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

ABSTRACT: OBJECTIVE:Hemoglobin ? (Hb ?) 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 ? regulates nitric oxide signaling, vascular reactivity, and blood pressure using a mimetic peptide of Hb ? to disrupt this interaction. APPROACH AND RESULTS:In silico modeling of Hb ? and eNOS identified a conserved sequence of interaction. By mutating portions of Hb ?, we identified a specific sequence that binds eNOS. A mimetic peptide of the Hb ? sequence (Hb ? X) was generated to disrupt this complex. Using in vitro binding assays with purified Hb ? and eNOS and ex vivo proximity ligation assays on resistance arteries, we have demonstrated that Hb ? X significantly decreased interaction between eNOS and Hb ?. Fluorescein isothiocyanate labeling of Hb ? X revealed localization to holes in the internal elastic lamina (ie, myoendothelial junctions). To test the functional effects of Hb ? X, we measured cyclic guanosine monophosphate and vascular reactivity. Our results reveal augmented cyclic guanosine monophosphate production and altered vasoconstriction with Hb ? X. To test the in vivo effects of these peptides on blood pressure, normotensive and hypertensive mice were injected with Hb ? X, which caused a significant decrease in blood pressure; injection of Hb ? X into eNOS(-/-) mice had no effect. CONCLUSIONS:These results identify a novel sequence on Hb ? that is important for Hb ?/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

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

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]

PMID: 25278292

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Project description:An increase in production of reactive oxygen species resulting in a decrease in nitric oxide bioavailability in the endothelium contributes to many cardiovascular diseases, and these reactive oxygen species can oxidize cellular macromolecules. Protein thiols are critical reducing equivalents that maintain cellular redox state and are primary targets for oxidative modification. We demonstrate endothelial NOS (eNOS) oxidant-induced protein thiyl radical formation from tetrahydrobiopterin-free enzyme or following exposure to exogenous superoxide using immunoblotting, immunostaining, and mass spectrometry. Spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) followed by immunoblotting using an anti-DMPO antibody demonstrated the formation of eNOS protein radicals, which were abolished by superoxide dismutase and L-NAME, indicating that protein radical formation was due to superoxide generation from the eNOS heme. With tetrahydrobiopterin-reconstituted eNOS, eNOS protein radical formation was completely inhibited. Using mass spectrometric and mutagenesis analysis, we identified Cys-908 as the residue involved in protein radical formation. Mutagenesis of this key cysteine to alanine abolished eNOS thiyl radical formation and uncoupled eNOS, leading to increased superoxide generation. Protein thiyl radical formation leads to oxidation or modification of cysteine with either disulfide bond formation or S-glutathionylation, which induces eNOS uncoupling. Furthermore, in endothelial cells treated with menadione to trigger cellular superoxide generation, eNOS protein radical formation, as visualized with confocal microscopy, was increased, and these results were confirmed by immunoprecipitation with anti-eNOS antibody, followed by immunoblotting with an anti-DMPO antibody. Thus, eNOS protein radical formation provides the basis for a mechanism of superoxide-directed regulation of eNOS, involving thiol oxidation, defining a unique pathway for the redox regulation of cardiovascular function.

Dataset Information

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

Publications

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

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