title: DL0805-2, a novel indazole derivative, relaxes angiotensin II-induced contractions of rat aortic rings by inhibiting Rho kinase and calcium fluxes.
abstract: OBJECTIVE
  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 Ca(2+) concentrations were detected with Fluo-3 AM.
  
  
  RESULTS
  Pretreatment with DL0805-2 (1-100 μmol/L) dose-dependently inhibited the constrictions of the aortic rings induced by a single dose of Ang II (10(-7) 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 μmol/L) suppressed Ang II-induced Ca(2+) 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 μmol/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.
  
  
  CONCLUSIONS
  DL0805-2 exerts a vasodilatory action in rat aortic rings through inhibiting the Rho/ROCK pathway and calcium fluxes.
authors.affiliation: 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.|Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
authors.forename: Tian-Yi
authors.initials: TY
authors.lastname: Yuan
authors.affiliation: 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.|Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
authors.forename: Yu-Cai
authors.initials: YC
authors.lastname: Chen
authors.affiliation: 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.|Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
authors.forename: Hui-Fang
authors.initials: HF
authors.lastname: Zhang
authors.affiliation: Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.affiliation: 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.
authors.forename: Xiao-Zhen
authors.initials: XZ
authors.lastname: Jiao
authors.affiliation: 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.
authors.forename: Ping
authors.initials: P
authors.lastname: Xie
authors.affiliation: Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
authors.forename: Lian-Hua
authors.initials: LH
authors.lastname: Fang
authors.affiliation: 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.|Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
authors.forename: Guan-Hua
authors.initials: GH
authors.lastname: Du
mesh_terms: D000199:Actins / Q000378:metabolism; D000804:Angiotensin II / Q000494:pharmacology*; D057912:Angiotensin II Type 2 Receptor Blockers / Q000737:chemistry; D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects* / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D066298:In Vitro Techniques; D007191:Indazoles / Q000737:chemistry / Q000494:pharmacology*; D008297:Male; D062105:Molecular Docking Simulation; D009119:Muscle Contraction / Q000187:drug effects; D009126:Muscle Relaxation / Q000187:drug effects; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000502:physiology; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation; D054645:Protein Phosphatase 1 / Q000378:metabolism; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014662:Vasoconstrictor Agents / Q000494:pharmacology*; D014665:Vasodilator Agents / Q000737:chemistry / Q000494:pharmacology*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors*
chemical_list: D000199:Actins; D057912:Angiotensin II Type 2 Receptor Blockers; D007191:Indazoles; D018994:Myosin Light Chains; C000598341:N-(1H-indazol-5-yl)-1-(4-methylbenzyl)pyrrolidine-3-carboxamide; D017382:Reactive Oxygen Species; D014662:Vasoconstrictor Agents; D014665:Vasodilator Agents; D000804:Angiotensin II; D054460:rho-Associated Kinases; C499850:Ppp1r12a protein, rat; D054645:Protein Phosphatase 1; D002118:Calcium