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

Abstract

Background: Nitrosation of a conserved cysteine residue at position 93 in the hemoglobin β chain (β93C) to form S-nitroso (SNO) hemoglobin (Hb) is claimed to be essential for export of nitric oxide (NO) bioactivity by the red blood cell (RBC) to mediate hypoxic vasodilation and cardioprotection.

Methods: To test this hypothesis, we used RBCs from mice in which 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.

Results: In an ex vivo model of cardiac ischemia/reperfusion injury, perfusion of a mouse heart with control RBCs (β93C) pretreated with an arginase inhibitor to facilitate export of RBC NO bioactivity improved cardiac recovery after ischemia/reperfusion injury, and the response was similar with β93A RBCs. Next, when human platelets were coincubated 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 pretreated 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 with a murine dorsal skin-fold window model. Exposure to acute systemic hypoxia caused vasodilatation, and the response was similar in β93C and β93A mice.

Conclusions: RBCs clearly have the fascinating ability to export NO bioactivity, but this occurs independently of SNO formation at the β93 cysteine of Hb.

Keywords: S-nitrosothiols; hemoglobins; nitric oxide; nitrite; nitrosation.

Figure 1.. Scheme of approaches used to study export of NO bioactivity by red blood cells.

Washed red blood cells (RBCs) from control mice (β93C) or mutant mice (β93A) lacking the cysteine-93 on the β-chain of hemoglobin were used in 3 model systems known to respond to export of RBC NO bioactivity: i) isolated aortic dilation or contraction in response to NO- or S-nitrosothiol treated RBCs, ii) isolated mouse hearts perfused with RBCs prior to ischemia reperfusion injury and measuring cardiac recovery, iii) RBC and nitrite-dependent inhibition of platelets activation, iv) in vivo assessment of hypoxic vasodilation.

Figure 2.. Effects of β93C and β93A RBCs on the recovery of cardiac function following ischemia-reperfusion

Hearts from wild type mice were given red blood cells (RBCs) from β93C mice incubated with vehicle (n=6) or nor-NOHA (n=5) or RBCs from β93A mice incubated with vehicle (n=5) or nor-NOHA (n=8). Nor-NOHA is an arginase inhibitor that induces export of NO bioactivity from RBCs. Data are shown as mean ± SEM. Significant differences between groups were analyzed using two-way ANOVA; **P < 0.01, ***P < 0.001. LVDP= Left Ventricular Developed Pressure.

Figure 3.. β93C is not required for RBC-mediated inhibition of platelet activation by nitrite

Murine red blood cells (RBCs) containing human hemoglobin β93C and mutant of β93C to β93A were used in the platelet activation assay at 10% hematocrit under partially deoxygenated conditions. Data show mean ± SEM, n=4. P values (*) were calculated using a paired t test: p< 0.03 between β93C with and without nitrite and p< 0.03 between β93A with and without nitrite.

Figure 4.. Responses of mouse aortae to hemolysates from of β93C and β93A RBCs or purified hemoglobin treated with NO gas

Hemolysates from red blood cells (RBCs) of β93C or β93A mice (A) or purified human ferrohemoglobin (B) were treated with NO gas in the absence of oxygen and then tested on phenylephrine (PE) and L-NAME (1 mM, used only in A) preconstricted mouse aortae. Non-treated human ferrohemoglobin (C) was used as control. Data show mean ± SEM. n=3–6.

Figure 5:. Effects of β93C and β93A on SNO-RBC dependent vasodilation

Panel A: S-nitrosothiol (SNO) concentrations normalized to heme in β93C and β93A red blood cells (RBCs) after reaction with S-nitroso cysteine. Data show total levels and levels after fractionating RBCs into high and low molecular weight (MWt) components. Data are mean ± SEM (n=3). P-values shown were calculated by unpaired t-test. Panel B and C: Rat aortic rings were incubated at 21% or 1% O₂ respectively, and vasodilation assessed in response to increasing concentrations of SNO-β93C RBCs and SNO-β93A RBCs. Data are mean ± SEM (n=3).

Figure 6:. In vivo effects of hypoxia on microvascular and hemodynamic parameters in β93C and β93A mice

β93C or β93A mice were exposed to global hypoxia and changes in arteriolar diameter (Panel A), mean arterial pressure (MAP, Panel B) and heart rate (HR, Panel C) were measured. Data show fold changes relative to normoxia and mean ± SEM (n=13–14). P value was not significant (NS) by unpaired t-test. Changes in MAP and Arterial diameter in response to hypoxia were significant in both groups of mice (p<0.001, students t test).