Inhibitory effects of AG490 on H₂O₂-induced TRPM2-mediated Ca²⁺entry

Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca²⁺-permeable channel that controls Ca²⁺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 Ca²⁺imaging and patch-clamp techniques. Treatment with AG490 almost completely blocked H₂O₂-induced increase in intracellular Ca²⁺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 Ca²⁺. 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 Ca²⁺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.