Laboratory of Cellular Pharmacology School of Pharmacy Aichi-Gakuin University 1-100 Kusumoto Chikusa, Nagoya 464-8650 Japan.
Pharmacol Res Perspect. 2016 Apr 7;4(3):e00232. doi: 10.1002/prp2.232. eCollection 2016 Jun.
The effects of diclofenac (Dic), an acetic acid derivative-type nonsteroidal anti-inflammatory drug, were examined on the function of transient receptor potential (TRP) melastatin (TRPM) 3 (TRPM3) in human embryonic kidney 293 cell-line (HEK293) cells with recombinant human TRPM3 isoforms (TRPM31325, TRPM3-3, TRPM3-9, and TRPM3-S) and in a neuroblastoma cell line human neuroblastoma IMR-32 cells (IMR-32 cells) derived from human peripheral neurons. TRPM3 responses evoked by pregnenolone sulfate (PregS) were effectively inhibited by Dic in a concentration-dependent manner in Ca(2+) measurement and electrophysiological assays. The apparent IC 50 for PregS-induced Ca(2+) response of TRPM31325, TRPM3-3, and TRPM3-9 was calculated to be 18.8, 42.5, and 7.1 μmol/L, respectively. The TRPM3-dependent Ca(2+) responses evoked by nifedipine, another TRPM3 agonist, were also significantly inhibited by Dic. In contrast, aceclofenac, an acetoxymethyl analog of Dic, had no effects on PregS-induced TRPM3 responses. Constitutive channel activity of TRPM3-S without TRPM3 agonists was substantially inhibited by Dic, ruling out the possibility of interaction of Dic against TRPM3 agonists to the channel binding sites. Moreover, Dic reversibly inhibited TRPM3 single-channel activity recorded in excised outside-out patches without affecting the channel conductance. In differentiated neuronal IMR-32 cells with endogenous TRPM3, Dic inhibited PregS-evoked Ca(2+) responses with an apparent IC 50 of 17.1 μmol/L. Taken together, our findings demonstrate that Dic inhibits human TRPM3 without interacting with the channel pore.
二氯芬酸（Dic）是一种醋酸衍生物型非甾体抗炎药，本研究旨在观察其对人胚肾 293 细胞系（HEK293）中重组人 TRPM3 同工型（TRPM31325、TRPM3-3、TRPM3-9 和 TRPM3-S）以及源自人外周神经元的神经母细胞瘤细胞系人神经母细胞瘤 IMR-32 细胞（IMR-32 细胞）中瞬时受体电位（TRP）melastatin（TRPM）3（TRPM3）功能的影响。在 Ca(2+)测量和电生理测定中，Dic 以浓度依赖的方式有效抑制孕烯醇酮硫酸盐（PregS）诱导的 TRPM3 反应。计算出 PregS 诱导的 TRPM31325、TRPM3-3 和 TRPM3-9 的 Ca(2+)反应的表观 IC 50 分别为 18.8、42.5 和 7.1 μmol/L。另一种 TRPM3 激动剂硝苯地平诱导的 TRPM3 依赖性 Ca(2+)反应也被 Dic 显著抑制。相比之下，Dic 的乙酰氧甲基类似物 aceclofenac 对 PregS 诱导的 TRPM3 反应没有影响。没有 TRPM3 激动剂的 TRPM3-S 组成型通道活性被 Dic 显著抑制，排除了 Dic 与 TRPM3 激动剂相互作用于通道结合位点的可能性。此外，Dic 可逆地抑制在分离的外面向外膜片记录的单个 TRPM3 通道活性，而不影响通道电导。在具有内源性 TRPM3 的分化神经元 IMR-32 细胞中，Dic 以 17.1 μmol/L 的表观 IC 50 抑制 PregS 诱导的 Ca(2+)反应。总之，我们的研究结果表明，Dic 抑制人 TRPM3 而不与通道孔相互作用。
