title: Cilostazol suppresses angiotensin II-induced vasoconstriction via protein kinase A-mediated phosphorylation of the transient receptor potential canonical 6 channel.
abstract: OBJECTIVE
  The goal of this study was to determine whether inhibition of transient receptor potential canonical (TRPC) channels underlies attenuation of angiotensin II (Ang II)-induced vasoconstriction by phosphodiesterase (PDE) 3 inhibition.
  
  
  RESULTS
  Pretreatment of rat thoracic aorta with cilostazol, a selective PDE3 inhibitor, suppressed vasoconstriction induced by Ang II but not that induced by KCl. The Ang II-induced contraction was largely dependent on Ca(2+) influx via receptor-operated cation channels. Cilostazol specifically suppressed diacylglycerol-activated TRPC channels (TRPC3/TRPC6/TRPC7) through protein kinase A (PKA)-dependent phosphorylation of TRPC channels in HEK293 cells. In contrast, we found that phosphorylation of TRPC6 at Thr69 was essential for the suppression of Ang II-induced Ca(2+) influx by PDE3 inhibition in rat aortic smooth muscle cells (RAoSMCs). Cilostazol specifically induced phosphorylation of endogenous TRPC6 at Thr69. The endogenous TRPC6, but not TRPC3, formed a ternary complex with PDE3 and PKA in RAoSMCs, suggesting the specificity of TRPC6 phosphorylation by PDE3 inhibition. Furthermore, inhibition of PDE3 suppressed the Ang II-induced contraction of reconstituted ring with RAoSMCs, which were abolished by the expression of a phosphorylation-deficient mutant of TRPC6.
  
  
  CONCLUSIONS
  PKA-mediated phosphorylation of TRPC6 at Thr69 is essential for the vasorelaxant effects of PDE3 inhibition against the vasoconstrictive actions of Ang II.
authors.affiliation: Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka, Japan.
authors.forename: Kinue
authors.initials: K
authors.lastname: Nishioka
authors.forename: Motohiro
authors.initials: M
authors.lastname: Nishida
authors.forename: Marina
authors.initials: M
authors.lastname: Ariyoshi
authors.forename: Zhong
authors.initials: Z
authors.lastname: Jian
authors.forename: Shota
authors.initials: S
authors.lastname: Saiki
authors.forename: Mayumi
authors.initials: M
authors.lastname: Hirano
authors.forename: Michio
authors.initials: M
authors.lastname: Nakaya
authors.forename: Yoji
authors.initials: Y
authors.lastname: Sato
authors.forename: Satomi
authors.initials: S
authors.lastname: Kita
authors.forename: Takahiro
authors.initials: T
authors.lastname: Iwamoto
authors.forename: Katsuya
authors.initials: K
authors.lastname: Hirano
authors.forename: Ryuji
authors.initials: R
authors.lastname: Inoue
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Kurose
mesh_terms: D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000201:enzymology; D020013:Calcium Signaling / Q000187:drug effects; D000077407:Cilostazol; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000201:enzymology; D009154:Mutation; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000201:enzymology; D058987:Phosphodiesterase 3 Inhibitors / Q000494:pharmacology*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D051381:Rats; D017207:Rats, Sprague-Dawley; D050052:TRPC Cation Channels / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D000074287:TRPC6 Cation Channel; D013777:Tetrazoles / Q000494:pharmacology*; D014162:Transfection; D014661:Vasoconstriction / Q000187:drug effects*; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D014665:Vasodilator Agents / Q000494:pharmacology*; D020741:rho GTP-Binding Proteins / Q000378:metabolism
chemical_list: D004075:Diglycerides; D058987:Phosphodiesterase 3 Inhibitors; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; D013777:Tetrazoles; C492146:Trpc6 protein, mouse; C492145:Trpc6 protein, rat; D014662:Vasoconstrictor Agents; D014665:Vasodilator Agents; D000804:Angiotensin II; D017868:Cyclic AMP-Dependent Protein Kinases; D020741:rho GTP-Binding Proteins; D000077407:Cilostazol