©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Direct Measurement of Nitric Oxide Generation in the Ischemic Heart Using Electron Paramagnetic Resonance Spectroscopy (*)

(Received for publication, August 26, 1994; and in revised form, September 27, 1994)

Jay L. Zweier (§) Penghai Wang Periannan Kuppusamy

From the Molecular and Cellular Biophysics Laboratories, Department of Medicine, Division of Cardiology, and the Electron Paramagnetic Resonance Center, The Johns Hopkins Medical Institutions, Johns Hopkins Bayview Medical Center, Baltimore, Maryland 21224

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Nitric oxide, NO, exerts numerous important regulatory functions in biological tissues and has been hypothesized to have a role in the pathogenesis of cellular injury in a number of diseases. It has been suggested that alterations in NO generation are a critical cause of injury in the ischemic heart. However, the precise alterations in NO generation which occur are not known, and there is considerable controversy regarding whether myocardial ischemia results in increased or decreased NO formation. Therefore, electron paramagnetic resonance studies were performed to directly measure NO in isolated rat hearts subjected to global ischemia, using the direct NO trap Fe-N-methyl-D-glucamine dithiocarbamate, which specifically binds NO giving rise to a characteristic triplet EPR spectrum with g = 2.04 and a(N) = 13.2 G. While only a small triplet signal was observed in normally perfused hearts, a 10-fold increase in this triplet EPR spectrum was observed after 30 min of ischemia indicating a marked increase in NO formation and trapping. Measurements were performed as a function of the duration of ischemia, and it was determined that with increased duration of ischemia NO formation and trapping was also increased. NO generation was inhibited by the nitric oxide synthase blocker, N-nitro-L-arginine methyl ester (L-NAME), suggesting that NO was generated via nitric oxide synthase. Blockade of NO generation with L-NAME resulted in more than a 2-fold increase in the recovery of contractile function in hearts reperfused after 30 min of global ischemia. Thus, ischemia causes a marked duration-dependent increase of NO in the heart which may in turn mediate postischemic injury.


INTRODUCTION

Over the last several years it has been demonstrated that the gaseous free radical nitric oxide, NO, is generated in biological cells and tissues and is of central importance in regulating a broad range of important biological functions(1) . In 1980 Furchgott and Zawadzki (2) demonstrated that the vascular relaxation induced by acetylcholine was dependent on the presence of the endothelium and that this effect was mediated by a labile factor termed endothelial-derived relaxing factor. It was subsequently hypothesized and then demonstrated that this endothelial-derived relaxing factor activity could be attributed to the formation of NO(3, 4, 5) . Its synthesis was first discovered in 1987 in macrophages and endothelial cells, and since that time, NO has been shown to have effects on target cells in many tissues(3, 4, 5, 6) . NO is now known to play an important role in blood pressure regulation, vascular tone, neural signaling, and immunological function(7, 8, 9) . It is known to induce the formation of the second messenger molecule cyclic GMP in both the generating and the target cells(10) . It has been demonstrated that there is an enzyme present in macrophages, endothelial cells, and neuronal cells which synthesizes NO from arginine(11, 12) . This enzyme, nitric oxide synthase, exists in two major forms with the macrophage enzyme differing substantially from the brain and endothelial forms both of which are quite similar, if not identical. There is evidence that NO may also have an important role as a mediator of tissue injury(13) .

It has been suggested that alterations in the generation of NO occur in tissues subjected to ischemia, and that these alterations result in altered endothelial function with altered tissue perfusion on subsequent reperfusion(14, 15) . In particular, there has been considerable controversy regarding the effect of ischemia on NO generation in the heart. Studies of endothelial function have been interpreted to suggest that the process of ischemia decreases NO generation, and that a loss of basal NO production is an important source of injury in hearts subjected to ischemia(14, 15) . Subsequently, other studies have been reported which demonstrate that inhibitors of nitric oxide synthase can protect against ischemic injury(16) . From these later studies it was suggested that NO may be involved in the process of tissue injury and that the production of NO may actually be increased by the process of ischemia, rather than decreased. Thus, indirect assessment of NO generation from measurements of organ function have resulted in considerable controversy and uncertainty regarding the effect of ischemia on NO generation in the heart.

While there has been a great need for techniques of directly measuring nitric oxide production in biological systems, there have been few, if any, techniques with sufficient sensitivity and specificity to provide quantitative measurements over the diverse range of physiological and pathophysiological applications of interest. Since NO is a free radical and reacts to form high affinity nitroso complexes with a variety of metal complexes and metalloproteins it has been proposed that the distinctive EPR spectra of these nitroso complexes could be used to serve as a quantitative measure of NO generation (17, 18, 19) . While measurement of nitroso-heme formation serves as an intrinsic trap providing a measure of NO generation, these complexes are labile in the presence of oxygen. The Fe-diethyldithiocarbamate complex has been proposed as a more stable and oxygen independent trap suitable for measuring NO generation in biological systems(20) . This complex has very limited solubility in water; therefore, recently the similar ferrous iron complex of N-methyl-D-glucamine dithiocarbamate (MGD),^1 FebulletMGD(2) (FebulletMGD), has been proposed as an oxygen-stable water soluble NO trap suitable for measuring nitric oxide in living tissues(21) .

In this study we have applied EPR spectroscopy to directly measure the effect of ischemia on the generation of NO in the heart. Using the NO trap FebulletMGD we observed that nitric oxide is markedly increased during ischemia with the formation of the characteristic FebulletMGDbulletNO triplet signal. This NO formation was largely blocked by inhibition of nitric oxide synthase and the resultant decrease in NO generation was associated with a marked improvement in the recovery of contractile function.


EXPERIMENTAL PROCEDURES

Isolated Heart Perfusion

Female Sprague-Dawley rats (250-300 g) were heparinized and anesthetized with intraperitoneal pentobarbital. The hearts were excised, the aorta was cannulated, and retrograde perfusion was initiated. Hearts were perfused at a constant pressure of 80 mm Hg using Krebs bicarbonate buffer (17 mM glucose, 120 mM sodium chloride, 25 mM sodium bicarbonate, 2.5 mM calcium chloride, 0.5 mM EDTA, 5.9 mM potassium chloride, and 1.2 mM magnesium chloride) bubbled with 95% O(2) and 5% CO(2) gas at 37 °C, as described previously(22) . A sidearm in the perfusion line allowed direct infusion of the FebulletMGD NO trap just proximal to the heart. In order to measure contractile function a latex balloon was inserted into the left ventricular cavity and connected to a pressure transducer via a hydraulic line and pressures recorded with a Gould RS4000 recorder. The balloon was initially inflated to achieve an end-diastolic pressure of 8-14 mm Hg. Left ventricular pressures, heart rate, and coronary flow were monitored throughout the period of perfusion. Hearts were rapidly frozen using liquid nitrogen cooled Wollenberger tongs. The frozen tissue was maintained at 77 K in liquid nitrogen and either ground or fractured to 1-2-mm particles and transferred to 3-mm EPR tubes. Alternatively, to minimize processing the tissue was fractured to 3-4-mm pieces and placed directly within the EPR finger Dewar. Similar spectra were observed with measurements in 3-mm tubes or directly in the EPR Dewar. The EPR tube or Dewar was filled to a sufficient height to fill the critical volume of the EPR cavity.

Materials

MGD was synthesized as described previously(23) . The N-methyl-D-glucamine and carbon disulfide required for this synthesis were purchased from Aldrich. S-Nitroso-N-acetylpenicillamine, was obtained from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). N-Nitro-L-arginine methyl ester, L-NAME, and all other reagents were obtained from Sigma. Fresh stock solutions of the FebulletMGD complex were prepared by addition ferrous ammonium sulfate to aqueous solutions of MGD at a ratio of 1:5. The final concentration of the complex used in the heart was 1 mM in Fe.

EPR Spectroscopy

EPR spectra were recorded at 77 K using a liquid nitrogen Dewar with a Bruker ER 300 spectrometer operating at X-band with 100 kHz modulation frequency and a TM110 cavity, as described previously(24) . The microwave frequency and magnetic field were precisely measured using an EIP 575 microwave frequency counter and Bruker ER 035M NMR gaussmeter. Relative quantitation of the free radical signals was performed by double integration.


RESULTS

In the absence of NO, no triplet EPR spectrum is observed from the FebulletMGD complex (Fig. 1A). After addition of the NO donor compound S-nitrosoacetylpenicillamine, a prominent triplet EPR spectrum is observed with a central g value of 2.04 and hyperfine splitting of 13.2 G (Fig. 1B).


Figure 1: EPR spectra of FebulletMGD in the presence and absence of NO. A, spectrum of a preparation of 1 mM Fe, 5 mM MGD in 50 mM HEPES buffer, pH 7.4. B, after incubation with a 2 mM concentration of the NO donor S-nitroso-N-acetylpenicillamine. Spectra were recorded at 77 K with microwave frequency of 9.316 GHz using 1.0 milliwatt microwave power and a modulation amplitude of 4.0 G. Each spectrum is a 60-s spectral acquisition of 100 G sweep width with a time constant of 0.32 s.



After cannulation and a 15-min period of control perfusion with equilibration of contractile function, developed pressures of 120 ± 10 mm Hg were observed, and hearts were then infused and loaded with a 1 mM concentration of the FebulletMGD complex (1 mM Fe, 5 mM MGD) for 5 min. The hearts were then immediately frozen or subjected to no flow ischemia and then frozen at 77 K. In normally perfused control hearts only a very weak spectrum of the triplet NObulletFebulletMGD complex was seen (Fig. 2A). In hearts subjected to ischemic durations of 30 min, however, a prominent triplet NO adduct signal appeared (Fig. 2B). In these hearts additional intrinsic signals were seen from the 1 electron reduced ubiquinone radical, centered at g = 2.004 and from ROO at g = 2.005, as well as from iron sulfur proteins at g = 1.94, as reported and assigned previously(24, 25) . These radical signals with absorption functions in the g = 2.008-2.000 region are located just up-field from the NO triplet and are seen as the positive deflections at the high field end of the spectra. In a series of 5 control and 5 hearts subjected to 30 min of ischemia, the intensity of the NO triplet was observed to be 10.5 ± 2.1-fold higher after 30 min of ischemia than in controls.


Figure 2: EPR spectra of NO trapping in hearts labeled with FebulletMGD. A, frozen tissue from a normally perfused heart. B, frozen tissue from a heart subjected to 30 min of ischemia. Spectra were recorded with a microwave frequency of 9.323 GHz using 1.0 milliwatt microwave power and a modulation amplitude of 4.0 G. Each spectrum was obtained from the sum of 20, 60-s spectral acquisitions of 100-G sweep width with a time constant of 0.32 s.



Further experiments were performed to determine the effect of the duration of ischemia on the magnitude of the NO signal. After 10 min of ischemia only a trace triplet signal was seen and after 20 min this signal increased. With 30 or 60 min of ischemia, further marked increases in this triplet signal were seen (Fig. 3). Relative quantitation of this signal was performed by double integration, and these results demonstrated that the magnitude of the triplet spectrum of trapped NO progressively increased as a function of the duration of ischemia (Fig. 4).


Figure 3: EPR spectra recorded from hearts subjected to varying duration of ischemia followed by freezing at 77 K. Hearts were labeled with FebulletMGD and spectra recorded as described in the legend to Fig. 2. A, 10 min of ischemia; B, 20 min of ischemia; C, 30 min of ischemia; D, 60 min of ischemia.




Figure 4: Graph of the effect of ischemic duration on the intensity of the observed triplet EPR signal of NObulletFebulletMGD.



In order to determine if the NO generation observed during ischemia was derived from nitric oxide synthase, experiments were performed in hearts treated with the blocker L-NAME. In hearts pretreated with a 1.0 mM concentration of L-NAME for at least 15 min prior to the onset of ischemia, a 70-80% decrease in the NObulletFebulletMGD signal was observed (Fig. 5). Thus, most of the NO generated and trapped during ischemia was generated by nitric oxide synthase.


Figure 5: Effect of the nitric oxide synthase blocker L-NAME on the EPR spectra of hearts labeled with FebulletMGD. A, spectrum of tissue frozen after 30 min of ischemia in the absence of the blocker. B, spectrum of tissue after 30 min of ischemia in the presence of 1.0 mML-NAME. Spectra were recorded as described in the legend to Fig. 2.



Further experiments were performed to determine if blocking nitric oxide generation in the ischemic heart would ameliorate or exacerbate the functional injury which occurs upon postischemic reperfusion. Hearts were subjected to 30 min of 37 °C global ischemia and 45 min of reperfusion with continuous measurement of contractile function and coronary flow. Eight hearts were studied, 4 untreated and 4 pretreated for 15 min with 1.0 mML-NAME. As expected in L-NAME-treated hearts, lower coronary flow was observed due to the loss of NO-induced vasodilation. Throughout the 45-min period of reflow the coronary flow was decreased by almost 2-fold (Fig. 6). This decrease in coronary flow in the L-NAME-treated hearts was highly significant with p < 0.01. In spite of this decreased coronary flow, much higher recovery of contractile function was observed throughout the period of reperfusion with more than a 2-fold increase in the recovery of left ventricular developed pressure (Fig. 7). This increase in left ventricular developed pressure was also highly significant with p < 0.01. Thus, inhibition of the marked increase in the NO formed during ischemia resulted in significantly improved recovery of contractile function in the postischemic heart.


Figure 6: Measurement of the recovery of coronary flow in untreated control hearts or hearts pretreated with the nitric oxide synthase blocker L-NAME, 1.0 mM. Hearts were subjected to 30 min of global ischemia followed by 45 min of reflow. Data are expressed as percent recovery of preischemic values. The lower values of coronary flow observed in L-NAME-treated hearts than in the untreated control hearts are consistent with the inhibition of NO generation by this blocker.




Figure 7: Measurement of the recovery of contractile function in untreated control hearts or hearts pretreated with the nitric oxide synthase blocker L-NAME, 1.0 mM. Hearts were subjected to 30 min of global ischemia followed by 45 min of reflow. Left ventricular developed pressures (LVDP) were measured before ischemia and after postischemic reflow. Data are expressed as percent recovery of preischemic values. Hearts treated with L-NAME exhibited significantly higher recovery of LVDP than in untreated control hearts.




DISCUSSION

Since the time that it was first demonstrated that biological cells and tissues generate nitric oxide, this free radical has been shown to exert a variety of important functions including modulation of vascular tone, neural signaling, and immune response(7, 8) . In the setting of ischemic injury NO could exert protective effects by increasing coronary flow or harmful effects with cellular injury resulting from the reaction with superoxide to form the reactive oxidant peroxynitrite(14, 15, 16) . Based on these opposite effects, different physiological studies of the ischemic and postischemic heart have been interpreted to suggest that NO generation may be markedly decreased or markedly increased.

In this study we have performed measurements to directly measure the alterations in NO generation which occur in the ischemic heart and to determine the functional consequences of this NO generation on the recovery of contractile function. Measurements of the magnitude of the triplet signal formed on trapping of NO by FebulletMGD were performed and enabled relative quantitation of the concentration of NO in the heart. It has recently been reported that the FebulletMGD complex is an ideal transition metal complex for the trapping and measurement of NO in living tissues and in vivo animals(21) . In our studies we also observed that the FebulletMGD complex was highly soluble in aqueous solution and that the NObulletFebulletMGD complex was stable and observable in aerobic solutions. A characteristic triplet spectrum was observed due to the coupling of the unpaired electron to the nitrogen nucleus of NO, which for the natural abundance isotope ^14N has nuclear spin, I = 1. This characteristic spectrum is centered at g = 2.04 with a nitrogen hyperfine coupling a(N) = 13.2 G. In our experiments we observed that this trap was non-toxic in the concentrations used and could be directly perfused into the heart without adverse effect. We observed that NO generation is markedly increased during ischemia as evidenced by the appearance of the characteristic triplet EPR signal on direct trapping with FebulletMGD. A progressive increase in the NO adduct was seen as a function of the duration of ischemia with more than a 10-fold increase after 30 min of ischemia. When hearts were pretreated with the specific nitric oxide synthase blocker, L-NAME, the generation of this NO triplet was decreased by 70-80%, demonstrating that the generation of NO was largely derived from nitric oxide synthase. In hearts in which NO generation was blocked with L-NAME, a marked cardioprotective effect was observed with more than a 2-fold increase in the recovery of contractile function. Thus, the marked increase in NO within the ischemic heart is associated with the process of postischemic injury. This apparent toxicity may be due to the reaction of NO with superoxide(13) .

There is considerable direct and indirect evidence that superoxide and superoxide-derived free radicals are generated in ischemic and reperfused myocardium. It has been demonstrated that superoxide dismutase treatment during ischemia and reperfusion can prevent functional injury and cell death(26, 27) . Direct and spin trapping EPR studies have provided direct evidence that oxygen free radicals are generated in the postischemic heart(24, 25) . Further studies have demonstrated that superoxide dismutase can block free radical generation and subsequent contractile dysfunction which occurs after postischemic reperfusion(22) . It has also been shown that reoxygenated endothelial cells give rise to a burst of free radical generation and suggested that the endothelial cell is an important site of free radical generation in the heart(28, 29) . In particular, it has been demonstrated that the superoxide generating enzyme xanthine oxidase is present within vascular endothelium of human, bovine, and rat aortic endothelial cells(28, 29, 30) . This enzyme is present in the rat heart and is responsible in part for the burst of radical generation which occurs during postischemic reperfusion(29) .

Thus, ischemia triggers both the generation of superoxide as well as increased amounts of NO. This increased production of NO and superoxide in ischemic and postischemic myocardium could result in the formation of the more potent oxidant peroxynitrite which is known to cause cellular injury(13) . These observations are consistent with the recent studies which have reported that treatment with nitric oxide synthase blockers can prevent postischemic injury (16) . While further studies will be required to fully understand the role of NO in the process of ischemic heart disease, the present study demonstrates that a large increase in the formation and accumulation of NO occurs during myocardial ischemia and this NO in turn contributes to the process of postischemic injury.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL-17655 and HL-38324. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by an Established Investigator Award from the American Heart Association. To whom correspondence should be addressed: Electron Paramagnetic Resonance Center, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Johns Hopkins Medical Institutions, Baltimore, MD 21224.

(^1)
The abbreviations used are: MGD, N-methyl-D-glucamine dithiocarbamate; L-NAME, N-nitro-L-arginine methyl ester.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.