Hemoglobin Interacts with Endothelial Nitric Oxide Synthase to Regulate Vasodilation in Human Resistance Arteries

ABSTRACT Background: In small arteries, constriction of vascular smooth muscle triggers local release of nitric oxide from the adjacent endothelial cell. This feedback vasodilation is a homeostatic mechanism that opposes vasoconstriction. Here, we investigate the role of endothelial alpha globin as a regulator of directed nitric oxide signaling across the myoendothelial junction. Methods: Human omental arteries 100-200µm in diameter were microdissected from omentum samples obtained during clinically indicated abdominal operations on NIH protocol 13-C-0176 (NCT01915225). Each artery was cannulated, perfused free of blood, and preserved for analysis or subjected to pressure myography. Preserved arteries underwent RNA extraction for gene expression; protein extraction for co-immunoprecipitation and Western blot; or immunostaining for multiphoton microscopy. Bio-layer interferometry quantified the binding of alpha globin to endothelial nitric oxide synthase (eNOS). Ex vivo pressure myography characterized arterial vasoreactivity before and after disruption of eNOS-Hb binding with an alpha globin mimetic peptide. Results: HBA1, HBA2, HBB, and NOS3 transcripts were abundant in RNA from the artery wall, and the blood cell gene SLC4A1 was not. Beta globin and eNOS co-immunoprecipitated with alpha globin in protein extracted from human omental artery segments, suggesting an eNOS-hemoglobin complex. Biolayer interferometry studies estimated alpha globin to bind to the oxidase domain of eNOS with an equilibrium dissociation constant of 1.3 x 10-6 M. Multiphoton microscopy of intact arteries revealed alpha globin, beta globin, and eNOS to co-localize within distinct punctates in a plane defined by the internal elastic lamina that separates endothelial cells from vascular smooth muscle. Forster resonance energy transfer confirmed close physical proximity of alpha globin to eNOS in situ. Omental arteries constricted to 39.1 ± 3.2 % of baseline diameter in response to phenylephrine. After treatment with an alpha globin mimetic peptide, the same arteries constricted to 64.6 ± 1.6% of baseline (p < 0.01). Inhibition of NOS with L-NAME restored vasoconstriction in the mimetic peptide-treated arteries to 41.9 ± 2.0% (p < 0.0001). Conclusion: Alpha globin and beta globin are expressed in the endothelium of human resistance arteries, form a complex with eNOS at the myoendothelial junction, and limit the release of nitric oxide triggered by alpha-1-adrenergic stimulation.