PT  - JOURNAL ARTICLE
AU  - Hausladen, Alfred
AU  - Qian, Zhaoxia
AU  - Zhang, Rongli
AU  - Premont, Richard T.
AU  - Stamler, Jonathan S.
TI  - <strong>Optimized S-nitrosohemoglobin synthesis in red blood cells to preserve hypoxic vasodilation via β<strong>Cys93</strong></strong>
AID  - 10.1124/jpet.122.001194
DP  - 2022 Jan 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - JPET-AR-2022-001194
4099  - http://jpet.aspetjournals.org/content/early/2022/05/05/jpet.122.001194.short
4100  - http://jpet.aspetjournals.org/content/early/2022/05/05/jpet.122.001194.full
AB  - 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. β-globin Cys93Ala (βCys93Ala) 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 RBCs in order to restore RBC function is not known. In particular, while methods have been developed to selectively S-nitrosylate βCys93 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 β-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 develop an optimized method that loads SNO in mouse RBCs to maintain vasodilation under hypoxia and show that loss of SNO loading in βCys93Ala 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-physiological 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 ββCys93Ala mutant RBCs that cannot carry S-nitrosothiol allosterically, providing genetic validation for the role of bCys93 in oxygen delivery.