Institute for Transformative Molecular Medicine (A.H., Z.Q., R.Z., R.T.P., J.S.S.), and Cardiovascular Research Institute (R.Z.), Case Western Reserve University School of Medicine, Cleveland, Ohio; and Harrington Discovery Institute (R.T.P., J.S.S.), University Hospitals Cleveland Medical Center, Cleveland, Ohio.
Institute for Transformative Molecular Medicine (A.H., Z.Q., R.Z., R.T.P., J.S.S.), and Cardiovascular Research Institute (R.Z.), Case Western Reserve University School of Medicine, Cleveland, Ohio; and Harrington Discovery Institute (R.T.P., J.S.S.), University Hospitals Cleveland Medical Center, Cleveland, Ohio
J Pharmacol Exp Ther. 2022 Jul;382(1):1-10. doi: 10.1124/jpet.122.001194. Epub 2022 May 5.
Classic physiology links tissue hypoxia to oxygen delivery through control of microvascular blood flow (autoregulation of blood flow). Hemoglobin (Hb) serves both as the source of oxygen and the mediator of microvascular blood flow through its ability to release vasodilatory S-nitrosothiol (SNO) in proportion to degree of hypoxia. -globin Cys93Ala (Cys93Ala) mutant mice deficient in S-nitrosohemoglobin (SNO-Hb) show profound deficits in microvascular blood flow and tissue oxygenation that recapitulate microcirculatory dysfunction in multiple clinical conditions. However, the means to replete SNO in mouse red blood cells (RBCs) to restore RBC function is not known. In particular, although methods have been developed to selectively S-nitrosylate Cys93 in human Hb and intact human RBCs, conditions have not been optimized for mouse RBCs that are used experimentally. Here we show that loading SNO onto Hb in mouse RBC lysates can be achieved with high stoichiometry and -globin selectivity. However, S-nitrosylation of Hb within intact mouse RBCs is ineffective under conditions that work well with human RBCs, and levels of metHb are prohibitively high. We developed an optimized method that loads SNO in mouse RBCs to maintain vasodilation under hypoxia and shows that loss of SNO loading in Cys93Ala mutant RBCs results in reduced vasodilation. We also demonstrate that differences in SNO/met/nitrosyl Hb stoichiometry can account for differences in RBC function among studies. RBCs loaded with quasi-physiologic amounts of SNO-Hb will produce vasodilation proportionate to hypoxia, whereas RBCs loaded with higher amounts lose allosteric regulation, thus inducing vasodilation at both high and low oxygen level. SIGNIFICANCE STATEMENT: Red blood cells from mice exhibit poor hemoglobin S-nitrosylation under conditions used for human RBCs, frustrating tests of vasodilatory activity. Using an optimized S-nitrosylation protocol, mouse RBCs exhibit hypoxic vasodilation that is significantly reduced in hemoglobin βCys93Ala mutant RBCs that cannot carry S-nitrosothiol allosterically, providing genetic validation for the role of βCys93 in oxygen delivery.
经典生理学将组织缺氧与通过控制微血管血流（血流自动调节）来输送氧气联系起来。血红蛋白（Hb）既是氧气的来源，也是通过其释放与缺氧程度成比例的血管舒张 S-亚硝基硫醇（SNO）来调节微血管血流的介质。缺乏 S-亚硝基血红蛋白（SNO-Hb）的β-球蛋白 Cys93Ala（Cys93Ala）突变小鼠表现出微血管血流和组织氧合的深刻缺陷，这些缺陷再现了多种临床情况下的微循环功能障碍。然而，补充小鼠红细胞（RBC）中的 SNO 以恢复 RBC 功能的方法尚不清楚。特别是，尽管已经开发了选择性 S-亚硝化为人 Hb 和完整人 RBC 的方法，但尚未针对实验中使用的小鼠 RBC 优化条件。在这里，我们表明可以用高化学计量和β-球蛋白选择性在小鼠 RBC 裂解物中加载 SNO。然而，在与人 RBC 配合良好的条件下，完整的小鼠 RBC 中的 Hb 无法有效地进行 S-亚硝化为，并且 metHb 的水平高得令人望而却步。我们开发了一种优化的方法，可在缺氧条件下将 SNO 加载到小鼠 RBC 中以维持血管舒张，并表明 Cys93Ala 突变 RBC 中 SNO 加载的丢失会导致血管舒张减少。我们还证明，SNO/met/nitrosyl Hb 化学计量比的差异可以解释不同研究中 RBC 功能的差异。用准生理量的 SNO-Hb 加载的 RBC 将产生与缺氧成比例的血管舒张，而用更高量加载的 RBC 会失去变构调节，从而在高氧和低氧水平下都诱导血管舒张。意义：在用于人 RBC 的条件下，来自小鼠的 RBC 表现出较差的血红蛋白 S-亚硝化为，这挫败了血管舒张活性的测试。使用优化的 S-亚硝化为方案，小鼠 RBC 表现出缺氧性血管舒张，在不能变构携带 SNO 的血红蛋白βCys93Ala 突变 RBC 中显着降低，为βCys93 在氧输送中的作用提供了遗传验证。
