Liaoning Provincial Key Laboratory of Biotechnology and Drug Discovery, School of Life Sciences, Liaoning Normal University, Dalian 116000, PR China; Laboratory medical college, Jilin Medical University, Jilin 132013, PR China.
Liaoning Provincial Key Laboratory of Biotechnology and Drug Discovery, School of Life Sciences, Liaoning Normal University, Dalian 116000, PR China.
Toxicol Appl Pharmacol. 2021 Jun 15;421:115543. doi: 10.1016/j.taap.2021.115543. Epub 2021 Apr 16.
Nimodipine is a clinically used dihydropyridine L-type calcium channel antagonist that effectively inhibits transmembrane Ca influx following the depolarization of smooth muscle cells, but the detailed effect on smooth muscle contraction is not fully understood. Ca-activated Cl channels (CaCCs) in vascular smooth muscle cells (VSMCs) may regulate vascular contractility. We found that nimodipine can inhibit transmembrane protein 16A (TMEM16A) activity in a concentration-dependent manner by cell-based fluorescence-quenching assay and short-circuit current analysis, with an IC value of ~5 μM. Short-circuit current analysis also showed that nimodipine prevented Ca-activated Cl current in both HT-29 cells and mouse colonic epithelia accompanied by significantly decreased cytoplasmic Ca concentrations. In the absence of extracellular Ca, nimodipine still exhibited an inhibitory effect on TMEM16A/CaCCs. Additionally, the application of nimodipine to CFTR-expressing FRT cells and mouse colonic mucosa resulted in mild activation of CFTR-mediated Cl currents. Nimodipine inhibited basolateral CCh-activated K channel activity with no effect on Na/K-ATPase activity. Evaluation of intestinal smooth muscle contraction showed that nimodipine inhibits intestinal smooth muscle contractility and frequency, with an activity pattern that was similar to that of non-specific inhibitors of CaCCs. In aortic smooth muscle, the expression of TMEM16A in thoracic aorta is higher than that in abdominal aorta, corresponding to stronger maximum contractility in thoracic aorta smooth muscle stimulated by phenylephrine (PE) and E. Nimodipine completely inhibited the contraction of aortic smooth muscle stimulated by E, and partially inhibited the contraction stimulated by PE. In summary, the results indicate that nimodipine effectively inhibits TMEM16A/CaCCs by reduction transmembrane Ca influx and directly interacting with TMEM16A, explaining the mechanisms of nimodipine relaxation of intestinal and aortic smooth muscle contraction and providing new targets for pharmacological applications.
尼莫地平是一种临床应用的二氢吡啶 L 型钙通道拮抗剂，能有效抑制平滑肌细胞去极化后跨膜 Ca 内流，但对平滑肌收缩的详细作用尚不完全清楚。血管平滑肌细胞（VSMCs）中的 Ca 激活 Cl 通道（CaCCs）可能调节血管收缩性。我们发现，尼莫地平可通过细胞荧光猝灭试验和短路电流分析，以浓度依赖方式抑制跨膜蛋白 16A（TMEM16A）的活性，IC 值约为 5 μM。短路电流分析还表明，尼莫地平可阻止 HT-29 细胞和小鼠结肠上皮细胞中的 Ca 激活的 Cl 电流，同时显著降低细胞质 Ca 浓度。在无细胞外 Ca 的情况下，尼莫地平仍对 TMEM16A/CaCCs 表现出抑制作用。此外，尼莫地平在 CFTR 表达的 FRT 细胞和小鼠结肠黏膜中的应用导致 CFTR 介导的 Cl 电流轻度激活。尼莫地平抑制基底外侧 CCh 激活的 K 通道活性，而对 Na/K-ATPase 活性无影响。评估肠道平滑肌收缩表明，尼莫地平抑制肠道平滑肌收缩性和频率，其活性模式与 CaCCs 的非特异性抑制剂相似。在主动脉平滑肌中，胸主动脉 TMEM16A 的表达高于腹主动脉，这与去甲肾上腺素（PE）和内皮素（E）刺激的胸主动脉平滑肌的最大收缩力更强相对应。尼莫地平完全抑制 E 刺激的主动脉平滑肌收缩，部分抑制 PE 刺激的收缩。综上所述，这些结果表明，尼莫地平通过减少跨膜 Ca 内流并直接与 TMEM16A 相互作用，有效抑制 TMEM16A/CaCCs，解释了尼莫地平松弛肠道和主动脉平滑肌收缩的机制，并为药理学应用提供了新的靶点。
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