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

AC Straub, JT Butcher, M Billaud… - … , and vascular biology, 2014 - Am Heart Assoc
AC Straub, JT Butcher, M Billaud, SM Mutchler, MV Artamonov, AT Nguyen, T Johnson…
Arteriosclerosis, thrombosis, and vascular biology, 2014Am Heart Assoc
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 …
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.
Am Heart Assoc