title: Protein kinase A signalling is involved in the relaxant responses to the selective β-oestrogen receptor agonist diarylpropionitrile in rat aortic smooth muscle in vitro.
abstract: OBJECTIVE
  The oestrogen receptor β (ERβ) selective agonist diarylpropionitrile (DPN) relaxes endothelium-denuded rat aorta, but the signalling mechanism is unknown. The aim of this study was to assess whether protein kinase A (PKA) signalling is involved in DPN action.
  
  
  METHODS
  cAMP was measured by radioimmunoassay, HSP20 phosphorylation by 2D gel electrophoresis with immunoblotting, and membrane potential and free cytosolic calcium by flow cytometry.
  
  
  RESULTS
  DPN increased cAMP content and hyperpolarised cell membranes over the same range of concentrations as it relaxed phenylephrine-precontracted aortic rings (10-300 µM). DPN-induced vasorelaxation was largely reduced by the PKA inhibitors Rp-8-Br-cAMPS (8-bromoadenosine-3', 5'-cyclic monophosphorothioate, Rp-isomer) and H-89 (N-(2-bromocynnamyl(amino)ethyl)-5-isoquinoline sulfonamide HCl) (-73%) and by the adenylate cyclase inhibitor MDL12330A (cis-N-(2-phenylcyclopentyl)-azacyclotridec-1-en-2-amine)) (-65.5%). Conversely, the PKG inhibitor Rp-8-Br-cGMP was inactive against DPN vasorelaxation. In aortic smooth muscle segments, DPN increased PKA-dependent HSP20 phosphorylation, an effect reversed by H-89. Relaxant responses to DPN were modestly antagonised (-23 to -48% reduction; n=12 per compound) by the potassium channel inhibitors iberiotoxin, PNU-37883A, 4-aminopyridine, or BaCl(2) . All four potassium channel inhibitors together reduced DPN relaxation by 86±9% (n=12) and fully blocked DPN hyperpolarisation.
  
  
  CONCLUSIONS
  ERβ-dependent relaxation of rat aortic smooth muscle evokes an adenylate cyclase/cAMP/PKA signalling pathway, likely activating the cystic fibrosis transmembrane conductance regulator chloride channel and at least four potassium channels.
authors.affiliation: Department of Physiology and Pharmacology, School of Medicine, Zaragoza, Spain.
authors.forename: Marta S
authors.initials: MS
authors.lastname: Valero
authors.forename: Desiree
authors.initials: D
authors.lastname: Pereboom
authors.forename: Silvia
authors.initials: S
authors.lastname: Barcelo-Batllory
authors.forename: Laia
authors.initials: L
authors.lastname: Brines
authors.forename: Ricardo P
authors.initials: RP
authors.lastname: Garay
authors.forename: José O
authors.initials: JO
authors.lastname: Alda
mesh_terms: D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D047629:Estrogen Receptor beta / Q000819:agonists* / Q000378:metabolism; D050886:HSP20 Heat-Shock Proteins / Q000378:metabolism; D007473:Ion Channels / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D009124:Muscle Proteins / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D011422:Propionates / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects; D014664:Vasodilation / Q000187:drug effects
chemical_list: C486182:2,3-bis(4-hydroxyphenyl)-propionitrile; D000067956:Adenylyl Cyclase Inhibitors; D047629:Estrogen Receptor beta; D050886:HSP20 Heat-Shock Proteins; D007473:Ion Channels; D009124:Muscle Proteins; D009570:Nitriles; D011422:Propionates; D047428:Protein Kinase Inhibitors; C087458:Hspb6 protein, rat; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases