Hemoglobin β93 Cysteine Is Not Required for Export of Nitric Oxide Bioactivity From the Red Blood Cell

Abstract

Nitrosation of a conserved cysteine residue at position 93 in the hemoglobin β-chain (β93C) to form S-nitroso hemoglobin (SNO-Hb) is claimed to be essential for export of NO bioactivity by the red blood cell (RBC) to mediate hypoxic vasodilation and cardioprotection. To test this hypothesis we used RBCs from mice where the β93 cysteine had been replaced with alanine (β93A) in a number of ex vivo and in vivo models suitable for studying export of NO bioactivity. In an ex vivo model of cardiac ischemia reperfusion (IR) injury, perfusion of a mouse heart with control RBCs (β93C) pre-treated with an arginase inhibitor to facilitate export of RBC NO bioactivity, improved cardiac recovery after IR injury and the response was similar with β93A RBCs. Next, when human platelets were co-incubated with RBCs and then deoxygenated in the presence of nitrite, export of NO bioactivity was detected as inhibition of ADP-induced platelet activation. This effect was the same in β93C and β93A RBCs. Moreover, vascular reactivity was tested in rodent aortas in the presence of RBCs pre-treated with S-nitrosocysteine, or with hemolysates or purified Hb treated with authentic NO to form nitrosyl(FeII)-Hb, the proposed precursor of SNO-Hb. SNO-RBCs or NO-treated Hb induced vasorelaxation, with no differences between β93C and β93A RBCs. Finally, hypoxic microvascular vasodilation was studied in vivo using a murine dorsal skin fold window model. Exposure to acute systemic hypoxia caused vasodilatation and the response was similar in β93C and β93A mice. RBCs clearly have the fascinating ability to export NO bioactivity but this occurs independently of SNO formation at β93 cysteine of Hb.