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Cardiovascular Research 1997 33(1):82-87; doi:10.1016/S0008-6363(96)00176-9
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Copyright © 1997, European Society of Cardiology

Activation of nitric oxide synthase by ischaemia in the perfused heart

Christophe Depré, Lionel Fiérain and Louis Hue*

Hormone and Metabolic Research Unit, University of Louvain Medical School, and International Institute of Cellular and Molecular Pathology, Avenue Hippocrate, 75, UCL-7529, B-1200 Brussels, Belgium

Received 7 March 1996; accepted 24 July 1996


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Recent data have indicated that the activity of nitric oxide synthase (NO synthase), the enzyme producing NO from L-arginine, could be modified by ischaemia. The aim of the present work was therefore to study whether ischaemia activated NO synthase. Methods: NO synthase activity was measured by the conversion of radioactive arginine into citrulline in extracts of isolated perfused rabbit hearts submitted to low-flow ischaemia and reperfusion. Results: When measured in heart homogenates, NO synthase activity was significantly increased during ischaemia. This activation was already detectable after 5 min of ischaemia and was maintained during the whole ischaemic period. After cell fractionation, NO synthase was recovered in cytosolic and membrane fractions. The increase in NO synthase activity by ischaemia was related to an activation of the cytosolic activity, while the membrane-bound NO synthase activity remained constant. Conclusion: NO synthase activity in the heart is rapidly stimulated by ischaemia and this stimulation is maintained during the whole ischaemic episode. This activation is found only in the cytosolic fraction, whereas the particulate activity is not affected by ischaemia.

KEYWORDS Myocardial ischemia; Nitric oxide; cGMP; Reperfusion; Rabbit, heart; Nitric oxide synthase


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Nitric oxide synthase catalyses the conversion of L-arginine into L-citrulline and nitric oxide (NO), a biological messenger involved in physiological processes, such as neurotransmission, control of blood pressure, clotting and immune responses (for reviews, see [1, 2]). In the cardiovascular system, NO acts as a vasodilating agent by relaxing vascular smooth muscle cells [3, 4]. It also decreases the contractility of the cardiac cell [5, 6]. NO synthase activity is known to be constitutively low in the normal heart. However, it may dramatically increase when the inducible isozyme of NO synthase is expressed as in inflammatory reactions or septic shock [7–9].

Little is known about the potential role of NO synthase in the ischaemic heart. However, several lines of evidence suggest that NO might be involved in the coronary autoregulation during ischaemia, since NO production from the coronary endothelium decreases vascular resistance in ischaemic heart [10, 11]. Moreover, some effects of NO are mediated through the production of cyclic GMP (cGMP)[12] and an increase in cGMP content has been described in the perfused rat heart submitted to no-flow ischaemia[13]. This increase was totally suppressed by inhibitors of NO synthase [13]. Finally, the addition of these inhibitors to the perfusate of rabbit hearts submitted to low-flow ischaemia decreased the ischaemic injury by stimulating glucose metabolism in these hearts [14]. This cardioprotection was lost when the hearts were perfused with L-arginine, a precursor of NO, or with sodium nitroprusside, a NO donor [14].

Taken together, these data suggest that ischaemia could increase the production of NO through activation of NO synthase. Therefore, in this study, we investigated the activity of NO synthase in perfused rabbit hearts submitted to low-flow ischaemia and reperfusion. We report here several properties of the constitutive heart NO synthase and its activation by ischaemia.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
The aim of this work was to study the effect of ischaemia on heart NO synthase activity. This section describes the methods used to obtain the samples, the homogenisation and fractionation procedures applied to the samples, and the assay of NO synthase activity and its validation.

2.1. Heart perfusion
NO synthase activity was measured in hearts perfused under normoxic conditions or submitted to different periods of low-flow ischaemia or reperfused. The protocol was the following. Hearts of anaesthetised (50 mg/kg Nembutal intravenously) male New-Zealand rabbits (1.7–2.5 kg) fed ad libitum were quickly removed and dropped in ice-cold buffered 0.15 mol/l NaCl, then perfused as described[14, 15]. Briefly, the hearts were retrogradely perfused at a constant flow of 5 ml/min per g wet wt with a Krebs-Henseleit buffer (1.5 mmol/l CaCl2) containing 5.5 mmol/l glucose and equilibrated with a 95% O2/5% CO2 gas phase. The left ventricular end-diastolic pressure (LVEDP) was set at 10 mmHg by filling a latex balloon inserted in the left ventricle. Each heart was paced at 180 bpm. Left ventricular developed pressure (LVDP), heart rate and coronary perfusion pressure (CPP) were recorded on line. After 30 min of perfusion in normoxic conditions (equilibration period), the flow was reduced to 0.20 ml/min per g wet wt and the perfusate was equilibrated with 95% air/5% CO2 to ensure low-flow ischaemia for a 60 min period. The hearts were maintained at 37°C by immersion in a thermostated reservoir filled with buffer. Ischaemic contracture, characterised by an increase in LVEDP, was measured as an index of irreversible damage[16]. The onset of ischaemic contracture was defined as the time at which LVEDP increased by more than 5 mmHg when compared to the preischaemic values. After 60 min of low-flow ischaemia, the flow rate and oxygen were restored to pre-ischaemic values and perfusion was continued for 15 min (reperfusion period). To measure NO synthase activity, hearts were freeze-clamped between aluminium tongs precooled in liquid nitrogen. The hearts were frozen at the end of the equilibration period, at several time points during low-flow ischaemia and after 15 min of reperfusion.

2.2. Preparation of cytosol and membrane fraction
Freeze-clamped heart samples were homogenised at 4°C with an Ultraturrax in 2 vol of homogenisation buffer containing 50 mM Tris (pH 7.4 at 4°C), 250 mM sucrose, 50 mM KCl, 5 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA, 1 mM benzamidine, 0.1 mM phenylmethyl-sulfonylfluoride (PMSF), as well as 1 µg/ml of pepstatin and leupeptin (all from Boehringer, excepted PMSF from Sigma). After centrifugation (10 000 x g for 15 min), the supernatant was used as such for assays of NO synthase. In some experiments the supernatant was further centrifuged (100 000 x g for 40 min) to separate the cytosol from the membrane fraction. The membrane fraction, which corresponded to the 100 000 x g pellet, was washed twice in the homogenisation buffer and finally resuspended in the same buffer. Before testing NO synthase activity, all samples were cleared of endogenous L-arginine by passing 5 ml of the preparation through 1 ml of Dowex AG-50W8 resin (200–400 mesh, Na+-form; Biorad), equilibrated with the homogenisation buffer [17].

2.3. NO synthase assay
NO synthase activity was measured by the formation of radioactive L-citrulline from 3H-labelled L-arginine as described previously [18]. Briefly, a 150 µl sample of the enzyme preparation (homogenate, cytosol or membrane fraction) was added to 75 µl of assay buffer containing 50 mM HEPES (pH 7.4 at 4°C), 0.2 mM NADPH, 0.5 mM CaCl2, 0.1 mM tetrahydrobiopterin (BH4, Calbiochem), 50 µM FAD, 50 µM FMN (Sigma) and 1 µg/ml calmodulin (Boehringer). The reaction was initiated by the addition of 25 µl of 0.5 mM L-[2,3,4,5-3H]arginine (Amersham) (50 µCi/nmol); for measurement of Km values, 25 µl of 10–250 µM L-arginine containing 2 µCi of radioactive L-arginine were added to obtain final concentrations of 1–25 µM L-arginine in the assay. The reaction was stopped by the addition of 0.15 ml of an ice-cold solution of 50 mM HEPES (pH 7.4) containing 10 mM L-citrulline as a carrier, 10 mM EGTA and 10 mM L-N-arginine methylester (L-NAME, Sigma), an inhibitor of NO synthase. The stopped reaction mixture was then applied on a column containing 1 ml of DOWEX 50W8 (Na+-form, 200–400 mesh) equilibrated in 50 mM HEPES at pH 7.0. Radioactive L-citrulline was eluted with 2 ml of the same buffer whereas L-arginine was retained on the column. We verified that at this pH the chromatographic separation of L-citrulline and L-arginine was observed as expected [9, 17, 18].

The test was linear for enzyme samples containing 0.4–1.3 mg of protein and for incubation periods up to 45 min at 37°C. Routinely, a 30 min incubation period was used. For each sample, the assay was run in duplicate and a blank value was obtained by adding the stop solution before the incubation.

In some experiments, the inhibitory effects of L-N-monomethylarginine (L-NMMA, Calbiochem) or aminoguanidine (Sigma), two inhibitors of NO synthase, were tested. The concentrations used are indicated in the text.

2.4. Other methods and expression of the results
The tissue content of L-arginine was measured in hearts perfused under normoxic conditions or after 15 min of ischaemia. Frozen heart samples were deproteinised in 10% perchloric acid and the deproteinised extracts were neutralised by KHCO3. The measurement of L-arginine concentration was performed after separation of the amino acids by anion exchange chromatography on an automatic aminoacid analyser routinely used for clinical studies (LKB alpha plus).

NO synthase activity is expressed as microunits (µU), i.e. pmol of L-citrulline formed per min, per mg of proteins, measured according to the Lowry method [19]. The number of hearts used in each experimental protocol is indicated in the legends to the figures. The data presented are the means ± s.e.m. for the indicated number of samples. The two-tailed Student's t-test was used for statistical comparisons when necessary and a value of P < 0.05 was considered as statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Effects of ischaemia on NO synthase activity in perfused rabbit hearts
Rabbit hearts were perfused, as described in the Methods, under normoxic conditions for 30 min (equilibration period) followed by low-flow ischaemia for 60 min and reperfusion in normoxic conditions for 15 min. We first established the physiological parameters of this model. At the end of the equilibration period, LVDP was 75 ± 8 mmHg and CPP was 40 ± 3 mmHg. During low-flow ischaemia, the hearts progressively developed an ischaemic contracture, the onset of which was observed at 25 ± 5 min. After 60 min of ischaemia, LVEDP reached a maximal value of 55 ± 5 mmHg. After 15 min of reperfusion, LVDP was 28 ± 5 mmHg (i.e., a functional recovery of 37% of the preischaemic values). These values are similar to those previously reported [14, 15] and correspond to the experimental model in which protection against ischaemic damage by inhibitors of NO synthase was previously observed [14].

NO synthase activity was first measured in homogenates prepared from hearts perfused for 30 min in normoxic conditions or 30 min normoxia followed by 15 min ischaemia. The effect of substrate (L-arginine) concentration on the rate of reaction was studied and the results are illustrated in Fig. GR1. From Lineweaver plots, we calculated a maximal activity (Vmax) of 0.70 ± 0.07 µU/mg of protein in normoxic hearts and of 1.05 ± 0.05 µU/mg of protein in the hearts submitted to 15 min period of ischaemia (P < 0.05). The apparent calculated Km values for L-arginine were the same in both conditions and equal to 8.9 ± 0.3 µM.


Figure 1
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  		Fig. GR1 Influence of the concentration of L-arginine on the activity of NO synthase. Left panel represents the NO synthase activity in normoxic (open symbols) and ischaemic hearts (filled symbols) in total heart homogenates. Right panel represents the corresponding values with the double-reciprocal linearised plot of Lineweaver and Burk for normoxic (open symbols) and ischaemic hearts (filled symbols). Each point represents the mean ± s.e.m. for 6 normoxic and 7 ischaemic hearts.

 
To compare this Km value to the concentration in intact tissues, we measured the tissue content of L-arginine in deproteinised extracts of hearts perfused in the same conditions as above (i.e., normoxia or 15 min ischaemia). The values (250 ± 20 nmol/g wet wt in normoxic and 340 ± 30 nmol/g wet wt in ischaemic hearts; n = 5; P < 0.05) correspond to 500–680 µM L-arginine assuming that 1 g wet wt contains about 0.5 ml of intracellular water. These concentrations largely exceed the apparent Km values for L-arginine in both groups.

The extracts from both normoxic and ischaemic hearts were then submitted to high-speed centrifugation in order to separate the cytosol from the membranes and NO synthase activity was measured in these two fractions. In normoxic hearts, NO synthase activity was recovered in both fractions (Fig. GR2). When measurements were performed on hearts submitted to 15 min ischaemia, the activity in the cytosolic fraction was doubled (Fig. GR2 A). The activity in the presence of a saturating concentration of L-arginine (50 µM) was 0.72 ± 0.07 and 1.40 ± 0.20 µU/mg of protein for normoxic and ischaemic hearts, respectively. The Km, calculated from Lineweaver plots, was 9.0 ± 0.4 µM and did not differ in the two conditions. No change in activity at 50 µM L-arginine (0.35 ± 0.05 µU/mg of protein) or Km (7.8 ± 0.5 µM) was seen in the membrane fraction (Fig. GR2 B).


Figure 2
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  		Fig. GR2 Influence of the concentration of L-arginine on the activity of NO synthase in cytosolic (panel A) and membrane fraction (panel B). Open symbols represent normoxic hearts and filled symbols represent hearts freeze-clamped after 15 min ischaemia. Each point represents the mean ± s.e.m. for 6 normoxic and 6 ischaemic hearts.

 
The time-course of activation of the cytosolic NO synthase during ischaemia was finally analysed. The activity of NO synthase present in cytosolic fractions prepared from hearts submitted to different periods of ischaemia was measured in the presence of a saturating concentration (50 µM) of L-arginine. Fig. GR3 shows that activation was already maximal after 5 min of ischaemia and was maintained during the whole period of 60 min of ischaemia, despite a decrease after 30 min of ischaemia. After 15 min of reperfusion, the activity was similar to the preischaemic values (Fig. GR3). No change in activity was observed in the membrane fraction at these different time points (0.34 ± 0.03, 0.30 ± 0.05 and 0.32 ± 0.07 µU/mg of protein before ischaemia and after 30 and 60 min of ischaemia, respectively).


Figure 3
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  		Fig. GR3 Time-course of cytosolic NO synthase activity before ischaemia (0–30 min), during ischaemia (from 30–90 min) and during reperfusion (from 90–105 min). Filled symbols represent NO synthase activity measured in the cytosolic fraction and the open symbol represents the corresponding activity in hearts perfused under normoxic conditions during 90 min. * P < 0.01 vs. corresponding values before ischaemia. Each point of the figure represents the mean±s.e.m. for 5–8 different hearts.

 
3.2. Characterisation of NO synthase activities
As the different isozymes of NO synthase are characterised by their dependence on calcium/calmodulin and by their sensitivity to analogues of L-arginine [20], the NO synthase activity of the two subcellular fractions prepared from heart homogenates was assessed in the presence of EGTA or in the presence of various concentrations of NO synthase inhibitors. The addition of 10 mM EGTA decreased the cytosolic activity by about 45% in both normoxic and ischaemic hearts, but reduced the membrane activity by 75% in these two groups. Both fractions were inhibited by L-NMMA (Fig. GR4), with an IC50 of 10–6 M and 10–4 M for the membrane-bound and the cytosolic activity, respectively. However, with aminoguanidine, considered as a specific inhibitor of inducible NO synthase[21, 22], the activity was not altered in either fraction (Fig. GR4), except for a slight inhibition (85 ± 5% of control activity) at high concentration (10–2 M) of the inhibitor. In both cases, the sensitivity to the inhibitor was similar when comparing normoxic and ischaemic hearts.


Figure 4
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  		Fig. GR4 Influence of L-NMMA (circles) and aminoguanidine (squares) on the inhibition of NO synthase in cytosolic fraction (open symbols) and membrane fraction (closed symbols). Each point represents the mean for 4 different hearts. For the sake of simplicity, error bars have not been included, the s.e.m. being less than 10% of the means.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Our data show that rabbit heart NO synthase is activated by ischaemia. Activation is rapid, persists during the whole ischaemic episode and disappears during reperfusion. We also show that NO synthase activity is present in both the cytosolic and the membrane fractions. Ischaemia only increased the cytosolic activity. To the best of our knowledge, this is the first study that demonstrates a kinetic modification of NO synthase induced by ischaemia in the heart. Our data correlate the increased production of NO in hypoxia [23–25] to a stimulation of the maximal activity of NO synthase.

4.1. NO synthase isozymes
Three isozymes of NO synthase (NOS) are known[9, 20]: the nNOS (neuronal, or type I), the iNOS (inducible, or type II) and the eNOS (endothelial, or type III) isozymes. They differ in several characteristic features, including their constitutive or inducible expression, their subcellular distribution and their dependence to calcium/calmodulin. The nNOS isozyme is a constitutive, soluble, calcium-dependent isozyme [18]. The eNOS isozyme shares the same properties, except that it is mainly bound to membranes, because of the presence of a consensus myristoylation site at the N-terminus [26]. A small part of eNOS activity is however detected in the soluble fraction[9]. In contrast to nNOS and eNOS, the iNOS isozyme is not constitutively expressed, but is induced by incubation of different cell types (as macrophages, hepatocytes or cardiomyocytes, for instance) with cytokines [27, 28]. The iNOS isozyme also differs from the other isozymes due to its independence of calcium/calmodulin [29] and to the fact that it is specifically inhibited by aminoguanidine[21, 22].

The range of activity that we found in our experiments is similar to the values reported previously for rat heart homogenates [30]. The activities present in cytosol and membrane fractions are constitutive and not inducible because they were present under basal conditions and because they were not inhibited by aminoguanidine, a specific inhibitor of iNOS. The membrane-bound activity is probably the eNOS isozyme since, like eNOS, it is bound to membranes, inhibited by EGTA, is calcium-dependent and sensitive to L-NMMA, but not to aminoguanidine. The cytosolic activity is partly calcium-independent and less sensitive to L-NMMA. It could correspond to nNOS, the cytosolic isozyme, although a stronger inhibition by EGTA would be expected. However, eNOS (or NOS III) has been reported to be the only isozyme constitutively expressed in isolated cardiomyocytes [31]. Therefore, as far as nNOS should be excluded, we conclude that the activity measured in the cytosol and activated by ischaemia represents the soluble fraction of the eNOS isozyme. Its lower sensitivity to EGTA and L-NMMA, when compared to membrane-bound activity, remains unexplained, however.

4.2. Activation of NO synthase by ischaemia
The changes in kinetic properties induced by ischaemia (i.e., increase in Vmax without change in Km for L-arginine) should result in an increased production of NO even in the presence of a saturating concentration of arginine. Therefore, our results explain the increase in cGMP that was observed in ischaemic hearts [13] and the recently reported stimulation of NO production in ischaemic hearts. Indeed, several reports have shown that NO production increases in the ischaemic and hypoxic heart [24, 25], as well as in coronary endothelium submitted to hypoxia [23]. Such stimulation of NO production seems to represent one of the major mediators of hypoxia-induced vasodilatation[32, 33], as well as exercise-induced vasodilatation in the presence of a flow-limiting stenosis [34].

The mechanism of the ischaemia-induced activation of NO synthase is not known. It does not correspond to an increased expression of inducible iNOS, because such activation is too fast to result from a change in protein content, and because the activity is not inhibited by aminoguanidine. The ischaemia-induced activation could result from an allosteric stimulation of the enzyme or (and) a covalent modification (e.g., after phosphorylation by a protein kinase). We have no direct evidence for any of these interpretations, although the experimental conditions of our assay (dilution of the extracts, addition of saturating concentrations of effectors) are more suitable to detect a stable change in activity as a result of covalent modification. Clearly, additional investigations are required to elucidate the mechanism of activation. It should however be borne in mind that oxygen also represents a substrate of this enzyme and its concentration is therefore crucial for enzyme activity. The Km of NO synthases for oxygen has been recently determined and ranges from 5 to 20 µM[35]. Therefore, the enzyme is saturated with oxygen in normoxic conditions, but NO production should fall in hypoxic conditions, when oxygen is limiting NO synthase activity. The opposite is observed [23–25]. The mechanism by which NO production is maintained or increased despite decreased oxygen concentration is presently unknown, and this underlines the complexity of the regulation of NO synthases. It could be related to a change in Km for oxygen in the ischaemia-activated enzymes. Another potential mechanism of activation of NO synthase during ischaemia could involve the activation of G-proteins (e.g., through the stimulation of {alpha}-adrenoreceptors at the onset of ischaemia).

Our recent observation [14] of the cardioprotection confered by inhibitors of NO synthase in rabbit hearts submitted to severe low-flow ischaemia suggests that NO plays an important role in the ischaemia-induced damage of cardiomyocytes. This protection has been proposed to be related to a stimulation of glucose uptake and metabolism[14]. Which step of glucose metabolism is directly or indirectly affected by NO is still unknown, although some indirect evidence indicates that glucose transport could be controlled by this biological effector.


   Acknowledgements
 
We are indebted to L. Maisin for technical assistance. C.D. is Research Assistant of the National Fund for Scientific Research (Belgium). This work was supported by research grants from the D.G. Higher Education and Scientific Research—French Community of Belgium, from the Fund for Medical Scientific Research (Belgium) and from the Interuniversity Poles of Attraction Programme initiated by the Belgian Federal Services.


   Notes
 
* Corresponding author. Tel. +32 2 7647529; fax +32 2 7627455. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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