title: Expression of guanylyl cyclase-A/atrial natriuretic peptide receptor blocks the activation of protein kinase C in vascular smooth muscle cells. Role of cGMP and cGMP-dependent protein kinase.
abstract: To understand the molecular mechanisms of cellular signaling of atrial natriuretic peptide (ANP), we have studied its effect on the enzymatic activity of endogenous and overexpressed protein kinase C (PKC) in rat thoracic aortic vascular smooth muscle (RTASM) cells. Angiotensin II (ANG II), endothelin-1 (ET-1), and 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated fourfold to fivefold PKC activity in PKC-alpha cDNA-transfected RTASM cells. However, pretreatment of these cells with ANP significantly inhibited the agonist-stimulated PKC activity in a dose-dependent manner. The inhibitory effect of ANP was more effective if cells were transfected with both PKC-alpha and guanylyl cyclase-A/atrial natriuretic peptide receptor (Npra) cDNAs. The agonist-stimulated PKC activity was also inhibited if RTASM cells were pretreated with cGMP analog 8-bromo-cGMP; however, the treatment of cells with a cAMP analog, dibutyryl-cAMP, did not show any discernible effect. The pretreatment of cells with Npra antagonist A-71915, significantly blocked the production of cGMP as well as the inhibitory effect of ANP on PKC activity. To further examine whether the antagonistic action of ANP and 8-bromo-cGMP on agonist-stimulated PKC activity were mediated through cGMP-dependent protein kinase (PKG), cells were treated with ANP or 8-bromo-cGMP and activators of PKC in the presence of KT-5823, a specific inhibitor of PKG. The treatment of cells with KT-5823 significantly attenuated the inhibitory effects of both ANP and 8-bromo-cGMP on agonist-stimulated PKC activity. The results from these studies provide strong evidence that ANP antagonizes the activation of PKC in RTASM cells, involving guanylyl cyclase-A receptor Npra and second messenger cGMP. Our data further support the notion that ANP acts as a negative mediator of signaling cross-talks between Npra and PKC in a cGMP-dependent manner, probably involving cGMP-dependent protein kinase in this process.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical College of Georgia, School of Medicine, Augusta 30912-2100, USA.
authors.forename: R
authors.initials: R
authors.lastname: Kumar
authors.forename: W A
authors.initials: WA
authors.lastname: Cartledge
authors.forename: T M
authors.initials: TM
authors.lastname: Lincoln
authors.forename: K N
authors.initials: KN
authors.lastname: Pandey
mesh_terms: D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000494:pharmacology*; D002273:Carcinogens / Q000494:pharmacology; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000494:pharmacology*; D019332:Endothelin-1 / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006162:Guanylate Cyclase / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D044005:Tetrahydroisoquinolines; D014162:Transfection
chemical_list: D002273:Carcinogens; D019332:Endothelin-1; D010446:Peptide Fragments; D044005:Tetrahydroisoquinolines; D000804:Angiotensin II; C078792:A 71915; D009320:Atrial Natriuretic Factor; D011493:Protein Kinase C; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C091715:atrial natriuretic factor receptor A; D006152:Cyclic GMP; D013755:Tetradecanoylphorbol Acetate