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Circulation. 1995;92:1911-1918

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(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

Christophe Depré, MD; Jean-Louis Vanoverschelde, MD; Jean-François Goudemant, PhD; Isabelle Mottet, MD; Louis Hue, MD, PhD

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.


*    Abstract
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 *Abstract
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 down arrowMethods
down arrowResults
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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


*    Introduction
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 up arrowAbstract
 *Introduction
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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.


*    Methods
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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 1Down 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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  		Table 1. Effect of NO Synthase Inhibitors and Other Agents on Cardiac Function During Normoxia, Ischemia, and Reperfusion

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.


*    Results
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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 1Up), 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 1Up) by an increase in end-diastolic pressure.

When the hearts were submitted to severe low-flow ischemia, they progressively developed a contracture (Fig 1Down), 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 1Down and Table 1Up). This effect was not observed at a vasoconstrictive concentration (100 µmol/L) of L-NMMA (Table 1Up). Protection against ischemic contracture also was observed with 1 µmol/L L-NAME, another inhibitor of NO synthase (Table 1Up). The protection was enantiomer-specific, as it was not observed with 1 µmol/L D-NMMA (Table 1Up), 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 1Up). Both L-arginine and SNP slightly decreased CPP by vasodilation (Table 1Up). 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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  		Figure 1. Plot shows time course of protection against ischemic contracture by L-NMMA. The time course of the development of ischemic contracture in control hearts (open circles) is compared with that in hearts perfused with 1 µmol/L L-NMMA (filled circles). The perfusion procedure is described in "Methods." Values are mean±SEM for at least six hearts in each group. *P<.01 vs corresponding control values.

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 1Up). 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 1Up). 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 1Up).

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 2Down), 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 1Up). 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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  		Table 2. Concentration of Hexose-Phosphates, cGMP, High-Energy Phosphates, Pi, and pHi in Control Hearts and in Hearts Perfused With 1 µmol/L L-NMMA

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 2Down 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 2Up) 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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  		Figure 2. 31P spectra of high-energy phosphates and Pi during ischemia and reperfusion. Example of 31P NMR spectra of controls (A) and hearts perfused with 1 µmol/L L-NMMA (B). The perfusion procedure was as for Table 1Up and is described in "Methods." Values obtained for ATP and PCr were comparable to those presented in Table 2Up, which were obtained by biochemical assays on deproteinized extracts. The chemical shift of Pi (the peak of which is represented by a vertical line) was used to estimate pHi; corresponding values are given in Table 2Up.

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 2Up). Similarly, the increase in Pi was less in the treated group (Table 2Up). During reperfusion without the inhibitor, the pretreated hearts presented better recovery of PCr and ATP content (Fig 2Up).

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 3Down, the onset of ischemia was rapidly followed by a marked increase both in glucose uptake (Fig 3ADown) and in lactate production (Fig 3BDown), 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 1Up and Table 1Up). 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 3ADown and 3BDown). This corresponded to the marked delay in the onset of the ischemic contracture (Fig 1Up). 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 3CDown).



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  		Figure 3. Plots show glucose uptake (A), lactate production (B), glycogen content (C), and detritiation rate of [3-3H]glucose (D) during ischemia. Controls (open circles) were compared with hearts perfused with 1 µmol/L L-NMMA (filled circles). Glycogen is expressed as glucose equivalents. Values are mean±SEM for n=6 in each group. *P<.01 vs corresponding control values.

The stimulation of glycolytic flux by L-NMMA was confirmed by a higher rate of detritiation of [3-3H]glucose (Fig 3DUp), 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 2Up) 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 4Down, 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 4Down), thus reinforcing the hypothesis that the functional protection is incident to the stimulation of exogenous glucose metabolism.



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  		Figure 4. Dose-response curve of protection against ischemic contracture and stimulation of glucose uptake by L-NMMA. Dose-response curve of the effect of L-NMMA on ischemic contracture (filled squares) and glucose uptake (open squares) is compared with the corresponding values in untreated controls (ctrl). Values are mean±SEM for n=5 in each group and were measured at the end of the 60-minute period of ischemia. *P<.01 vs corresponding control values.


*    Discussion
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*Discussion
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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 2Up).

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 2Up and Fig 3Up). 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 3Up and Table 2Up) 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 2Up). 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.


*    Selected Abbreviations and Acronyms
 
8-Br-cGMP = 8-bromo-cGMP
CPP = coronary perfusion pressure
D-NMMA = D-N-monomethylarginine
L-NAME = L-N-arginine methylester
L-NMMA = L-N-monomethylarginine
LVDP = left ventricular developed pressure
LVEDP = left ventricular end-diastolic pressure
NMR = nuclear magnetic resonance
NO = nitric oxide
PCr = phosphocreatine
pHi = intracellular pH
Pi = inorganic phosphate
SNP = sodium nitroprusside


*    Acknowledgments
 
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.


*    References
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*References
 

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