Suzuki Hiroka, Hatano Noriyuki, Muraki Yukiko, Itoh Yuka, Kimura Satoko, Hayashi Hidetoshi, Onozaki Kikuo, Ohi Yoshiaki, Haji Akira, Muraki Katsuhiko
Laboratory of Cellular Pharmacology, School of Pharmacy, Aichi-Gakuin University, Nagoya, Japan;
Department of Drug Metabolism and Disposition, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan;
Am J Physiol Cell Physiol. 2014 Aug 15;307(4):C384-94. doi: 10.1152/ajpcell.00182.2013. Epub 2014 Jun 18.
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.
瞬时受体电位锚蛋白1（TRPA1）是一种Ca(2+)可渗透的非选择性阳离子通道，在神经元和非神经元细胞中表达，在急性和炎性疼痛中起重要作用。在此，我们表明，一种NADPH氧化酶（NOX）抑制剂二苯碘鎓（DPI）在表达人TRPA1的人胚肾细胞（HEK-TRPA1）和人成纤维样滑膜细胞中作为TRPA1激活剂发挥作用。以0.03 - 10 μM的浓度应用DPI可在HEK-TRPA1细胞中以浓度依赖性方式诱导Ca(2+)反应。该Ca(2+)反应被选择性TRPA1拮抗剂HC-030031（HC）有效阻断。相比之下，DPI对表达TRPV1-V4或TRPM8的HEK细胞没有影响。其他四种NOX抑制剂，阿朴吗啡（APO）、VAS2870（VAS）、白花丹素和2-乙酰吩噻嗪，也在HEK-TRPA1细胞中诱导Ca(2+)反应，该反应可被HC预处理抑制。在存在5 mM谷胱甘肽的情况下，对DPI的Ca(2+)反应有效降低。此外，TRPA1中半胱氨酸621的突变显著抑制了DPI诱导的Ca(2+)反应，而它不抑制APO和VAS诱导的反应。在具有外向型和内向型配置的切除膜片中，DPI诱导了通道活性。新霉素的内部应用显著抑制了DPI诱导的内向电流。在具有TRPA1的炎性滑膜细胞中，DPI引发了对HC敏感的Ca(2+)反应。在小鼠中，足底注射DPI引起疼痛相关反应，该反应可被预先给予HC抑制。综上所述，我们的研究结果表明，DPI和其他NOX抑制剂在不介导NOX的情况下激活人TRPA1。
