(Circulation. 1995;92:1911-1918.)
© 1995 American Heart Association, Inc.
Articles |
Protection Against Ischemic Injury by Nonvasoactive Concentrations of Nitric Oxide Synthase Inhibitors in the Perfused Rabbit Heart
From the Hormone and Metabolic Research Unit, International Institute of Cellular and Molecular Pathology, University of Louvain Medical School, Brussels, Belgium (C.D., J.-L.V., L.H.), and the Laboratory of Experimental NMR, Department of Medical Imaging, University of Louvain Medical School, Louvain-la-Neuve, Belgium (J.-F.G., I.M.).
Correspondence to Prof L. Hue, HORM Unit, ICP-UCL 7529, Avenue Hippocrate, 75, B-1200 Brussels, Belgium. E-mail hue@horm.ucl.ac.be.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background The functional and metabolic effects of inhibitors of nitric oxide (NO) synthase on ischemic hearts have not been investigated. This work was designed to perform such a study in isolated perfused rabbit hearts submitted to low-flow ischemia.
Methods and Results After a 30-minute equilibration period, the hearts were submitted to low-flow ischemia for 60 minutes followed by reperfusion for 30 minutes. Functional and metabolic parameters were followed in hearts perfused with or without inhibitors of NO synthase or NO precursors, which were added 15 minutes before ischemia but were absent during reperfusion. Ischemic contracture was delayed and reduced in hearts perfused with 1 µmol/L L-N-monomethylarginine (L-NMMA) or 1 µmol/L L-N-arginine methylester, two inhibitors of NO synthase, but not with D-N-monomethylarginine, the inactive enantiomer of L-NMMA. The protection was suppressed by addition to the perfusate containing L-NMMA of 1 mmol/L L-arginine or 0.1 mmol/L sodium nitroprusside but not by addition of 10 µmol/L 8-bromo cGMP, a cGMP analogue. The functional protection by 1 µmol/L L-NMMA was related to a stimulation of glycolysis from exogenous glucose and a preservation of the glycogen stores. This resulted in a better maintenance of high-energy phosphates and a lower acidosis as measured by 31P nuclear magnetic resonance spectroscopy. During reperfusion, functional recovery was more than doubled, and enzyme release was halved in L-NMMA–treated hearts compared with controls. The functional and metabolic protection was maximal at 1 nmol/L to 1 µmol/L L-NMMA, ie, below the vasoactive concentrations of the inhibitor.
Conclusions Nonvasoactive concentrations of NO synthase inhibitors protect the heart against ischemic damage; this relates to a stimulation of glycolysis from exogenous glucose.
Key Words: glucose • ischemia
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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NO is a biological messenger synthesized from L-arginine by the enzyme NO synthase (see References 1 and 2 for review). It was first described in the endothelium as mediating the effects of vasodilators on vascular smooth muscle cells.3 4 5 NO also might be involved in the regulation of the inotropic state of the cardiac cell,6 although conflicting results have been reported concerning its effect on contractility.7 8 A myocardial isozyme of NO synthase is induced in endotoxemia9 and has been implicated in decreased contractility observed in septic shock.10 Moreover, the negative inotropic effect of cytokines is prevented by low (10 µmol/L) concentrations of L-NMMA, a specific inhibitor of NO synthase.11 It is not known if the main biological effects of NO in heart are due to its release either from the endothelium, followed by its diffusion to the cardiomyocyte, or from the cardiomyocyte itself, then acting as an autocrine system.
Several effects of NO on the myocardium are mediated by the production of cGMP,1 and, accordingly, perfusion of rat hearts with NO synthase inhibitors decreased cGMP concentration.12 cGMP in turn may act on cGMP-dependent protein kinase or cAMP phosphodiesterase.13 However, several cGMP-independent effects of NO have been reported as the production of free radicals,14 which is considered to be a major factor of reperfusion injury after myocardial ischemia. In smooth muscle cells, NO directly stimulates calcium-dependent potassium channels15 and Na+/K+-ATPase.16
A thorough study of the effects of NO or NO synthase inhibitors on heart function and metabolism during ischemia has not been carried out, and the importance of NO synthase activity during ischemia and reperfusion is controversial. L-NMMA has been reported to reduce infarct size in rabbit when infused before ischemia.17 This beneficial effect was interpreted as resulting from "preconditioning" as a result of a state of mild vasoconstriction. Conversely, L-arginine, the substrate of NO synthase, when added before reperfusion reduced the infarct size by limiting reperfusion injury.18 19 Our hypothesis to explain these conflicting results is that NO synthase inhibitors protect when added before the ischemic episode, whereas L-arginine protects when added before reperfusion. Therefore, we investigated the functional and metabolic effects of NO synthase inhibitors during ischemia. To evaluate the functional and metabolic consequences of the presence of the inhibitors during the ischemic episode on the subsequent reperfusion, the latter was performed with or without the inhibitors. We used a model of low-flow ischemia in which glucose metabolism can be continuously recorded together with functional parameters. To simplify the metabolic situation, fatty acids were not added to the perfusate because they interfere with glucose metabolism and cannot be oxidized during severe ischemia. Because vasoconstriction (a major effect of NO synthase inhibitors) could worsen the functional and metabolic state of ischemic hearts, it was decided to use nonvasoactive concentrations of inhibitors and to study their effects on heart function and metabolism during ischemia.
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Methods |
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Perfusion Protocol
Male New Zealand White rabbits (weight, 1.5 to 2.0 kg) fed ad libitum were anesthetized with pentobarbital (50 mg/kg IV). The heart was quickly removed and dropped in ice-cold buffered 0.15 mol/L NaCl. The aorta was cannulated and the heart perfused retrogradely, as described previously,20 at a constant flow of 5 mL/min per gram wet weight with a Krebs-Henseleit buffer (1.5 mmol/L CaCl2) containing 5.5 mmol/L glucose as sole substrate and equilibrated with a 95% O2/5% CO2 gas phase. The pulmonary artery was cannulated to measure the coronary output and to sample the coronary effluent. A latex balloon was inserted into the left ventricle, and LVEDP was set at 10 mm Hg by filling the balloon with physiological saline. Each heart was paced at 180 beats per minute. LVDP, heart rate, dP/dt, and CPP were recorded on-line. After 30 minutes of equilibration, the flow was reduced to 0.25 mL/min per gram wet weight for 60 minutes, and the perfusate was equilibrated with 95% air/5% CO2 to ensure tissue hypoxia during this period. These conditions correspond to a 90% to 95% reduction of the normal flow and have been established previously to induce irreversible ischemic damage.20 21 The hearts were maintained at 37°C by immersion in a thermostated reservoir filled with buffer. Hearts then were reperfused for 30 minutes with the same buffer as during the preischemic episode. These control hearts were compared with hearts that were perfused with different effectors of the NO pathway during the last 15 minutes of equilibration and the 60 minutes of ischemia. The following conditions were tested: (1) inhibition of NO synthase by addition of different concentrations of L-NMMA (Calbiochem) or L-NAME (Sigma), (2) the enantiomer specificity of NO synthase by addition of D-NMMA (Calbiochem), (3) stimulation of endogenous NO production by addition of L-arginine (Sigma), (4) production of NO by addition of SNP (Roche) as an exogenous source of NO, and (5) addition of a cGMP analogue, 8-Br-cGMP (Sigma). Each product was diluted in buffer; at the concentrations used (below mmol/L), these additions did not affect the pH of the buffer. None of these test compounds were present during the 30-minute reperfusion except when indicated.
Since the duration of the whole experimental protocol could
last up to
120 minutes, the stability of the model under normoxic conditions was
confirmed by the measurement of LVDP and CPP throughout the experiment.
The values at 90 and 120 minutes were not significantly different from
the control values, which are given in Table 1
and were
measured at 30 minutes. Similarly, the concentration of ATP, PCr, and
glycogen were the same at 30 and 90 minutes of perfusion under normoxic
conditions (not shown).
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Analytical Procedure
Before ischemia and at the indicated
times of the
ischemic episode, the left ventricles were rapidly dissected
with scissors and, while still perfused, they were freeze-clamped
between precooled (liquid nitrogen) aluminium tongs. The frozen left
ventricles were weighed, then powdered with a pestle in a mortar filled
with liquid nitrogen, and deproteinized by homogeneization in 3 vol
of 10% perchloric acid with an Ultraturrax. ATP, PCr, hexose
6-phosphates (ie, glucose 6-phosphate and fructose 6-phosphate) and
fructose 1,6-bisphosphate were measured enzymatically22 in
neutralized extracts. cGMP was measured as described.12
Glucose equivalents of glycogen were measured after digestion with
amyloglucosidase in a separate alkaline extract neutralized with acetic
acid.23
Glucose and lactate were measured enzymatically22 both in the perfusate and in the coronary effluent. Glucose uptake and lactate release during ischemia were calculated by multiplying the difference of concentration between the perfusate and the effluent by the flow rate. Glycolytic flux through phosphofructo-1-kinase during ischemia was measured by the rate of detritiation of [3-3H]glucose (2 µCi/100 mL of perfusate, Amersham) added 15 minutes before ischemia. Separation of tritiated glucose from tritiated water was performed by chromatography on Dowex AG1-X8 (200-400 mesh, borate form, Bio-Rad), as described previously.24 25
The release of creatine kinase (CK) and lactate dehydrogenase (LDH) were measured22 in samples taken every 2 minutes from the coronary effluent. The total amount released was calculated by multiplying enzyme activity in the effluent by the flow rate.
NMR Spectroscopy
31P spectra were obtained by NMR
spectroscopy as
follows. The hearts were placed into a Brucker Biospec spectrometer
(4.7 T) containing a home-built thermostated probe and were
submitted to the same experimental protocol as described above. The
buffer used in these experiments was phosphate free. The data were
recorded at 81 MHz using a 60° pulse delivered at 1.5-second
intervals with a 6000-Hz spectral width. Spectra resulted from the
addition of 120 scans. The Fourier transforms were performed after zero
filling and exponential multiplication (line broadening, 20 Hz).
Methylene phosphonic acid was used as an external reference for
absolute quantification of the metabolites. Estimation of peak areas
was performed by fitting in the time domain. These areas were scaled
according to saturation factor calculated from the longitudinal
relaxation time of the phosphorous compounds. The chemical shift of
Pi was used to estimate the value of
pHi.26
Statistical Analysis
All the values are mean±SEM for
at least five different hearts
and are expressed as per gram of dry weight. The heart dry weight over
wet weight ratios were similar in all groups; this ratio was 13±1%
after the 30-minute equilibration period and 11.5±1% after 60-minute
ischemia. Unpaired two-tailed Student's t test
and ANOVA with Bonferroni correction for repeated comparisons were used
to determine statistically significant differences (P<.05)
between groups.
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Results |
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Effects of NO Synthase Inhibitors on Physiological Parameters
The effects of different concentrations of L-NMMA on CPP and LVDP were first tested to estimate the vasoconstrictive capacity of the inhibitor. Perfusion with 1 µmol/L L-NMMA did not affect CPP or LVDP during the 30-minute equilibration period (Table 1
), nor did it affect the dP/dt (controls,
1690±100;
L-NMMA–treated, 1675±90 mm Hg/min). These values were not
modified
after 90 minutes of perfusion (values not shown). The lack of effect on
CPP indicated that, at this concentration, the inhibitor
was devoid of any major vasoconstrictive effect on the
coronary vasculature. In contrast, perfusion with 100 µmol/L
L-NMMA markedly increased CPP by vasoconstriction and slightly affected
LVDP (Table 1) by an increase in end-diastolic
pressure.
When the hearts were submitted to severe low-flow ischemia,
they progressively developed a contracture (Fig 1), the
amplitude of which is related to the ischemic
damage.27 Perfusion with 1 µmol/L L-NMMA delayed the
time of onset of ischemic contracture (defined as the time at
which end-diastolic pressure increased by more than 5
mm Hg when compared with preischemic value) and decreased
its amplitude by more than 50% (Fig 1 and Table
1). This effect was
not observed at a vasoconstrictive concentration (100
µmol/L) of L-NMMA (Table 1). Protection against ischemic
contracture also was observed with 1 µmol/L L-NAME, another
inhibitor of NO synthase (Table 1). The protection was
enantiomer-specific, as it was not observed with 1 µmol/L D-NMMA
(Table 1), which does not inhibit NO synthase.28
Moreover,
L-NMMA did not decrease ischemic contracture when perfused
together with 1 mmol/L L-arginine, the substrate of NO
synthase, or with 100 µmol/L SNP, a direct NO donor (Table
1). Both
L-arginine and SNP slightly decreased CPP by vasodilation
(Table 1). Protection also was lost when 1 µmol/L L-NMMA
was
present before but not during the ischemic episode (not
shown), therefore suggesting that the protection conferred by 1
µmol/L L-NMMA cannot be explained by a "preconditioning"
effect, as previously suggested.17
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After the 60-minute
period of low-flow ischemia, the hearts
were reperfused at the initial flow rate and with oxygen but without
L-NMMA, and their functional recovery was measured. L-NMMA–pretreated
hearts recovered 75±4%, whereas control hearts recovered only
35±3%
of their preischemic function, as measured by LVDP (Table 1).
Similarly, the recovery of dP/dt was significantly improved in
pretreated hearts (1015±75 in controls versus 1500±100 mm Hg/min
in
L-NMMA–pretreated hearts; P<.05). CK and LDH were measured
in the coronary effluent during the first 15 minutes of
reperfusion to evaluate the cellular damage. The release of both
enzymes was reduced by 50% in 1 µmol/L L-NMMA–pretreated hearts
(365±60 versus 145±30 U/g for CK; 50±9 versus 25±7
U/g for LDH in
control and treated hearts, respectively; P<.01).
The
improvement of functional recovery was the same whether the hearts
were pretreated with 1 µmol/L L-NAME or 1 µmol/L L-NMMA (Table
1).
However, in hearts pretreated with vasoconstrictive
concentrations (100 µmol/L) of L-NMMA, no protection against
ischemic contracture was observed, and the recovery was similar
to that of control hearts. This also was the case for hearts pretreated
either with 1 µmol/L D-NMMA or with 1 µmol/L
L-arginine or 100 µmol/L SNP together with 1 µmol/L
L-NMMA (Table 1).
As described in "Methods," all the hearts were reperfused with buffer devoid of any test compounds. However, to assess the functional effect of NO synthase inhibitors not only during ischemia but also during reperfusion, some hearts were perfused with 1 µmol/L L-NMMA both during ischemia and reperfusion. In this case, although the inhibitors did confer protection against ischemic contracture as expected, the functional recovery was, however, severely altered (30±5% of preischemic LVDP) because of a marked increase of the LVEDP during reperfusion (45±5 mm Hg at the end of reperfusion).
Relation Between cGMP and Cardioprotection During
Ischemia
Perfusion with 1 µmol/L L-NMMA decreased cGMP
concentration
(Table 2), as expected from previous
experiments.12 To test whether cardioprotection could be
related to this decrease, 10 µmol/L 8-Br-cGMP, a cGMP analogue,
was added together with 1 µmol/L L-NMMA. Under these conditions,
8-Br-cGMP did not prevent the protective effect of L-NMMA on
contracture, although it did decrease CPP (Table 1). However,
when
8-Br-cGMP was present during the reperfusion period together
with 1 µmol/L L-NMMA, it suppressed the increase of
diastolic pressure observed in hearts reperfused with 1
µmol/L L-NMMA alone (see above). Under this peculiar condition, ie,
when both 1 µmol/L L-NMMA and 10 µmol/L 8-Br-cGMP were
present at reperfusion, the functional recovery was 78±5%
of preischemic values. The latter results are in agreement with
the protective effect of L-arginine during
reperfusion.18 19
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Effects of NO Synthase Inhibitors on High-Energy
Phosphates
The mechanism by which low concentrations of L-NMMA play a
protective role during ischemia may involve an increased
production and/or a decreased consumption of high-energy
phosphates. Therefore, control hearts and hearts treated with 1
µmol/L L-NMMA were studied by 31P NMR spectroscopy during
low-flow ischemia and reperfusion following the same
experimental protocol. The values for ATP and PCr obtained by
31P NMR were the same as those measured by biochemical
methods in deproteinized extracts. Fig 2 shows that the
spectra recorded during the equilibration period were very similar
in both groups. The fact that the concentrations of PCr, ATP, and
Pi were the same in both groups after 30 minutes (Table
2)
or 90 minutes (not shown) of perfusion under normoxic conditions
further confirmed the absence of vasoconstriction in hearts perfused
with 1 µmol/L L-NMMA.
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During low-flow ischemia, hearts treated with
1 µmol/L
L-NMMA presented a slower decrease in PCr, ATP, and
pHi as measured by the chemical shift of
Pi, and the values reached after 60 minutes of
ischemia were consistently higher in the treated hearts
than in the controls (Table 2). Similarly, the increase in
Pi was less in the treated group (Table 2). During
reperfusion without the inhibitor, the pretreated hearts
presented better recovery of PCr and ATP content (Fig 2).
Effects of NO Synthase Inhibitors on Glucose
Metabolism
Because glucose was the only energy-providing substrate in
the
preparation and because of the importance of glycolysis during
ischemia, glucose metabolism was investigated to
explain the energetic modifications brought about by L-NMMA. As shown
in Fig 3, the onset of ischemia was rapidly
followed by a marked increase both in glucose uptake (Fig 3A)
and in
lactate production (Fig 3B), corresponding to the
well-known "Pasteur effect." In control hearts, this
enhancement was transient and started to fall between 15 and 30 minutes
after the onset of ischemia, which corresponded to the time of
onset of ischemic contracture (Fig 1 and Table
1). In contrast,
glucose uptake and lactate production reached higher values in
hearts treated with L-NMMA, and this difference was maintained
throughout the entire period of ischemia (Fig 3A and
3B). This
corresponded to the marked delay in the onset of the ischemic
contracture (Fig 1). Maintenance of a higher rate of exogenous
glucose uptake during ischemia also was accompanied by a
decreased rate of glycogen breakdown in L-NMMA–treated hearts as
compared with controls (Fig 3C).
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The stimulation of
glycolytic flux by L-NMMA was confirmed by a higher
rate of detritiation of [3-3H]glucose (Fig
3D), which
measures the flux through phosphofructo-1-kinase, the first committed
step of glycolysis. Moreover, measurement of the changes in hexose
phosphate concentration brought about by L-NMMA during ischemia
(Table 2) indicated that the concentration of hexose
6-phosphates but
not fructose 1,6-bisphosphate was increased in the treated group. This
confirms that glucose transport and phosphorylation
also were stimulated, and it suggests that phosphofructo-1-kinase
became rate limiting for the overall glycolytic flux.
Dose-Dependent Effects of NO Synthase
Inhibitors
We investigated the range of concentrations of L-NMMA
that protected against ischemic contracture and stimulated
glucose uptake. As shown in Fig 4, an effect of L-NMMA
on both parameters was detected with concentrations as low
as 0.1 nmol/L, ie, several orders of magnitude lower than the
concentrations at which vasoconstriction was observed in this model (10
µmol/L). The protection was maximal in the 10-9 to
10-6 mol/L range of concentrations of L-NMMA. It was not
observed with higher concentrations, at which L-NMMA was found to
increase CPP. The dose-response curve for the stimulation of
glucose uptake by L-NMMA was the mirror image of the protection against
ischemic contracture (Fig 4), thus reinforcing the hypothesis
that the functional protection is incident to the stimulation of
exogenous glucose metabolism.
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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Our results show that perfusion of rabbit hearts with low concentrations of L-NMMA, a specific inhibitor of NO synthase, decreased the ischemic damage during low-flow ischemia and, if removed at reperfusion, induced a better functional recovery and less cellular damage.
The protection presented the following characteristic features: (1) it was not observed with concentrations of L-NMMA that induced vasoconstriction, (2) it was abolished by an excess of L-arginine or SNP, (3) it appeared to be independent from cGMP despite the fact that L-NMMA decreased cGMP concentrations, and (4) it was related to a stimulation of glycolysis during ischemia, thus allowing better preservation of energetic resources. These points are discussed below.
Dose-Dependent Effects of NO Synthase
Inhibitors
The beneficial effect of NO synthase inhibitors was
observed at concentrations (1 nmol/L to 1 µmol/L) of the
inhibitors known to be devoid of any
vasoconstrictive effect,29 30
whereas the protection was lost when L-NMMA was used at
concentrations (100 µmol/L) inducing vasoconstriction. It could be
argued that despite the absence of vasoconstrictive
effect on coronary arteries, low doses of L-NMMA could
redistribute the flow through microcirculation without changing CPP and
so create regional ischemia. However, there is no element to
postulate such an effect. As shown by NMR spectroscopy, both PCr and
Pi—the most sensitive indices of
ischemia—were similar to the values of the controls under
normoxic conditions. Moreover, LVDP and dP/dt would have been affected
if regional ischemia had occured. The absence of a
"preconditioning effect" further excludes that protection during
ischemia was a result of the vasoconstrictive
effect of the inhibitor.
Besides the endothelial isozyme of NO synthase, both constitutive and inducible forms have been described in cardiomyocytes.9 The constitutive form has been involved in the control of the inotropic state of the heart6 ; however, very little is known about this myocardial isozyme. The activity of the enzyme in the heart is lower than in most other organs,31 and its kinetic properties are unknown. Obviously, a complete study of this enzyme and its sensitivity toward the inhibitors is required to identify the NO synthase isozyme whose inhibition protects the heart during ischemia.
Mode of Action of NO Synthase Inhibitors
The functional and
metabolic protection conferred by
low doses of inhibitors of NO synthase suggests that their
effects were mediated by a decrease in NO production. The
evidence for the latter is indirect. The direct demonstration of such a
change would be difficult to obtain because the activity of heart NO
synthase is very low. The sensitive method of NO detection by
hemoglobin, which has been used successfully to measure NO in the
coronary effluent of normoxic hearts,32 cannot be
applied to ischemic conditions because of the
oxygen-carrying hemoglobin. Our repeated attempts to detect NO
production during ischemia by a less sensitive
method33 have so far failed (not shown). Therefore, we
have no direct evidence for an inhibition in situ of NO synthase by the
concentrations of inhibitors used, and we cannot exclude
the possibility that the inhibitors were mediating their
effects independently of NO synthase. However, our data show that both
the NO precursor L-arginine and the NO donor SNP prevent
protection by 1 µmol/L L-NMMA. These results confirm our hypothesis
that the protection conferred by NO synthase inhibitors is
mediated by a decrease in NO production. This is further
supported by the decrease in cGMP concentration observed after L-NMMA
addition to the perfusate (Table 2).
Effect of cGMP During Ischemia and
Reperfusion
The protection against ischemic contracture conferred by
NO synthase inhibitors during low-flow ischemia
was not antagonized by 8-Br-cGMP, suggesting that cardioprotection
was not cGMP dependent. Among the cGMP-independent effects that have
been reported so far, the production of free
radicals14 might be relevant to ischemia and
reperfusion. NO also was found to stimulate ion channels15
and Na+/K+-ATPase16 in
smooth muscle cells. Whether this is applicable to the heart remains to
be demonstrated. Finally, NO can specifically inhibit
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by ADP
ribosylation of the enzyme.34 35 36 This
observation is
certainly relevant to the control of glycolysis (see discussion
below) and to the protection conferred by NO synthase
inhibitors.
It can be argued that the concentration of 8-Br-cGMP used in this study was too low to reach the cardiomyocytes. However, the impairment of functional recovery that was observed when the inhibitors were present during reperfusion could be antagonized by the cGMP analogue. There is thus a cGMP-dependent effect that is in agreement with recent reports demonstrating the beneficial effect of NO donors or L-arginine during reperfusion.18 19
Control of ATP Concentration
It has been proposed that
ischemic contracture results
from an elevation of intracellular calcium concentration and/or ATP
depletion.37 Since we have no measurement of intracellular
calcium, we can only comment on ATP. Measurements of ATP and PCr by
both NMR spectroscopy and enzyme assays indicated that high-energy
phosphates were better maintained in the ischemic hearts
treated with the inhibitors. In agreement with a previous
report,37 the delay of onset of contracture afforded by
L-NMMA is related to lower ATP depletion.
The better preservation of
ATP in the ischemic hearts treated
with L-NMMA could be due to an increased production resulting
from a stimulation of glycolysis, to a decreased utilization, or a
combination of both. Although our results show a clear stimulation of
glycolysis, the other hypothesis should not be neglected. Indeed, ATP
and PCr concentrations were already higher in treated hearts than in
controls after 5 minutes of ischemia, a time at which
glycolysis was maximally stimulated in both groups (Table 2 and
Fig 3).
In addition, during the first minutes of ischemia, glycogen
breakdown was smaller in treated than in control hearts, further
suggesting that a decreased ATP consumption should be taken into
consideration. This could suggest that NO synthase
inhibitors not only may stimulate ATP production by
glycolysis but also decrease to some extent the activity of the
ATP-consuming reactions in the heart.
Effects of NO Synthase Inhibitors on Glucose
Metabolism During Ischemia
The stimulation of glycogen breakdown and
glycolytic flux by
ischemia has been extensively studied in several experimental
models.38 39 In our model, glucose uptake and
glycolysis
were increased threefold to fourfold during the first 15 minutes of
ischemia, corresponding to the "Pasteur effect." Such an
enhancement of glucose metabolism was transient, and during
the second part of ischemia, glucose utilization progressively
decreased. The change in glucose uptake correlated with the development
of ischemic contracture. This biphasic situation was not
observed in hearts treated with low concentrations of the
inhibitors. The treated hearts displayed a sustained
glucose uptake and metabolism, and their glycogen content
was better preserved than in control hearts. These results can be
compared with previous studies in which perfusion with high
extracellular glucose improved the postischemic functional
recovery.40 41 42 43 In our
study, however, glucose
concentration was kept at physiological values,
indicating that inhibition of NO synthase stimulated glucose uptake and
metabolism through glycolysis, leading to a sparing effect
of glycogen stores.
Regarding the control of glycolysis during low-flow
ischemia, the biphasic response in the control hearts and the
effect of the NO synthase inhibitors will be discussed
separately. The early response, ie, stimulation of glycolysis by
hypoxia, results from a concerted stimulation of both glucose
transport and phosphofructo-1-kinase.44 45 This
stimulation of phosphofructo-1-kinase most probably results from
changes in the concentration of "regulatory" metabolites: a
decrease in the concentration of inhibitors such as ATP and
citrate and an increase in the concentration of the stimulators AMP and
Pi. The mechanism of stimulation of glucose transport is
not known. During the second phase of ischemia, glycolysis was
progressively inhibited in the control hearts. Inhibition of
phosphofructo-1-kinase by a drop in pHi and inhibition of
GAPDH by lactate have been proposed to explain this
situation.21 However, the progressive decrease in glucose
uptake and hexose 6-phosphate concentrations (Fig 3 and Table
2) that
is observed in control hearts during the second phase of
ischemia does not support this previous interpretation. Rather,
these changes indicate that glucose transport was progressively
inhibited and became rate limiting. If phosphofructo-1-kinase and/or
GAPDH had been rate limiting, ie, more inhibited than glucose
transport, then the concentration of metabolites upstream from the
inhibited step, ie, the concentration of hexose 6-phosphates, should be
increased. This was not the case (Table 2). We therefore
propose that
the primary event in the inhibition of glycolysis that occurred during
the second phase of ischemia in our model is the inhibition of
glucose transport; the inhibition of phosphofructo-1-kinase and GAPDH,
if any, are only secondary events.
Concerning the stimulation of glycolysis by NO synthase inhibitors, we propose that it is mediated by an increased glucose transport. Under this condition, the flux through phosphofructo-1-kinase is relatively smaller than through the transporter, and hence hexose-6-phosphates accumulate. Concerning the mechanism involved in the stimulation of glucose transport by NO synthase inhibitors, we can only say that it is independent of cGMP.
A recent study46 demonstrated that NO synthase inhibitors allow a better functional recovery after no-flow ischemia in the Langendorff-perfused rat heart. The hypothesis proposed by the authors is that these inhibitors prevent the formation of free radicals from NO. This hypothesis could explain the increase of glucose uptake by the inhibitors, as free radicals have been shown to induce glycolytic inhibition.47 However, despite a clear-cut protective effect of NO synthase inhibitors during ischemia, free radical production mainly occurs at the onset of reperfusion.48
As far as we know, this is the first study of the effects of NO synthase inhibitors on the metabolism of the ischemic heart in vitro. Whether the cardioprotection reported here can be applicable to the clinical situation remains to be demonstrated.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported in part by the National Fund for Medical Research (Belgium), the Belgian Programme on Interuniversity Poles of Attraction initiated by the Belgian Federal Services of Scientific, Technical, and Cultural Affairs, and the D.G. Higher Education and Scientific Research–French Community of Belgium. C.D. is Research Assistant of the National Fund for Scientific Research (Belgium). J.-F.G. and I.M. are supported by Grant ARC: 91/96-146 (Belgium). We thank G. Rousseau and K. Veitch for their continued interest and fruitful comments.
Received February 27, 1995; revision received April 18, 1995; accepted May 3, 1995.
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TopAbstractIntroductionMethods Results Discussion References |
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