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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.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.
SUBMITTER: Straub AC
PROVIDER: S-EPMC4239174 | biostudies-literature | 2014 Dec
REPOSITORIES: biostudies-literature
Straub Adam C AC Butcher Joshua T JT Billaud Marie M Mutchler Stephanie M SM Artamonov Mykhaylo V MV Nguyen Anh T AT Johnson Tyler T Best Angela K AK Miller Megan P MP Palmer Lisa A LA Columbus Linda L Somlyo Avril V AV Le Thu H TH Isakson Brant E BE
Arteriosclerosis, thrombosis, and vascular biology 20141002 12
<h4>Objective</h4>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.<h4>Approach and results</h4>In silico modeling of Hb α and eNOS identified a conserved sequence of interaction. By mutating por ...[more]