Impact of NO on ET-1- and AM-induced inotropic responses: potentiation by combined administration

¹ Department of Pharmacology and Toxicology, Biocenter Oulu, 90014 University of Oulu, Finland; ² First Department of Medicine, Semmelweis University of Medicine, 1089 Budapest, Hungary; and ³ Department of Medicine, Christchurch Hospital, 8001 Christchurch, New Zealand

	ABSTRACT

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We characterize herein the impact of myocardial nitric oxide (NO) synthesis on the inotropic response to two cardioactive peptides, endothelin-1 (ET-1) and adrenomedullin (AM). In the isolated perfused rat heart preparation, intracoronary infusion of AM (0.03 and 1 nmol/l) and ET-1 (0.08 and 1 nmol/l) for 30 min induced a dose-dependent, gradual increase in developed tension, the maximal responses being equal. Inhibition of myocardial NO synthase (NOS) by N-nitro-L-arginine methyl ester (L-NAME; 300 µmol/l) enhanced the inotropic response to ET-1 at a concentration of 1 nmol/l; meanwhile, the effect of AM was not augmented significantly. The inotropic response to simultaneous administration of low, equipotent doses of AM (0.03 nmol/l) and ET-1 (0.08 nmol/l) was significantly smaller than that of either peptide alone. This depressed response was more than overcome by concomitant administration of L-NAME. In conclusion, this study reveals that the maximal inotropic response to ET-1 can be augmented by inhibition of myocardial NOS, whereas it has only a minor impact on the effect of AM. The inotropic response to combined administration of low doses of AM and ET-1 is substantially suppressed by endogenous NO, whereas the individual effects of the peptides at these doses are not the subject of secondary modulation by NO.

myocardial contractility; N-nitro-L-arginine methyl ester; perfused rat heart

	INTRODUCTION

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IN ADDITION TO THE INFLUENCE of the autonomic nervous system and mechanical loading of ventricular muscle, there are a number of substances synthesized in the myocardium that might alter contractility in an autocrine/paracrine manner (42, 52). The potent vasoconstrictor peptide endothelin-1 (ET-1), originally isolated from the supernatant of endothelial cells (54), is synthesized also by cardiac myocytes (45) and has specific receptors in the heart (1, 39). In vitro studies have shown that ET-1 is the most potent inotropic substance on a molar basis identified so far (18, 22). More recently, adrenomedullin (AM), a vasodilating peptide belonging to the calcitonin gene-related peptide family (23), has also been reported to have specific binding sites in the myocardium (31). Furthermore, AM is actively produced by cardiac myocytes (16, 49) and nonmyocytes (16, 44), indicating the existence of a potential autocrine/paracrine regulatory loop. Indeed, in intact animals AM has a strong cardiostimulatory effect in addition to its hypotensive action (34). In this regard, we reported recently that AM has a direct positive inotropic effect in the isolated perfused rat heart preparation (46, 47). Both ET-1 (19) and AM (39) possess a unique, slowly developing but sustained inotropic effect that clearly differs from the responses to -adrenergic agonists, which develop rapidly, usually over a matter of seconds (22, 47), suggesting a potentially different role in the regulation of cardiac contractility.

Nitric oxide (NO) is a free radical gas that mediates endothelium-dependent vasodilation (10, 33). NO synthesis has also been shown to contribute to the vasodilator action of AM (13) and to oppose ET-1-induced vasoconstriction (7, 25). A growing body of evidence suggests that NO synthesis occurs also in the myocardium, contributing importantly to the regulation of cardiac contractility (12, 21). Two isoforms of NO synthase (NOS), neuronal (nNOS, Ref. 53) and endothelial (eNOS, Ref. 41), constitutively catalyze the formation of NO from L-arginine in the heart. The third isoform, inducible NOS (iNOS), is expressed only under pathophysiological conditions (6). Interestingly, myocardial NO production has been shown to attenuate the inotropic responses to -adrenergic agonists both in vitro (2, 8, 50) and in vivo (20). More recently, however, it was reported that inotropic responses to -adrenergic stimulation were preserved in mice deficient in eNOS (51). These conflicting observations prompted us to determine whether myocardial NOS can modulate the inotropic responses to substances other than -adrenergic agonists. We were particularly interested in the autocrine/paracrine cardioactive factors ET-1 and AM, which possess potent, unique inotropic effects differing qualitatively from -adrenergic agonists. Furthermore, although a number of locally synthesized substances have been reported to have profound effects on cardiac contractility (42, 52), little information is available on interactions among these factors. Therefore, our second objective was to study inotropic responses to combined administration of AM and ET-1 in the isolated perfused rat heart preparation.

	MATERIALS AND METHODS

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Drugs. Drugs used were rat adrenomedullin-(1---50) (Phoenix Pharmaceuticals), endothelin-1 (Peninsula Laboratories), N-nitro-L-arginine methyl ester (L-NAME, Sigma), and bosentan (generously supplied by Dr. Martine Clozel, Hoffmann-La Roche).

Animals. Male Sprague-Dawley rats (n = 96, weighing 242 ± 2 g) from the colony of the Center of Experimental Animals at the University of Oulu were used. The rats were housed in plastic cages in a room with a controlled humidity of 40% and a temperature of 22°C. A 12:12-h light-dark environmental light cycle was maintained. The experimental design was approved by the Animal Use and Care Committee of the University of Oulu. The study protocol conforms with the European convention for the protection of vertebrate animals used for experimental and other scientific purposes.

Isolated perfused rat heart preparation. The isolated perfused rat heart preparation used in this study was similar to that described previously (27, 47). Briefly, rats were decapitated and hearts were quickly removed and arranged for retrograde perfusion by the Langendorff technique. The hearts were perfused with a modified Krebs-Henseleit bicarbonate buffer, pH 7.40, equilibrated with 95% O₂-5% CO₂ at 37°C. The composition of the buffer was (in mmol/l) 113.8 NaCl, 22.0 NaHCO₃, 4.7 KCl, 1.2 KH₂PO₄, 1.1 MgSO₄, 2.5 CaCl₂, and 11.0 glucose.

Contractile force (apicobasal displacement) was obtained by connecting a force displacement transducer (Grass Instruments, FT03) to the apex of the heart at an initial preload stretch of 2 g. Heart rate was counted from contractions by the Grass tachograph and was increased 15-20% above the spontaneous beating frequency by using a Grass stimulator (model S88, 11 V, 0.5 ms) to avoid any secondary effects caused by changes in heart rate. Variations in perfusion pressure arising from changes in coronary vascular resistance were measured by a pressure transducer (Micron Instruments, MP-15) situated on a side arm of the aortic cannula. All recordings were made using a Grass 7DA polygraph. Each experiment was started by perfusing the hearts for 60 min (equilibration period) using a flow rate of 7 ml/min with a peristaltic pump (Minipuls 3, model 312). To diminish the basal NO synthesis, which is regulated by the level of vascular shear stress (24), the flow rate was then decreased to the final experimental rate of 5.5 ml/min, including the flow rate of the infusion cannula as described previously (27, 47).

Experimental design. A 10-min control period was followed by addition of vehicle, AM or ET-1, into the aortic perfusion cannula as a continuous infusion via an infusion pump (Skyelectronics, Secan PSA 55) at a rate of 0.5 ml/min for 30 min. All hearts were used for one experiment only, and the study was conducted in a controlled and randomized manner, i.e., vehicle and substances were run concomitantly and randomly. Preliminary experiments were performed to determine the maximal responses to AM and ET-1 and also concentrations of the peptides resulting in clearly submaximal but equal responses. L-NAME (300 µmol/l), a NOS inhibitor, and bosentan (1 µmol/l), an ET_A/B receptor antagonist, were infused alone and in combination with AM and/or ET-1. These concentrations of L-NAME [P. Taskinen, O. Vuolteenaho, and H. Ruskoaho (unpublished data) and others (32)] and bosentan (27) are known to be effective in the Langendorff preparation.

Statistics. Results are presented as means ± SE. The data were analyzed with two-way ANOVA for repeated measurements. The statistical differences between two groups for one parameter were determined with Student's t-test. Differences were considered statistically significant at the level of P < 0.05.

	RESULTS

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Effects of AM and ET-1 on myocardial contractility. Initially, we compared the effects of AM and ET-1 on cardiac contractility under the same experimental conditions. Infusion of vehicle alone did not significantly alter the contractile parameters. Administration of AM (0.03 and 1 nmol/l) and ET-1 (0.08 and 1 nmol/l) induced dose-dependent increases in developed tension (Fig. 1, Table 1). The maximal responses to AM and ET-1 at the concentration of 1 nmol/l were equal. Moreover, AM at 0.03 nmol/l elicited an equivalent inotropic effect to ET-1 at 0.08 nmol/l (Fig. 1, Table 1). Based on the present results AM appears to be one of the most potent endogenous inotropic substances yet identified.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effect of adrenomedullin (AM) and endothelin-1 (ET-1) on developed tension (DT) in isolated perfused, paced rat hearts. After a control period, as shown by the arrow, vehicle or peptides were added to the perfusion fluid for 30 min. Results are expressed as a percent change vs. baseline values. Each point is the mean ± SE from 6 to 9 separate experiments run on different isolated rat hearts. Number of experiments and baseline values of contractility in each group are presented in Table 1. * F = 20, P < 0.001 AM vs. vehicle.  F = 31.8, P < 0.001 ET-1 vs. vehicle.  F = 21.4, P < 0.001 vs. vehicle and F = 6.1, P = 0.005 vs. 0.08 nmol/l ET-1. § F = 45.9, P < 0.001 vs. vehicle and F = 11.6, P < 0.001 vs. 0.03 nmol/l AM for drug and time interaction by 2-way ANOVA.

View this table: [in this window] [in a new window]   Table 1.   Changes in developed tension in the experimental groups

In agreement with previous reports (22, 47), both peptides elicited a unique, slowly developing effect, but an apparent difference in the time profiles could be observed (Fig. 1). The mean time to reach the maximum response was 25.4 ± 1.3 min with AM and 15.7 ± 2.7 min with ET-1 (P < 0.01). In view of the known potent inotropic action of ET-1 and because the response to AM developed in a relatively sluggish manner, we addressed the possibility that the inotropic effect of AM might be mediated by ET-1 by infusing the peptides in the presence or absence of the ET_A/B receptor antagonist bosentan. As expected, the ET-1-induced increase in developed tension was abolished by simultaneous administration of 1 µmol/l bosentan, but the response to AM remained unchanged (Table 1). Thus the inotropic action of AM was not apparently dependent on stimulation of endogenous ET-1 or its receptors.

Influence of inhibition of myocardial NOS on ET-1- and AM-induced positive inotropic responses. When L-NAME, an inhibitor of NO synthase, was infused alone into the coronary circulation, the contractile parameters remained constant (Fig. 2, Table 1), indicating that the impact of NO on basal contractility is minimal under our experimental conditions. In contrast, the early phase of the inotropic response to 1 nmol/l ET-1 was markedly augmented by L-NAME (Fig. 2A, Table 1). The inotropic effect of AM at 1 nmol/l was unchanged by inhibition of NOS, although there appeared to be a trend toward an enhanced effect with the lower dose of AM (Fig. 2B, Table 1, not significant).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   A: effect of nitric oxide synthase (NOS) inhibition on the positive inotropic response to ET-1. After a control period, as shown by the arrow, ET-1 was infused alone or in combination with L-NAME (300 µmol/l) to the perfusion fluid for 30 min. B: effect of NOS inhibition on the positive inotropic response to AM. After a control period, as shown by the arrow, AM was infused alone or in combination with L-NAME (300 µmol/l) to the perfusion fluid for 30 min. Results in both panels are expressed as a percent change vs. baseline values. Each point is the mean ± SE from 6 to 9 separate experiments run on different isolated hearts. Number of experiments and baseline values of contractility in each group are presented in Table 1. * F = 4.62, P = 0.004 vs. ET-1 (first 15 min of infusion) for drug and time interaction by 2-way ANOVA.

Combined effect of AM and ET-1 on myocardial contractility: modulation by NO. Equipotent doses of AM and ET-1 (0.03 and 0.08 nmol/l, respectively), which induced submaximal inotropic responses, were chosen to permit identification of a potential additive effect of them. Unexpectedly, combined administration of AM and ET-1 induced a significantly smaller increase in developed tension than either peptide alone (Fig. 3, Table 1). Although the individual responses to the peptides at these doses were not increased significantly by L-NAME treatment (Fig. 2), we tested whether NO was responsible for the observed attenuated effect induced by simultaneous infusion of AM and ET-1. Indeed, concomitant administration of L-NAME more than reversed the depressed inotropic response to the two peptides, showing that the degree of suppression elicited by NO was potentiated (Fig. 3, Table 1). When bosentan was added to this combination in the presence of L-NAME, the response was significantly decreased, suggesting that ET receptors are essential in the potentiation effect (data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Interaction of AM and ET-1 in the isolated perfused, paced rat heart. After a control period, as shown by the arrow, combination of submaximal concentrations of AM (0.03 nmol/l) and ET-1 (0.08 nmol/l) was added to the perfusion fluid in the presence or absence of NOS inhibitor L-NAME (300 µmol/l) for 30 min. Results are expressed as a percent change vs. baseline values. Each point is the mean ± SE from 6 to 9 separate experiments run on different isolated hearts. Number of experiments and baseline values of contractility in each group are presented in Table 1. * F = 2.93, P = 0.005 vs. AM.  F = 5.66, P < 0.001 vs. ET-1.  F = 14.8, P < 0.001 vs. AM plus ET-1 for drug and time interaction by 2-way ANOVA.

Effect of AM, ET-1, and L-NAME on other hemodynamic parameters. The initial resting tension (2.0 ± 0.03 g) of the perfused hearts was not significantly affected by AM and ET-1 at the lower dose or by L-NAME. At the higher concentration (1 nmol/l), ET-1-induced a slight increase in resting tension to 2.6 ± 0.06 g (P < 0.001 vs. vehicle). The average heart rate, maintained by atrial pacing, was 309 ± 2 beats/min with no significant differences between the experimental groups.

The mean perfusion pressure of the control hearts, generated by constant coronary flow, was initially 32.1 ± 0.9 and 32.8 ± 1.0 mmHg after 30 min of vehicle infusion. There were no significant differences in baseline values between the experimental groups. Overall, changes in perfusion pressure were small but the expected and characteristic vascular effects of AM and ET-1 were observed. Despite near maximum dilatation of the coronary arteries induced by the relatively low coronary flow rate, AM slightly but significantly decreased the perfusion pressure from 30.9 ± 0.9 to 29.2 ± 1.0 mmHg and from 32.9 ± 1.7 to 28.4 ± 1.2 mmHg, at doses of 0.03 and 1 nmol/l, respectively (P < 0.001, AM vs. vehicle). ET-1 by contrast increased perfusion pressure but only at the higher dose (from 31.4 ± 0.6 to 36.9 ± 1.7 mmHg, P < 0.001, ET-1 1 nmol/l vs. vehicle). L-NAME alone had no effect, but it significantly enhanced the vasoconstrictor effect of ET-1 at the higher dose, the increase in perfusion pressure becoming apparent after 10 min and reaching the maximum at 30 min (from 33.0 ± 0.8 to 60.4 ± 6.4 mmHg, P < 0.001).

	DISCUSSION

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The role of NO as a regulator of myocardial contractility has been disputed very recently by Vandecasteele et al. (51), reporting that mice deficient in eNOS have unaltered responses to -adrenergic agonists compared with the nontransgenic littermates, although several previous well-performed studies have shown that the positive inotropic effect induced by -adrenergic agonists is augmented by inhibition of NO synthesis (2, 20, 50). Our results show for the first time that the inotropic response to ET-1 is also limited by myocardial NO in that L-NAME enhanced responsiveness to ET-1 most obvious at the higher dose administered. These data support the hypothesis that NO plays an important role in the modulation of inotropic responses.

In view of the rapid onset of the enhancement by NOS inhibition, it is most likely due to decreased activity of eNOS because this is the subtype constitutively expressed in the myocardium (12, 21, 41) and the detectability of iNOS gene expression has been shown to demand at least 6 h of stimulation both in cardiac myocytes (5) and in microvascular endothelial cells (4). In the heart, eNOS has been detected in endocardial (40) and in microvascular (35) endothelium but also in cardiac myocytes (2, 3), where it seems to be the predominant subtype. It has been proven that, in endothelial cells, a transient increase in intracellular Ca²⁺ concentration by a receptor agonist initiates the activation of eNOS via Ca²⁺-calmodulin complex that disrupts the heteromeric complex of eNOS and caveolin, a protein attaching eNOS to the caveolae of the plasma membrane (9). Similarly, ET-1 binds to the ET_B receptor on endothelial cells inducing an increase in intracellular Ca²⁺ that in complex with calmodulin leads to the activation of NO synthesis (15). ET-1 has also been shown to elevate the Ca²⁺ concentration of cardiac myocytes (28) but it remains unclear whether this is followed by activation of myocyte eNOS. nNOS, which recently has been reported to be present in the cardiac sarcoplasmic reticulum (53), may also contribute to the net myocardial NO synthesis.

Recently, Ikenouchi et al. (17) suggested that AM exerts a negative inotropic effect with rapid onset mediated via the L-arginine-NO pathway in isolated adult rabbit ventricular myocytes. In contrast, the maximal AM-induced elevation in developed tension of intact heart in this study was not altered by L-NAME, and there was minimal effect at the lower dose, suggesting that stimulation of NO synthesis by AM per se is of minor importance in the rat myocardium under the conditions of our study. In addition to the different experimental conditions, the differences in regulation of eNOS activity between two species may explain the inconsistency between these two studies. Although the present data indicate that the inotropic responses to ET-1 and AM are differentially modulated by myocardial NOS, the physiological significance of the finding will require additional studies.

Of particular interest was our finding that equally potent submaximal concentrations of ET-1 and AM together induced a lesser inotropic response than either peptide alone. We further showed that this depressed effect of combined administration was more than overcome by concomitant infusion of L-NAME, suggesting that the attenuation was due to enhanced synthesis of NO, whereas the individual responses to the peptides at these doses were not the subject of secondary modulation by NO. To our knowledge, this potentiation represents a new phenomenon in the regulation of cardiac contractility by NO (Fig. 4). Because the Ca²⁺-calmodulin pathway is the only one reported to activate eNOS and because both AM and ET-1 are known to have an influence on intracellular Ca²⁺ metabolism (15, 28, 43, 47), it can be presumed that their simultaneous administration potentiates NO production via manipulation of cardiac myocyte or endothelial cell Ca²⁺ concentrations.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   A diagram describing the hypothesis of mutual relationships of AM and ET-1 on nitric oxide synthesis and regulation of myocardial contractility. +, Stimulation; , inhibition. The thickness of the arrows represents the strength of the action.

In conclusion, we have shown that myocardial NO modulates differentially the inotropic effects of ET-1 and AM in the isolated rat heart: inhibition of myocardial NOS augments the inotropic effect of ET-1, whereas it has only a minor impact on the effect of AM. Combined administration of submaximal concentrations of AM and ET-1 appears to potentiate the myocardial synthesis of NO, leading to a lesser inotropic response than either peptide induces alone.

Perspectives

ET-1 has been shown to significantly increase the contractility of normal cardiomyocytes but exerts a negative inotropic effect on cells isolated from failing ventricular myocardium (48). Very recently, MacCarthy et al. (26) showed that inhibition of ET_A receptors significantly impairs the contractile function of normal human left ventricle but has no effect in patients with dilated cardiomyopathy. Previously, the responses of failing myocardium to -adrenergic agonists have been reported to be blunted, most likely due to excessive production of NO (11). It is tempting to speculate that the unresponsiveness of failing myocardium to ET-1 may also be explained, at least partially, by the same mechanism, because the data presented here show that the inotropic effect of ET-1 on the healthy heart is controlled by NO. In contrast to ET-1, the early evidence suggests that the cardiostimulatory effect of AM may be preserved in experimental congestive heart failure (29, 36). These findings may reflect that AM-induced inotropic effect is not blunted by NO under those pathophysiological conditions, but this hypothesis should be addressed in future studies. Experimental studies have shown that after an early rise (30), cardiac NO production diminishes after the transition to cardiac failure (37). Recently, Heymes et al. (14) reported a positive correlation between eNOS gene expression and indexes of left ventricular performance in failing human heart. The stimulated release of NO was shown to improve left ventricular dysfunction, suggesting that NO may exert beneficial hemodynamic effects through maintenance of the Frank-Starling response (14). Therefore the interaction of AM and ET-1 to potentiate the activity of NOS needs to be assessed also in failing myocardium characterized by increased levels of AM (19) and ET-1 (38). The present results emphasize that the inotropic state seems not to be simply determined by the quantitative balance between positive and negative inotropic substances but also by the mutual, complex interactions of them. Thus the outcome of overall myocardial performance following a combined inotropic intervention is not necessarily predictable based on prior documentation of responses to the individual inotropic agents used.

	ACKNOWLEDGEMENTS

This work was supported by the Medical Research Council, Academy of Finland, the Sigrid Juselius Foundation, the Finnish Heart Research Foundation, Emil Aaltonen Foundation, Ida Montin Foundation, the Hungarian Research Foundation (OTKA: F26487) and the Ministry of Health of Hungary (ETT: T-11 083/97).

	FOOTNOTES

Address for reprint requests and other correspondence: H. Ruskoaho, Dept. of Pharmacology and Toxicology, Faculty of Medicine, Univ. of Oulu, PO Box 5000, 90014 University of Oulu, Finland (E-mail: heikki.ruskoaho{at}oulu.fi).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 1 October 1999; accepted in final form 28 February 2000.

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