title: Regulation of cardiac nitric oxide signaling by nuclear β-adrenergic and endothelin receptors.
abstract: At the cell surface, βARs and endothelin receptors can regulate nitric oxide (NO) production. β-adrenergic receptors (βARs) and type B endothelin receptors (ETB) are present in cardiac nuclear membranes and regulate transcription. The present study investigated the role of the NO pathway in the regulation of gene transcription by these nuclear G protein-coupled receptors. Nitric oxide production and transcription initiation were measured in nuclei isolated from the adult rat heart. The cell-permeable fluorescent dye 4,5-diaminofluorescein diacetate (DAF2 DA) was used to provide a direct assessment of nitric oxide release. Both isoproterenol and endothelin increased NO production in isolated nuclei. Furthermore, a β3AR-selective agonist, BRL 37344, increased NO synthesis whereas the β1AR-selective agonist xamoterol did not. Isoproterenol increased, whereas ET-1 reduced, de novo transcription. The NO synthase inhibitor l-NAME prevented isoproterenol from increasing either NO production or de novo transcription. l-NAME also blocked ET-1-induced NO-production but did not alter the suppression of transcription initiation by ET-1. Inhibition of the cGMP-dependent protein kinase (PKG) using KT5823 also blocked the ability of isoproterenol to increase transcription initiation. Furthermore, immunoblotting revealed eNOS, but not nNOS, in isolated nuclei. Finally, caged, cell-permeable isoproterenol and endothelin-1 analogs were used to selectively activate intracellular β-adrenergic and endothelin receptors in intact adult cardiomyocytes. Intracellular release of caged ET-1 or isoproterenol analogs increased NO production in intact adult cardiomyocytes. Hence, activation of the NO synthase/guanylyl cyclase/PKG pathway is necessary for nuclear β3ARs to increase de novo transcription. Furthermore, we have demonstrated the potential utility of caged receptor ligands in selectively modulating signaling via endogenous intracellular G protein-coupled receptors.
authors.affiliation: Department of Biochemistry, Université de Montréal, Montreal, Quebec H3T 1J4, Canada.
authors.forename: George
authors.initials: G
authors.lastname: Vaniotis
authors.forename: Irina
authors.initials: I
authors.lastname: Glazkova
authors.forename: Clémence
authors.initials: C
authors.lastname: Merlen
authors.forename: Carter
authors.initials: C
authors.lastname: Smith
authors.forename: Louis R
authors.initials: LR
authors.lastname: Villeneuve
authors.forename: David
authors.initials: D
authors.lastname: Chatenet
authors.forename: Michel
authors.initials: M
authors.lastname: Therien
authors.forename: Alain
authors.initials: A
authors.lastname: Fournier
authors.forename: Artavazd
authors.initials: A
authors.lastname: Tadevosyan
authors.forename: Phan
authors.initials: P
authors.lastname: Trieu
authors.forename: Stanley
authors.initials: S
authors.lastname: Nattel
authors.forename: Terence E
authors.initials: TE
authors.lastname: Hébert
authors.forename: Bruce G
authors.initials: BG
authors.lastname: Allen
mesh_terms: D000818:Animals; D019332:Endothelin-1 / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D011804:Quinolines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D011943:Receptors, Adrenergic, beta / Q000235:genetics / Q000378:metabolism*; D017466:Receptors, Endothelin / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D019332:Endothelin-1; D011804:Quinolines; D011943:Receptors, Adrenergic, beta; D017466:Receptors, Endothelin; D009569:Nitric Oxide; C000470:EEDQ; D007545:Isoproterenol; D019331:NG-Nitroarginine Methyl Ester
keywords: AC; ATP; CYP; DAF-2; DNA; DTT; EEDQ; ET-1; ETB; ETR; Endothelin receptors; G protein-coupled receptor; GC; GPCR; ISO; N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; NFκB; NO; NOS; Nitric oxide; Nuclear membranes; Nup62; PBS; PI3K; PKB; PKG; PMSF; PTX; Protein kinase G; RNA; TCA; TX-100; Transcription; Triton X-100; UTP; adenosine triphosphate; adenylyl cyclase; cET-1; cGMP; caged ET-1; cyanopindolol; cyclic guanosine 3′,5′-monophosphate; deoxyribonucleic acid; diaminofluorescein-2; dithiothreitol; endothelin 1; endothelin receptor; endothelin type B receptor; guanylyl cyclase; isoproterenol; nitric oxide; nitric oxide synthase; nuclear factor kappa-light-chain-enhancer of activated B-cells; nucleoporin 62; pertussis toxin; phenylmethanesulfonylfluoride; phosphate buffered saline; phosphoinositide 3-kinase; protein kinase B; protein kinase G; qPCR; quantitative real-time polymerase chain reaction; ribonucleic acid; trichloroacetic acid; uridine 5(′) triphosphate; α-adrenergic receptor; αAR; β-Adrenergic receptors; β-adrenergic receptor; βAR