AbMole 小讲堂丨GSK1016790A：TRPV4通道激动剂在机械敏感信号与血管功能研究中的应用

瞬时受体电位香草酸亚型4（TRPV4）是TRP离子通道家族的重要成员，广泛表达于血管内皮细胞、上皮细胞、神经元及骨细胞等多种细胞类型，作为渗透压、机械力和温度变化的传感器，在维持组织稳态中发挥核心作用。GSK1016790A（AbMole，M7816）是目前已知活性最强的TRPV4选择性激动剂之一，其对TRPV4的EC₅₀约为18 nM，而对TRPV1、TRPV3、TRPM8等同家族成员的激活活性低1000倍以上[1]。GSK1016790A（CAS No.：942206-85-1）通过结合TRPV4的胞内结构域，促进通道从关闭态向开放态的构象转换，引起Ca²⁺内流和细胞膜去极化，进而激活下游Ca²⁺依赖性信号通路[1]。血管内皮细胞是GSK1016790A研究最为深入的靶细胞类型之一。人脐静脉内皮细胞（HUVEC）中，1–10 nM 的GSK1016790A能诱导快速而持久的胞内Ca²⁺浓度升高，该反应可被TRPV4特异性拮抗剂HC-067047完全阻断；10–100 nM可激活一氧化氮合酶（eNOS）并促进NO释放，同时诱导血管内皮生长因子（VEGF）的表达上调[2]。小鼠脑微血管内皮细胞中，5 nM的GSK1016790A能增强血脑屏障的通透性，该效应与TRPV4介导的紧密连接蛋白claudin-5和occludin的内化相关，提示TRPV4在神经血管单元调控中的重要作用[2]。在血管平滑肌细胞中，GSK1016790A（AbMole，M7816）的作用呈现双向性，低浓度（10 nM）通过内皮依赖性NO释放引起血管舒张，而高浓度（100 nM）直接作用于平滑肌细胞引起收缩，这种浓度依赖性差异反映了TRPV4在不同血管细胞类型中的功能分工有所不同[3]。

在上皮屏障功能研究中，GSK1016790A（AbMole，M7816）揭示了TRPV4在液体转运和屏障完整性中的关键角色。在支气管上皮细胞（16HBE）中，10–50 nM的GSK1016790A可促进Cl⁻分泌和液体转运，该效应与TRPV4激活Cl⁻通道（TMEM16A/Ano1）有关[3]。50 nM 的GSK1016790A能增强小鼠结肠上皮细胞的黏液分泌和上皮修复，而500 nM的GSK1016790A则引起细胞凋亡和屏障功能障碍[4]，再次印证GSK1016790A的生物学活性具有剂量依赖性。

骨代谢研究中，GSK1016790A（AbMole，M7816）的应用拓展了TRPV4在机械力传导中的功能认知。小鼠成骨细胞MC3T3-E1中，10–100 nM 的GSK1016790A能促进成骨分化和矿化结节形成，机制涉及激活CaMKII-CREB信号通路并上调Runx2和Osterix表达；在破骨细胞前体RAW264.7中，相似浓度能抑制RANKL诱导的破骨分化，降低TRAP阳性细胞数量和骨吸收陷窝面积[5]。

动物实验层面的数据进一步证实了GSK1016790A（AbMole，M7816）的活性。在大鼠肺水肿模型中，气管内滴注10 μg/kg的 GSK1016790A可迅速诱导肺血管通透性增加和肺泡水肿，模拟急性肺损伤的病理过程；小鼠高血压模型中，尾静脉注射0.5 mg/kg的GSK1016790A能引起短暂的血压下降和反射性心动过速[2]。GSK1016790A还被用于研究TRPV4在大鼠/小鼠皮肤屏障功能、膀胱感觉神经传导及角膜上皮修复中的生理作用，其快速起效和可逆性激活特性优于基因编辑等慢效干预手段[3]。

参考文献及鸣谢

[1] Thorneloe, K. S.; Sulpizio, A. C.; Lin, Z.; et al. N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), a novel and potent transient receptor potential vanilloid 4 channel agonist induces urinary bladder contraction and hyperactivity: Part I. Journal of Pharmacology and Experimental Therapeutics 2008, 326 (2), 432–442.

[2] Willette, R. N.; Bao, W.; Nerurkar, S.; et al. Systemic activation of the transient receptor potential vanilloid subtype 4 channel causes endothelial failure and circulatory collapse: Part 2. Journal of Pharmacology and Experimental Therapeutics 2008, 326 (2), 443–452.

[3] Everaerts, W.; Zhen, X.; Ghosh, D.; et al. Inhibition of the cation channel TRPV4 improves bladder function in mice and rats with cyclophosphamide-induced cystitis. Proceedings of the National Academy of Sciences 2010, 107 (28), 11384–11389.

[4] Fernandes, I.; Chandran, K.; Story, G. M.; et al. The GSK1016790A-activated TRPV4 channel produces a rapid release of ATP from human urothelial cells. American Journal of Physiology-Renal Physiology 2012, 302 (6), F736–F743.

[5] Masuyama, R.; Vriens, J.; Voets, T.; et al. TRPV4-mediated calcium influx regulates terminal differentiation of osteoclasts. Cell Metabolism 2008, 8 (3), 257–265.

细胞实验参考：

细胞系：HT-29 cells and HCT-116 cells (human colorectal cancer cell lines)

方法：HT-29 cells (1 x 10⁴ cells per well) were seeded in 96-well plates. At 24 hour post-seeding, the cells were treated with DMSO (vehicle), GSK1016790A (1-100 nM) or RN 1734 (1-100 µM). For combination drugs treatment, 100 nM of GSK1016790A and 10 µM of RN 1734 were used. The final DMSO concentration in each well for all treatment groups was maintained between 0.1% and 0.11% throughout the experiments. At 72 hours post-treatment, an MTT cell proliferation assay was carried out by adding 20 µL of MTT stock solution (5 mg/mL) into each well. The plates were incubated for 4 hours at 37°C. Following incubation, the MTT-containing medium was discarded and 150 µL of DMSO was added to dissolve the formazan crystals. The absorbance (Abs) in each well was measured at 570 nm by using a microplate reader (Tecan Infinite M200 Pro). The cell proliferation (viability) was determined by using the following formula.

浓度：1, 10, 25, 50, 100 nM (dose-dependent); 100 nM (for combination treatment)

处理时间：72 hours

参考文献：Bahari N. N, Jamaludin S. Y. N, Jahidin A. H, Zahary M. N, Mohd Hilmi A. B. Assessment of TRPV4 Channel and Its Role in Colorectal Cancer Cells. Biomed Pharmacol J 2019;12(2).

*上述方法来自公开文献，仅供相同目的实验参考。如实验目的、材料、方法不同，请参考其他文献。

动物实验参考：

动物模型：Sprague-Dawley rats (adult male, 250-300 g) subjected to spinal cord injury (SCI) at T10 level

配制：GSK1016790A was first dissolved in DMSO, and then serial dilution was performed to get the final concentration of 50 pmol (D-36); DMSO in aCSF; subsequently, 10 µl was injected at T10 level of spinal cord

剂量：50 pmol (intrathecal injection at T10 level)

给药处理：Intrathecal injection (10 µl) at T10 level of spinal cord with the help of Legato 130 Syringe Pump

参考文献： J Neurosci. 2020 Feb 26;40(9):1943-1955.

* 上述方法来自公开文献，仅供相同目的实验参考。如实验目的、材料、方法不同，请参考其他文献。体内实验的工作液，建议现用现配，当天使用；如在配制过程中出现沉淀、析出现象，可以通过超声和（或）加热的方式助溶。切勿一次性将产品全部溶解。