051:Transient Receptor Potential Channels; D020032:Tyrphostins; C095512:alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide; D006861:Hydrogen Peroxide; D011065:Poly(ADP-ribose) Polymerases; C507904:JAK1 protein, human; C507924:JAK2 protein, human; D053613:Janus Kinase 1; D053614:Janus Kinase 2; D019311:Staurosporine
keywords: AG490; Janus kinase 2; Oxidative stress; TRPA1; TRPM2