Hemoglobin α/eNOS coupling at myoendothelial junctions is required for nitric oxide scavenging during vasoconstriction

Arterioscler Thromb Vasc Biol. 2014 Dec;34(12):2594-600. doi: 10.1161/ATVBAHA.114.303974. Epub 2014 Oct 2.


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.

Approach and 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.

Keywords: endothelial cells; endothelial nitric oxide synthase; hemoglobin α; nitric oxide.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Blood Pressure / physiology
  • Cells, Cultured
  • Computer Simulation
  • Conserved Sequence
  • Endothelial Cells / metabolism
  • Humans
  • Intercellular Junctions / physiology
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Myocytes, Smooth Muscle / metabolism
  • Nitric Oxide / metabolism*
  • Nitric Oxide Synthase Type III / deficiency
  • Nitric Oxide Synthase Type III / genetics
  • Nitric Oxide Synthase Type III / metabolism*
  • Protein Interaction Domains and Motifs
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Signal Transduction
  • Vascular Resistance / physiology
  • Vasoconstriction / physiology*
  • alpha-Globins / chemistry
  • alpha-Globins / genetics
  • alpha-Globins / metabolism*


  • Recombinant Proteins
  • alpha-Globins
  • Nitric Oxide
  • Nitric Oxide Synthase Type III
  • Nos3 protein, mouse