title: Urocortin 2 stimulates nitric oxide production in ventricular myocytes via Akt- and PKA-mediated phosphorylation of eNOS at serine 1177.
abstract: Urocortin 2 (Ucn2) is a cardioactive peptide exhibiting beneficial effects in normal and failing heart. In cardiomyocytes, it elicits cAMP- and Ca(2+)-dependent positive inotropic and lusitropic effects. We tested the hypothesis that, in addition, Ucn2 activates cardiac nitric oxide (NO) signaling and elucidated the underlying signaling pathways and mechanisms. In isolated rabbit ventricular myocytes, Ucn2 caused concentration- and time-dependent increases in phosphorylation of Akt (Ser473, Thr308), endothelial NO synthase (eNOS) (Ser1177), and ERK1/2 (Thr202/Tyr204). ERK1/2 phosphorylation, but not Akt and eNOS phosphorylation, was suppressed by inhibition of MEK1/2. Increased Akt phosphorylation resulted in increased Akt kinase activity and was mediated by corticotropin-releasing factor 2 (CRF2) receptors (astressin-2B sensitive). Inhibition of phosphatidylinositol 3-kinase (PI3K) diminished both Akt as well as eNOS phosphorylation mediated by Ucn2. Inhibition of protein kinase A (PKA) reduced Ucn2-induced phosphorylation of eNOS but did not affect the increase in phosphorylation of Akt. Conversely, direct receptor-independent elevation of cAMP via forskolin increased phosphorylation of eNOS but not of Akt. Ucn2 increased intracellular NO concentration ([NO]i), [cGMP], [cAMP], and cell shortening. Inhibition of eNOS suppressed the increases in [NO]i and cell shortening. When both PI3K-Akt and cAMP-PKA signaling were inhibited, the Ucn2-induced increases in [NO]i and cell shortening were attenuated. Thus, in rabbit ventricular myocytes, Ucn2 causes activation of cAMP-PKA, PI3K-Akt, and MEK1/2-ERK1/2 signaling. The MEK1/2-ERK1/2 pathway is not required for stimulation of NO signaling in these cells. The other two pathways, cAMP-PKA and PI3K-Akt, converge on eNOS phosphorylation at Ser1177 and result in pronounced and sustained cellular NO production with subsequent stimulation of cGMP signaling.
authors.affiliation: Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, Illinois;
authors.forename: Stefanie
authors.initials: S
authors.lastname: Walther
authors.affiliation: Institute of Pharmacology and Clinical Pharmacy, Biochemical and Pharmacological Centre Marburg, Philipps-University of Marburg, Marburg, Germany;
authors.forename: Florentina
authors.initials: F
authors.lastname: Pluteanu
authors.affiliation: Department of Cardiology and Pneumology, University Medicine Göttingen, Göttingen, Germany;
authors.forename: Susanne
authors.initials: S
authors.lastname: Renz
authors.affiliation: Institute of Pharmacology and Clinical Pharmacy, Biochemical and Pharmacological Centre Marburg, Philipps-University of Marburg, Marburg, Germany;
authors.forename: Yulia
authors.initials: Y
authors.lastname: Nikonova
authors.affiliation: Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, Illinois;
authors.forename: Joshua T
authors.initials: JT
authors.lastname: Maxwell
authors.affiliation: Molecular Neuroendocrinology Group of the Max Planck Institute for Experimental Medicine, Göttingen, Germany, and Specialized Neuroscience Research Program 2, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii; Division of Endocrinology, Department of Internal Medicine, Shanghai Ninth People's Hospital of Shanghai Jiaotong University, Shanghai, China;
authors.forename: Li-Zhen
authors.initials: LZ
authors.lastname: Yang
authors.affiliation: Institute of Pharmaceutical Sciences, Pharmacology and Toxicology, Karl-Franzens-University of Graz, Graz, Austria.
authors.forename: Kurt
authors.initials: K
authors.lastname: Schmidt
authors.affiliation: Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, Illinois;
authors.forename: Joshua N
authors.initials: JN
authors.lastname: Edwards
authors.affiliation: Division of Cardiology, Medical University of Graz, Graz, Austria;
authors.forename: Paulina
authors.initials: P
authors.lastname: Wakula
authors.affiliation: Ludwig-Boltzmann-Institute for Translational Heart Failure Research, Graz, Austria; Institute of Biophysics, Medical University of Graz, Graz, Austria;
authors.forename: Klaus
authors.initials: K
authors.lastname: Groschner
authors.affiliation: Department of Cardiology and Pneumology, University Medicine Göttingen, Göttingen, Germany; Clinic and Policlinic for Internal Medicine II, University Clinics Regensburg, Regensburg, Germany; and.
authors.forename: Lars S
authors.initials: LS
authors.lastname: Maier
authors.affiliation: Molecular Neuroendocrinology Group of the Max Planck Institute for Experimental Medicine, Göttingen, Germany, and Specialized Neuroscience Research Program 2, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii; Sanford Burnham Medical Research Institute, La Jolla, California.
authors.forename: Joachim
authors.initials: J
authors.lastname: Spiess
authors.affiliation: Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, Illinois;
authors.forename: Lothar A
authors.initials: LA
authors.lastname: Blatter
authors.affiliation: Division of Cardiology, Medical University of Graz, Graz, Austria; Ludwig-Boltzmann-Institute for Translational Heart Failure Research, Graz, Austria;
authors.forename: Burkert
authors.initials: B
authors.lastname: Pieske
authors.affiliation: Institute of Pharmacology and Clinical Pharmacy, Biochemical and Pharmacological Centre Marburg, Philipps-University of Marburg, Marburg, Germany; jens.kockskaemper@staff.uni-marburg.de.
authors.forename: Jens
authors.initials: J
authors.lastname: Kockskämper
mesh_terms: D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011817:Rabbits; D018019:Receptors, Corticotropin-Releasing Hormone / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D054832:Urocortins / Q000378:metabolism*
chemical_list: C101531:CRF receptor type 2; D018019:Receptors, Corticotropin-Releasing Hormone; D054832:Urocortins; D009569:Nitric Oxide; D012694:Serine; D000242:Cyclic AMP; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D017868:Cyclic AMP-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D006152:Cyclic GMP
keywords: cardiac myocyte; eNOS phosphorylation; nitric oxide; urocortin