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

BackgroundIn 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. MethodsHuman omental arteries 100-200{micro}m 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. ResultsHBA1, 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). ConclusionAlpha 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/21255004v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@bcff32org.highwire.dtl.DTLVardef@16698borg.highwire.dtl.DTLVardef@16b458borg.highwire.dtl.DTLVardef@1a2ea07_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract. Hemoglobin binds to endothelial NOS in the myoendothelial junction where it regulates the diffusion of nitric oxide that is produced in response to alpha-1-adrenergic signaling. Phenylephrine (PE) engages alpha-1-adrenergic receptors (1) on vascular smooth muscle cells triggering an influx of calcium (Ca++) and activating myosin light chain kinase (MLCK) to constrict muscle fibers ({approx}) and constrict the artery. Calcium enters the endothelial cell via putative gap junctions where it activates endothelial nitric oxide synthase (NOS) via calmodulin (CM). Nitric oxide (NO) produced by NOS can diffuse into the smooth muscle cell where it activates myosin light chain phosphatase (MLCP) via soluble guanylate cyclase and cGMP (both not shown) to relax smooth muscle fibers and dilate the artery. Hemoglobin () bound to NOS prevents the diffusion of NO into the smooth muscle cell likely by catalyzing the reaction with oxygen (O2) to produce nitrate (NO3-), an anion that cannot diffuse across the cell membrane. When hemoglobin is displaced from NOS with a mimetic peptide (not shown), NO diffusion increases and counteracts vasoconstriction; when NOS is inhibited by L-NAME, the mimetic peptide has no effect. Thus hemoglobin limits the diffusion of NO across the myoendothelial junction in the setting of alpha-1-adrenergic stimulation. C_FIG