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

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. cAMP was measured by radioimmunoassay, HSP20 phosphorylation by 2D gel electrophoresis with immunoblotting, and membrane potential and free cytosolic calcium by flow cytometry. 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. 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.