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Updated: Apr 23, 2026
En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries
Published on: February 25, 2016
Hemoglobin α/eNOS coupling at myoendothelial junctions is required for nitric oxide scavenging during
Adam C Straub1, Joshua T Butcher1, Marie Billaud1
1From 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.
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.
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