Regulation of cardiac nitric oxide signaling by nuclear β-adrenergic and endothelin receptors.
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.
['Vaniotis|George|G|', 'Glazkova|Irina|I|', 'Merlen|Clémence|C|', 'Smith|Carter|C|', 'Villeneuve|Louis R|LR|', 'Chatenet|David|D|', 'Therien|Michel|M|', 'Fournier|Alain|A|', 'Tadevosyan|Artavazd|A|', 'Trieu|Phan|P|', 'Nattel|Stanley|S|', 'Hébert|Terence E|TE|', 'Allen|Bruce G|BG|']
D000818:Animals D019332:Endothelin-1 D007545:Isoproterenol D008297:Male D032383:Myocytes, Cardiac D019331:NG-Nitroarginine Methyl Ester D009569:Nitric Oxide D011804:Quinolines D051381:Rats D017207:Rats, Sprague-Dawley D060888:Real-Time Polymerase Chain Reaction D011943:Receptors, Adrenergic, beta D017466:Receptors, Endothelin D015398:Signal Transduction
Nitric oxide Nitric oxide Endothelin receptors Endothelin receptors βAR NO NO β-Adrenergic receptors β-Adrenergic receptors ETB Nuclear membranes Transcription G protein-coupled receptor isoproterenol ET-1 PKG caged ET-1 guanylyl cyclase AC ATP CYP DAF-2 DNA DTT EEDQ ETR GC GPCR ISO N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline NFκB NOS Nup62 PBS PI3K PKB PMSF PTX Protein kinase G RNA TCA TX-100 Triton X-100 UTP adenosine triphosphate adenylyl cyclase cET-1 cGMP cyanopindolol cyclic guanosine 3′,5′-monophosphate deoxyribonucleic acid diaminofluorescein-2 dithiothreitol endothelin 1 endothelin receptor endothelin type B receptor 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 receptor