title: Nitric oxide regulates cardiac intracellular Na⁺ and Ca²⁺ by modulating Na/K ATPase via PKCε and phospholemman-dependent mechanism.
abstract: In the heart, Na/K-ATPase regulates intracellular Na(+) and Ca(2+) (via NCX), thereby preventing Na(+) and Ca(2+) overload and arrhythmias. Here, we test the hypothesis that nitric oxide (NO) regulates cardiac intracellular Na(+) and Ca(2+) and investigate mechanisms and physiological consequences involved. Effects of both exogenous NO (via NO-donors) and endogenously synthesized NO (via field-stimulation of ventricular myocytes) were assessed in this study. Field stimulation of rat ventricular myocytes significantly increased endogenous NO (18 ± 2 μM), PKCε activation (82 ± 12%), phospholemman phosphorylation (at Ser-63 and Ser-68) and Na/K-ATPase activity (measured by DAF-FM dye, western-blotting and biochemical assay, respectively; p<0.05, n=6) and all were abolished by Ca(2+)-chelation (EGTA 10mM) or NOS inhibition l-NAME (1mM). Exogenously added NO (spermine-NONO-ate) stimulated Na/K-ATPase (EC50=3.8 μM; n=6/grp), via decrease in Km, in PLM(WT) but not PLM(KO) or PLM(3SA) myocytes (where phospholemman cannot be phosphorylated) as measured by whole-cell perforated-patch clamp. Field-stimulation with l-NAME or PKC-inhibitor (2 μM Bis) resulted in elevated intracellular Na(+) (22 ± 1.5 and 24 ± 2 respectively, vs. 14 ± 0.6mM in controls) in SBFI-AM-loaded rat myocytes. Arrhythmia incidence was significantly increased in rat hearts paced in the presence of l-NAME (and this was reversed by l-arginine), as well as in PLM(3SA) mouse hearts but not PLM(WT) and PLM(KO). We provide physiological and biochemical evidence for a novel regulatory pathway whereby NO activates Na/K-ATPase via phospholemman phosphorylation and thereby limits Na(+) and Ca(2+) overload and arrhythmias. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes".
authors.affiliation: Cardiovascular Division, King's College London, The Rayne Institute, St. Thomas' Hospital, London, UK.
authors.forename: Davor
authors.initials: D
authors.lastname: Pavlovic
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Hall
authors.forename: Erika J
authors.initials: EJ
authors.lastname: Kennington
authors.forename: Karen
authors.initials: K
authors.lastname: Aughton
authors.forename: Andrii
authors.initials: A
authors.lastname: Boguslavskyi
authors.forename: William
authors.initials: W
authors.lastname: Fuller
authors.forename: Sanda
authors.initials: S
authors.lastname: Despa
authors.forename: Donald M
authors.initials: DM
authors.lastname: Bers
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Shattock
mesh_terms: D000200:Action Potentials; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004558:Electric Stimulation; D006352:Heart Ventricles / Q000166:cytology; D066298:In Vitro Techniques; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D032383:Myocytes, Cardiac / Q000502:physiology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D051744:Protein Kinase C-epsilon / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D000099211:Phospholamban
chemical_list: D002118:Calcium; D002135:Calcium-Binding Proteins; D008565:Membrane Proteins; D019331:NG-Nitroarginine Methyl Ester; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D010750:Phosphoproteins; D051744:Protein Kinase C-epsilon; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D000099211:Phospholamban; C069141:phospholemman
keywords: 2,3-butanedione monoxime; ARVM; Arrhythmia; BDM; Bis; EGTA; FXYD-1; GC; N(G)-nitro-l-arginine methyl ester; NO; Nitric oxide; PKC; PLB; PLM; Phospholemman; Protein kinase C; Sodium pump; VASP; VF; adult rat ventricular myocytes; bisindolylmaleimide; ethylene glycol tetraacetic acid; guanylate cyclase; l-NAME; nitric oxide; phospholamban; phospholemman; protein kinase C; vasodilatory protein; ventricular fibrillation