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eNOS coupling at myoendothelial junctions is required for nitric oxide scavenging during vasoconstriction
Abstracts/Nitric Oxide 42 (2014) 99–153
P181. Reduced nitric oxide bioavailability contributes to endothelial dysfunction in a murine model of β-thalassemia http://dx.doi.org/10.1016/j.niox.2014.09.124 Jonas T. Sertorio, Christina Mucci, Adam C. Straub, Charles Mc Tiernan, Mark T. Gladwin Vascular Medicine Institute, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA
Objective/purpose: Nitric oxide (NO) promotes vasodilation and inhibits both platelet aggregation and leukocyte adhesion. However, under conditions of intravascular hemolysis, NO reacts with cell-free oxyhemoglobin via a fast dioxygenation reaction (NO scavenging) to form methemoglobin and nitrate, which limits NO bioavailability and promotes endothelial dysfunction, platelet activation and vasculopathy. Red blood cell (RBC) microparticles can also scavenge NO at a rate comparable with cell-free hemoglobin. In hematologic disorders, such as thalassemia, there is increased RBC hemolysis and RBC microparticle formation. In this study we investigated the systemic effects of NO consumption by RBC microparticles and cellfree hemoglobin in a mouse model of β-thalassemia intermedia (th3/+). Methods: Wild type control C57BL/6 (WT) and age and gender-matched th3/+ mice (n = 10 per group), were euthanized, blood collected and complete blood count assessed (Hemavet). Quantification of RBC microparticles and reticulocytes in whole blood was performed immediately after blood collection using a flow cytometer (BD Fortessa). RBC microparticles were defined as glycophorin A-PE+ and annexin V-FITC+ events and were further characterized by size. The ability of plasma with cell-free hemoglobin and RBC microparticles to scavenge NO was measured with a validated NO consumption assay in a NO chemiluminescence analyzer (Sievers-GE). Vascular reactivity was assessed in isolated thoracodorsal arteries (TDA) with a pressure myograph (DMT). Results: Compared with WT, th3/+ mice had significantly higher levels of RBC microparticles in whole blood (as % of microparticles from total RBCs; th3/+ 2.73 ± 0.28% vs. WT 0.75 ± 0.15%, P < 0.05), higher levels of reticulocytes (as % of RBC; th3/+ 36 ± 2.62% vs. WT 11.98 ± 1.39%).) and higher levels of plasma NO consumption (th3/+ 27 ± 1.29 μM vs. WT 15.68 ± 1.08 μM). Vasodilatory responses to acetylcholine in TDA pre-constricted with phenylephrine were compromised in th3/+ mice compared with WT controls, which demonstrates endothelial cell dysfunction. Conclusion: Both cell-free hemoglobin and RBC microparticles likely contribute to endothelial cell dysfunction in a murine model of β-thalassemia. Future studies will evaluate the hypothesis that these pathways lead to pulmonary hypertension and will be enhanced by surgical splenectomy. Keywords: Beta thalassemia; Red blood cell microparticles; Cell-free hemoglobin; Nitric oxide; Endothelial cell dysfunction.
P183. Modulation of cytoglobin nitrite reductase rates by distal histidine mutations http://dx.doi.org/10.1016/j.niox.2014.09.125 Courtney Sparacino-Watkins a, Venkata Ragireddy b, Mark Gladwin a, Jesus Tejero b a Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh b University of Pittsburgh
Cytoglobin (Cgb) is a recently discovered six-coordinate heme globin with high sequence and structural similarity to hemoglobin and myoglobin but with unknown function. We have previously studied the nitrite reductase activity of another six-
141
coordinated globin, neuroglobin (Ngb). Our prior work indicates that removal of the distal histidine in Ngb causes a permanent five-coordinated heme iron and increases reactivity toward nitrite. In order to study the role of the distal histidine in Cgb, we generated the mutants H81A, H81L, H81Q and H81W. We studied the reduction of nitrite to NO of wild-type Cgb and mutants by UV-visible spectroscopy. During the reaction, the deoxy (FeII) cgb species oxidizes forming Met (FeIII) Cgb and NO that binds to the ferrous heme producing the nitrosyl (FeII– NO) complex. We carry out the reaction in the presence of dithionite that reduces the FeIII species back to FeII, thereby monitoring the change of FeII to FeIINO. Mutations of Cgb His81 cause in all cases a significant increase in nitrite reduction rates. The observed bimolecular constants are: 1.22 M−1 s−1 (WT); 10.7 M−1 s−1 (H81W); 76 M−1 s−1 (H81L); 210 M−1 s−1 (H81Q) and 1100 M−1 s−1 (H64A). The increase in rate correlates better with the decrease in size of the side chain; this suggests that the rate-limiting factor for nitrite reduction is the access of nitrite into the heme pocket. In summary, we show that the reaction of Cgb with nitrite is regulated by the distal histidine binding to the heme and the restriction of exogenous ligands into the heme pocket. The rates observed for the Cgb His81 mutants are remarkably similar to those observed for the analogous residue in Ngb (His64) but different from the similar residue in myoglobin/hemoglobin. This indicates that Ngb can be used as a model for studies on Cgb and other six coordinate proteins. Keywords: Cytoglobin; Neuroglobin; Nitrite; Nitric oxide.
P185. Hemoglobin α/eNOS coupling at myoendothelial junctions is required for nitric oxide scavenging during vasoconstriction http://dx.doi.org/10.1016/j.niox.2014.09.126 Adam Straub a, Anh Nguyen a, Stephanie Mutchler a, Brant Isakson b a University of Pittsburgh b University of Virginia
Rationale: Hb α and eNOS form a macromolecular complex at myoendothelial junctions; however the functional role of this interaction remains undefined. Objective: To test if direct coupling of eNOS and Hb α regulates NO signaling, vascular reactivity and blood pressure using a mimetic peptide of Hb α to disrupt this interaction. Methods 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 interaction. Utilizing 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. FITC-labeling of Hb α X revealed localization to holes in the internal elastic lamina (i.e., myoendothelial junctions). To test the functional effects of Hb α X, we measured cGMP and vascular reactivity. Our results reveal augmented cGMP 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. Conclusion: 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. Keywords: Nitric oxide; Hemoglobin; eNOS; Blood pressure.
P181. Reduced nitric oxide bioavailability contributes to endothelial dysfunction in a murine model of β-thalassemia http://dx.doi.org/10.1016/j.niox.2014.09.124 Jonas T. Sertorio, Christina Mucci, Adam C. Straub, Charles Mc Tiernan, Mark T. Gladwin Vascular Medicine Institute, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA
Objective/purpose: Nitric oxide (NO) promotes vasodilation and inhibits both platelet aggregation and leukocyte adhesion. However, under conditions of intravascular hemolysis, NO reacts with cell-free oxyhemoglobin via a fast dioxygenation reaction (NO scavenging) to form methemoglobin and nitrate, which limits NO bioavailability and promotes endothelial dysfunction, platelet activation and vasculopathy. Red blood cell (RBC) microparticles can also scavenge NO at a rate comparable with cell-free hemoglobin. In hematologic disorders, such as thalassemia, there is increased RBC hemolysis and RBC microparticle formation. In this study we investigated the systemic effects of NO consumption by RBC microparticles and cellfree hemoglobin in a mouse model of β-thalassemia intermedia (th3/+). Methods: Wild type control C57BL/6 (WT) and age and gender-matched th3/+ mice (n = 10 per group), were euthanized, blood collected and complete blood count assessed (Hemavet). Quantification of RBC microparticles and reticulocytes in whole blood was performed immediately after blood collection using a flow cytometer (BD Fortessa). RBC microparticles were defined as glycophorin A-PE+ and annexin V-FITC+ events and were further characterized by size. The ability of plasma with cell-free hemoglobin and RBC microparticles to scavenge NO was measured with a validated NO consumption assay in a NO chemiluminescence analyzer (Sievers-GE). Vascular reactivity was assessed in isolated thoracodorsal arteries (TDA) with a pressure myograph (DMT). Results: Compared with WT, th3/+ mice had significantly higher levels of RBC microparticles in whole blood (as % of microparticles from total RBCs; th3/+ 2.73 ± 0.28% vs. WT 0.75 ± 0.15%, P < 0.05), higher levels of reticulocytes (as % of RBC; th3/+ 36 ± 2.62% vs. WT 11.98 ± 1.39%).) and higher levels of plasma NO consumption (th3/+ 27 ± 1.29 μM vs. WT 15.68 ± 1.08 μM). Vasodilatory responses to acetylcholine in TDA pre-constricted with phenylephrine were compromised in th3/+ mice compared with WT controls, which demonstrates endothelial cell dysfunction. Conclusion: Both cell-free hemoglobin and RBC microparticles likely contribute to endothelial cell dysfunction in a murine model of β-thalassemia. Future studies will evaluate the hypothesis that these pathways lead to pulmonary hypertension and will be enhanced by surgical splenectomy. Keywords: Beta thalassemia; Red blood cell microparticles; Cell-free hemoglobin; Nitric oxide; Endothelial cell dysfunction.
P183. Modulation of cytoglobin nitrite reductase rates by distal histidine mutations http://dx.doi.org/10.1016/j.niox.2014.09.125 Courtney Sparacino-Watkins a, Venkata Ragireddy b, Mark Gladwin a, Jesus Tejero b a Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh b University of Pittsburgh
Cytoglobin (Cgb) is a recently discovered six-coordinate heme globin with high sequence and structural similarity to hemoglobin and myoglobin but with unknown function. We have previously studied the nitrite reductase activity of another six-
141
coordinated globin, neuroglobin (Ngb). Our prior work indicates that removal of the distal histidine in Ngb causes a permanent five-coordinated heme iron and increases reactivity toward nitrite. In order to study the role of the distal histidine in Cgb, we generated the mutants H81A, H81L, H81Q and H81W. We studied the reduction of nitrite to NO of wild-type Cgb and mutants by UV-visible spectroscopy. During the reaction, the deoxy (FeII) cgb species oxidizes forming Met (FeIII) Cgb and NO that binds to the ferrous heme producing the nitrosyl (FeII– NO) complex. We carry out the reaction in the presence of dithionite that reduces the FeIII species back to FeII, thereby monitoring the change of FeII to FeIINO. Mutations of Cgb His81 cause in all cases a significant increase in nitrite reduction rates. The observed bimolecular constants are: 1.22 M−1 s−1 (WT); 10.7 M−1 s−1 (H81W); 76 M−1 s−1 (H81L); 210 M−1 s−1 (H81Q) and 1100 M−1 s−1 (H64A). The increase in rate correlates better with the decrease in size of the side chain; this suggests that the rate-limiting factor for nitrite reduction is the access of nitrite into the heme pocket. In summary, we show that the reaction of Cgb with nitrite is regulated by the distal histidine binding to the heme and the restriction of exogenous ligands into the heme pocket. The rates observed for the Cgb His81 mutants are remarkably similar to those observed for the analogous residue in Ngb (His64) but different from the similar residue in myoglobin/hemoglobin. This indicates that Ngb can be used as a model for studies on Cgb and other six coordinate proteins. Keywords: Cytoglobin; Neuroglobin; Nitrite; Nitric oxide.
P185. Hemoglobin α/eNOS coupling at myoendothelial junctions is required for nitric oxide scavenging during vasoconstriction http://dx.doi.org/10.1016/j.niox.2014.09.126 Adam Straub a, Anh Nguyen a, Stephanie Mutchler a, Brant Isakson b a University of Pittsburgh b University of Virginia
Rationale: Hb α and eNOS form a macromolecular complex at myoendothelial junctions; however the functional role of this interaction remains undefined. Objective: To test if direct coupling of eNOS and Hb α regulates NO signaling, vascular reactivity and blood pressure using a mimetic peptide of Hb α to disrupt this interaction. Methods 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 interaction. Utilizing 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. FITC-labeling of Hb α X revealed localization to holes in the internal elastic lamina (i.e., myoendothelial junctions). To test the functional effects of Hb α X, we measured cGMP and vascular reactivity. Our results reveal augmented cGMP 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. Conclusion: 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. Keywords: Nitric oxide; Hemoglobin; eNOS; Blood pressure.