Effects of S-Nitrosation and Cross-Linking of Hemoglobin on Hypoxic Pulmonary Vasoconstriction in Isolated Rat Lungs
Free hemoglobin (Hb) and red blood cells augment hypoxic pulmonary vasoconstriction (HPV) by scavenging nitric oxide (NO). S-nitrosation of Hb (SNO-Hb) may confer vasodilatory properties by allowing release of NO during deoxygenation and/or by interaction with small-molecular weight thiols. Likewise, cross-linking of free Hb may limit its vasoconstrictive effect by preventing abluminal movement of the molecule. We compared the effects of free SNO-Hb and Hb intramolecularly cross-linked at the β-cysteine 93 residue [Bis(maleidophenyl)-polyethylene glycol2000HbA (Bis-Mal-PEGHb)] to those of free oxyHb on pulmonary artery pressure (PAP), HPV, and exhaled NO (eNO) in isolated, perfused rat lungs. Ventilation of lungs with anoxic gas for 5 minutes reduced perfusate Po2 to 11±1.0 Torr. Addition of SNO-Hb or Bis-Mal-PEGHb (100 μmol/L) to buffer perfusate increased normoxic PAP and augmented HPV in similar magnitude as free oxyHb, but had no effect on eNO. Addition of the allosteric modulator inositol hexaphosphate to increase Hb P50 and the thiol glutathione (GSH) to allow removal of NO from Hb via transnitrosation to the perfusate did not reduce augmentation of HPV by SNO-Hb or increase eNO. GSH resulted in an ≈50% reduction in perfusate [S-nitrosothiol], in association with an increase in perfusate [metHb]. Free SNO-Hb is a net NO scavenger and pulmonary vasoconstrictor in this model, although thiol-mediated release of NO from SNO-Hb does occur. However, release of NO from SNO-Hb was not influenced by deoxygenation-mediated allosteric changes in Hb across a broad range of oxyHb saturation. Cross-linking of Hb does not limit its pulmonary vasoconstrictor effects.
The rapid oxidation of nitric oxide (NO) by oxyhemoglobin (oxyHb) to form methemoglobin (metHb) and nitrate acts to limit the magnitude and duration of the vasorelaxant effects of NO.1 In the pulmonary circulation, this is manifest as increased pulmonary vascular resistance (PVR) and augmentation of hypoxic pulmonary vasoconstriction (HPV) by red blood cells (RBCs) and free Hb.2–8⇓⇓⇓⇓⇓⇓
An additional reaction of NO with Hb is S-nitrosation at the β-cysteine 93 (β-cys93) residue to form S-nitrosoHb (SNO-Hb).9 This reaction is also reversible, particularly in the presence of low-molecular weight thiols such as glutathione (GSH),9,10⇓ and/or under the allosteric influence of deoxygenation.9,11,12⇓⇓ This dynamic, oxygen-linked property of SNO-Hb has led to the theory that Hb may act as a carrier of NO from the lung to the peripheral circulation, where it is released on deoxygenation of Hb. That free and intraerythrocytic SNO-Hb results in systemic and cerebral vasodilation supports this hypothesis.9,11⇓
Recently, we showed that free SNO-Hb is a potent a vasoconstrictor in the pulmonary circulation.6 In addition, we were unable to demonstrate an allosteric effect of deoxygenation on NO release from SNO-Hb, although this observation was limited by the low P50 of free SNO-Hb and the inability to reduce perfusate Po2 below ≈38 Torr despite ventilation with anoxic gas in this model.
In the present work, we again studied the effects of SNO-Hb on PVR and HPV. However, we expanded on our previous work by using a different animal model (isolated, perfused rat lungs), which allowed a reduction in the perfusate Po2 to ≈10 Torr during anoxic ventilation, a level that should promote deoxygenation of SNO-Hb. In addition, we studied both lower and higher concentrations of free Hb and compared the vasoconstrictive potential of SNO-Hb to Hb that is intramolecularly cross-linked at the β-cys93 residue, which precludes SNO-Hb formation and dissociation of the Hb tetramer.
Materials and Methods
The protocol was approved by the Animal Care Committee of the Veterans Affairs Puget Sound Health Care System.
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
All Hb products (oxyHb, SNO-Hb, and the cross-linked Hb bis(maleidophenyl)-polyethylene glycol2000HbA [Bis-Mal-PEGHb]) were synthesized from human blood (donated by the investigators of the present study; see also online data supplement).
Measurement of Synthesized SNO-Hb and Perfusate Samples
Synthesized SNO-Hb and perfusate [SNO] were quantified using the I3− chemiluminescent assay and by HPLC-ionization mass spectrometry (see online data supplement).
To check for Hb tetramer dissociation, perfusate samples containing SNO-Hb were obtained from a representative experiment at 5-minute intervals. Nondenaturing gel electrophoresis was performed by mixing samples with loading buffer (5 μL) and running in Invitrogen 4% to 20% Tris-Glycine gel, followed by staining with Blue-code.
For measurement of metHb in fresh perfusate samples, we used the method described by Winterbourn.13 Details are provided in the online data supplement.
Determination of Oxygen Binding Affinities of Hb and SNOHb
Oxygen binding curves of Hb and SNO-Hb were determined using a tonometer as previously described14 (see online data supplement).
Please see the online data supplement for details of the preparation of isolated, perfused rat lungs (ATL, Kent, Wash), and description of the model.
Effect of Free Hb on PAP and HPV
After an initial hypoxic challenge with buffer, the lungs were entered into one of three primary groups based on the particular modification of free Hb added to the perfusate: (1) OxyHb, (2) SNO-Hb, and (3) Bis-Mal-PEGHb. An identical protocol was followed for each experiment as follows: during normoxic ventilation, free Hb was added to the perfusate to achieve a concentration of 100 μmol/L (Hb concentrations are expressed as tetrameric Hb units). After a 5-minute stabilization period, the lungs were subjected to two consecutive 5-minute hypoxic challenges (FIO2 0) for 5 minutes, separated by 5 minutes of normoxic ventilation, at which time data were again recorded. A subset of experiments were also performed with free oxyHb and SNO-Hb at lower concentrations (400 nmol/L to 16 μmol/L) using the same protocol.
Effect of Inositol Hexaphosphate (IHP) and GSH on Augmentation of HPV by Hb and on Perfusate [SNO]
To determine whether inorganic phosphates and low molecular weight thiols affect the response of the pulmonary circulation to free Hb, IHP (100 μmol/L) followed by GSH (5 mmol/L) were added to perfusates containing either 100 μmol/L oxyHb or SNO-Hb during normoxic ventilation. Each addition was followed in 5 minutes by a hypoxic challenge as described above. Five minutes after each intervention (IHP, GSH, or hypoxia) a perfusate sample was obtained and immediately frozen for later analysis of perfusate [SNO].
Effect of SNO-Glutathione (GSNO) on HPV and Perfusate and Exhaled NO
In a final series of experiments, and after measurement of perfusate [SNO] from the above experiments, the effects of GSNO on HPV and exhaled NO were assessed. After initial stabilization and perfusion with buffer, the NOS inhibitor Nω-nitro-l-arginine (L-NA) (100 μmol/L) was added to the perfusate. After 5 additional minutes, a series of three hypoxic challenges were performed, before and after addition of GSNO to the perfusate to produce concentrations of 5 and 20 μmol/L. These concentrations were chosen due to their similarity to the observed fall in perfusate [SNO] after hypoxic ventilation and addition of GSH to the perfusate, respectively (see Results and Figure 4). A fourth hypoxic challenge was performed after addition of oxyHb (100 μmol/L) and additional GSNO (20 μmol/L) to the perfusate.
Statistical analysis was performed using the computer software package StatView (Abacus Concepts). Data are presented as mean±SEM. Variables before and after addition of free Hb, GSH, or IHP to the perfusate were compared using a paired t test. Group comparisons were performed using 1-way analysis of variance (ANOVA) at individual time points, or repeated measures ANOVA for group differences over time. The Bonferroni/Dunn correction for multiple comparisons was performed where appropriate. A value of P<0.05 was accepted as statistically significant.
The P50 of synthesized stock SNO-Hb was 5.5 Torr and increased to 8.3 Torr in the presence of IHP. The P50 of SNO-Hb diluted in perfusate was similar (Figure 1). Perfusate Po2 was 11.1±1.0 Torr after 5 minutes of anoxic ventilation in a pooled group of experiments from all series (n=19). This Po2 should result in a Hb saturation change of ≈10% for SNO-Hb and 35% for SNO-Hb in the presence of IHP (Figure 1).
Gel electrophoresis of perfusate samples containing SNO-Hb revealed a predominance of a single band at ≈60 kDa, suggesting the absence of significant Hb tetramer dissociation (dimerization) (Figure 2).
Effect of Free Hb on PAP and HPV
Addition of free oxyHb, Bis-Mal-PEGHb, or SNO-Hb to buffer perfusate at a concentration of 100 μmol/L resulted in small increases in normoxic PAP (Figure 3A) and augmentation of HPV after 5 minutes of anoxic ventilation (Figure 3B). Likewise, the peak hypoxic pressure attained (13.1±2.1, 10.2±0.9, and 12.4±1.9 Torr, respectively) and the time to development of peak HPV (185.4±12.0, 184.6±16.4, and 209.3±12.4 seconds, respectively) did not differ significantly between groups. Addition of free oxyHb or SNO-Hb at low concentrations (400 nmol/L to 16 μmol/L) did not significantly change HPV in comparison to the response with buffer (Figure 4).
Effect of IHP and GSH on Augmentation of HPV by Hb and on Perfusate [SNO]
Addition of IHP to perfusate containing free SNO-Hb did not reduce augmentation of HPV in comparison to oxyHb plus IHP (P=0.89) (Figure 5). Likewise, addition of excess GSH to perfusate containing free Hb and IHP did not reduce augmentation of HPV by either oxyHb (P=0.31) or SNO-Hb (P=0.75) (Figure 5).
Total perfusate [SNO] was 42.5±5.2 μmol/L ≈15 minutes after addition of SNO-Hb (Figure 6A). Perfusate [SNO] fell by 1.3±0.6 μmol/L per min over 5 minutes of anoxic ventilation, and by 0.2±0.5 μmol/L per min during 5 minutes of normoxic ventilation; rates that were not significantly different (P=0.24, 2-tailed paired t test). However, addition of 5 mmol/L GSH to the perfusate during normoxic ventilation resulted in a greater than 50% reduction in perfusate [SNO] over 5 minutes, from 33.7±2.2 to 16.2±3.0 μmol/L, a rate of decline of 3.5±0.8 μmol/L per min (P=0.05, pre versus post GSH rates). During anoxic ventilation plus GSH, the rate of [SNO] decline fell to 0.8±0.1 μmol/L per min (P=0.03, normoxia versus hypoxia). (Figure 6A). Addition of GSH to perfusate resulted in a greater than 50% increase in the percentage of metHb (Figure 6B). The stability of perfusate SNO before and after treatment of samples with KCN and K3Fe(CN)6 suggested that no nitrosyl(heme)Hb was formed in the perfusate.
Effect of GSNO on PAP and HPV
Addition of 5 μmol/L GSNO to buffer perfusate containing L-NA (100 μmol/L) had no significant effect on normoxic PAP (Figure 7A). However, 20 μmol/L GSNO reduced PAP compared with L-NA alone and 5 μmol/L GSNO (P<0.05). Addition of 100 μmol/L oxyHb plus 20 μmol/L GSNO to the perfusate reversed the effect of GSNO on PAP (Figure 7A). Inhibition of NOS by L-NA augmented HPV (Figure 7B). GSNO also inhibited HPV, with no effect of GSNO at a concentration of 5 μmol/L, a reduction of HPV at a concentration of 20 μmol/L, and a reversal of the effect of 20 μmol/L GSNO by oxyHb (100 μmol/L) (Figure 7B).
Exhaled NO in Isolated, Perfused Rat Lungs
Exhaled NO during normoxic ventilation in lungs perfused with buffer before addition of Hb was 2.1±0.3 ppb (n=18). The response of eNO to various interventions is shown in Figure 8. Addition of SNP (10 and 100 μmol/L) to the perfusate increased eNO slightly. However, eNO was not affected by addition of Hb (any modification) to the perfusate, inhibition of NOS by L-NA, hypoxia (anoxic ventilation), or addition of GSNO (5 and 20 μmol/L) to the perfusate (Figure 8A). Furthermore, addition of SNO-Hb plus IHP followed by GSH to the perfusate had no effect on eNO (Figure 8B).
Hb, NO, and the Pulmonary Circulation
We and others have previously shown that free Hb and RBCs promote pulmonary vasoconstriction and augment HPV.2–5,7,8,15,16⇓⇓⇓⇓⇓⇓⇓ Recently, we verified that these effects were due to scavenging of NO by Hb, particularly when Hb is in the free form.6 These findings are not surprising given the high affinity and rapid reaction rate of NO with Hb, and the inhibition of the vascular relaxant effect of NO by free Hb.17–20⇓⇓⇓ They are also consistent with the observed effect of free Hb products on systemic blood pressure (hypertension).21,22⇓
In addition to the oxidation and addition reactions of Hb with NO, a third, reversible reaction involving S-nitrosation of the β-cys93 residue to form SNO-Hb may be of physiological importance, thus making the relationship between Hb, NO, and vascular tone more complex than previously appreciated. The detection of an arterial-venous gradient in SNO-Hb9 and evidence that administered SNO-Hb reduces systemic blood pressure9,11⇓ and increases cerebral blood flow11 has led to the hypothesis that Hb is an active regulator of vascular tone. In this theory, allosteric changes in Hb during oxygenation and deoxygenation promote uptake of NO by Hb in the lung and release of NO in the peripheral tissues, with the resulting systemic vasodilation acting to maximize oxygen delivery to hypoxic tissues.11
The effect of SNO-Hb on the pulmonary circulation has not been defined in vivo. In the isolated, perfused lung model, in the absence of a membrane oxygenator in the perfusion circuit, SNO-Hb added to the perfusate should release NO and promote vasodilation if the above, proposed theory holds true. The release of NO from SNO-Hb should be further augmented by hypoxic ventilation and deoxygenation of Hb, and by the presence of a low-molecular weight thiol in the perfusate to facilitate transnitrosation.9,10⇓ In an earlier study in isolated, perfused rabbit lungs, however, we found that SNO-Hb was a potent vasoconstrictor that augmented HPV to the same degree as oxyHb.6 Unfortunately, we were unable to reduce the perfusate Po2 to less than ≈38 Torr due to the relatively large size of the perfusion circuit. Because the affinity of free SNO-Hb for oxygen is high, with a P50 of less than 10 Torr,12,14⇓ deoxygenation of Hb may not have occurred, and an allosteric effect of deoxygenation on NO release from SNO-Hb could not be disproved. In addition, the very low concentration of SNO-Hb in the perfusate (4 μmol/L) may have resulted in dissociation of Hb tetramers (dimerization), thus eliminating the allosteric influence of deoxygenation.23,24⇓
In the present study, isolated perfused, rat lungs were subjected to anoxic ventilation. This resulted in a profound reduction in perfusate Po2 (11.1±1.0 Torr after 5 minutes of anoxic ventilation). These conditions predict 10% to 35% desaturation of SNO-Hb given the measured P50 of 5.5 to 8.3 Torr in the absence and presence of IHP (Figure 1), and it is unlikely that an arteriolar intraluminal Po2 less than 11 Torr is achievable under normal physiological conditions.25 According to the hypothesis that deoxygenation of SNO-Hb promotes NO release under the experimental conditions used in these studies, NO release from SNO-Hb should occur with concomitant conversion of free Hb from a vasoconstrictor to a vasodilator. However, SNO-Hb addition to the perfusate resulted in pulmonary vasoconstriction during normoxia and augmentation of HPV (Figure 3) to the same degree as oxyHb. Augmentation of HPV by SNO-Hb occurred in both the absence (Figure 3) and presence (Figure 5) of IHP and GSH, and with a perfusate SNO-Hb concentration high enough to preclude dimerization (100 μmol/L). Furthermore, hypoxia did not accelerate the rate of decline of perfusate S-nitrosothiol concentration, and in fact, appeared to slow this rate in the presence of GSH (Figure 6). These data suggest that allosteric influences as induced by Hb deoxygenation on the release of NO from free SNO-Hb are limited and are consistent with previous reports on SNOHb and GSH interactions.14,26⇓
Although dissociation of Hb tetramers into dimers is a potential problem when working with dilute Hb solutions, the perfusate Hb concentration of 100 μmol/L in the present study is sufficient to preclude this concern.23 In addition, IHP reduces dimerization by stabilizing T state hemoglobin. The sigmoidal shape of the oxyHb dissociation curve (Figure 1) suggests the presence of tetrameric Hb and preserved allosteric activity in both the absence and presence of IHP. Finally, gel electrophoresis of perfusate samples containing SNO-Hb provides direct evidence against significant dissociation of Hb tetramers in the present study (Figure 2).
We were unable to show either release of appreciable free NO (as measured in the exhaled gas) or GSNO formation from SNO-Hb. This is remarkable in light of the large fall in perfusate [SNO] on addition of GSH to perfusate containing SNO-Hb (Figure 6). Because our assay measures total S-nitrosothiol concentration (primarily SNO-Hb and GSNO), any changes in the rate of SNO loss may represent changes in the stability of GSNO in the perfusate. However, in our previous study, where increased sample volume allowed us to partition perfusate [SNO] into components, GSNO was not present as a stable intermediate unless the oxidizing capacity of Hb was completely removed (100% metHb formation).6 Thus, the rate of perfusate [SNO] loss in the present experiments is likely representative of the rate of SNO loss from Hb, with GSNO formed as a rapidly oxidized intermediate. This assumption is supported by the large increase in metHb after GSH addition (Figure 6). Although we cannot stoichiometrically account for all of the reduction in perfusate SNO, it appears that the majority is lost to heme oxidation of NO and is thereby unavailable to produce vasorelaxant effects.
There was a large discrepancy between the predicted and measured SNO in the initial perfusate samples. The loss of SNO between addition of SNO-Hb to the perfusate and the first perfusate sample measurement may be due to several mechanisms. The average time between addition of SNO-Hb to the perfusate and the first perfusate sample was ≈15 minutes. In the absence of GSH, perfusate SNO falls at a rate of ≈1 μmol/L per min, thus explaining the loss of ≈15 μmol/L SNO. However, it is possible that contact between SNO-Hb and thiols in the vascular endothelial layer (or contributed to the perfusate by the vascular endothelium) accelerated the loss of perfusate SNO (GSH increases the rate of loss of SNO to ≈3.5 μmol/L per min). Unfortunately, we did not take perfusate samples immediately after addition of SNO-Hb and cannot corroborate the above hypothesis.
There are several reasons for not using intact RBCs in this study. First, there is interest in developing blood substitutes, which led to the development of Hb cross-linked at the β-cysteine 93 residue (see following discussion). Second, we were specifically interested in the behavior of free SNO-Hb in our model, as others have proposed that free SNO-Hb is a vasodilator in the systemic circulation.9,11⇓ The use of free Hb in lieu of intact RBCs is also useful to isolate the specific effect of Hb on the circulation, as the RBC membrane acts to modulate this effect.7 Intact erythrocytes containing SNO-Hb may in fact behave quite differently than free SNO-Hb both in vitro and in vivo. Further investigation will be necessary to explore the effect of intraerythrocytic SNO-Hb on the pulmonary circulation.
A relatively dilute concentration (100 μmol/L) of Hb was chosen for study for several reasons. (1) This concentration is sufficient and necessary to produce a vascular response in isolated rat lungs, as is shown by previous work and the present data (Figures 3 and 4⇑). (2) Other investigators have shown that free Hb in approximate plasma concentrations of 3 to 20 micromolar results in systemic vasoconstriction, whereas free SNO-Hb in this range results in vasodilation in vivo.9,11⇓ (3) Physiological concentrations of free Hb result in intense pulmonary vasoconstriction and pulmonary edema (personal observations). It is also important to note that the perfusate concentration of S-nitrosothiol contributed by Hb in our model is far greater than that measured in rat blood (≈300 nanomolar) or human blood (200 nanomolar or less).9,27⇓
Hb Modification and HPV
Efforts to produce a free Hb-based oxygen carrier (HBOC) have been complicated by the reaction between Hb and NO, manifesting experimentally and clinically as vasoconstriction and systemic hypertension.1,21,22⇓⇓ These effects can be reduced by intra- and intermolecular cross-linking and conjugation of the Hb molecule to prevent abluminal movement.28,29⇓
The effects of modified HBOCs on the pulmonary circulation have not been rigorously studied, and there are no reports of the effects of these products on HPV to our knowledge. In the present study, we measured the effects of an intramolecularly cross-linked HBOC, Bis-Mal-PEGHb, on the pulmonary circulation during normoxia and hypoxia. Cross-linking of Hb outside the central cavity (at the β-cys93 residues) results in a product with mildly increased oxygen affinity and retained responsiveness to allosteric effectors in comparison to native HbA.30 An additional aspect of this Hb product is that nitrosation of the β-cys93 residue is precluded, thus providing for a unique comparison with SNO-Hb and normal oxyHb.
As depicted in Figures 1 and 2⇑, Bis-Mal-PEGHb increased normoxic PAP and augmented HPV to a similar degree as oxyHb and SNO-Hb. Although the difference in HPV response during perfusion with buffer or buffer containing Bis-Mal-PEGHb did not quite achieve statistical significance (P=0.06), the difference in HPV between the three Hb products was not significantly different by ANOVA (P=0.68 to 0.91). These data suggest that simple intramolecular cross-linking of the Hb molecule does not reduce its pulmonary vasoconstrictor effects. Further work is necessary to determine if this product maintains systemic vasoconstrictor properties in addition to its pulmonary effects.
Exhaled NO in the Rat Lung
We found that the concentration of exhaled NO was low in the rat lung in comparison to other species and was surprisingly resistant to manipulation of perfusate conditions (Figures 6 and 7⇑). Specifically, eNO did not change significantly with addition of free Hb, nonselective NOS inhibition, or hypoxic ventilation (Figure 6). This is in contrast to other species, including rabbits, pigs, dogs, and humans, whereby similar manipulations result in a fall in eNO.8,31–38⇓⇓⇓⇓⇓⇓⇓⇓ Our findings are similar to those of other investigators, who have documented eNO concentrations of ≈1 to 5 ppb from intact rats and isolated rat lungs, in addition to insensitivity to NOS inhibition and excess NOS substrate (l-arginine).39–44⇓⇓⇓⇓⇓ The lack of change in eNO is in contrast to the observed vascular effects seen with addition of free Hb to the perfusate (Figure 2), NOS inhibition, and addition of GSNO to the perfusate. The reason for the insensitivity of the exhaled gas to detect changes in NO production and/or concentration in rat lungs are unclear, although Stitt et al42 have shown that rat lung tissue has considerable capacity to absorb NO. This capacity may limit the ability of the exhaled gas to reflect changes in blood or perfusate concentrations and likewise endothelial production of NO in the rat.
Of note is that an increase in eNO was detectable after addition of the NO donor SNP to the perfusate, although similar changes were not observed with addition of GSNO (Figure 6). GSNO at a concentration of 20 μmol/L did attenuate HPV, an effect that was reversed by addition of oxyHb. This suggests that GSNO exerts vascular effect by release of free NO, and again reflects the dichotomy between vascular effects and exhaled concentrations of NO in the rat lung.
Using an isolated, perfused, rat lung model, we have shown that free SNO-Hb and intramolecularly cross-linked Hb have pulmonary vasoconstrictor effects similar to that of free oxyHb. NO release from SNO-Hb in this model is dependent on thiol addition and independent of oxygen tension and allosteric effectors. The NO released from free SNO-Hb appears to be rapidly inactivated by reaction with heme, thus preventing measurable vascular effects. This does not preclude release of SNO and vasodilation by intact RBCs containing SNO-Hb, however. Exhaled gas from the rat lung is insensitive to changes in NO production and concentration that are associated with vascular effects, thus limiting the utility of this measurement as a tool for studying pulmonary vascular physiology.
This work was supported by NIH grants HL03796-01 and HL45571.
Original received September 18, 2001; resubmission received February 22, 2002; revised resubmission received August 22, 2002; accepted August 26, 2002.
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