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Am J Physiol Heart Circ Physiol 286: H985-H991, 2004; doi:10.1152/ajpheart.00115.2003
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Role of myoglobin as a scavenger of cellular NO in myocardium

Ulrike Kreutzer and Thomas Jue

Department of Biological Chemistry, University of California Davis, Davis, California 95616

Submitted 4 February 2003 ; accepted in final form 20 October 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent studies have detected a 1H nuclear magnetic resonance (NMR) reporter signal of metmyoglobin (metMb) during bradykinin stimulation of an isolated mouse heart. The observation has led to the hypothesis that Mb reacts with cellular nitric oxide (NO). However, the hypothesis depends on an unequivocal detection of metMb signals in vivo. In solution, nitrite oxidization of Mb produces a characteristic set of paramagnetically shifted 1H NMR signals. In the upfield spectral region, MbO2 and MbCO exhibit the {gamma}CH3 Val E11 signals at –2.8 and –2.4 ppm, respectively. In the same spectral region, nitrite oxidation of Mb produces a set of signals at –3.7 and –4.7 ppm at 35°C. Previous studies have confirmed the visibility of metMb signals in perfused rat myocardium. With bradykinin infusion, perfusion pressure and rate-pressure product decrease, consistent with endogenous NO formation. However, neither myocardial O2 consumption nor high-energy phosphate levels, as reflected in the 31P NMR signals, show any significant change. Bradykinin still triggers a similar physiological response even in the presence of CO that is sufficient to inhibit 86% Mb. In all cases, the 1H NMR spectra from perfused rat myocardium reveal no metMb signals. The results suggest that bradykinin-induced NO does not interact significantly with cellular Mb to produce an NMR-detectable quantity of metMb in the perfused rat myocardium. As a consequence, the experiments cannot confirm the intriguing proposal that Mb acts as a cellular NO scavenger.

nuclear magnetic resonance; heart; oxidative phosphorylation; bioenergetics; respiration

THE PRESENCE OF MYOGLOBIN (Mb) in myocytes has always raised questions about its functional role as just a O2 storage protein or a facilitator of O2 diffusion (1, 35, 37). The high concentration of Mb in marine animals certainly buffers tissue hypoxemia during a dive or apnea, but in a beating rat heart, the Mb O2 store can prolong normal heart function for only a few seconds (4, 13, 26). Investigators have also posited that Mb facilitates O2 transport in the cell because the low PO2 environment regulating cellular function would confer a potential advantage of oxymyoglobin (MbO2) versus free O2 diffusion (35). Yet Mb inactivation with CO or nitrite has yielded mixed results. Some experiments have detected an altered metabolic (met) response on Mb inactivation; others, however, have found no significant change in respiration or high-energy phosphate state (6, 12, 32). Physical measurements under in vivo conditions have yet to confirm definitively that Mb diffusion can compete effectively with free O2 diffusion (15, 33). These observations have stimulated researchers to consider other functional roles of Mb.

Recent studies have reported the detection of a metmyoglobin (metMb) reporter signal in perfused isolated mouse hearts during bradykinin stimulation (8). Because bradykinin stimulates endogenous nitric oxide (NO) production, which can subsequently oxidize Mb, the observation suggests that Mb reacts with cellular NO and acts as an NO scavenger (8). Such a novel role for Mb has attracted exuberant endorsements and indirectly supports the role of NO in regulating respiration (3, 10, 24). However, at first glance, both the low cellular level of NO and an active Mb reductase appear to militate against a significant presence of cellular metMb and therefore any nuclear magnetic resonance (NMR)-detectable signals. Clearly, corroborative NMR experiments must confirm the detection of metMb in vivo to underpin the novel NO scavenging role for Mb.

We have investigated the interaction of NO synthesis and Mb oxidation in the well-defined perfused rat heart model. On nitrite oxidation, the characteristic set of metMb signals appears in the 1H NMR spectra, consistent with previous in vitro and in vivo observations (5, 22). The upfield reporter metMb signals appear at –3.7 and –4.7 ppm at 35°C (5, 22). These peaks are distinct from the MbO2 and MbCO signal of the {gamma}CH3 Val E11 at –2.8 and –2.4 ppm, respectively, and report on the oxidation of the heme Fe (5, 12, 21). With bradykinin infusion up to 1 µM, perfusion pressure and rate pressure product (RPP) decrease, consistent with the formation of cellular NO. However, myocardial O2 consumption (MO2) and high-energy phosphate levels, as reflected in the 31P NMR spectra, show no statistically significant change. Even in the presence of 86% CO, inhibition of Mb bradykinin still elicits a similar physiological response without any corresponding decrease in respiration or high-energy phosphate levels. In all cases, the 1H NMR spectra reveal no detectable metMb signals. With nitrite infusion, Mb becomes oxidized and reveals the characteristic metMb signals, in perfect agreement with previous studies (5). The experimental results then suggest that either bradykinin stimulates a negligible amount of NO production in the myocardium or that the steady-state metMb pool does not rise to an NMR-detectable level in the perfused rat myocardium.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal preparation and heart perfusion. The animal care procedures followed the guidelines of the University of California at Davis's institutional animal care review committee. The procedure for rat heart perfusion at 35°C was performed as previously described (4). Male Sprague-Dawley rats (350–400 g) were anesthetized by an intraperitoneal injection of pentobarbital sodium (65 mg/kg) and heparinized (1,000 U/kg). The heart was quickly isolated and placed in ice-cold buffer solution until aortic cannulation. The heart was then perfused in Langendorff mode with a Krebs-Henseleit buffer containing (in mM) 118 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.8 CaCl2, 23 NaHCO3, 1.2 MgSO4, and 15 glucose. The perfusate was maintained at 35°C with a circulating water bath (Lauda MT-3) and temperature-jacketed reservoirs and tubings. A peristaltic pump (Rainin Rabbit) maintained a constant, nonrecirculating perfusion flow. Perfusion pressure was measured with a pressure transducer (Medex) via a Y-connection in the aortic cannula. After the heart had stabilized the perfusate flow rate was adjusted to result in a mean perfusion pressure of 87 mmHg, which corresponds to a constant flow rate of ~19 ml/min. A saline-filled latex balloon inserted in the left ventricle monitored the heart rate (HR) and left ventricular (LV) pressure via a second pressure transducer. The balloon volume was adjusted to give an end-diastolic pressure of 6–8 mmHg. RPPs were calculated from HR times the LV developed pressure (LVDP). The perfusate was first gassed with 95% O2-5% CO2 and then passed through a home-built oxygenator equilibrated with the desired gas mixture. The oxygenator contained 50 ft of gas-permeable Silastic tubing (0.058 in ID, 0.077 in OD; Dow Corning) wrapped around a heat exchanger. The perfusate was passed through a 5- and 0.45-µm Millipore filter. Pressure transducers as well as O2 electrode were connected to a Biopac recording system.

Perfusate O2 measurement. Perfusate flowing from the pulmonary artery formed a bath in the NMR tube, in which the heart was placed and was subsquently isolated with a Teflon plug with holes to permit perfusate overflow. Approximately 50% of the perfusate was withdrawn via a Tygon catheter inserted at the level of the pulmonary artery at a constant rate of 10 ml/min. The remaining 50% was withdrawn above the Teflon plug as overflow.

A meter (model 5300, Yellow Springs Instrument) monitored the outflow perfusate O2 concentration with the oxygen electrode (model 5331, Yellow Springs Instrument) in a temperature-jacketed chamber. The value was subsequently corrected for O2 loss in the ~3 m tubing [Tygon S50HL 2.4 mm ID/4 mm OD (inflow); 1.6/4.8 mm (outflow)].

The correction was based on parallel experiments with exactly the same perfusion equipment arrangement, experimental conditions, and tubing length, which were employed in the NMR experiments. No heart was connected to the inflow catheter. As the perfusate oxygen tension varied with different mixtures of 95% O2-5% CO2 and 95% N2-5% CO2, two sets of measurements mapped the O2 at the oxygen electrode and at the catheter tip of the inflow and outflow lines, respectively (n = 6). The sampled data at the oxygen chambers were plotted against the data at the catheter tips, yielding a linear calibration curve for both the inflow (y = 0.92 x + 14, r > 0.99) and outflow (y = 1.15x – 24, r > 0.99), where y = PO2 at the catheter tip and x = PO2 at the chamber. The measured values were corrected according to these calibrations and are designated in the study as the arterial and venous PO2.

MO2 was calculated then from the arterial and venous oxygen content and the flow rate. For control hearts, the MO2 was 28.2 + 3.0 µmol/min g dry wt (n = 6).

Nitrite oxidation of Mb. A 5/1 stoichiometry of sodium nitrite (Aldrich) to Mb will immediately oxidize all MbO2 to metMb in vitro. The reaction is characterized by the disappearance of the MbO2 {alpha}- and {beta}-bands at 581 and 543 nm, respectively, and the appearance of the broad metMb bands at 505 and 635 nm (Agilent model 8452A spectrophotometer). Parallel 1H NMR and perfused heart experiments confirm Mb oxidization under these conditions because the characteristic set of metMb signals appear as MbO2 signals disappear, thereby confirming the conversion of all MbO2 to metMb (5).

Infusion protocol. After a 20- to 30-min control period, bradykinin was infused through a Y connector in the inflow perfusion to reach 10–7 or 10–6 M concentration in the arterial perfusate. 1H and 31P spectra were acquired during the control, 20-min bradykinin infusion, and the reperfusion period. At the end of the experiment, 40 mM NaNO2 was infused to confirm the detection of the metMb signals (5).

For CO equilibration experiments, a gas flow controller (model 400, Teledyne Hastings) introduced CO into the oxygenator to achieve a final gas mixture of 85% O2-5% CO2-10% N2 or 10% CO. After the control period, in which the heart was perfused with O2-saturated buffer, the gas mixture was switched to N2 for 20 min then to CO. After 20 min of CO perfusion, 10–7 M bradykinin was infused for 20 min.

NMR. A DRX 400-MHz Bruker spectrometer was used to record 1H/31P signals with a 20-mm 1H-{X} probe. A modified 1331 binomial pulse sequence suppressed the H2O line and centered the excitation at –3.2 ppm, between the MbO2 Val E11 resonance at –2.8 ppm and the metMb reporter signal at –3.7 ppm (18, 21). The 1H 90° pulse was 70 µs, calibrated against the perfusate H2O signal. Observing the MbO2 signal required a 40-ms acquisition time. The spectral width was set at 8,065 Hz; the data size was 512 points. Three thousand transients were averaged for a typical 1H spectrum, requiring ~3 min of signal accumulation. The free induction decays were zero filled to 2K and multiplied by an exponential-Gaussian window function. A spline fit then smoothed the baseline. All spectral lines were referenced as 4.65 ppm at 35°C, which was in turn calibrated against sodium-3-(trimethylsilyl)propionate-2,2,3,3-d4. For the 31P spectra, a typical control spectrum used a 45° pulse angle, a 0.5-s repetition time, and 256 scans/block (~2.5 min). The 31P 90° pulse was 72 µs, calibrated against a 0.1 M phosphate solution. Spectral width was set at 6,494 Hz; the data size was 4K. Free induction decays were zero-filled to 8K and apodized with an exponential function. The 31P signals were referenced to phosphocreatine as 0 ppm and integrated to obtain relative concentrations. 1H NMR spectra of horse heart (Sigma) metMb solutions in D2O were recorded with a 5 mm 1H[13C] probe with a 1H 90° pulse of 6.5 µs using identical 1H acquisition parameters.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The distinct 1H NMR signals of solution state metMb are shown in Fig. 1. Figure 1A shows the upfield spectra of horse heart metMb obtained with a broadband excitation pulse. Four distinct upfield signals appear at –3.7, –4.7, –6.1, and at –7.0 ppm at 35°C. These unassigned signals exhibit a characteristic temperature dependence associated with a paramagnetic interaction and must arise from either the heme groups or amino acid residues interacting with the heme Fe (22). Specifically, the two metMb reporter signals at –3.7 and –4.7 ppm appear in the region where the {gamma}CH3 Val E11 signals of MbCO and MbO2 at –2.4 and –2.8 ppm would resonate. With a semiselective excitation centered at –3.2 ppm, the spectra reveal clearly the metMb signal at –3.7 and –4.7 ppm (Fig. 1B). These two signals serve then to identify the presence of metMb in tissue.



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  		Fig. 1. Nonselective and selective excitation of 1H nuclear magnetic resonance (NMR) signals of horse heart metmyoglobin (metMb) in D2O. A: nitrite oxidation of MbO2 to metMb results in the appearance of four metMb signals at –3.7, –4.7, –6.1, and –7.0 ppm at 35°C. The two temperature-dependent signals at –3.7 and –4.7 ppm resonate in the same spectral region where the {gamma}CH3 Val E11 signal of MbO2 at –2.8 ppm would appear. B: a semiselective binomial-selective excitation frequency centered at –3.2 ppm suppresses the water signal and is required for in vivo investigation of perfused myocardium. The excitation selectivity detects clearly the in phase metMb signal at –3.7 ppm and has sufficient bandwidth to detect the second metMb peak at –4.7 ppm as well. However, it has insufficient excitation bandwidth to detect the signals at –6.1 and –7.0 ppm.

 

With the perfused heart under normoxic, bradykinin infusion, and reperfusion conditions, the NMR spectra and physiological monitors map the alterations in the Mb oxidation states as well as cellular function. Under control condition, the 1H NMR spectra show the distinct signal of the Mb Val E11 signal at –2.8 ppm. The RPP and MO2 are consistent with previous reports (see Table 1). After a 20-min control period, the heart received 10–7 M bradykinin in the arterial perfusate. The physiological response is shown in Fig. 2. Although the control HR 255 ± 38 shows no significant difference after bradykinin infusion, 262 ± 32 beats/min, the LVDP drops from 118 ± 7 to 100 ± 6 mmHg, which results in a RPP change from 30 ± 4 x 103 to 26 ± 3 x 103 mmHg/min. The perfusion pressure also decreases from 87 ± 6 to 71 ± 4 mmHg. The decrease in perfusion pressure and RPP mirror the literature reported action of bradykinin, mediated purportedly by NO formation (2, 7, 27). No perturbation appears in the 31P spectra. No significant alteration in MO2 occurs. The data are listed in Table 1. Titrating the infused bradykinin from 10–9 to 10–6 M shows perfusion pressure and RPP response reaches a steady state between 10–8 and 10–7 M. Further increase to 10–6 M bradykinin does not produce any further alteration in the myocardial response. At all concentrations of bradykinin, no metMb signals appear in the 1H spectra from the perfused myocardium.


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  		Table 1. Myocardial function with bradykinin stimulation

 


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  		Fig. 2. The histograms of physiological changes in perfusion pressure (PP; A), rate-pressure product (RPP; B), and oxygen consumption (C) with (open bar) and without (solid bar) infusion of bradykinin: PP and RPP decrease significantly by 18% and 12%, respectively. Oxygen consumption shows no significant change. Data are means ± SD, n = 6. *P < 0.05, significant difference vs. control.

 

Figure 3 reveals the bradykinin interaction in the presence of CO. A buffer saturated with 10% N2-85% O2-5% CO2 serves as the control condition because the Mb inactivation experiment uses 10% CO saturated buffer to inhibit 86% of Mb. The histograms of perfusion pressure, RPP, and MO2 show a small but significant alteration with the infusion of CO to saturate MbCO at 86%. The physiological response is consistent with previous experimental observations (12, 34). With 86% Mb inactivated by CO, bradykinin still decreases significantly perfusion pressure and RPP. The data are listed in Table 2.



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  		Fig. 3. The histograms of physiological changes in PP (A), RPP (B), and oxygen consumption (C) on infusion with CO and CO with bradykinin. Striped bars = 10% N2 control, open bars = 10% CO, solid bars = 10% CO + 10–7 M bradykinin. PP and RPP show a slight but significant decrease with CO. With bradykinin, the perfusion pressure and RPP decrease even further. Oxygen consumption decreases slightly with CO but does not change with the addition of bradykinin. Data are means ± SD, n = 6. *P < 0.05, Significant difference vs. N2 control; {dagger}P < 0.05, significant difference vs. CO.

 

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  		Table 2. Myocardial function with CO-inactivated Mb during bradykinin stimulation

 

Figure 4 shows the corresponding 1H NMR spectra of Mb in the presence of bradykinin and CO. Under control conditions, 95% O2-5% CO2 the signal from the Val E11 of Mb is clearly detectable (see Fig. 4A). On 10–7 M bradykinin infusion, the spectra display no spectral change in the peak from MbO2. No metMb signals appear (see Fig. 4B). Even with 10–6 M infused bradykinin, no metMb signal appears. Figure 4C shows the spectra after introduction of a gas mixture of 85%O2-10% CO-5% CO2 and bradykinin. The MbCO peak at –2.4 ppm emerges. Bradykinin does not affect either the MbCO or the residual MbO2 Val E11 signal and still does not produce any metMb signal. After reperfusion with oxygenated buffer to remove the CO and to restore fully the MbO2 peak, a perfusion with 40 mM nitrite buffer induces Mb oxidation to produce the characteristic metMb signals at –3.7 and –4.7 ppm (Fig. 4D).



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  		Fig. 4. 1H NMR spectra of isolated rat heart perfused with 95% O2-5% CO2 (A), 95% O2-5% CO2 and 10–7 M bradykinin (B), 85% O2-5% CO2-10% CO and 10–7 M bradykinin (C), and 95% O2-5% CO2 and 40 mM nitrite (D). Under normoxic conditions, the Mb {gamma}CH3 Val E11 signal appears at –2.8 ppm, reflecting the control myocardial oxygenated state. With bradykinin, the physiological response is consistent with nitric oxide (NO) production. However, no changes appear in the 1H spectra. With 10% CO, the CO displaces the MbO2 and saturates 86% of the Mb. Bradykinin infusion still produces an NO-induced change in the physiological response. However, the {gamma}CH3 Val E11 MbCO signal at –2.4 ppm remains constant. On reperfusion with 95% O2-5% CO2 and 40 mM nitrite, nitrite oxidizes Mb to metMb, revealing the two characteristic peaks at –3.7 and 4.7 ppm.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Bradykinin-induced oxidation of Mb. Recent studies (8) have observed the presence of a metMb signal on bradykinin stimulation of perfused mouse heart and have ascribed a novel NO-mediated Fe(II) to Fe(III) oxidation of cellular Mb. The observation has led to a newly proposed role for Mb as an NO scavenger, which has generated much exuberance (3). In the solution state, the oxidation of MbO2 to metMb produces a characteristic spectral change in the upfield region. In particular, two reporter signals at –3.7 and –4.7 ppm 35°C identify the presence of metMb (Fig. 1). Solutions studies (22) have not assigned these peaks, which do not originate from any heme group. Integration analysis shows that the peaks correspond to the three-proton intensity. However, the line width of the metMb signal at –3.7 ppm is 60% broader than the Val E11 signal of MbO2 at –2.8 ppm (5).

Even with selective excitation, both metMb reporter signals at –3.7 and –4.7 ppm must appear in the spectra, consistent with previous experimental findings (5, 22). Figure 1 illustrates the upfield spectral region of MbO2 and metMb, obtained from broadband and selective excitation. In response to bradykinin stimulation, the cell produces NO, which triggers vasodilation, as reflected in the perfusion pressure and LVDP. Indeed, bradykinin elicits a significant drop in perfusion pressure (from 87 to 71 mmHg) and in RPP (30 to 26 mmHg/min) and LVDP (from 118 to 100 mmHg). HR remains unchanged (255 and 262 min–1). However, no significant concomitant decrease in MO2 occurs. Infusing bradykinin at the physiological 10–7 M dosage appears to saturate the cell receptors. Increasing stepwise the bradykinin concentration from 10–7 to 10–6 M produces no dose-dependent alteration in the physiological response. The observation is consistent with NO production, as described in previous reports (8, 16, 25).

Despite the presence of NO, the observed 1H NMR spectra from perfused rat myocardium do not reveal any metMb signals, even when the heart receives the pharmacological dose of 10–6 M bradykinin. An infusion of nitrite to oxidize cellular Mb confirms that the NMR has the sensitivity to detect the metMb signals, consistent with previous observations (22). Clearly, in perfused rat myocardium the experimental data do not show any significant interaction of Mb with NO to produce a detectable amount of metMb. Given the 15/1 signal to noise (S/N) of the Val E11 Mb signal at –2.8 ppm, corresponding to ~200 µM Mb, the NMR technique has the threshold sensitivity to detect a 10% saturated MbO2 (2/1 S/N) and can quantitatively evaluate changes in 20% MbO2 (4/1 S/N) However, the paramagnetic relaxation of metMb broadens the –3.7 ppm peak line by 60%, relative to Val E11 signal. Consequently, a completely oxidized cellular metMb of 200 µM will yield only a 9/1 S/N. A threshold detectable limit of 2/1 S/N will require the presence of at least 20% metMb or 40 µM, whereas a quantitative analysis will require 40% metMb or 80 µM.

Detectability of NO interaction with Mb. In light of the experimental results of this study, the feasibility of detecting an NO-induced metMb signal in myocardium requires a reexamination. The cellular NO concentration depends on the net NO formation and degradation kinetics, such as the NO synthase (NOS) and Mb reductase reactions. Although much uncertainty still surrounds the parameters required to conduct a rigorous kinetics analysis, in vitro data still yield an estimate of the steady-state concentration of NO to produce an NMR detectable metMb signal and the time required to reach a particular steady-state concentration.

The analysis depends on an ideal limiting case: no Mb reductase activity and NO degradation pathways exist. The steady state approximates the equilibrium state, which seems reasonable in light of the rapid kinetics of Mb binding to O2 and NO and the subsequent oxidation of Mb to metMb. The cellular Mb is 200 µM and is completely NMR visible (14, 17). Finally, the basal O2 concentration is >10 µM (19, 20). The O2 concentration assumption arises from perfused or in situ rat myocardium experiments, where NMR has the sensitivity to measure 10% deoxy Mb and yet cannot detect the proximal hisitdyl NH signal of deoxy Mb signals under normoxic conditions. That suggests the myocyte basal cellular PO2 saturates >90% of the MbO2 or a cellular PO2 threshold concentration of ~10 µM, given the Mb p50 of 2.4 Torr (3.2 µM) at 37°C (4, 18, 19, 28).

In vitro kinetics studies show that NO binds to Mb with ~230,000 times higher affinity than O2 at a dissociation constant (KDNO/KDO2 = 4 x 10–6 µM/9 x 10–1 µM), Table 3 (11, 28). Given the resting O2 concentration as 10 µM, the NO concentration associated with a 1/1 ratio of MbNO/MbO2 is then 10 µM/2.3 x 105 = 4.3 x 10–5 µM. If all of the MbNO reacts to produce the end product metMb, then the required NO is also 4.3 x 10–5 µM. To produce a threshold detectable signal, reflecting 20% metMb or metMb/MbO2 at 1/4 ratio, requires an equilibrated, free steady-state NO concentration of 8 x 10–6 µM. During bradykinin stimulation, local NO levels can rise to 8 x 10–1 µM (23).


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  		Table 3. Properties of Mb and NO in myocyte

 

However, the cellular MbO2 concentration is 200 µM, and a 1/1 mixture would mean that 100 µM of Mb must exist as either MbNO or MbO2. Because NOS is the only source of NO formation in the cell, it must synthesize at least 100 µM of NO to bind Mb and then maintain a free NO concentration at 4.3 x 10–5 µM to reach metMb/MbO2 at a 1:1 ratio. Malinski and colleagues (23) used a microsensor technique to determine the bradykinin-induced NO formation rate. They reported a NO formation rate of 0.04 µM/s in rabbit aorta at 100 µm distance from an endothelial cell (23). Assuming this NO formation rate with no competing degradation reaction, the cell NO kinetics will require 2,500 s or 42 min for NO to reach a steady-state level, sufficient to produce 100 µM of MbNO. Even at the NO concentration required to achieve the threshold detection 1:4 ratio of metMb/MbO2, the reaction requires 1,000 s or 17 min.

In the cell, the ideal limiting conditions of the analysis certainly do not hold. NO oxidation of Mb must take much longer because many reactions compete against the formation of steady-state NO and metMb: NO has a very short half-life and reacts via other metabolic pathways. Mb reductase reduces metMb at a rate of 0.5 µM/s, 10 times faster than the NO production rate (see Table 3) (26). Finally, NO reacts with Mb in a complex set of reactions: it can react with Fe(II) as well as Fe(III) Mb and involves different reaction intermediates, which argue against a simple interpretation of NO reaction with Mb in vivo (9).

Although the above analysis has several uncertainties and will require further experimental data on NO production, Mb reductase activity, and local NO level to substantiate, the competing enzyme reactions in the cell and the NO formation time to oxidize Mb to metMb would tend to militate against an NMR-detectable concentration of metMb in the cell, consistent with the results reported in this present study.

CO and NO interaction. If NO reacts significantly with cellular Mb to produce metMb, then introducing CO into the perfusate to inhibit Mb function might mask the Mb function as an NO scavenger (3, 12). The extent of the interference depends upon the cellular NO concentration. In perfused rat heart studies, CO can inactivate Mb without altering respiration, when the MbCO/MbO2 ratio is <4/1 (12). Given the cellular O2 concentration of ~10 µM and the MbCO/MbO2 partition coefficient of 36, the cell must contain 1.1 µM CO to achieve a MbCO/MbO2 ratio of 4/1. If the cell can attain a steady state, 1.1 x 10–4 µM of NO in the presence of 1.1 µM CO the MbNO/MbCO will reach a 1:1 ratio, based on the MbCO/MbNO partition coefficient of 9,000 (11, 34). Table 3 shows that the cellular NO concentration can reach 10–2 µM, which would yield an MbNO/MbCO ratio of 82/1. In effect, the presence of sufficient CO to inhibit 80% of Mb function but not respiration does not appear to significantly impair any imputed NO scavenging function of Mb, if NO reaches 10–2 µM. However, if NO does not reach that level, CO can certainly affect Mb scavenging of NO. However, that condition would raise the other perspective in the present analysis: bradykinin cannot induce sufficient NO to form a detectable metMb signal. Any purported role of Mb in NO scavenging becomes moot.

The potential CO interference with Mb function as a postulated NO scavenger has led some investigators to dismiss peremptorily the validity of any CO inactivation experiments to assess Mb function (3). That perspective, however, overlooks the published data consistent with present study's results. CO inactivation of Mb does not produce any significant functional or MO2 degradation over a broad range of physiological conditions in the rat myocardium, including ischemia and postischemic reperfusion (12, and Y. Chung, S. J. Huang, and A. Glabe, unpublished observations). The initial decline in the myocardial RPP with very low CO concentration appears to originate from the direct CO binding to guanylate cyclase rather than any binding with myoglobin. Neither RPP nor MO2 shows a dose-dependent relationship with CO until MbCO saturation >75% (12). The observation is consistent with the observation that O2 has a higher binding affinity to cytochrome oxidase than CO (36).

If as asserted, MbCO can no longer scavenge NO, then NO would bind to cytochrome oxidase and reduce the respiration rate, according to the paradigm of NO regulation of respiration (10, 24). Yet, up to 86% Mb inhibition with CO in perfused rat myocardium, MO2 remains constant (12). In the presence of CO inactivation of Mb, bradykinin, however, still exerts the identical physiological alteration associated with NO formation. Although exogenous CO inactivates 86% of Mb, neither CO nor MbCO impairs the bradykinin-induced myocardial and vascular response to NO. The results are consistent with the view that CO inhibition of Mb does not appear to modulate any NO interaction with cytochrome oxidase, resulting in a decrease in respiration. Alternatively, Mb does not participate significantly in scavenging cellular NO in the perfused myocardium, consistent with the current study's experimental results.

NO modulation of respiration. A respiratory control model has recently emerged to posit a role of NO in modulating mitochondrial energy generation (10, 24). In that model, the competitive binding of NO and O2 serves to regulate cytochrome oxidase activity and therefore energy generation. Clearly, if Mb binds NO, it would modulate the available NO/O2 ratio and could control respiration (8). However, the present study has not detected any metMb signal during bradykinin stimulation, although the physiological response indicates the presence of NO. In the presence of exogenous CO sufficient to saturate 86% of Mb, bradykinin still elicits the NO-induced response. The undetected metMb signals under well oxygenated as well as under CO inactivation conditions imply that Mb does not appear to interact significantly with NO. In conclusion, under the reported experimental conditions, the results have not confirmed the findings of Flogel et al. (12) and therefore the hypothesis that Mb functions as a bioscavenger of NO.


   ACKNOWLEDGMENTS
 
GRANTS

This study was supported by National Institute of General Medical Sciences Grant GM-58688 (to T. Jue), the University of California Tobacco-Related Disease Research Program 8KT0042, and American Heart Association Western States 0265319Y (to U. Kreutzer).


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Jue, Dept. of Biological Chemistry, Univ. of California, Davis, CA 95616-8635 (E-mail: tjue{at}ucdavis.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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