Inhibitory effects of AG490 on H2O2-induced TRPM2-mediated Ca(2+) entry.
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.
['Shimizu|Shunichi|S|', 'Yonezawa|Ryo|R|', 'Hagiwara|Tamio|T|', 'Yoshida|Takashi|T|', 'Takahashi|Nobuaki|N|', 'Hamano|Satoshi|S|', 'Negoro|Takaharu|T|', 'Toda|Takahiro|T|', 'Wakamori|Minoru|M|', 'Mori|Yasuo|Y|', 'Ishii|Masakazu|M|']
D001562:Benzimidazoles D015220:Calcium Channels D020013:Calcium Signaling D004791:Enzyme Inhibitors D016166:Free Radical Scavengers D057809:HEK293 Cells D006801:Humans D006861:Hydrogen Peroxide D053613:Janus Kinase 1 D053614:Janus Kinase 2 D009419:Nerve Tissue Proteins D018384:Oxidative Stress D018408:Patch-Clamp Techniques D000067856:Poly(ADP-ribose) Polymerase Inhibitors D011065:Poly(ADP-ribose) Polymerases D011728:Pyridones D017382:Reactive Oxygen Species D011994:Recombinant Proteins D019311:Staurosporine D000074025:TRPA1 Cation Channel D050053:TRPM Cation Channels D050051:Transient Receptor Potential Channels D020032:Tyrphostins D020298:U937 Cells
AG490 TRPM2 Janus kinase 2 Oxidative stress TRPA1