Background:Current paradigms suggest that nitric oxide (NO) produced by endothelial cells (ECs) through 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 RBC eNOS compared with EC eNOS for vascular hemodynamics and nitric oxide 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 2 founder lines carrying a floxed eNOS (eNOS^flox/flox) for Cre-inducible knockout (KO), and 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 in ECs (EC eNOS KO or EC eNOS KI mice). Vascular function, hemodynamics, and nitric oxide 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, and increased systolic and diastolic blood pressure. RBC eNOS KO mice showed no alterations in acetylcholine-mediated dilation or flow-mediated dilation but were hypertensive. Treatment with the nitric oxide synthase inhibitor N^γ-nitro-l-arginine methyl ester further increased blood pressure in RBC eNOS KOs, demonstrating that eNOS in both ECs and RBCs contributes to blood pressure regulation. Although 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 than in EC eNOS KOs. Reactivation of eNOS in ECs or RBCs rescues the hypertensive phenotype of the eNOS^inv/inv mice, whereas 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.

中文翻译：

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红细胞和内皮 eNOS 独立调节循环一氧化氮代谢物和血压


背景：当前的范例表明，内皮细胞（EC）通过血管壁中的内皮一氧化氮合酶（eNOS）产生的一氧化氮（NO）是血流和血压的主要调节剂。然而，红细胞 (RBC) 也携带具有催化活性的 eNOS，但其作用存在争议且仍未明确。本研究旨在阐明红细胞 eNOS 与 EC eNOS 相比对血管血流动力学和一氧化氮代谢的功能意义。方法：我们通过使用细胞特异性 Cre 诱导基因生成 eNOS 的组织特异性功能丧失和功能获得模型失活或重新激活。我们创建了 2 个带有 floxed eNOS (eNOS ^flox/flox ) 的创始系，用于 Cre 诱导型敲除 (KO)，以及带有失活 floxed/反向外显子 (eNOS ^inv/inv ) 的基因构建体，用于 Cre 诱导型敲入 (KI) ），分别允许在红系细胞（RBC eNOS KO 或 RBC eNOS KI 小鼠）或 EC（EC eNOS KO 或 EC eNOS KI 小鼠）中靶向删除或重新激活 eNOS。比较离体和体内的血管功能、血流动力学和一氧化氮代谢。结果：EC eNOS KOs 表现出主动脉对乙酰胆碱的扩张反应显着受损、血流介导的扩张丧失以及收缩压和舒张压升高。 RBC eNOS KO 小鼠在乙酰胆碱介导的扩张或血流介导的扩张方面没有表现出任何改变，但患有高血压。使用一氧化氮合酶抑制剂N ^γ -硝基- L -精氨酸甲酯治疗进一步增加了 RBC eNOS KO 中的血压，表明 EC 和 RBC 中的 eNOS 都有助于血压调节。 尽管EC eNOS KO和RBC eNOS KO都具有较低的血浆亚硝酸盐和硝酸盐浓度，但红细胞eNOS KO中红细胞中结合的NO水平低于EC eNOS KO。 EC 或 RBC 中 eNOS 的重新激活可挽救 eNOS ^inv/inv小鼠的高血压表型，而仅在 RBC eNOS KI 小鼠中结合的 NO 水平得以恢复。 结论：这些数据表明 EC 和 RBC 中的 eNOS 对血压的影响独立体内平衡。