A nitric oxide processing defect of red blood cells created by hypoxia: deficiency of S-nitrosohemoglobin in pulmonary hypertension

Abstract

The mechanism by which hypoxia [low partial pressure of O(2) (pO(2))] elicits signaling to regulate pulmonary arterial pressure is incompletely understood. We considered the possibility that, in addition to its effects on smooth muscle, hypoxia may influence pulmonary vascular tone through an effect on RBCs. We report that exposure of native RBCs to sustained hypoxia is accompanied by a buildup of heme iron-nitrosyl (FeNO) species that are deficient in pO(2-)governed intramolecular transfer of NO to cysteine thiol, yielding a deficiency in the vasodilator S-nitrosohemoglobin (SNO-Hb). S-nitrosothiol (SNO)-deficient RBCs produce impaired vasodilator responses in vitro and exaggerated pulmonary vasoconstrictor responses in vivo and are defective in oxygenating the blood. RBCs from hypoxemic patients with elevated pulmonary arterial pressure (PAP) exhibit a similar FeNO/SNO imbalance and are thus deficient in pO(2)-coupled vasoregulation. Chemical restoration of SNO-Hb levels in both animals and patients restores the vasodilator activity of RBCs, and this activity is associated with improved oxygenation and lower PAPs.

Figures

Fig. 1.

Sustained hypoxia impairs production of SNO-Hb. (A) SNO-Hb yield in venous RBCs exposed to room air either immediately (0 min) or 30 min after acquisition. (B) SNO-Hb yield from NO/deoxyHb mixtures (Hb[FeNO]) (pO2 < 1 mm Hg; 1 μM NO) aerated at varying times after incubation. (C) EPR spectra of Hb[FeNO] mixtures held at low pO2 (HbO2 saturation ≈ 45%) for varying periods. (D) SNO yield in normal RBCs oxygenated at pO2 = 38 mmHg, pO2 = 66 mmHg, or pO2 = 150 mmHg (room air).

Fig. 2.

Influence of SNO-Hb depletion on RBC-dependent hypoxic pressor responses (HPV) in isolated perfused lungs. (A and B) HPV in the presence of RBCs either depleted of SNO-Hb or replete in SNO-Hb (≈10 nM final concentration). Representative tracings are depicted in A, and mean (±SEM) of n = 5 experiments is shown in B. (C) Influence of Hb or SNO-Hb (1 μM) HPV (pO2 = 25 mmHg) in isolated perfused lungs.

Fig. 3.

Influence of RBC-SNO repletion in vivo or ex vivo on hemodynamics and gas exchange in pigs. (A) Temporal correlation of changes in SNO-Hb level (Top), alveolar-arterial (A-a) pO2 difference (Middle), and mean PVR (Bottom) in pigs inhaling the SNO-generating gas ENO (100 ppm, 15 min). SNO/Hb, mol of SNO per mol of Hb (tetramer). Baseline mean (±SEM) pO2 and pCO2 were 91 (±3) and 38 (±2) mm Hg, respectively. (B) RBC-mediated improvement in A-a pO2 difference in pigs after i.v. infusion of either SNO-depleted or SNO-replete porcine RBCs (P < 0.05). Baseline mean (±SEM) pO2 and pCO2 were 97 (±5) and 38 (±1) mm Hg in the group receiving SNO-depleted RBCs, and 99 (±5) and 38 (±1) mm Hg in those receiving SNO-replete RBCs.

Fig. 4.

RBC-NO levels, RBC function, and PAP in patients. (A) Levels of SNO-Hb and Hb[FeNO] in arterial blood of normal human subjects and PAH patients. NO/Hb, mol of NO per mol of Hb (tetramer). *, P < 0.05 vs. normal patients; n = 5-8. (B) SNO content of blood presented as the percentage of total NO bound to Hb. (C) Increased FeNO/SNO signature of PAH. (D) Inverse correlation between RBC-NO levels and PAP. Hb-NO levels (expressed as mol of NO per mol of Hb tetramer) and baseline PAP in 14 patients. (E and F) Vasorelaxation (percent change in initial tension) by RBCs from normal subjects and from PAH patients. (E) Actual tracings of the hypoxia-mediated vasodilator response by RBCs of deendothelialized pulmonary artery [in the presence and absence of the guanylate cyclase inhibitor ODQ [1H(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one] (1 μM)] and intact aortic rings. (F) Mean aortic ring results (±SEM) expressed as the relative area under the time-tension curve (AUC) or as the peak decrease in tension; data are from seven individuals in each group. Hypoxia-mediated vasodilation by RBCs is impaired in PAH. *, P < 0.05 vs. normal subjects.

Fig. 5.

In vivo RBC-SNO repletion by ENO inhalation in PAH patients. (A-C) Increases in total Hb-bound NO (A) and SNO-Hb (B and C) were seen in every patient treated with ENO inhalation (70 ppm, 10 min). (D) Hb[FeNO] did not change during ENO inhalation. (E) ENO normalized the FeNO/SNO ratio. *, significant difference vs. baseline by paired t test (A and B).

Fig. 6.

In vivo restoration of RBC bioactivity by ENO inhalation in PAH patients. RBC relaxation in PAH patients before and after ENO (A), and individual (B) and mean data from eight patients (C) are shown. RBC-induced vasorelaxation was enhanced by ENO inhalation (70 ppm, 10 min). *, P < 0.05 relative to baseline, which did not differ significantly from normal RBCs.

Fig. 7.

Effects of ENO on hemodynamics and oxygenation in PAH patients. Changes in PVR (A), PAP (B), systemic vascular resistance (C), and arterial pO2 (D) in response to inhaled ENO at ≈1.5, 10, or 70 ppm (0.0025%, 0.025%, or 0.125%, 10 min each) are shown. Data represent the mean ± SEM from 5-10 patients. *, significant difference vs. baseline by mixed procedures (in A, B, and D).