(Received for publication, August 26, 1994; and in revised form, September 27, 1994)
From the
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
= 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.
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),
Fe
MGD
(Fe
MGD), 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 FeMGD we observed that nitric oxide
is markedly increased during ischemia with the formation of the
characteristic Fe
MGD
NO 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.
In the absence of NO, no triplet EPR spectrum is observed
from the FeMGD 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
FeMGD 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 FeMGD 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 NO
Fe
MGD
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 FeMGD. 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 FeMGD 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
NOFe
MGD.
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 NOFe
MGD
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
FeMGD. 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.
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 FeMGD were performed and
enabled relative quantitation of the concentration of NO in the heart.
It has recently been reported that the Fe
MGD 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 Fe
MGD complex was highly
soluble in aqueous solution and that the NO
Fe
MGD 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
N has nuclear spin, I = 1. This
characteristic spectrum is centered at g = 2.04 with a nitrogen
hyperfine coupling a
= 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 Fe
MGD. 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.