title: Hemoglobin α/eNOS coupling at myoendothelial junctions is required for nitric oxide scavenging during vasoconstriction.
abstract: OBJECTIVE
  Hemoglobin α (Hb α) and endothelial nitric oxide synthase (eNOS) form a macromolecular complex at myoendothelial junctions; the functional role of this interaction remains undefined. To test if coupling of eNOS and Hb α regulates nitric oxide signaling, vascular reactivity, and blood pressure using a mimetic peptide of Hb α to disrupt this interaction.
  
  
  RESULTS
  In silico modeling of Hb α and eNOS identified a conserved sequence of interaction. By mutating portions of Hb α, we identified a specific sequence that binds eNOS. A mimetic peptide of the Hb α sequence (Hb α X) was generated to disrupt this complex. Using in vitro binding assays with purified Hb α and eNOS and ex vivo proximity ligation assays on resistance arteries, we have demonstrated that Hb α X significantly decreased interaction between eNOS and Hb α. Fluorescein isothiocyanate labeling of Hb α X revealed localization to holes in the internal elastic lamina (ie, myoendothelial junctions). To test the functional effects of Hb α X, we measured cyclic guanosine monophosphate and vascular reactivity. Our results reveal augmented cyclic guanosine monophosphate production and altered vasoconstriction with Hb α X. To test the in vivo effects of these peptides on blood pressure, normotensive and hypertensive mice were injected with Hb α X, which caused a significant decrease in blood pressure; injection of Hb α X into eNOS(-/-) mice had no effect.
  
  
  CONCLUSIONS
  These results identify a novel sequence on Hb α that is important for Hb α/eNOS complex formation and is critical for nitric oxide signaling at myoendothelial junctions.
authors.affiliation: From the Department of Pharmacology and Chemical Biology (A.C.S.) and Heart, Lung, Blood and Vascular Medicine Institute (A.C.S., A.T.N., M.P.M.), University of Pittsburgh, PA; Robert M. Berne Cardiovascular Research Center (J.T.B., M.B., S.M.M., T.J., A.K.B., B.E.I.), Department of Molecular Physiology and Biophysics (M.B., M.V.A., A.V.S., B.E.I.), Deparment of Pediatrics (L.A.P.), Department of Chemistry (L.C.), and Department of Medicine (T.H.L.), University of Virginia, Charlottesville.
authors.forename: Adam C
authors.initials: AC
authors.lastname: Straub
authors.affiliation: From the Department of Pharmacology and Chemical Biology (A.C.S.) and Heart, Lung, Blood and Vascular Medicine Institute (A.C.S., A.T.N., M.P.M.), University of Pittsburgh, PA; Robert M. Berne Cardiovascular Research Center (J.T.B., M.B., S.M.M., T.J., A.K.B., B.E.I.), Department of Molecular Physiology and Biophysics (M.B., M.V.A., A.V.S., B.E.I.), Deparment of Pediatrics (L.A.P.), Department of Chemistry (L.C.), and Department of Medicine (T.H.L.), University of Virginia, Charlottesville.
authors.forename: Joshua T
authors.initials: JT
authors.lastname: Butcher
authors.affiliation: From the Department of Pharmacology and Chemical Biology (A.C.S.) and Heart, Lung, Blood and Vascular Medicine Institute (A.C.S., A.T.N., M.P.M.), University of Pittsburgh, PA; Robert M. Berne Cardiovascular Research Center (J.T.B., M.B., S.M.M., T.J., A.K.B., B.E.I.), Department of Molecular Physiology and Biophysics (M.B., M.V.A., A.V.S., B.E.I.), Deparment of Pediatrics (L.A.P.), Department of Chemistry (L.C.), and Department of Medicine (T.H.L.), University of Virginia, Charlottesville.
authors.forename: Marie
authors.initials: M
authors.lastname: Billaud
authors.affiliation: From the Department of Pharmacology and Chemical Biology (A.C.S.) and Heart, Lung, Blood and Vascular Medicine Institute (A.C.S., A.T.N., M.P.M.), University of Pittsburgh, PA; Robert M. Berne Cardiovascular Research Center (J.T.B., M.B., S.M.M., T.J., A.K.B., B.E.I.), Department of Molecular Physiology and Biophysics (M.B., M.V.A., A.V.S., B.E.I.), Deparment of Pediatrics (L.A.P.), Department of Chemistry (L.C.), and Department of Medicine (T.H.L.), University of Virginia, Charlottesville.
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Mutchler
authors.affiliation: From the Department of Pharmacology and Chemical Biology (A.C.S.) and Heart, Lung, Blood and Vascular Medicine Institute (A.C.S., A.T.N., M.P.M.), University of Pittsburgh, PA; Robert M. Berne Cardiovascular Research Center (J.T.B., M.B., S.M.M., T.J., A.K.B., B.E.I.), Department of Molecular Physiology and Biophysics (M.B., M.V.A., A.V.S., B.E.I.), Deparment of Pediatrics (L.A.P.), Department of Chemistry (L.C.), and Department of Medicine (T.H.L.), University of Virginia, Charlottesville.
authors.forename: Mykhaylo V
authors.initials: MV
authors.lastname: Artamonov
authors.affiliation: From the Department of Pharmacology and Chemical Biology (A.C.S.) and Heart, Lung, Blood and Vascular Medicine Institute (A.C.S., A.T.N., M.P.M.), University of Pittsburgh, PA; Robert M. Berne Cardiovascular Research Center (J.T.B., M.B., S.M.M., T.J., A.K.B., B.E.I.), Department of Molecular Physiology and Biophysics (M.B., M.V.A., A.V.S., B.E.I.), Deparment of Pediatrics (L.A.P.), Department of Chemistry (L.C.), and Department of Medicine (T.H.L.), University of Virginia, Charlottesville.
authors.forename: Anh T
authors.initials: AT
authors.lastname: Nguyen
authors.affiliation: From the Department of Pharmacology and Chemical Biology (A.C.S.) and Heart, Lung, Blood and Vascular Medicine Institute (A.C.S., A.T.N., M.P.M.), University of Pittsburgh, PA; Robert M. Berne Cardiovascular Research Center (J.T.B., M.B., S.M.M., T.J., A.K.B., B.E.I.), Department of Molecular Physiology and Biophysics (M.B., M.V.A., A.V.S., B.E.I.), Deparment of Pediatrics (L.A.P.), Department of Chemistry (L.C.), and Department of Medicine (T.H.L.), University of Virginia, Charlottesville.
authors.forename: Tyler
authors.initials: T
authors.lastname: Johnson
authors.affiliation: From the Department of Pharmacology and Chemical Biology (A.C.S.) and Heart, Lung, Blood and Vascular Medicine Institute (A.C.S., A.T.N., M.P.M.), University of Pittsburgh, PA; Robert M. Berne Cardiovascular Research Center (J.T.B., M.B., S.M.M., T.J., A.K.B., B.E.I.), Department of Molecular Physiology and Biophysics (M.B., M.V.A., A.V.S., B.E.I.), Deparment of Pediatrics (L.A.P.), Department of Chemistry (L.C.), and Department of Medicine (T.H.L.), University of Virginia, Charlottesville.
authors.forename: Angela K
authors.initials: AK
authors.lastname: Best
authors.affiliation: From the Department of Pharmacology and Chemical Biology (A.C.S.) and Heart, Lung, Blood and Vascular Medicine Institute (A.C.S., A.T.N., M.P.M.), University of Pittsburgh, PA; Robert M. Berne Cardiovascular Research Center (J.T.B., M.B., S.M.M., T.J., A.K.B., B.E.I.), Department of Molecular Physiology and Biophysics (M.B., M.V.A., A.V.S., B.E.I.), Deparment of Pediatrics (L.A.P.), Department of Chemistry (L.C.), and Department of Medicine (T.H.L.), University of Virginia, Charlottesville.
authors.forename: Megan P
authors.initials: MP
authors.lastname: Miller
authors.affiliation: From the Department of Pharmacology and Chemical Biology (A.C.S.) and Heart, Lung, Blood and Vascular Medicine Institute (A.C.S., A.T.N., M.P.M.), University of Pittsburgh, PA; Robert M. Berne Cardiovascular Research Center (J.T.B., M.B., S.M.M., T.J., A.K.B., B.E.I.), Department of Molecular Physiology and Biophysics (M.B., M.V.A., A.V.S., B.E.I.), Deparment of Pediatrics (L.A.P.), Department of Chemistry (L.C.), and Department of Medicine (T.H.L.), University of Virginia, Charlottesville.
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Palmer
authors.affiliation: From the Department of Pharmacology and Chemical Biology (A.C.S.) and Heart, Lung, Blood and Vascular Medicine Institute (A.C.S., A.T.N., M.P.M.), University of Pittsburgh, PA; Robert M. Berne Cardiovascular Research Center (J.T.B., M.B., S.M.M., T.J., A.K.B., B.E.I.), Department of Molecular Physiology and Biophysics (M.B., M.V.A., A.V.S., B.E.I.), Deparment of Pediatrics (L.A.P.), Department of Chemistry (L.C.), and Department of Medicine (T.H.L.), University of Virginia, Charlottesville.
authors.forename: Linda
authors.initials: L
authors.lastname: Columbus
authors.affiliation: From the Department of Pharmacology and Chemical Biology (A.C.S.) and Heart, Lung, Blood and Vascular Medicine Institute (A.C.S., A.T.N., M.P.M.), University of Pittsburgh, PA; Robert M. Berne Cardiovascular Research Center (J.T.B., M.B., S.M.M., T.J., A.K.B., B.E.I.), Department of Molecular Physiology and Biophysics (M.B., M.V.A., A.V.S., B.E.I.), Deparment of Pediatrics (L.A.P.), Department of Chemistry (L.C.), and Department of Medicine (T.H.L.), University of Virginia, Charlottesville.
authors.forename: Avril V
authors.initials: AV
authors.lastname: Somlyo
authors.affiliation: From the Department of Pharmacology and Chemical Biology (A.C.S.) and Heart, Lung, Blood and Vascular Medicine Institute (A.C.S., A.T.N., M.P.M.), University of Pittsburgh, PA; Robert M. Berne Cardiovascular Research Center (J.T.B., M.B., S.M.M., T.J., A.K.B., B.E.I.), Department of Molecular Physiology and Biophysics (M.B., M.V.A., A.V.S., B.E.I.), Deparment of Pediatrics (L.A.P.), Department of Chemistry (L.C.), and Department of Medicine (T.H.L.), University of Virginia, Charlottesville.
authors.forename: Thu H
authors.initials: TH
authors.lastname: Le
authors.affiliation: From the Department of Pharmacology and Chemical Biology (A.C.S.) and Heart, Lung, Blood and Vascular Medicine Institute (A.C.S., A.T.N., M.P.M.), University of Pittsburgh, PA; Robert M. Berne Cardiovascular Research Center (J.T.B., M.B., S.M.M., T.J., A.K.B., B.E.I.), Department of Molecular Physiology and Biophysics (M.B., M.V.A., A.V.S., B.E.I.), Deparment of Pediatrics (L.A.P.), Department of Chemistry (L.C.), and Department of Medicine (T.H.L.), University of Virginia, Charlottesville. brant@virginia.edu.
authors.forename: Brant E
authors.initials: BE
authors.lastname: Isakson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001794:Blood Pressure / Q000502:physiology; D002478:Cells, Cultured; D003198:Computer Simulation; D017124:Conserved Sequence; D042783:Endothelial Cells / Q000378:metabolism; D006801:Humans; D007365:Intercellular Junctions / Q000502:physiology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014655:Vascular Resistance / Q000502:physiology; D014661:Vasoconstriction / Q000502:physiology*; D055542:alpha-Globins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D055542:alpha-Globins; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse
keywords: endothelial cells; endothelial nitric oxide synthase; hemoglobin α; nitric oxide