DL0805-2, a novel indazole derivative, relaxes angiotensin II-induced contractions of rat aortic rings by inhibiting Rho kinase and calcium fluxes  

作　　者：Tian-yi YUAN Yu-cai CHEN Hui-fang ZHANG Li LI Xiao-zhen JIAO Ping XIE Lian-hua FANG Guan-hua DU 

机构地区：[1]State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China [2]Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing100050, China

出　　处：《Acta Pharmacologica Sinica》2016年第5期604-616,共13页中国药理学报（英文版）

摘　　要：Aim： DL0805-2 [N-（1H-indazol-5-yl）-1-（4-methylbenzyl） pyrrolidine-3-carboxamide] is a DL0805 derivative with more potent vasorelaxant activity and lower toxicity. This study was conducted to investigate the vasorelaxant mechanisms of DL0805-2 on angiotensin II （Ang II）- induced contractions of rat thoracic aortic rings in vitro. Methods： Rat thoracic aortic rings and rat aortic vascular smooth muscle cells （VSMCs） were pretreated with DL0805-2, and then stimulated with Ang II. The tension of the aortic rings was measured through an isometric force transducer. Ang II-induced protein phosphorylation, ROS production and F-actin formation were assessed with Western blotting and immunofluorescence assays. Intracellular free Ca2~ concentrations were detected with Fluo-3 AM. Results＂ Pretreatment with DL0805-2 （1-100 pmol/L） dose-dependently inhibited the constrictions of the aortic rings induced by a single dose of Ang II （10T mol/L） or accumulative addition of Ang II （10^-10-10^-7 mol/L）. The vasodilatory effect of DL0805-2 was independent of endothelium. In the aortic rings, pretreatment with DL0805-2 （1, 3, and 10 pmol/L） suppressed Ang II-induced Ca2~ influx and intracellular Ca^2＋ mobilization, and Ang II-induced phosphorylation of two substrates of Rho kinase （MLC and MYPT1）. In VSMCs, pretreatment with DL0805-2 （1, 3, and 10 pmol/L） also suppressed Ang II-induced Ca^2＋ fluxes and phosphorylation of MLC and MYPT1. In addition, pretreatment with DL0805-2 attenuated ROS production and F-actin formation in the cells. Conclusion： DL0805-2 exerts a vasodilatory action in rat aortic rings through inhibiting the Rho/ROCK pathway and calcium fluxes.Aim： DL0805-2 [N-（1H-indazol-5-yl）-1-（4-methylbenzyl） pyrrolidine-3-carboxamide] is a DL0805 derivative with more potent vasorelaxant activity and lower toxicity. This study was conducted to investigate the vasorelaxant mechanisms of DL0805-2 on angiotensin II （Ang II）- induced contractions of rat thoracic aortic rings in vitro. Methods： Rat thoracic aortic rings and rat aortic vascular smooth muscle cells （VSMCs） were pretreated with DL0805-2, and then stimulated with Ang II. The tension of the aortic rings was measured through an isometric force transducer. Ang II-induced protein phosphorylation, ROS production and F-actin formation were assessed with Western blotting and immunofluorescence assays. Intracellular free Ca2~ concentrations were detected with Fluo-3 AM. Results＂ Pretreatment with DL0805-2 （1-100 pmol/L） dose-dependently inhibited the constrictions of the aortic rings induced by a single dose of Ang II （10T mol/L） or accumulative addition of Ang II （10^-10-10^-7 mol/L）. The vasodilatory effect of DL0805-2 was independent of endothelium. In the aortic rings, pretreatment with DL0805-2 （1, 3, and 10 pmol/L） suppressed Ang II-induced Ca2~ influx and intracellular Ca^2＋ mobilization, and Ang II-induced phosphorylation of two substrates of Rho kinase （MLC and MYPT1）. In VSMCs, pretreatment with DL0805-2 （1, 3, and 10 pmol/L） also suppressed Ang II-induced Ca^2＋ fluxes and phosphorylation of MLC and MYPT1. In addition, pretreatment with DL0805-2 attenuated ROS production and F-actin formation in the cells. Conclusion： DL0805-2 exerts a vasodilatory action in rat aortic rings through inhibiting the Rho/ROCK pathway and calcium fluxes.

关 键 词：DL0805-2 angiotensin II VASORELAXATION Rho/ROCKs Ca^2＋ fluxes rat aortic ring vascular smooth muscle cells 

分 类 号：Q257[生物学—细胞生物学] Q813