title: Anti-hypertrophic and anti-oxidant effect of beta3-adrenergic stimulation in myocytes requires differential neuronal NOS phosphorylation.
abstract: BACKGROUND
  Stimulation of β3-adrenoreceptors (β3-AR) blunts contractility and improves chronic left ventricular function in hypertrophied and failing hearts in a neuronal nitric oxide synthase (nNOS) dependent manner. nNOS can be regulated by post-translational modification of stimulatory phosphorylation residue Ser1412 and inhibitory residue Ser847. However, the role of phosphorylation of these residues in cardiomyocytes and β3-AR protective signaling has yet to be explored.
  
  
  OBJECTIVE
  We tested the hypothesis that β3-AR regulation of myocyte stress requires changes in nNOS activation mediated by differential nNOS phosphorylation.
  
  
  RESULTS
  Endothelin (ET-1) or norepinephrine induced hypertrophy in rat neonatal ventricular cardiomyocytes (NRVMs) was accompanied by increased β3-AR gene expression. Co-administration of the β3-AR agonist BRL-37433 (BRL) reduced cell size and reactive oxygen species (ROS) generation, while augmenting NOS activity. BRL-dependent augmentation of NOS activity and ROS suppression due to NE were blocked by inhibiting nNOS (L-VNIO). BRL augmented nNOS phosphorylation at Ser1412 and dephosphorylation at Ser847. Cells expressing constitutively dephosphorylated Ser1412A or phosphorylated Ser847D nNOS mutants displayed reduced nNOS activity and a lack of BRL modulation. BRL also failed to depress ROS from NE in cells with nNOS-Ser847D. Inhibiting Akt decreased BRL-induced nNOS-Ser1412 phosphorylation and NOS activation, whereas Gi/o blockade blocked BRL-regulation of both post-translational modifications, preventing enhancement of NOS activity and ROS reduction. BRL resulted in near complete dephosphorylation of Ser847 and a moderate rise in Ser1412 phosphorylation in mouse myocardium exposed to chronic pressure-overload.
  
  
  CONCLUSIONS
  β3-AR regulates myocardial NOS activity and ROS via activation of nNOS involving reciprocal changes in phosphorylation at two regulatory sites. These data identify a novel and potent anti-oxidant and anti-hypertrophic pathway due to nNOS post-translational modification that is coupled to β3-AR receptor stimulation.
authors: Department of Medicine, Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors: Vabren L
authors: VL
authors: Watts
authors: Fernando M
authors: FM
authors: Sepulveda
authors: Oscar H
authors: OH
authors: Cingolani
authors: Alice S
authors: AS
authors: Ho
authors: Xiaolin
authors: X
authors: Niu
authors: Rosa
authors: R
authors: Kim
authors: Karen L
authors: KL
authors: Miller
authors: Koenraad
authors: K
authors: Vandegaer
authors: Djahida
authors: D
authors: Bedja
authors: Kathleen L
authors: KL
authors: Gabrielson
authors: Gerald
authors: G
authors: Rameau
authors: Brian
authors: B
authors: O'Rourke
authors: David A
authors: DA
authors: Kass
authors: Lili A
authors: LA
authors: Barouch
mesh_terms: D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D002478:Cells, Cultured; D004983:Ethanolamines / Q000494:pharmacology; D047468:Immunoprecipitation; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D032342:Muscle Cells / Q000187:drug effects / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism*; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species; D022702:Receptors, Adrenergic, beta-3 / Q000378:metabolism*
chemical_list: D000318:Adrenergic beta-Agonists; D000975:Antioxidants; D004983:Ethanolamines; D017382:Reactive Oxygen Species; D022702:Receptors, Adrenergic, beta-3; C057368:BRL 37344; D052248:Nitric Oxide Synthase Type I
keywords: Beta3-adrenergic receptors; Heart failure; Hypertrophy; Neuronal nitric oxide synthase; Reactive oxygen species
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