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Nitric Oxide Transport in Blood: A Third Gas in the Respiratory Cycle

Allan Doctor, Jonathan S. Stamler

10.1002/cphy.c090009

Source: Volume 1, Issue 1, January 2011

Published online: January 2011

Full Article on Wiley Online Library



Abstract

The trapping, processing, and delivery of nitric oxide (NO) bioactivity by red blood cells (RBCs) have emerged as a conserved mechanism through which regional blood flow is linked to biochemical cues of perfusion sufficiency. We present here an expanded paradigm for the human respiratory cycle based on the coordinated transport of three gases: NO, O2, and CO2. By linking O2 and NO flux, RBCs couple vessel caliber (and thus blood flow) to O2 availability in the lung and to O2 need in the periphery. The elements required for regulated O2‐based signal transduction via controlled NO processing within RBCs are presented herein, including S‐nitrosothiol (SNO) synthesis by hemoglobin and O2‐regulated delivery of NO bioactivity (capture, activation, and delivery of NO groups at sites remote from NO synthesis by NO synthase). The role of NO transport in the respiratory cycle at molecular, microcirculatory, and system levels is reviewed. We elucidate the mechanism through which regulated NO transport in blood supports O2 homeostasis, not only through adaptive regulation of regional systemic blood flow but also by optimizing ventilation‐perfusion matching in the lung. Furthermore, we discuss the role of NO transport in the central control of breathing and in baroreceptor control of blood pressure, which subserve O2 supply to tissue. Additionally, malfunctions of this transport and signaling system that are implicated in a wide array of human pathophysiologies are described. Understanding the (dys)function of NO processing in blood is a prerequisite for the development of novel therapies that target the vasoactive capacities of RBCs. © 2011 American Physiological Society. Compr Physiol 1:541‐568, 2011.

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Figure 1. Figure 1.

Local vascular reflexes support maintenance of O2 delivery to tissue in the setting of progressive hypoxia. In a classic paper 244, Guyton demonstrated regional autoregulation of systemic blood flow in normal dogs (following spinal anesthesia) by observing variation in blood flow during constant pressure blood perfusion of the femoral artery, while reducing the hemoglobin oxygen saturation (Hb So2%) from 100% to 0% in the perfusing blood. (A) Stepwise reduction in Hb So2% caused a progressive increase in blood flow through the leg. (B) These data demonstrate that autoregulation of blood flow occurs at a local level and this regulation serves to improve oxygen supply when blood oxygen content falls. In addition, effects on blood flow were replicated by injecting partially deoxygenated versus oxygenated red blood cells into the artery, demonstrating that effects could be elicited during arteriovenous transit (<1 s). Reproduced with permission from 244.
Figure 2. Figure 2.

Red blood cells (RBCs) transduce regional O2 gradients in tissue to control nitric oxide (NO) bioactivity in plasma by trapping or delivering NO groups as a function of hemoglobin (Hb) O2 saturation. (A) In this fashion, circulating NO groups are processed by Hb into the highly vasoactive (thiol‐based) NO congener, S‐nitrosothiol (SNO). By exporting SNOs as a function of Hb deoxygenation, RBCs precisely dispense vasodilator bioactivity in direct proportion to regional blood flow lack. (B) O2 delivery homeostasis requires biochemical coupling of vessel tone to environmental cues that matches perfusion sufficiency to metabolic demand. Because oxy‐ and deoxy‐Hb process NO differently (see text), allosteric transitions in Hb conformation afford context‐responsive (O2‐coupled) control of NO bioavailability, thereby linking the sensor and effector arms of this system. Specifically, Hb conformation governs the equilibria among deoxy‐HbFeNO (A; NO sink), SNO‐oxy‐Hb (B; NO store), and acceptor thiols including the membrane protein SNO‐AE‐1 (C; bioactive NO source). Direct SNO export from RBCs or S‐transnitrosylation from RBCs to plasma thiols (D) or to endothelial cells directly (not shown) yields vasoactive SNOs, which influence resistance vessel caliber and close this signaling loop. Thus, RBCs either trap (A) or export (D) NO groups to optimize blood flow. (C) NO processing in RBCs (A and B) couples vessel tone to tissue pO2; this system subserves hypoxic vasodilation in the arterial periphery and thereby calibrates blood flow to regional tissue hypoxia. See Figures 10 and 11 and related discussion for more details.
Figure 3. Figure 3.

Hemoglobin oxygen saturation (Hb So2%) exerts coordinated governance of red blood cell (RBC) S‐nitrosothiol (SNO) content, RBC vasoactivity, and human peripheral blood flow. (A) Human blood gas measurements of SNO and O2 64. RBCs with (black) or without (red) added extracellular glutathione (GSH) were deoxygenated under inert gas. The natural logarithm of SNO content in RBCs (SNO RBC) is linearly related to Hb So2. GSH accelerated SNO RBC decay, consistent with O2‐linked export of NO groups to extraerythrocytic thiols. (B) RBCs induce graded relaxation of systemic arteries (aortic ring bioassay) that is inversely related to Hb So2 across the physiological range, recapitulating hypoxic vasodilation (HVD) 196. Hb So2 spanned from red (oxy, >90% Hb So2) to blue (deoxy, <40% Hb So2). (C) Leg vascular conductance increases as blood O2 content falls (HVD) 99. Hb So2 and thus arterial blood O2 content were manipulated by CO exposure ± varying Fio2 (fraction of inspired O2; hypoxia versus hyperoxia). Neither vascular conductance nor blood flow correlated with blood pO2 per se. Note the similarity between observations in panels A, B, and C and Guyton's original observations 244 (Fig. 1). Panel (C) Reproduced with permission from 99.
Figure 4. Figure 4.

Hypoxic vasodilation (1% O2) by native (untreated) human red blood cells (RBCs) of mouse aortic segments requires neither eNOS nor intact endothelium 62. (A) Hypoxiacoupled vasodilation by RBCs is preserved despite endothelial denudation, inhibition of NOS activity with L‐NAME, or eNOS deletion (eNOS‐/‐). RBC‐induced vasorelaxation was greatly attenuated by the guanylate cyclase inhibitor ODQ. (n = 9 to 34; * P < 0.05). (B, C) Representative tracings from the vascular ring preparation show that removal of endothelium eliminates acetylcholine (ACh)‐induced vasorelaxation but has no effect on RBC‐induced vasorelaxation [N.B. vessel rings are precontracted with phenylephrine (PE)].
Figure 5. Figure 5.

Hypoxic vasodilation (HVD) by native human red blood cells (RBCs) of rabbit aortic segments is S‐nitrosothiol (SNO) based 62. (A) RBC‐induced vasorelaxation was potentiated by cysteine and by N‐acetylcysteine but was unaffected by nitrite (NO2−) or the prostaglandin synthesis inhibitors indomethacin and aspirin (all agents added 2 min before RBCs) (n = 5 to 11; * P < 0.05). (B‐D), Representative tracings illustrate the augmentation of relaxation by cysteine and the attenuation of relaxation by S‐nitroso‐hemoglobin depletion (∼80% depletion). (E) Addition of NO2− to the bath (even at supraphysiologic concentrations of 1 μM) before (NO2− → RBC), simultaneously with the addition of RBCs (RBC + NO2−), or near the termination of RBC‐induced vasorelaxation (RBC → NO2−), had no discernible effect on the magnitude or duration of RBC‐induced HVD.
Figure 6. Figure 6.

Conformation‐specific binding between hemoglobin (Hb) and the red blood cell (RBC) membrane protein AE‐1 affords O2 responsive control of NO trapping 111. (A) Under oxygenated conditions (normoxic RBC, at left), the RBC membrane constitutes a significant barrier to NO entry via tight association between the submembrane cytoskeleton and the cytoplasmic domain of the band 3 (AE‐1) membrane protein (ankyrin successfully competes with oxy‐Hb for binding to AE‐1). Upon RBC deoxygenation (hypoxic RBC), oxy‐Hb R‐state is converted to deoxy‐Hb T‐state, which now successfully competes with ankyrin for the AE‐1 cytoplasmic domain (affinity for AE‐1 ranks as follows: deoxy‐Hb > ankyrin > oxy‐Hb). Proximal apposition of heme to the membrane and diminished cytoskeleton‐membrane interaction allows increased NO entry and affords intraerythrocytic Hb greater access to extraerythrocytic NO. Moreover, if deoxy‐Hb encounters high concentrations of NO, “super‐T” Hb‐α‐Fe(II)NO may form [in which NO bound to the α‐subunit disrupts normal heme‐globin linkage, locking Hb in the deoxy‐ or T‐conformation 211,312; see text for details]. (B) Increasing the proportion of intraerythrocytic T‐state Hb [by forming either deoxy‐Hb or α‐Fe(II)NO, both of which are T‐state tetramers and bind avidly to AE‐1] increases NO consumption by intact RBCs, as measured by a competition assay. In this plot, increased consumption in treated vs. control RBCs appears as an increased KRBC/KRBC control ratio (y‐axis) (+ P < 0.05, n = 4 to 8). (C) Increased NO uptake by NO‐pretreated hypoxic RBCs correlates with the formation of α‐Fe(II)NO (i.e., “super‐T” Hb) (+ P < 0.05, n = 4 for each sample). Reproduced with permission from 111.
Figure 7. Figure 7.

O2 exerts allosteric control over hemoglobin (Hb) S‐nitrosylation 149. (A) O2 loading accelerates S‐nitrosylation of Hb. Rates of Hb S‐nitrosylation by S‐nitrosothiol (SNO)‐cysteine are faster in the oxy conformation [HbFe(II)O2] than in the deoxy state [HbFe(II)]. (B) Deoxygenation accelerates denitrosylation of S‐nitroso‐hemoglobin (SNO‐Hb). Rates of SNO decomposition (and transnitrosylative transfer) are much faster in the deoxy conformation [SNO‐HbFe(II)] than in the oxy state [SNO‐HbFe(II)O2]. The decomposition of SNO‐HbFe(II) is further accelerated by the presence of excess glutathione (GSH) (Note: parallel to data in Figure 3). Within the dead time of the assay system (∼15 s), a major fraction of SNO‐HbFe(II) was converted to S‐nitroso‐glutathione (dashed line).
Figure 8. Figure 8.

Crystal structures and models of S‐nitrosothiol hemoglobin (SNO‐Hb). A crystal structure of R‐state SNO‐Hb has been published by Arnone and colleagues 38. SNO‐Hb did not crystallize in T‐state, consistent with thermodynamic predictions of instability [Note: subsequent work by Zhao and Houk suggests that SNO‐Cysβ93 in Hb crystals is a radical species 315]. Structural models of Hb and SNO‐Hb demonstrate that positioning of Cysβ93 and SNO‐βCys93 is dependent upon Hb conformation, consistent with crystallographic findings 262. (A) Deoxy‐Hb: Cysβ93 is exposed on the protein surface, above the external His146‐Asp94 salt bridge; Tyrβ145 packs alongside its side chain. The γ‐sulfur of Cysβ93 (yellow) is accessible to solvent in a cavity formed by the COOH‐terminus of β‐helix F, the COOH‐terminus of the β‐subunit, and helix C of the α2‐subunit. The α‐ and β‐carbons of Cysβ93 are gray. (B) Oxy‐Hb: Cysβ93 rotates in and away from solvent (and the broken salt bridge); Tyrβ145 is positioned over the top of its side chain. The γ‐sulfur of Cysβ93 is buried below its β‐carbon (gray) and is further shielded by structural changes. (C) SNO‐deoxy‐Hb: In the T‐structure, SNO is highly exposed to solvent (favoring NO group donation). The SNO is positioned either above or below the His146‐Asp94 salt bridge; if positioned up, the sulfur (yellow) and nitrogen (blue) are exposed, and if positioned down, the sulfur and oxygen (red) are exposed. (D) SNO‐oxy‐Hb: In the R‐structure, SNO is protected from solvent (disfavoring NO group donation). The NO group (blue and red) is accommodated behind the β‐heme, under Tyrβ145. The β‐carbon (gray) buries the sulfur (yellow). The SNO is also shielded by the backbone and side chain conformations.
Figure 9. Figure 9.

O2‐dependent variation in S‐nitrosothiol hemoglobin (SNO‐Hb) (▪) and Hb[FeNO] (□) demonstrate the association between Hb conformation and intramolecular heme → thiol migration of NO groups in red blood cells 196. (A‐D) Moles NO per mole Hb tetramer in arterial and mixed venous blood from humans breathing either 21% O2 at 1 ATA (absolute atmospheres) (A and C), 21% O2 at 0.56 ATA (equivalent to ∼12% O2 at 1 ATA) (B), or 100% O2 at 3 ATA (D). Total Hb‐bound NO equals the sum of the two bars for each condition. These data demonstrate O2‐dependent shuttling of Hb‐bound NO groups between heme and βCys93. (E and F) SNO content of blood Hb, presented as the fraction of NO‐Hb (% SNO), correlates with Hb O2 saturation (E) but not with pO2 (F). * P < 0.05, paired t‐test, indicates amounts of SNO‐Hb significantly different from zero; ** P < 0.05, paired t‐test, indicates a significant arterial‐venous difference in SNO‐Hb. Data represent the mean (s.e.m.) from 8‐11 individuals. See also Figure 3 showing similar results in human blood across a wide range of Hb O2 saturations.
Figure 10. Figure 10.

pO2‐regulated export of NO groups from red blood cells (RBCs) can occur via NO group transfer from S‐nitrosothiol hemoglobin (SNO‐Hb) to an extraerythrocytic thiol reactant 223. Circulatory transit was simulated for human whole blood in a thin‐film tonometer (under 5% CO2, balance N2, pH 7.4) after spiking the sample with the nonnative, low‐mass thiol, N‐acetylcysteine (NAC, 100 μM in plasma). The concentration of S‐nitroso‐N‐acetylcysteine (SNOAC) that formed in plasma was measured by mass spectrometry and confirmed by mass labeling with 15N. The conversion of extraerythrocytic NAC to SNOAC correlated with RBC O2 and SNO content. Serum SNOAC formed as a function of Hb So2. (A) Liquid chromatogram demonstrating coelution of RBC‐generated SNOAC with the 15N‐labeled SNOAC standard. (B) Mass spectrum demonstrating paired signals from RBC‐generated SNOAC and the 15N‐labeled SNOAC standard (m/z 194). (C) Extraerythrocytic SNOAC concentration follows oxy‐Hb desaturation (by co‐oximetry). “♦” ‐ extraerythrocytic SNOAc values indexed (as %) to amount measured when Hb So2 = 56%. (D) RBC SNO content (black) decreased in tandem with Hb O2 desaturation (dashed blue). Note that as SNO content in RBCs fell, extraerythrocytic SNOAC (yellow symbols) accumulated. Note also that SNOAC levels were below the limits of detection when the Hb O2 saturation was above 80%. These data indicate that the O2‐responsive vasoactivity demonstrated for human RBCs (see Fig. 3) can involve transfer of NO groups by RBCs to extraerythrocytic low‐mass thiols.
Figure 11. Figure 11.

The red blood cell (RBC) membrane furnishes a platform for O2‐regulated NO processing and export 226. (A) NO group ligation (Fe‐ or S‐) and disposition (cytosol or membrane) after exposure of intact RBCs to NO. Note that FeNO (representing HbFeNO) is predominantly cytosolic, whereas S‐nitrosothiol (SNO) is largely membrane associated. (B) The intrinsic membrane protein AE‐1 (band 3 protein) is trans‐S‐nitrosylated by S‐nitroso‐hemoglobin. SNO content of AE‐1 immunoprecipitates derived either from inside‐out RBC membrane vesicles (IOVs) incubated with free (B1) or sepharose‐bound SNO‐Hb (B2) or from membrane extracts of RBCs treated with NO (B3). Note that the cytoplasmic domain of AE‐1 contains two reactive cysteine thiols, which are exposed on the outer surface of RBC‐derived IOVs. (C) Membrane SNO is necessary for RBC‐mediated vasodilation. Representative traces display tension generated by aortic rings at high or low (hypoxic) pO2. At 95% O2, RBC IOVs S‐nitrosylated by Hb [as in (B)] above elicit relaxation, whereas NO‐treated intact RBCs [as in (A) above] elicit contraction. At ∼1% O2 (representative of tissue pO2), NO‐treated RBCs elicit relaxation, which is abrogated by the AE‐1 inhibitor DIDS. Addition of membranes derived from NO‐treated RBCs produces relaxation, whereas an equivalent aliquot of cytosol (containing HbFeNO) elicits contraction. (D) Cartoon illustrating a juxta‐membrane Hb population in which R → T transition promotes docking of deoxy‐Hb (via the β‐cleft or 2,3‐DPG binding cavity) to the cytoplasmic domain of AE‐1. Concomitantly, NO is transferred from βCys93 to a cysteine thiol within AE‐1. In addition, R‐to‐T transition may facilitate a shift in the intraerythrocytic equilibrium between SNO‐Hb and S‐nitroso‐glutathione (GSNO).
Figure 12. Figure 12.

O2‐responsive generation of S‐nitrosothiols (SNOs) from red blood cells (RBCs) signals the ventilatory response to hypoxia 178. After implantation of a cannula proximal to the brainstem nucleus tractus soliatarius (NTS), minute ventilation () was monitored in conscious rats, using whole‐body plethysmography. (A) during (shaded) and following a brief period of hypoxia demonstrated the characteristic hypoxic ventilatory response. (B) Injection into the NTS (at arrow) of the low‐mass SNO S‐nitrosocysteinylglycine (CGSNO, 1 nM) resulted in a marked increase in , with onset and decay characteristics identical to those observed in (A). (C) Injection of the low‐mass fraction from deoxygenated blood‐derived plasma (at arrow, black tracing) recapitulates the increase observed in (A) and (B); this response was absent when the low‐mass fraction from oxygenated blood was injected (gray). (D) Glutathione (GSH) reacts with deoxygenated but not oxygenated blood to form S‐nitroso‐glutathione (GSNO). Liquid chromatography/mass spectrometry was performed on plasma from blood to which 400 μM of GSH had been added. The GSNO peak (eluted at 5.82 min; blue) in the deoxygenated blood‐derived plasma was attenuated after ultraviolet treatment (violet), demonstrating SNO ligation and is not evident in the oxygenated blood‐derived plasma (red). These observations indicate that deoxygenated but not oxygenated blood reacts with reduced thiol species to form SNO (also see Fig. 8). (E) Mean differences in before and after the injection of the deoxygenated fractions (black) analyzed in (D); oxygenated fractions (gray) had no effect. Reproduced with permission from 178.


Figure 1.

Local vascular reflexes support maintenance of O2 delivery to tissue in the setting of progressive hypoxia. In a classic paper 244, Guyton demonstrated regional autoregulation of systemic blood flow in normal dogs (following spinal anesthesia) by observing variation in blood flow during constant pressure blood perfusion of the femoral artery, while reducing the hemoglobin oxygen saturation (Hb So2%) from 100% to 0% in the perfusing blood. (A) Stepwise reduction in Hb So2% caused a progressive increase in blood flow through the leg. (B) These data demonstrate that autoregulation of blood flow occurs at a local level and this regulation serves to improve oxygen supply when blood oxygen content falls. In addition, effects on blood flow were replicated by injecting partially deoxygenated versus oxygenated red blood cells into the artery, demonstrating that effects could be elicited during arteriovenous transit (<1 s). Reproduced with permission from 244.



Figure 2.

Red blood cells (RBCs) transduce regional O2 gradients in tissue to control nitric oxide (NO) bioactivity in plasma by trapping or delivering NO groups as a function of hemoglobin (Hb) O2 saturation. (A) In this fashion, circulating NO groups are processed by Hb into the highly vasoactive (thiol‐based) NO congener, S‐nitrosothiol (SNO). By exporting SNOs as a function of Hb deoxygenation, RBCs precisely dispense vasodilator bioactivity in direct proportion to regional blood flow lack. (B) O2 delivery homeostasis requires biochemical coupling of vessel tone to environmental cues that matches perfusion sufficiency to metabolic demand. Because oxy‐ and deoxy‐Hb process NO differently (see text), allosteric transitions in Hb conformation afford context‐responsive (O2‐coupled) control of NO bioavailability, thereby linking the sensor and effector arms of this system. Specifically, Hb conformation governs the equilibria among deoxy‐HbFeNO (A; NO sink), SNO‐oxy‐Hb (B; NO store), and acceptor thiols including the membrane protein SNO‐AE‐1 (C; bioactive NO source). Direct SNO export from RBCs or S‐transnitrosylation from RBCs to plasma thiols (D) or to endothelial cells directly (not shown) yields vasoactive SNOs, which influence resistance vessel caliber and close this signaling loop. Thus, RBCs either trap (A) or export (D) NO groups to optimize blood flow. (C) NO processing in RBCs (A and B) couples vessel tone to tissue pO2; this system subserves hypoxic vasodilation in the arterial periphery and thereby calibrates blood flow to regional tissue hypoxia. See Figures 10 and 11 and related discussion for more details.



Figure 3.

Hemoglobin oxygen saturation (Hb So2%) exerts coordinated governance of red blood cell (RBC) S‐nitrosothiol (SNO) content, RBC vasoactivity, and human peripheral blood flow. (A) Human blood gas measurements of SNO and O2 64. RBCs with (black) or without (red) added extracellular glutathione (GSH) were deoxygenated under inert gas. The natural logarithm of SNO content in RBCs (SNO RBC) is linearly related to Hb So2. GSH accelerated SNO RBC decay, consistent with O2‐linked export of NO groups to extraerythrocytic thiols. (B) RBCs induce graded relaxation of systemic arteries (aortic ring bioassay) that is inversely related to Hb So2 across the physiological range, recapitulating hypoxic vasodilation (HVD) 196. Hb So2 spanned from red (oxy, >90% Hb So2) to blue (deoxy, <40% Hb So2). (C) Leg vascular conductance increases as blood O2 content falls (HVD) 99. Hb So2 and thus arterial blood O2 content were manipulated by CO exposure ± varying Fio2 (fraction of inspired O2; hypoxia versus hyperoxia). Neither vascular conductance nor blood flow correlated with blood pO2 per se. Note the similarity between observations in panels A, B, and C and Guyton's original observations 244 (Fig. 1). Panel (C) Reproduced with permission from 99.



Figure 4.

Hypoxic vasodilation (1% O2) by native (untreated) human red blood cells (RBCs) of mouse aortic segments requires neither eNOS nor intact endothelium 62. (A) Hypoxiacoupled vasodilation by RBCs is preserved despite endothelial denudation, inhibition of NOS activity with L‐NAME, or eNOS deletion (eNOS‐/‐). RBC‐induced vasorelaxation was greatly attenuated by the guanylate cyclase inhibitor ODQ. (n = 9 to 34; * P < 0.05). (B, C) Representative tracings from the vascular ring preparation show that removal of endothelium eliminates acetylcholine (ACh)‐induced vasorelaxation but has no effect on RBC‐induced vasorelaxation [N.B. vessel rings are precontracted with phenylephrine (PE)].



Figure 5.

Hypoxic vasodilation (HVD) by native human red blood cells (RBCs) of rabbit aortic segments is S‐nitrosothiol (SNO) based 62. (A) RBC‐induced vasorelaxation was potentiated by cysteine and by N‐acetylcysteine but was unaffected by nitrite (NO2−) or the prostaglandin synthesis inhibitors indomethacin and aspirin (all agents added 2 min before RBCs) (n = 5 to 11; * P < 0.05). (B‐D), Representative tracings illustrate the augmentation of relaxation by cysteine and the attenuation of relaxation by S‐nitroso‐hemoglobin depletion (∼80% depletion). (E) Addition of NO2− to the bath (even at supraphysiologic concentrations of 1 μM) before (NO2− → RBC), simultaneously with the addition of RBCs (RBC + NO2−), or near the termination of RBC‐induced vasorelaxation (RBC → NO2−), had no discernible effect on the magnitude or duration of RBC‐induced HVD.



Figure 6.

Conformation‐specific binding between hemoglobin (Hb) and the red blood cell (RBC) membrane protein AE‐1 affords O2 responsive control of NO trapping 111. (A) Under oxygenated conditions (normoxic RBC, at left), the RBC membrane constitutes a significant barrier to NO entry via tight association between the submembrane cytoskeleton and the cytoplasmic domain of the band 3 (AE‐1) membrane protein (ankyrin successfully competes with oxy‐Hb for binding to AE‐1). Upon RBC deoxygenation (hypoxic RBC), oxy‐Hb R‐state is converted to deoxy‐Hb T‐state, which now successfully competes with ankyrin for the AE‐1 cytoplasmic domain (affinity for AE‐1 ranks as follows: deoxy‐Hb > ankyrin > oxy‐Hb). Proximal apposition of heme to the membrane and diminished cytoskeleton‐membrane interaction allows increased NO entry and affords intraerythrocytic Hb greater access to extraerythrocytic NO. Moreover, if deoxy‐Hb encounters high concentrations of NO, “super‐T” Hb‐α‐Fe(II)NO may form [in which NO bound to the α‐subunit disrupts normal heme‐globin linkage, locking Hb in the deoxy‐ or T‐conformation 211,312; see text for details]. (B) Increasing the proportion of intraerythrocytic T‐state Hb [by forming either deoxy‐Hb or α‐Fe(II)NO, both of which are T‐state tetramers and bind avidly to AE‐1] increases NO consumption by intact RBCs, as measured by a competition assay. In this plot, increased consumption in treated vs. control RBCs appears as an increased KRBC/KRBC control ratio (y‐axis) (+ P < 0.05, n = 4 to 8). (C) Increased NO uptake by NO‐pretreated hypoxic RBCs correlates with the formation of α‐Fe(II)NO (i.e., “super‐T” Hb) (+ P < 0.05, n = 4 for each sample). Reproduced with permission from 111.



Figure 7.

O2 exerts allosteric control over hemoglobin (Hb) S‐nitrosylation 149. (A) O2 loading accelerates S‐nitrosylation of Hb. Rates of Hb S‐nitrosylation by S‐nitrosothiol (SNO)‐cysteine are faster in the oxy conformation [HbFe(II)O2] than in the deoxy state [HbFe(II)]. (B) Deoxygenation accelerates denitrosylation of S‐nitroso‐hemoglobin (SNO‐Hb). Rates of SNO decomposition (and transnitrosylative transfer) are much faster in the deoxy conformation [SNO‐HbFe(II)] than in the oxy state [SNO‐HbFe(II)O2]. The decomposition of SNO‐HbFe(II) is further accelerated by the presence of excess glutathione (GSH) (Note: parallel to data in Figure 3). Within the dead time of the assay system (∼15 s), a major fraction of SNO‐HbFe(II) was converted to S‐nitroso‐glutathione (dashed line).



Figure 8.

Crystal structures and models of S‐nitrosothiol hemoglobin (SNO‐Hb). A crystal structure of R‐state SNO‐Hb has been published by Arnone and colleagues 38. SNO‐Hb did not crystallize in T‐state, consistent with thermodynamic predictions of instability [Note: subsequent work by Zhao and Houk suggests that SNO‐Cysβ93 in Hb crystals is a radical species 315]. Structural models of Hb and SNO‐Hb demonstrate that positioning of Cysβ93 and SNO‐βCys93 is dependent upon Hb conformation, consistent with crystallographic findings 262. (A) Deoxy‐Hb: Cysβ93 is exposed on the protein surface, above the external His146‐Asp94 salt bridge; Tyrβ145 packs alongside its side chain. The γ‐sulfur of Cysβ93 (yellow) is accessible to solvent in a cavity formed by the COOH‐terminus of β‐helix F, the COOH‐terminus of the β‐subunit, and helix C of the α2‐subunit. The α‐ and β‐carbons of Cysβ93 are gray. (B) Oxy‐Hb: Cysβ93 rotates in and away from solvent (and the broken salt bridge); Tyrβ145 is positioned over the top of its side chain. The γ‐sulfur of Cysβ93 is buried below its β‐carbon (gray) and is further shielded by structural changes. (C) SNO‐deoxy‐Hb: In the T‐structure, SNO is highly exposed to solvent (favoring NO group donation). The SNO is positioned either above or below the His146‐Asp94 salt bridge; if positioned up, the sulfur (yellow) and nitrogen (blue) are exposed, and if positioned down, the sulfur and oxygen (red) are exposed. (D) SNO‐oxy‐Hb: In the R‐structure, SNO is protected from solvent (disfavoring NO group donation). The NO group (blue and red) is accommodated behind the β‐heme, under Tyrβ145. The β‐carbon (gray) buries the sulfur (yellow). The SNO is also shielded by the backbone and side chain conformations.



Figure 9.

O2‐dependent variation in S‐nitrosothiol hemoglobin (SNO‐Hb) (▪) and Hb[FeNO] (□) demonstrate the association between Hb conformation and intramolecular heme → thiol migration of NO groups in red blood cells 196. (A‐D) Moles NO per mole Hb tetramer in arterial and mixed venous blood from humans breathing either 21% O2 at 1 ATA (absolute atmospheres) (A and C), 21% O2 at 0.56 ATA (equivalent to ∼12% O2 at 1 ATA) (B), or 100% O2 at 3 ATA (D). Total Hb‐bound NO equals the sum of the two bars for each condition. These data demonstrate O2‐dependent shuttling of Hb‐bound NO groups between heme and βCys93. (E and F) SNO content of blood Hb, presented as the fraction of NO‐Hb (% SNO), correlates with Hb O2 saturation (E) but not with pO2 (F). * P < 0.05, paired t‐test, indicates amounts of SNO‐Hb significantly different from zero; ** P < 0.05, paired t‐test, indicates a significant arterial‐venous difference in SNO‐Hb. Data represent the mean (s.e.m.) from 8‐11 individuals. See also Figure 3 showing similar results in human blood across a wide range of Hb O2 saturations.



Figure 10.

pO2‐regulated export of NO groups from red blood cells (RBCs) can occur via NO group transfer from S‐nitrosothiol hemoglobin (SNO‐Hb) to an extraerythrocytic thiol reactant 223. Circulatory transit was simulated for human whole blood in a thin‐film tonometer (under 5% CO2, balance N2, pH 7.4) after spiking the sample with the nonnative, low‐mass thiol, N‐acetylcysteine (NAC, 100 μM in plasma). The concentration of S‐nitroso‐N‐acetylcysteine (SNOAC) that formed in plasma was measured by mass spectrometry and confirmed by mass labeling with 15N. The conversion of extraerythrocytic NAC to SNOAC correlated with RBC O2 and SNO content. Serum SNOAC formed as a function of Hb So2. (A) Liquid chromatogram demonstrating coelution of RBC‐generated SNOAC with the 15N‐labeled SNOAC standard. (B) Mass spectrum demonstrating paired signals from RBC‐generated SNOAC and the 15N‐labeled SNOAC standard (m/z 194). (C) Extraerythrocytic SNOAC concentration follows oxy‐Hb desaturation (by co‐oximetry). “♦” ‐ extraerythrocytic SNOAc values indexed (as %) to amount measured when Hb So2 = 56%. (D) RBC SNO content (black) decreased in tandem with Hb O2 desaturation (dashed blue). Note that as SNO content in RBCs fell, extraerythrocytic SNOAC (yellow symbols) accumulated. Note also that SNOAC levels were below the limits of detection when the Hb O2 saturation was above 80%. These data indicate that the O2‐responsive vasoactivity demonstrated for human RBCs (see Fig. 3) can involve transfer of NO groups by RBCs to extraerythrocytic low‐mass thiols.



Figure 11.

The red blood cell (RBC) membrane furnishes a platform for O2‐regulated NO processing and export 226. (A) NO group ligation (Fe‐ or S‐) and disposition (cytosol or membrane) after exposure of intact RBCs to NO. Note that FeNO (representing HbFeNO) is predominantly cytosolic, whereas S‐nitrosothiol (SNO) is largely membrane associated. (B) The intrinsic membrane protein AE‐1 (band 3 protein) is trans‐S‐nitrosylated by S‐nitroso‐hemoglobin. SNO content of AE‐1 immunoprecipitates derived either from inside‐out RBC membrane vesicles (IOVs) incubated with free (B1) or sepharose‐bound SNO‐Hb (B2) or from membrane extracts of RBCs treated with NO (B3). Note that the cytoplasmic domain of AE‐1 contains two reactive cysteine thiols, which are exposed on the outer surface of RBC‐derived IOVs. (C) Membrane SNO is necessary for RBC‐mediated vasodilation. Representative traces display tension generated by aortic rings at high or low (hypoxic) pO2. At 95% O2, RBC IOVs S‐nitrosylated by Hb [as in (B)] above elicit relaxation, whereas NO‐treated intact RBCs [as in (A) above] elicit contraction. At ∼1% O2 (representative of tissue pO2), NO‐treated RBCs elicit relaxation, which is abrogated by the AE‐1 inhibitor DIDS. Addition of membranes derived from NO‐treated RBCs produces relaxation, whereas an equivalent aliquot of cytosol (containing HbFeNO) elicits contraction. (D) Cartoon illustrating a juxta‐membrane Hb population in which R → T transition promotes docking of deoxy‐Hb (via the β‐cleft or 2,3‐DPG binding cavity) to the cytoplasmic domain of AE‐1. Concomitantly, NO is transferred from βCys93 to a cysteine thiol within AE‐1. In addition, R‐to‐T transition may facilitate a shift in the intraerythrocytic equilibrium between SNO‐Hb and S‐nitroso‐glutathione (GSNO).



Figure 12.

O2‐responsive generation of S‐nitrosothiols (SNOs) from red blood cells (RBCs) signals the ventilatory response to hypoxia 178. After implantation of a cannula proximal to the brainstem nucleus tractus soliatarius (NTS), minute ventilation () was monitored in conscious rats, using whole‐body plethysmography. (A) during (shaded) and following a brief period of hypoxia demonstrated the characteristic hypoxic ventilatory response. (B) Injection into the NTS (at arrow) of the low‐mass SNO S‐nitrosocysteinylglycine (CGSNO, 1 nM) resulted in a marked increase in , with onset and decay characteristics identical to those observed in (A). (C) Injection of the low‐mass fraction from deoxygenated blood‐derived plasma (at arrow, black tracing) recapitulates the increase observed in (A) and (B); this response was absent when the low‐mass fraction from oxygenated blood was injected (gray). (D) Glutathione (GSH) reacts with deoxygenated but not oxygenated blood to form S‐nitroso‐glutathione (GSNO). Liquid chromatography/mass spectrometry was performed on plasma from blood to which 400 μM of GSH had been added. The GSNO peak (eluted at 5.82 min; blue) in the deoxygenated blood‐derived plasma was attenuated after ultraviolet treatment (violet), demonstrating SNO ligation and is not evident in the oxygenated blood‐derived plasma (red). These observations indicate that deoxygenated but not oxygenated blood reacts with reduced thiol species to form SNO (also see Fig. 8). (E) Mean differences in before and after the injection of the deoxygenated fractions (black) analyzed in (D); oxygenated fractions (gray) had no effect. Reproduced with permission from 178.

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Further Reading
 1. Singel DJ, Stamler JS. Chemical physiology of blood flow regulation by red blood cells: Role of nitric oxide and S‐nitrosohemoglobin. Annu Rev Physiol 67: 99–145, 2005.
 2. Stamler JS, Singel DJ, Loscalzo J. Biochemistry of nitric oxide and its redox‐activated forms. Science 258: 1898–1902, 1992.
 3. Gaston B, Singel D, Doctor A, Stamler JS. S‐Nitrosothiol signaling in respiratory biology. Am J Respir Crit Care Med 173: 1186–1193, 2006.
 4. Zimmet JM, Hare JM. Nitroso‐redox interactions in the cardiovascular system. Circulation 114: 1531–1544, 2006.
 5. Hess DT, Matsumoto A, Kim SO, Marshall HE, Stamler JS. Protein S‐nitrosylation: Purview and parameters. Nat Rev Mol Cell Biol 6: 150–166, 2005.
 6. Lima B, Forrester MT, Hess DT, Stamler JS. S‐Nitrosylation in cardiovascular signaling. Circ Res. 106: 633–46, 2010.

Further Reading
1.      Singel DJ and Stamler JS. Chemical Physiology of Blood Flow Regulation by Red Blood Cells: Role of Nitric Oxide and S-Nitrosohemoglobin. Annual Review of Physiology 67: 99-145,2005.
2.      Stamler JS, Singel DJ and Loscalzo J. Biochemistry of nitric oxide and its redox-activated forms. Science 258: 1898-1902, 1992.
3.      Gaston B, Singel D, Doctor A and Stamler JS. S-Nitrosothiol Signaling in Respiratory Biology. Am J Respir Crit Care Med 173: 1186-1193,2006.
4.      Zimmet JM and Hare JM. Nitroso-Redox Interactions in the Cardiovascular System. Circulation 114: 1531-1544, 2006.
5.      Hess DT, Matsumoto A, Kim SO, Marshall HE and Stamler JS. Protein S-Nitrosylation: Purview and Parameters. Nat Rev Mol Cell Biol 6:150-166,2005.
6.      Lima B, Forrester MT, Hess DT, Stamler JS. S-nitrosylation in cardiovascular signaling. Circ Res. 106:633-46, 2010.


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Article Title: Nitric Oxide Transport in Blood: A Third Gas in the Respiratory Cycle
 

How to Cite

Allan Doctor, Jonathan S. Stamler. Nitric Oxide Transport in Blood: A Third Gas in the Respiratory Cycle. Compr Physiol 2011, 1: 541-568. doi: 10.1002/cphy.c090009