Effects of S-Nitrosation of Hemoglobin on Hypoxic Pulmonary Vasoconstriction and Nitric Oxide Flux STEVEN DEEM, MARK T. GLADWIN, JOHN T. BERG, MARK E. KERR, and ERIK R. SWENSON Departments of Anesthesiology and Medicine (Pulmonary and Critical Care), University of Washington and Puget Sound Veterans Affairs Health Care System, Seattle, Washington; and Department of Critical Care Medicine, National Institutes of Health, Bethesda, Maryland
Free hemoglobin (Hb) augments hypoxic pulmonary vasoconstriction (HPV), ostensibly by scavenging nitric oxide (NO). However, recent evidence suggests that Hb that is S-nitrosated may act as an NO donor and vasodilator. We studied the effects of oxyHb, Hb that is chemically modified to prevent heme binding or oxidation of NO (cyanometHb), and Hb that is S-nitrosated (SNO-Hb and SNOcyanometHb) on HPV, expired NO (eNO), and perfusate S-nitrosothiol (SNO) concentration in isolated, perfused rabbit lungs. Perfusate containing either 4 M oxyHb or SNO-Hb increased normoxic pulmonary artery pressure (Ppa), augmented HPV dramatically, and resulted in an 80% fall in eNO in comparison to perfusion with buffer, whereas 4 M cyanometHb or SNO-cynanometHb had no effect on these variables. Excess glutathione (GSH) added to perfusate containing SNO-Hb resulted in a 20 to 40% fall in the perfusate SNO concentration, with a concomitant increase in metHb content, without affecting Ppa, HPV, or eNO. In conclusion, free Hb augments HPV by scavenging NO, an effect that is not prevented by S-nitrosation. NO released from SNO-Hb in the presence of GSH does not produce measurable vascular effects in the lung or changes in eNO because of immediate oxidation and metHb formation.
Red blood cells (RBCs) and free hemoglobin (Hb) augment hypoxic pulmonary vasoconstriction (HPV) in isolated, perfused lungs, a phenomenon that appears to be related to the ability of hemoglobin (Hb) to scavenge nitric oxide (NO) from the pulmonary circulation (1–6). The evidence of mechanism has been largely circumstantial, however, and based on the following observations. (1) In vitro and in vivo work, which suggests that the primary reaction of oxyHb with NO is that of oxidation, with metHb and nitrate formation and a resulting reduction in available free NO (7, 8). (2) Addition of RBCs or free Hb to buffer perfusate of isolated lungs, or induction of insovolemic anemia in whole animals, results in a fall in expired NO (eNO) (4, 5, 9, 10). (3) Administration of inhaled NO inhibits HPV (11). (4) Inhibition of NO synthase (NOS) augments HPV (4, 5, 12). However, Hb has additional properties that may affect HPV, including oxidant activity and cytotoxic effects on endothelial cells that may also result in a reduction of NO production (13, 14), and a strong affinity for carbon monoxide, a compound that also has the capacity to inhibit HPV (15). In addition to the classic oxidation reaction of oxyHb with NO to form metHb and nitrate, NO reacts with deoxyHb to form nitrosyl(heme)Hb (16–18). Although conventional thought holds that these are the primary reactions of NO and Hb, and that the predominant role of Hb in NO biology is that of scavenger
(Received in original form July 31, 2000 and in revised form November 9, 2000) Supported by Grants HL-03796 and HL-45571 from the National Heart, Lung, and Blood Institute. Correspondence and requests for reprints should be addressed to Steven Deem, M.D., Dept. of Anesthesiology, Box 359724, Harborview Medical Center, Seattle, WA 98104. E-mail:
[email protected] Am J Respir Crit Care Med Vol 163. pp 1164–1170, 2001 Internet address: www.atsjournals.org
and inhibitor of NO effect, it has recently been proposed that a third reaction of NO with Hb is important. S-nitrosation of Hb at the highly conserved -cysteine 93 (-cys93) residue to form S-nitroso-Hb (SNO-Hb) may confer vasodilatory properties on Hb via the dynamic release of NO during deoxygenation (19). This theory postulates that in peripheral tissues where O2 tension is low, deoxygenation of Hb (R→T transition) results in allosteric structural changes that destabilize the S-NO bond. The NO released, either in the form of free NO or as transnitrosated thiols like SNO-glutathione (GSNO), promotes vasodilation with enhanced local blood flow and O2 delivery. This theory is therefore at odds with the classic paradigm of Hb solely as an NO scavenger. Although the effect of SNO-Hb on the pulmonary circulation has not been studied, SNO-Hb might attenuate HPV if uptake of NO by -cys93 was reduced because of failure of reoxygenation of Hb during alveolar transit. The aim of the current study was to test the effects of free human Hb in various modifications on NO flux and vascular tone in isolated, perfused rabbit lungs. We hypothesized that treatment of Hb to make cyanometHb to prevent heme-binding of NO or metHb formation would prevent augmentation of HPV and reduction of eNO by Hb. In addition, we hypothesized that: (1) S-nitrosation of Hb (SNO-Hb) would attenuate the vasoconstrictive properties of free Hb; (2) release of free NO from SNO-Hb (either spontaneously, under the allosteric effect of deoxygenation and promoted by organic phosphates [2,3 diphosphoglycerate or inosine hexaphosphate], or in the presence of excess thiol) would be reflected in the expired gas (eNO) and in a fall in the perfusate S-nitrosothiol concentration.
METHODS The protocol was approved by the Animal Care Committee of the Puget Sound Veterans Affairs Health Care System.
Reagents All reagents were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise noted.
Hb Preparation OxyHb. Fresh human blood was washed, hemolysed, and dialyzed. The total Hb concentration was measured by conversion to cyanomethemoglobin (24). Aliquots were frozen at ⫺80⬚ C. Prior to experimental use, the percentages of oxy-and metHb were determined by the method of Winterbourn (20). SNO-HB. S-nitroso-N-acetylpenicillamine (SNAP) was synthesized as previously described (21). OxyHb was dialyzed against 1% borate buffer (pH, 9.2) containing 0.1 mM DTPA for 4 h at 4⬚ C. SNAP was added to the oxyHb in a ratio of 2 mM SNAP/50 M Hb. The reaction was allowed to continue for 40 min in the dark at 4⬚ C and then dialyzed against 6 L of 3 mM PBS (pH, 7.4) containing 0.1 mM DTPA for 6 h at 4⬚ C with three buffer changes. Mass spectroscopy (22) verified S-nitrosation of the -cys93 residue. Chemiluminescence (24) revealed formation of approximately 1.4 moles of SNO- per Hb tetramer for SNO-Hb. MetHb percentage of both SNO-Hb and oxyHb was less than 1%.
Deem, Gladwin, Berg, et al.: S-nitrosation of Hb on HPV and Expired NO CyanometHb and SNO-cyanometHb. KCN and K3Fe(CN6) were added to purified oxyHb at 5-fold excess and incubated at 4⬚ C for 30 min. The mixture was dialyzed (10,000 MW cutoff) overnight against 6 L of 3 mM PBS (pH, 7.4 at 4⬚ C) with three buffer changes. Reaction with SNAP was performed as described above and the I3 ⫺ chemiluminescent assay (24) revealed formation of approximately 0.72 to 0.99 moles of SNO- per SNO-cyanometHb tetramer, depending on the sample tested.
Ozone-based Chemiluminescent Detection of Nitrosyl(heme)hemoglobin (HbFe IINO) and S-nitroshemoglobin in Perfusate Samples The method for the measurement of nitrite and S-nitrosothiols by reaction with I3⫺ to release NO gas was applied to lung perfusate samples (22–24). This assay demonstrates linear sensitivity for both SNO-Hb and HbFeIINO from 0.001 to 100% (22, 24). To determine whether the NO was released from SNO-hemoglobin and then autocaptured by the heme to form HbFeIINO, perfusate samples were reacted with and without 0.2 M KCN and K3Fe(CN)6, which selectively removes the NO from heme while preserving the S-nitrosothiol bond (24).
Chemiluminescent Measurement of Perfusate SNO-glutathione (GSNO) Released from SNO-Hb Because the I3⫺ reductant used to measure SNO-Hb also measures GSNO and nitrite (but not nitrate), the injection of perfusate samples into this reductant quantitatively measures both GSNO and SNO-Hb. In order to distinguish SNO-Hb from GSNO and/or nitrite released from SNO-Hb, the perfusate samples were reacted in the I3⫺ reductant before and after passage through a sephadex G25 sizing column, which will separate the hemoglobin from the low molecular weight S-nitrosothiol fraction (GSNO) and nitrite.
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quence without addition of IHP or GSH (time-control group), with data recording and perfusate sampling at the appropriate intervals. In another SNO-Hb subgroup, 2,3 diphosphoglycerate (2,3 DPG) (200 M) was added to free Hb before addition to the perfusate. After an initial hypoxic challenge, GSH was added to the perfusate in excess and another hypoxic challenge performed. To control for any direct effects of IHP and GSH on Ppa, HPV, and eNO, a group of lungs underwent repeated hypoxic challenges before and after addition of these effectors alone to the perfusate. In a final group of rabbits, the sensitivity of our system to detect the appearance of NO released in the perfusate in the expired gas was tested. Lungs were perfused with Hb-free perfusate and ventilated with normoxic, nomacarbic gas, and the NO donors sodium nitroprusside or GSNO were added to the perfusate in increasing concentrations of 0.1, 1, and 10 M. Expired NO was measured at 5 min and at its peak. NO was allowed to return to baseline (or exhibit stability over 5 min) prior to addition of the next higher dose or alternate NO donor. Hemodynamic data were used in the analysis only if Ppa returned to within 30% of baseline after a hypoxic challenge for all interventions.
Statistics Statistical analysis was performed using the computer software package StatView (Abacus Concepts, Berkely, CA). 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 one-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 when appropriate. A p ⬍ 0.05 was accepted as statistically significant.
RESULTS
Methemoglobin concentrations in fresh lung perfusate samples were measured by the method of Winterbourn (20).
A total of 61 experiments were performed, with nine experiments excluded from analysis because of technical difficulties or the development of gross pulmonary edema or refractory pulmonary hypertension during the study.
Experimental Preparation
Perfusate PO2 during Normoxic and Hypoxic Ventilation
The isolated, perfused, rabbit lung model used for these experiments has been described in detail elsewhere (5). In all experiments, initial perfusion was with buffered Krebs-Henseleit solution, and 20 to 30 min of perfusion and normoxic ventilation was followed by a 5-min hypoxic challenge (FIO2, 0.05; FICO2, 0.05; balance, N2). HPV was defined as the change in pulmonary artery pressure (Ppa) with hypoxia from the immediate normoxic baseline.
During normoxic ventilation, perfusate PO2 in all studies was 147.3 ⫾ 1.3 mm Hg. After 5 min of ventilation with hypoxic gas (FIO2, 0.05), perfusate PO2 fell to 64.5 ⫾ 1.8 mm Hg, and after 5 min of anoxic ventilation (FIO2, 0), to 37.9 ⫾ 1.7 mm Hg. An additional 5 min of anoxic ventilation did not result in further reduction in the perfusate PO2.
Experimental Protocol
Effects of Free Hb on Pulmonary Artery Pressure and HPV
After the initial hypoxic challenge with buffer perfusion, the lungs were entered into one of four primary groups based on the particular modification of free Hb added to the perfusate: (1) oxyHb, (2) SNOHb, (3) cyanometHb, (4) SNO-cyanometHb. 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 4 M. After a 5-min stabilization period, data were recorded and perfusate samples taken for later analysis. The lung was then subjected to a 5-min hypoxic challenge (FIO2 0.05), at which time data were again recorded and samples taken. After another 5-min normoxic stabilization period, recording was repeated and either inosine hexaphosphate (IHP) (4 M), which binds to tetrameric Hb in a molar ratio of 1:1, or reduced glutathione (GSH) in excess (100 M) was added to the perfusate. After 5 min, the hypoxic challenge was repeated as described. A third hypoxic challenge was performed following the above procedure after addition of the alternate effector (IHP or GSH) to the perfusate. Because perfusate PO2 did not fall below approximately 65 mm Hg after 5 min of hypoxic ventilation at FIO2 0.05, a final “anoxic” challenge was performed in a subset of experiments in all groups. Preparations were ventilated with anoxic, normocarbic ventilatory gas for 10 min, and then returned to normoxia for 5 min for a final set of measurements. In a subset of experiments to which SNO-Hb was added to the perfusate, repeated hypoxic challenges were performed in the above se-
Baseline normoxic pulmonary artery pressure during buffer perfusion did not differ between the four major groups (OxyHb, n ⫽ 11; SNO-Hb, n ⫽ 22; CyanometHb, n ⫽ 5; SNO-CyanometHb, n ⫽ 5) (Figure 1). However, addition of oxyHb or SNO-Hb to the perfusate resulted in a small but statistically significant increase in Ppa during normoxia (p ⬍ 0.05 versus buffer, both groups), whereas addition of cyanometHb or SNO-cyanometHb had no effect on normoxic Ppa (Figure 1). HPV during perfusion with buffer was weak in all groups, with the change in Ppa after 5 min of hypoxia less than or equal to approximately 1 mm Hg in all groups (Figure 2). Addition of oxyHb or SNO-Hb to the perfusate resulted in dramatic augmentation of HPV compared with buffer perfusion (p ⬍ 0.05, both groups), whereas cyanometHb or SNO-cyanometHb had no effect on HPV (Figure 2). HPV during oxyHb perfusion was not significantly different from that during SNO-Hb perfusion. Addition of SNO-Hb plus 2,3 DPG to the perfusate also resulted in augmentation of HPV (⌬Ppa 12.5 ⫾ 1.6 mm Hg, p ⬍ 0.01 versus buffer perfusion) (n ⫽ 4). Sequential addition of IHP followed by GSH to perfusates containing oxyHb or SNO-Hb was associated with a statistically nonsignificant increase in HPV (Table 1). Likewise, ven-
Perfusate Methemoglobin Concentration
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TABLE 1. HYPOXIC PULMONARY VASOCONSTRICTION (HPV)* AFTER SEQUENTIAL ADDITION OF FREE Hb, IHP, AND GSH TO BUFFER PERFUSATE Intervention Group OxyHb, n ⫽ 7 SNO-Hb, n ⫽ 8 CyanometHb, n ⫽ 4 SNO-cyanometHb, n ⫽ 4
Hb (4 M)
IHP (4 M)
GSH (100 M)
Anoxia†
3.3 ⫾ 1.0 2.3 ⫾ 0.4 0.6 ⫾ 0.4 1.0 ⫾ 0.5
5.6 ⫾ 3.7 9.0 ⫾ 3.3 1.2 ⫾ 0.4 1.2 ⫾ 0.9
6.3 ⫾ 1.7 18.3 ⫾ 5.7 1.4 ⫾ 0.4 1.6 ⫾ 1.1
7.8 ⫾ 1.5 16.7 ⫾ 3.4 2.2 ⫾ 0.9 1.0 ⫾ 0.5
Definition of abbreviations: GSH ⫽ glutathione; Hb ⫽ hemoglobin; IHP ⫽ inosine hexaphosphate. * The change in Ppa (mm Hg) from the previous normoxic baseline after 5 min of hypoxic ventilation (FIO2 0.05). † Ventilation at FIO2 0 for 5 min in the presence of Hb, IHP, and GSH.
Figure 1. Normoxic pulmonary artery pressure (Ppa) before and after addition of four modifications of free Hb (4 M) to the perfusate of lungs perfused with Krebs-Henseleit buffer. *p ⬍ 0.05 versus buffer perfusion. OxyHb (n ⫽ 8), SNO-Hb (n ⫽ 20), CyanometHb and SNOcyanometHb (n ⫽ 5).
tion of 10 M an increase in eNO of 12.0 ⫾ 1.3 ppb (p ⫽ 0.0008). Addition of GSNO to reach perfusate concentrations of 0.1 and 1 M resulted in no significant change in eNO after 5 min, however, whereas 10 M GSNO resulted in an increase in eNO of 6.4 ⫾ 1.0 ppb (p ⫽ 0.003).
tilation with anoxic gas in the presence of IHP and GSH for 5 min did not change HPV in comparison with hypoxic ventilation in either oxyHb or SNO-Hb groups (Table 1). In lungs perfused with either cyanometHb or SNO-cyanometHb, addition of IHP followed by GSH to the perfusate had no effect on HPV (Table 1). Addition of GSH alone to perfusates containing SNO-Hb (n ⫽ 5) or SNO-Hb plus 2,3 DPG (n ⫽ 4) likewise had no significant effect on normoxic Ppa or HPV (data not shown). Addition of IHP, GSH, or their combination to the perfusate had no effect on normoxic Ppa or HPV in buffer-perfused lungs (n ⫽ 5, data not shown), and addition of GSH and/or IHP to the perfusate had no effect on normoxic Ppa with any of the Hb preparations.
Effects of Hb Modification on Expired NO during Normoxic and Hypoxic Conditions
Sensitivity of Expired Gas to Detect Changes in Perfusate NO Concentration
Addition of SNP to the perfusate to reach a concentration of 0.1 M resulted in an increase in eNO after 5 min of 1.0 ⫾ 0.4 ppb (p ⫽ 0.09); a concentration of 1 M resulted in an increase in eNO of 5.4 ⫾ 0.7 ppb (p ⫽ 0.002), and a concentra-
Figure 2. The change in pulmonary artery pressure (⌬Ppa) from normoxic baseline during ventilation with hypoxic gas (hypoxic pulmonary vasoconstriction, HPV) before and after addition of four modifications of free Hb (4 M) to the perfusate of lungs perfused with KrebsHenseleit buffer. *p ⬍ 0.05 versus buffer perfusion. OxyHb (n ⫽ 8), SNO-Hb (n ⫽ 20), CyanometHb and SNO-cyanometHb (n ⫽ 5).
Expired NO during normoxic ventilation did not differ between the four major groups during buffer perfusion (Figure 3). After 5 min of hypoxic ventilation, eNO fell by 5 to 7 ppb, or 10 to 15% of baseline, in all groups during buffer perfusion. Addition of either oxyHb or SNO-Hb to the perfusate resulted in an approximately 80% decrease in eNO (p ⬍ 0.001 versus buffer perfusion) during normoxic ventilation (Figure 3). CyanometHb had no significant affect on eNO, but eNO increased by 1.8 ⫾ 0.7 ppb after addition of SNO-cyanometHb to the perfusate (p ⫽ 0.05). (Figure 3). Expired NO during normoxic ventilation was significantly different between oxyHb and SNO-Hb groups versus cyanometHb and SNO-cyanometHb groups (p ⬍ 0.001, all comparisons). During ventilation with hypoxic gas (FIO2, 0.05), eNO fell by 10 to 12% in the presence of all Hb modifications (Figure 4). Ventilation with anoxic gas (FIO2, 0) in the presence of both IHP and GSH in the perfusate resulted in a further reduction
Figure 3. Expired NO from isolated lungs ventilated with normoxic gas before and after addition of four modifications of free Hb (4 M) to buffer perfusate. *p ⬍ 0.001 versus buffer perfusion; †p ⬍ 0.001 versus CyanometHb and SNO-cyanometHb. OxyHb (n ⫽ 8), SNO-Hb (n ⫽ 20), CyanometHb and SNO-cyanometHb (n ⫽ 5).
Deem, Gladwin, Berg, et al.: S-nitrosation of Hb on HPV and Expired NO
Figure 4. Percentage change in expired NO from normoxia after 5 min of ventilation with hypoxic (FIO2, 0.05) or anoxic gas from lungs perfused with buffer containing one of four modifications of free Hb (4 M). Perfusate during anoxic ventilation also contained IHP (4 M) and GSH (100 M). OxyHb (n ⫽ 8), SNO-Hb (n ⫽ 11), CyanometHb and SNOcyanometHb (n ⫽ 5). *p ⬍ 0.001, anoxia versus hypoxia.
in eNO, an effect that was not dependent on Hb modification, including S-nitrosation (SNO-Hb) (Figure 4). Addition of GSH or IHP alone to the perfusate had no significant effect on eNO during either normoxic or hypoxic ventilation in any Hb group; the data for SNO-Hb are represented in Figure 5. Neither GSH nor IHP changed eNO when added to buffer perfusate (data not shown). Effects of Time, Hypoxia and/or GSH on Perfusate S-nitrosothiol and metHb Concentrations
The S-nitrosothiol concentration at baseline in perfusates containing SNO-Hb was 3.6 ⫾ 0.6 M in the time-control group (n ⫽ 5), and 4.4 ⫾ 0.2 M in the study group to which IHP and GSH were added (n ⫽ 9), a difference that was not statistically significant. For clarity, the S-nitrosothiol concentrations over time are presented as the percentage of baseline S-nitrosothiol concentration at individual time points (Figure 6). In the timecontrol group there was a gradual and linear decrease in perfusate [SNO] that was independent of hypoxia. A similar pattern was seen in the study group over the first four time points,
Figure 5. Expired NO preaddition and postaddition of IHP or GSH to buffer perfusate containing SNO-Hb (4 M). Preaddition and postaddition normoxic values and hypoxic values were recorded after 5 min of ventilation at FIO2, 0.21, or FIO2, 0.05, respectively.
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Figure 6. The perfusate concentration of S-nitrosothiol ([SNO]) as a percentage of the baseline value over time in the perfusate from isolated lungs perfused with buffer containing SNO-Hb (4 M) or SNOcyanometHb (4 m). Lungs were subjected to three 5-min hypoxic challenges. IHP (4 M) followed by GSH (100 M) were added to the perfusate of study lungs (SNO-Hb, n ⫽ 9, or SNO-cyanometHb, n ⫽ 4), whereas time-control lungs perfused with SNO-Hb (n ⫽ 5) were followed over time, without IHP or GSH addition. *p ⫽ 0.008, study versus control groups. p ⬍ 0.0001, study versus control and SNO-cyanometHb groups over time.
and the rate of decline in perfusate [SNO] was unaffected by addition of IHP (Figure 6). However, addition of GSH to the perfusate in the study group resulted in an approximately 30% fall in [SNO] (p ⫽ 0.008, study versus time-control groups after GSH, and p ⬍ 0.0001, study versus time-control groups by repeated-measures ANOVA). Hypoxia after the combination of GSH and IHP in the perfusate resulted in no further acceleration of perfusate [SNO] decline (Figure 6). In a subset of study experiments to which GSH was added to the perfusate prior to IHP (n ⫽ 4), a reduction in perfusate [SNO] of 33 ⫾ 4% after addition of GSH was seen (p ⫽ 0.007, pre-GSH versus post-GSH; data not shown). Treatment of perfusate samples with KCN/K3Fe(CN)6 to displace NO from nitrosyl-heme bonds revealed nearly identical perfusate [SNO] concentrations to those prior to treatment, suggesting that the decrease in perfusate [SNO] over time and with GSH was not explained by intramolecular transfer of NO from -cys93 to heme. Perfusate [SNO] in the presence of SNO-cyanometHb (n ⫽ 4) fell more slowly than in either of the SNO-Hb groups (Figure 6). As expected, hypoxic ventilation in the absence or presence of IHP had no effect on perfusate [SNO] in the presence of SNO-cyanometHb. Addition of GSH to the perfusate did not affect perfusate [SNO] in this group, suggesting that either the NO was not released or was released as GSNO that could not subsequently react with the ferric-CN bound heme. When the perfusate samples were reanalyzed after passage through a sephadex G25 column to remove GSNO, total perfusate [SNO] fell by 45 ⫾ 4% after addition of GSH to the perfusate (p ⬍ 0.05, pre-GSH versus post-GSH). These data suggest that GSH removes NO from the -cys93 residue to form GSNO, and it remains in this form in the absence of the oxidizing potential of oxyheme. The percentage metHb was similar at baseline (5 min after addition of Hb to the perfusate) in both study and time-control groups to which SNO-Hb was added to the perfusate, but lower in perfusates containing oxyHb (Figure 7). In addition, the rate of increase in percentage metHb was faster in both
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Figure 7. The percentage of metHb of total Hb from isolated lungs perfused with buffer containing SNO-Hb (4 M) or oxyHb (4 M). Lungs were subjected to three 5-min hypoxic challenges. IHP (4 M) followed by GSH (100 M) were added to the perfusate of study lungs (SNO-Hb, n ⫽ 9) and lungs perfused with oxyHb (n ⫽ 5). Time-control lungs perfused with SNO-Hb (n ⫽ 5) were followed over time, without IHP or GSH addition. *p ⬍ 0.05, oxyHb versus study group; † p ⬍ 0.05, control versus study group; p ⫽ 0.001, study versus control and oxyHb groups over time.
groups containing SNO-Hb compared with oxyHb (Figure 7). In the SNO-Hb groups, metHb levels increased at approximately the same rate until after addition of GSH to the perfusate in the study group, at which time metHb levels increased disproportionately in this group (p ⬍ 0.05, study versus control groups after GSH) (Figure 7). The difference between all groups over time was highly significant by repeated-measures ANOVA (p ⫽ 0.001).
DISCUSSION This study has revealed several new findings regarding the interactions between Hb, NO, and pulmonary vascular reactivity. First, this work provides direct evidence that Hb increases pulmonary vascular tone and augments HPV by reducing available NO through heme-NO interactions. Second, we have shown that S-nitrosation does not ameliorate the increase in pulmonary vascular tone associated with free Hb, and neither does it prevent the reduction in eNO seen when free Hb is added to cell-free perfusate, suggesting dominance of heme-NO scavenging over NO donor properties. Third, we have been unable to confirm an allosteric effect of hypoxia on NO release by extracellular SNO-Hb, although we have documented a reduction in perfusate S-nitrosothiols when GSH is added to SNO-Hb. This reduction in perfusate S-nitrosothiol is not measurable in either expired NO or in terms of vascular effects, however, and our data suggest that much of the NO released from SNOHb via GSH is converted immediately to metHb and nitrate. In addition, GSH releases NO from R-state Hb (SNO-Hb, SNOcyanometHb), suggesting that thiol chemistry rather than allosteric structural transitions, are active in this system. Discussion of the Model
The isolated, perfused lung model is well-established for investigation of the pulmonary circulation, and provides the opportunity to study and control multiple variables over a relatively short period of time. In particular, this model allows control of alveolar and perfusate PO2 and ready measurement
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of the pulmonary physiologic response to alveolar hypoxia, i.e., vasoconstriction. In addition, the isolated lung model allows measurement of free NO in the expired gas, and S-nitrosothiols, including SNO-Hb, in the perfusate during interventions that are predicted to either reduce NO or promote release of NO from Hb. If SNO-Hb releases significant amounts of free NO, either spontaneously or in response to hypoxia and/or the presence of thiols, free NO should be readily detectable in the expired gas because of the closed nature of the perfusion circuit. In this sense, the induction of alveolar hypoxia in our model mimics the physiologic situation in the tissues where hemoglobin is desaturated during capillary passage, and directly tests the perfusion-metabolism matching function of SNO-Hb. We have shown that addition of NO scavengers (oxyHb, Figure 3) to the perfusate in our model results in a reduction in expired NO, and addition of NO donors to the perfusate results in an increase in expired NO. This is particularly true for SNP, where a 1 M perfusate concentration resulted in an increase in eNO of approximately 5 ppb. On the other hand, changes in eNO were not evident until the perfusate concentration of GSNO was 10 M, which is likely due to the high stability of the SNO-glutathione bond. These data support the ability of the expired gas to reflect changes in perfusate/vascular free NO, but suggest that in the presence of significant GSNO formation changes in eNO would be unlikely. The direct response of the pulmonary circulation to hypoxia is vasoconstriction. This differs from the systemic vasculature, where hypoxia results in vasodilation. This effect necessitates the use of pharmacologic preconstriction to test the effects of vasodilators on aortic rings, including the putative vasodilator SNO-Hb, in the presence of hypoxia (25, 26). In fact, it has been argued that earlier work demonstrating vasodilatory properties of SNO-Hb may have been due to a direct hypoxic effect, rather than to NO delivery by Hb (25). The vasoconstrictive effect of hypoxia in the pulmonary circulation thus offers a unique opportunity to study the vasodilator properties of SNO-Hb. Hb Augments HPV by Scavenging NO
We and others have previously shown the profound ability of free Hb and intact RBCs to enhance HPV (1–6). That this effect is related to the ability of Hb to “scavenge” available NO, and thus provide an environment favorable to vasoconstriction, seems likely. In the current study, we undertook a unique approach to investigate the relationship between Hb, NO, and pulmonary vascular reactivity. Chemical modification of Hb to form cyanometHb prevents NO scavenging by either nitrosyl-heme addition or metHb formation. In our model, the lack of effect of cyanometHb on either eNO, normoxic Ppa, or HPV in contrast to the marked reduction in eNO, and increased Ppa and HPV in the presence of oxyHb provides strong evidence for a mechanistic relationship. SNO-Hb Is a Net Scavenger of NO and a Vasoconstrictor in the Pulmonary Circulation
In 1996, Jia and colleagues (19) made a surprising proposal regarding the interaction between Hb and NO. On the basis of the observation of interspecies conservation of the -cysteine 93 residue of Hb and other findings from their laboratory, and they suggested that Hb is more than a mere scavenger of NO, that in fact it acts as a carrier of NO from lung to peripheral tissue. Under their hypothesis, S-nitrosation of the -cysteine 93 residue occurs under allosteric control, being favored by binding of oxygen to Hb in the lung. In peripheral tissues, the transition of Hb from the R (oxy) to T (deoxy) state results in
Deem, Gladwin, Berg, et al.: S-nitrosation of Hb on HPV and Expired NO
unloading of NO from SNO-Hb, a process that is favored in the presence of thiols such as GSH. By this process, Hb facilitates the delivery of oxygen to tissue by unloading NO in vessels where PO2 is lowest, resulting in increased blood flow and improved oxygen delivery. Indeed, work from the same laboratory has shown that free SNO-Hb, in contrast to oxyHb, has the potential to reduce systemic blood pressure at predicted plasma concentrations both similar to (19) and higher than (26) those used in our study, and to increase cerebral blood flow (26). Allosteric regulation of NO binding and release at the -cys93 residue remains controversial based on the high O2 affinity of SNO-Hb, which suggests that release of NO could occur only under conditions of extreme hypoxia (21, 27). This is particularly true when SNO-Hb is in the free form, where the P50 of free SNO-Hb is less than 10 mm Hg (21, 27, 28). However, Jia and colleagues (19) and Stamler and colleagues (26) have reported evidence of vasodilation in rat systemic and cerebral vessels in response to venous injection of free SNO-Hb, suggesting that in vivo, either deoxygenation is unimportant to release of NO from SNO-Hb or that the P50 of free SNO-Hb is higher in vivo than in vitro. The very high affinity of heme for NO (Ka ⫽ 107M⫺1s⫺1) should also preclude reactions of NO with -cys93 and scavenge any NO released from this site. It has been proposed that small thiols such as glutathione ferry the NO to and from the cysteine residue, protecting it from heme scavenging. This idea is supported by the observation that the release of NO from SNO-Hb is enhanced in the presence of reduced glutathione (GSH) (19, 25). However, GSH appears to promote release of NO from SNO-Hb under both oxy and deoxy conditions, suggesting that thiol chemistry predominates over R↔T transition in regulating binding and release of NO at the -cys93 residue (21, 25). To date, there have been no published data examining the effect of SNO-Hb on the pulmonary circulation. Although the potential effect of SNO-Hb on HPV is not clear, it is possible that alveolar hypoxia might impair the uptake or promote release of NO at the -cys93 residue, thus providing a relative inhibitory effect on HPV and a reduction in the delivery of NO to systemic tissues. On the other hand, this effect may be counterbalanced by the increased capacity of venous blood to inhibit NO, ostensibly through increased nitrosyl-heme formation (16, 29). We have shown that free SNO-Hb has no apparent vasodilatory effects in the pulmonary circulation. In fact, SNOHb acts as a pulmonary vasoconstrictor at least as potent as oxyHb, increasing Ppa during both normoxic and hypoxic conditions (Figures 1 and 2). Promotion of NO release from SNOHb by addition of the organic phosphates 2,3 DPG or IHP, or by addition of excess thiol in the form of GSH, did not reduce the vasoconstrictive effects of SNO-Hb (Table 1). Indeed, these interventions were associated with a trend towards greater pulmonary vasoconstriction, an effect that may have been timedependent, but one which does not support a vasodilatory role for SNO-Hb. In keeping with the lack of even relative pulmonary vasodilation with SNO-Hb, we were unable to detect evidence of significant free NO release by SNO-Hb as reflected in the eNO (Figures 3–5). The lack of change in expired NO occurred despite a gradual reduction in the perfusate nitrosothiol concentration over time. The fall in perfusate nitrosothiol appeared to be independent of allosteric effects in the form of hypoxia, but it was enhanced in the presence of GSH. However, even in association with a 30% reduction in nitrosothiol concentration (an absolute quantity of approximately 250 nmoles) after ad-
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dition of GSH to SNO-Hb, the eNO did not change significantly (Figures 5 and 6). Of note, this fall in nitrosothiol could generate nearly 1,000 ppm NO in the expired gas over the 10min period that nitrosothiol fell in the perfusate if free NO were released. In addition, because the assay includes nitrosothiols in the form of GSNO, these data suggest that if transnitrosation is the mechanism by which GSH facilitates removal of NO from SNO-Hb, the intermediary GSNO is shortlived. The apparent contradiction between the lack of change in eNO and the fall in perfusate nitrosothiols may be explained by at least two mechanisms. First, it is possible that NO released from SNO-Hb is adsorbed in biologic membrane because of the high partition coefficient for NO in lipid. The second explanation for the lack of correlation between eNO, perfusate nitrosothiols, and HPV relates to the observed increase in metHb that virtually mirrors the fall in perfusate SNO (Figures 6 and 7). These data suggest that NO released from SNO-Hb, even when this release is facilitated by GSH, is rapidly oxidized to form metHb and nitrate. Indeed, metHb levels increased much more slowly in perfusates containing oxyHb than in those containing SNO-Hb (Figure 7), an observation that supports the notion that metHb is formed as NO is released from SNO groups on Hb. In addition, when autooxidation and heme nitrosylation are blocked (SNO-cyanometHb), GSH addition appears to result in GSNO and/or nitrite formation, with an attendant fall in perfusate SNO-Hb concentration. Our data are consistent with those of Wolzt and colleagues (25) who found that addition of GSH to SNO-Hb resulted in minimal GSNO formation in vitro. They hypothesized that GSH removed free NO from SNO-Hb with the simultaneous formation of a mixed disulfide bond (GS-HbO2), and HNO as an intermediate. These investigators also showed that the vasorelaxant properties of SNO-Hb in preconstricted rat aortic rings were inhibited by low concentrations of oxyHb. Our findings are consistent with this latter observation, in that we show apparent “auto-scavenging” of NO released from SNOHb by either intramolecular or intermolecular transfer of NO and resulting metHb formation. Of note, when GSH was added to SNO-cyanometHb, our data suggest that NO is removed from Hb with attendant formation of GSNO or HNO (which would react with water to form nitrite). This conclusion is supported by the observation that when perfusate samples were passed through a sizing column to remove GSNO and nitrite, a 45% fall in perfusate S-nitrosothiol concentration after addition of GSH to the perfusate occurred, in contrast to the minimal change observed in total perfusate S-nitrosothiol after addition of GSH (Figure 6). These data suggest that, in the presence of oxyhemoglobin, GSH releases NO (as GSNO or nitrite) from the -cys93 residue, whereupon it is oxidized to form nitrate and metHb. Lack of an Allosteric Effect on NO Release from SNO-Hb
Also in keeping with the findings of Wolzt and colleagues (25), we were unable to document an allosteric effect of oxygen binding on NO release from SNO-Hb. This is likely due to the increased oxygen affinity of SNO-Hb (21, 27), which prevents deoxygenation of (and unloading of NO from) SNO-Hb unless PO2 is very low (21). This effect is magnified when Hb is extra-erythrocytic: The P50 of free Hb is less than 10 mm Hg, and falls further as Hb is S-nitrosated (21, 28). The perfusate PO2 after 5 min of ventilation with FIO2 0.05, was approximately 65 mm Hg in our model, and fell to approximately 37 mm Hg after 5 min of ventilation with anoxic gas. This PO2 would likely be too high to result in significant deoxygenation
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of either free oxyHb or SNO-Hb. However, when the organic phosphates 2,3 DPG and IHP were added to the perfusate to increase the P50 and Hb, release of NO from SNO-Hb during hypoxic or anoxic ventilation was still not demonstrable. IHP has been reported to increase P50 of SNO-Hb by 15-fold (21, 28), an increase that should have allowed for deoxygenation of Hb in our model. However, because of the low Hb concentration in the perfusate, we were unable to measure P50 directly. Thus, it remains possible that insufficient deoxygenation occurred in our model to allow an allosteric effect of NO release from SNO-Hb. It has also been suggested that low Hb concentrations promote the dissociation of Hb tetramers to dimers, an effect that would prevent an allosteric transition during deoxygenation (28). The addition of inorganic phosphates to free Hb and intermittent cycles of hypoxia should have limited dimerization in our model, however. The lack of an observed allosteric effect of oxygenation on NO release from SNO-Hb in our model does not preclude such an effect when Hb is intra-erythrocytic. Given the considerably higher P50 of intracellular Hb, deoxygenation of Hb is possible at more moderate levels of hypoxia. Further studies using intact RBCs containing SNO-Hb will be necessary to address this issue. The fall in perfusate S-nitrosothiol over time, and as enhanced by the presence of GSH during normoxia in the SNO-Hb experiments (Figure 6), suggests that an allosteric transformation of Hb is not necessary for removal of NO from -cys93. This is supported by similar findings in the SNO-cyanometHb experiments, where an allosteric change in Hb is prohibited. We were unable to document whether an allosteric transformation of Hb enhances GSH-facilitated removal of NO from SNO-Hb. It is also unclear whether a similar situation would exist inside the intact RBC, where GSNO may reside in relative equilibrium with SNO-Hb, and where allosteric changes in Hb may be necessary to shift the balance in favor of GSNO formation. In the pulmonary circulation, free SNO-Hb is a vasoconstrictor and net scavenger of free nitric oxide, and augments HPV as potently as Hb that is not S-nitrosated. This appears to be due to the strong affinity of NO for heme, with rapid oxidation of NO and metHb formation as NO is released from the -cys93 residue of Hb. Whether encapsulation of Hb within the RBC membrane confers NO donor and pulmonary vasodilator properties on SNO-Hb is open to question, and will require further study. Acknowledgment : The writers would like to thank Meg Pease-Fye and Lewis Pannell for assistance in performing mass spectroscopy and chemiluminescence measurements, and Rakesh Patel, Ph.D. for advice on the synthesis of SNO-Hb.
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