Red Blood Cell and Endothelial eNOS Independently Regulate Circulating Nitric Oxide Metabolites and Blood Pressure

Abstract

Background: Current paradigms suggest that nitric oxide (NO) produced by endothelial cells (ECs) via endothelial nitric oxide synthase (eNOS) in the vessel wall is the primary regulator of blood flow and blood pressure. However, red blood cells (RBCs) also carry a catalytically active eNOS, but its role is controversial and remains undefined. This study aimed to elucidate the functional significance of red cell eNOS compared to EC eNOS for vascular hemodynamics and NO metabolism. Methods: We generated tissue-specific "loss-" and "gain-of-function" models for eNOS by using cell-specific Cre-induced gene inactivation or reactivation. We created two founder lines carrying a floxed eNOS (eNOS flox/flox ) for Cre-inducible knock out (KO), as well as gene construct with an inactivated floxed/inverted exon (eNOS inv/inv ) for a Cre-inducible knock in (KI), which respectively allow targeted deletion or reactivation of eNOS in erythroid cells (RBC eNOS KO or RBC eNOS KI mice) or endothelial cells (EC eNOS KO or EC eNOS KI mice). Vascular function, hemodynamics, and NO metabolism were compared ex vivo and in vivo . Results: The EC eNOS KOs exhibited significantly impaired aortic dilatory responses to acetylcholine, loss of flow-mediated dilation (FMD), and increased systolic and diastolic blood pressure. RBC eNOS KO mice showed no alterations in acetylcholine-mediated dilation or FMD but were hypertensive. Treatment with the NOS inhibitor L-NAME further increased blood pressure in RBC eNOS KOs, demonstrating that eNOS in both ECs and RBCs contributes to blood pressure regulation. While both EC eNOS KOs and RBC eNOS KOs had lower plasma nitrite and nitrate concentrations, the levels of bound NO in RBCs were lower in RBC eNOS KOs as compared to EC eNOS KOs. Crucially, reactivation of eNOS in ECs or RBCs rescues the hypertensive phenotype of the eNOS inv/inv mice, while the levels of bound NO were restored only in RBC eNOS KI mice. Conclusions: These data reveal that eNOS in ECs and RBCs contribute independently to blood pressure homeostasis.

Therapeutic potential of colchicine in cardiovascular medicine: a pharmacological review

“…The main vasodilator is nitric oxide (NO). Endothelial cells produce NO via endothelial nitric oxide synthase (eNOS) in the vessel wall [ 63 ], which hence regulates blood pressure and blood flow and it also inhibits oxidation of LDL [ 18 , 64 ]. Endothelial cells adhere to circulating leukocytes via vascular cell adhesion molecule-1 (VCAM-1) on their cell surface [ 65 ].…”

Hypoxic vasodilatory defect and pulmonary hypertension in mice lacking hemoglobin β-cysteine93 S-nitrosylation

“… 32 , 33), which has often been assumed to be responsible for the full effect but without empirical evidence. Moreover, it had not been previously possible to discern the role of endothelium versus RBCs because endothelial NOS (eNOS) inhibition reduces levels of SNO-Hb and RBC SNO ( 13 , 34 ). We conclude that vasodilation following occlusion is evidently mediated by both RBCs and endothelium, the former stimulated by hypoxia ( 35 ) and the latter by shear.…”

“…The new work on RBC eNOS also deserves mechanistic comment. The authors of that report ( 34 ) seem to favor a role for nitrite in blood pressure lowering by RBC eNOS, but their data show otherwise. eNOS deletion from RBCs does not reduce nitrite or overall levels of NO metabolites in RBCs.…”

“…An exciting new report for a role of RBC eNOS in control of blood pressure ( 34 ) merits comment in this regard. Whereas eNOS in RBCs contributes to the overall blood pressure–lowering effect of NO, it plays no role in FMD, as shown in the same study ( 34 ). By contrast, Hb βCys93 plays a major role in “FMD” (this report) but has no role in blood pressure control ( 7 , 23 ).…”

“…Only heme-NO levels are apparently lower in eNOS –/– RBCs, which is well rationalized by lower amounts of NO produced, as NO binds heme directly. In addition, eNOS generates SNOs including SNO proteins, which may lower blood pressure, and SNO levels in plasma and RBCs are altered in mice with RBC-specific eNOS deletion ( 34 ). Thus, SNOs derived from RBC eNOS mice likely contribute to the circulating SNO pool, which mediates blood pressure lowering, just as SNOs are central to all NO bioactivity ( 27 , 35 ).…”