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. 2000 Jan 15;522(Pt 2):311–320. doi: 10.1111/j.1469-7793.2000.00311.x

Inhibition of nitric oxide synthase augments the positive inotropic effect of nitric oxide donors in the rat heart

Gerhard Müller-Strahl *, Karin Kottenberg *, Heinz-Gerd Zimmer *, Eike Noack *, Georg Kojda *
PMCID: PMC2269760  PMID: 10639106

Abstract

  1. In this investigation we studied the effects of nitric oxide on contractility and heart rate in normal saline-perfused rat hearts where shear stress-induced endothelial NO synthesis substantially contributes to total cardiac NO production. In addition, we sought to estimate the concentrations of exogenous NO producing inotropic effects.

  2. We investigated the effects of glyceryl trinitrate (GTN), S-nitroso-d,l-penicillamine (SNAP), sodium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolat (DEA/NO), and DEA/NO in the presence of the NO synthase inhibitor Nω-nitro-l-arginine (L-NA) in constant-flow-perfused spontaneously beating rat Langendorff hearts and in rat working hearts.

  3. In Langendorff hearts, GTN (10 nm to 100 μm, n = 32) induced a positive inotropic response that plateaued at 1 μm GTN with a maximal rate of increase of left ventricular pressure during ventricular contraction (+dP/dtmax) of 6.33 ± 2.56 % (n = 11, P < 0.5). Similarly, both spontaneous NO donors (0.1 nm to 1 μm, corresponding to approximately 0.03-0.3 μm NO) induced a positive inotropic response of 10.6 ± 3.1 % (SNAP; n = 15, P < 0.05) and 11.5 ± 2.7 % (DEA/NO, n = 15, P < 0.05).

  4. The positive inotropic effect of SNAP and DEA/NO progressively declined from 1 μm to 100 μm of the NO donors (corresponding to approximately 0.3-30 μm NO).

  5. In the isolated working rat heart, 0.1 μm DEA/NO induced an increase of +dP/dtmax of 7.5 ± 2.5 % (n = 9, P < 0.05). Inhibition of NO synthase by L-NA produced a 4-fold increase in this effect of DEA/NO.

  6. We suggest that physiological NO concentrations support myocardial performance. In normal rat hearts the positive inotropic effect of NO appears to be almost maximally exploited by the endogenous NO production.

During the last two decades nitric oxide emerged as an important endogenous mediator in the cardiovascular system. NO activates soluble guanylate cyclase to produce cGMP (Ahlner et al. 1991). Pharmacological effects of organic nitrates are also mediated by NO which is released after enzymatic degradation of these drugs. In contrast, experimentally used NO donors such as SNAP or DEA/NO are capable of releasing NO spontaneously (Ahlner et al. 1991; Keefer et al. 1996).

There are numerous investigations on the vascular effects of NO, while its action on myocardial contractility has rarely been studied and has yielded contradictory results: early investigations showed a positive inotropic effect of nitrovasodilators such as glyceryl trinitrate (GTN) or sodium nitroprusside in isolated cardiac muscle preparations from cats, guinea-pigs and humans and similar results have been obtained in the dog in vivo (Raff et al. 1970; Strauer, 1971; Korth, 1975; Diamond et al. 1977). In contrast, more recent studies provided evidence for a negative inotropic effect of exogenous NO in rat isolated cardiomyocytes and rabbit papillary muscle and of sodium nitroprusside in humans in vivo (Brady et al. 1993; Ishibashi et al. 1993; Paulus et al. 1994).

There is also evidence for a role of endogenous NO in the regulation of myocardial performance. Expression of endothelial and inducible NO synthase (NOS) occurs in cardiomyocytes of different species including man (Schulz et al. 1992; Balligand et al. 1994, 1995). Again, the reported effects of endogenous NO on myocardial contractility are contradictory: pharmacological inhibition of NOS in normal hearts of rat and dog induced a reduction of myocardial contractile function (Klabunde et al. 1991, 1992; Gardiner et al. 1991; Lechevalier et al. 1994; Kojda et al. 1997; Zappellini et al. 1997), while other investigations in myocardial preparations of hamster, rat and dog produced opposite results (Hare et al. 1995; Finkel et al. 1995; Kaye et al. 1996).

Recent results from studies on isolated rat cardiomyocytes suggested that NO alters the contractility of isolated cardiomyocytes in a biphasic manner (Kojda et al. 1996). Low concentrations of NO increased the contractility of these cells, while high concentrations had the opposite effect. Both of these changes were shown to be dependent on cGMP. A similar biphasic effect of NO also occurs in cat papillary muscle (Mohan et al. 1996) and cGMP has been shown to biphasically alter L-type calcium current in ventricular myocytes (Ono & Trautwein, 1991; Shirayama & Pappano, 1996). It has also been shown that endogenous NO augments the Frank-Starling response (Prendergast et al. 1997). According to these results we speculated that in the normal heart endogenous NO production supports myocardial contractility. We also sought to estimate the concentration ranges of NO that produce positive and negative inotropic effects in isolated heart preparations. To accomplish this, we investigated the inotropic action of the organic nitrate GTN, the spontaneous NO donors SNAP and DEA/NO in isolated constant-flow-perfused spontaneously beating Langendorff hearts of normal young Wistar rats. In addition, the inotropic effects of DEA/NO were examined in an isolated working heart preparation before and after suppression of endogenous NO production.

METHODS

Constant-flow Langendorff preparation

This study was performed with a total of 60 isolated hearts of male Wistar rats at an age of 3–4 months and a mean (±s.e.m.) body weight of 338 ± 6 g. The mean wet weight of the hearts was 1.02 ± 0.02 g. Immediately after killing the animals by cervical dislocation the hearts were rapidly excised, prepared by the technique of Langendorff and perfused at a constant pressure of 110 cmH2O with an oxygenated (95 % O2, 5 % CO2) Krebs-Henseleit buffer (pH 7.4, 37°C) of the following composition (mM): Na+ 143.07, K+ 5.87, Ca2+ 1.60, Mg2+ 1.18, Cl− 125.96, HCO3− 25.00, H2PO4− 1.18, SO42− 1.18 and glucose 5.05. Heart rate, left ventricular peak pressure (LVP), +dP/dtmax (maximal increase of left ventricular pressure per time unit during ventricular contraction) and -dP/dtmax (maximal decrease of left ventricular pressure per time unit during ventricular relaxation) were measured with a manometer connected to a small balloon, which was filled with 50 % ethanol and inserted into the left ventricle via the mitral valve. Coronary perfusion pressure (CPP) was measured with a tip manometer placed near the aortic valve. Both manometers were connected to a computer (imc, Meßsysteme GmbH, Berlin, Germany) to provide on-line recording of data. The oxygen content of the buffer was measured using an electrode (Radiometer, Willich, Germany) and maintained at 650 mmHg.

Hearts were beating spontaneously and the pulmonary artery was cannulated to measure coronary flow (CF). After preparation and equilibration (30 min) of the hearts at constant-pressure Langendorff perfusion, all experiments were done at constant volume perfusion. The constant flow rate was adapted to the CF measured under constant-pressure Langendorff conditions. The mean rate of the constant CF was 9.8 ± 0.2 ml min−1 g−1 (n = 60). A constant proportion of 10 % of this flow rate was applied by means of a double perfusion pump using a 50 ml syringe connected to a catheter placed in the aorta near the aortic valve. The other syringe of the perfusion pump was used to infuse the drugs diluted in Krebs-Henseleit buffer. Drug application was performed by switching from the Krebs-Henseleit buffer-containing syringe to the syringe containing the drug dissolved in Krebs-Henseleit buffer. This system guaranteed that application of the drugs was not associated with variations in CF. First application of a drug was initiated after a second equilibration period (30 min).

Experimental protocol

SNAP solution (100 μm) preincubated for 30 min at 37°C (SNAP control solution), different concentrations of noradrenaline (NA), DEA/NO, SNAP, GTN and N-acetyl-d,l-penicillamine (NAP) were applied. A 15 min washout period with buffer followed each drug application. In 6 hearts a concentration- response curve for NA (10 nM to 5 μm) was performed. In the other 54 hearts a repeated infusion of 0.1 μm NA was used to investigate the response to adrenergic stimulation. After the second infusion of NA the experiments were performed according to the following experimental protocols. To avoid a destabilisation of the heart preparation and a possible development of a nitrate tolerance it was necessary to restrict the duration of the experiments to a maximum of 2.5 h and the number of GTN applications to a maximum of three.

In 7 hearts SNAP (0.1 and 1 nM) followed by a SNAP solution that was preincubated for 30 min at 37°C (100 μm) and followed by NAP (10 and 100 μm) was infused. Neither the preincubated SNAP solution nor NAP showed an effect on the measured parameters. In 15 hearts GTN (1–100 nM), SNAP (10 and 100 nM) and DEA/NO (0.1 nM to 0.1 μm) were infused. In 11 hearts GTN (1 and 10 μm) and SNAP (1–10 μm) were infused. In 8 hearts DEA/NO (1–100 μm) was infused. In 6 hearts GTN (100 μm) was infused. In 7 hearts 0.005 % (v/v) dimethylsulfoxide (704 nM) was infused. In 3 of these hearts NaNO3 (100 μm) and in 4 of these hearts NaNO2 (100 μm) were infused. NaNO3, NaNO2 and dimethylsulfoxide had no effect on any of the measured parameters.

There was a delay time from the onset of the drug infusion to exposure of the hearts of approximately 30 s. The effects of the drugs were maximal within 2 min of exposure of the hearts. The effects were measured at that time and are related to the directly preceding equilibration values. In case of the repeated NA infusion we measured the effects of the second infusion. The total duration of the experiments at constant-flow perfusion was less than 2 h for 19 hearts and less than 2.5 h for 41 hearts (see Table 1).

Table 1.

Effect of the duration of the experiment and of 0.1 μm noradrenaline on myocardial function of rat hearts during constant-flow perfusion

Condition n Time (h) CPP (mmHg) +dP/dtmax (mmHg s−1) −dP/dtmax (mmHg s−1) LVP (mmHg) Heart rate (beats min−1)
Equilibration 60 0.5 75.6 ± 4.5 +2140 ± 115 −1341 ± 80 60.9 ± 3.1 262 ± 6
Noradrenaline 54 1.0 77.7 ± 4.8 +3302 ± 261* −3203 ± 207* 90.9 ± 6.9* 288 ± 5*
Equilibration 60 1.5 89.8 ± 5.3* +2163 ± 145 −1347 ± 109 61.8 ± 3.8 264 ± 5
Equilibration 41 2.0 96.5 ± 5.9* +2172 ± 145 −1316 ± 99 68.4 ± 4.6 243 ± 6*
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Values for coronary perfusion pressure (CPP), +dP/dtmax, −dP/dtmax, left ventricular peak pressure (LVP) and heart rate were measured at the indicated time points as described in Methods. The effects of the second noradrenaline infusion in all hearts except those used for the concentration–response curve are presented. The duration of 41 experiments exceeded 2.0 h but not 2.5 h. All values are expressed as means ± s.e.m. of n individual hearts. Significant differences to the values obtained at the first equilibration period are indicated

*

(P < 0.05, paired t test).

Isolated working heart preparation

Hearts of 9 female Wistar rats (2–3 months old, 221.5 ± 6.8 g) were examined. Mean heart weight was 0.79 ± 0.06 g. Animals were narcotised in ether and thoracotomised. The heart was excised and arrested in ice-cold saline until the retrograde perfusion with Krebs-Henseleit buffer set in via an aortic cannula. To complete this preparation a pulmonary vein and pulmonary artery cannula were added. The remaining afferent vessels to the left and right of the heart were ligated. Switching to the working mode was established by guiding the perfusion medium through the left atrial cannula to the left heart (7.35 mmHg filling pressure) which ejected the fluid against a hydrostatic load of 58.8 mmHg. Pre- and afterload of the preparation was kept constant throughout the experiment in order to exclude load-induced changes of contractility.

Coronary effluate could be collected at the pulmonary artery cannula. Aortic and coronary flow were measured with an automated gravimetric method. Ejected fluid did not recirculate. In this preparation coronary vessels were perfused at a constant pressure. Control experiments (n = 21) revealed that coronary vasorelaxation with adenosine did not alter cardiac contractility. While adenosine (50 μm) increased CF from 9.7 ± 0.4 to 14.6 ± 0.8 ml g−1 min−1 (P < 0.05), CO (from 64.3 ± 4 to 63.1 ± 3.2 ml g−1 min−1), +dP/dtmax (from 3549 ± 101 to 3846 ± 111 mmHg s−1), -dP/dtmax (from 2985 ± 96 to 3064 ± 108) and left ventricular maximal pressure (LVP, from 93.8 ± 1.4 to 94.7 ± 1.2 mmHg) remained unchanged. Thus, neither an enhanced supply with oxygen nor the Gregg phenomenon (Gregg, 1963) modifies cardiac contractility in this preparation. A second consequence of the condition of constant afterload is that changes in cardiac contractility are less reflected in changes of LVP but rather in changes of rate of contraction (dP/dtmax) or relaxation (-dP/dtmax) or in changes of CO. Left ventricular pressure was measured with a micro-tip pressure transducer (1.4 French, SPR-600, Millar Instruments, Houston, TX, USA). Data were recorded and analysed continuously and selected fractions were transferred to a computer at a sampling frequency of 1 kHz for further analysis. Infusion of DEA/NO (1 μm) and adenosine (50 μm) into the left atrial cannula was accomplished with a high precision pump at a rate of 1 % CO.

In addition to the nine runs done on hearts of female Wistar rats, five were done on hearts of male rats of the same age but slightly higher body weight (253.0 ± 3.74 g). Heart weight of the male animals was not significantly different from the female control group (P > 0.48)

Experimental protocol

The experimental protocol consisted of an initial 15 min for the stabilisation of the spontaneous heart activity followed by a first application of DEA/NO. Then the perfusion medium was changed to one containing the NOS inhibitor L-NA (0.1 mM). After another 15 min of adaptation, adenosine was given to produce a selective and NO-independent increase of CF. Five minutes after withdrawal of adenosine CF regressed to control values. Following this, DEA/NO was administered a second time.

Permission for this study was provided by the regional government (O45/87) and the experiments were performed according to the guidelines for the use of experimental animals as given by ‘Deutsches Tierschutzgesetz’ and to the ‘Guide for the Care and Use of Laboratory Animals’ of the US National Institute of Health.

Measurement of NO

The concentration of the NO release from DEA/NO and SNAP was determined in Krebs-Henseleit buffer using a polarographic method (ISO-NO-electrode, WPI, Berlin, Germany). The maximal concentration of NO measured at initial concentrations of 10 μm DEA/NO and 10 μm SNAP at 37°C in the presence of 150 mmHg of oxygen was 3384 ± 24 nM (n = 3) and 492.3 ± 4.3 nM (n = 3), respectively. These peak NO concentrations occurred after 90 s. In the presence of 700 mmHg oxygen tension, the peak NO concentration measured at 10 μm DEA/NO was slightly but significantly smaller (2538 ± 112 nM, n = 3).

Substances and solutions

DEA/NO was a gift from Professor Dr L. Keefer, National Cancer Institute, Frederick, MD, USA and SNAP was synthesised as described previously (Kojda et al. 1996). An aqueous GTN solution was purchased from Pohl-Boskamp GmbH & Co., Hohenlockstedt, Germany. All other chemicals were from Sigma, Deisenhofen, Germany, or Merck, Darmstadt, Germany in analytical grade.

One millilitre of the stock solution of GTN contained 1 mg of GTN (4.404 mM) and 49 mg of glucose monohydrate. Stock solutions of NA (10 mM) were prepared in distilled water. Stock solutions of SNAP (0.2 M) and NAP (0.2 M) were prepared in dimethylsulfoxide. Stock solutions of DEA/NO (10 mM) were prepared in sodium hydroxide solution (pH = 12). All stock solutions were freshly prepared each day, protected from daylight, kept on ice and diluted with Krebs-Henseleit buffer as required immediately before infusion into the hearts. In some experiments the stock solution of SNAP was diluted to 1 mM with Krebs-Henseleit buffer and then incubated for 30 min at 37°C before infusion into the hearts (final concentration 100 μm). All concentrations indicated in the figures and tables are expressed as final concentrations in the perfusion buffer.

Statistics

All data were analysed by standard computer programs (SAS PC Software 6.04, Graph Pad Prism 2.01 and SPSS 6.1.3) and are expressed as mean values and standard errors of the mean (s.e.m.). Half-maximal effective concentrations of NA were calculated from the respective concentration-response curves using a non-linear regression analysis based on a sigmoidal shape of the curve and are given in -logM (pD2 values). Significant differences were evaluated using either Student's paired two-tailed t test or ANOVA and a P value below 0.05 was considered significant.

RESULTS

Constant-flow Langendorff preparation

The baseline values of the parameters of contractility are shown in Table 1. There was no change in contractile activity during the experimental period indicating stable preparations. By contrast, after 1.5 h of constant-flow perfusion a significant increase in CPP was observed and after 2 h heart rate was slightly reduced (Table 1). All drugs elicited significant changes of the parameters of contractility, while coronary perfusion pressure and heart rate were not consistently altered. Original recordings of changes of LVP induced by application of the drugs are shown in Fig. 1. The hearts showed a normal concentration-dependent response to NA with a submaximal reaction at 0.1 μm NA. A similar submaximal response was observed after repeated infusion of 0.1 μm NA in the other hearts (Table 1). The corresponding half-maximal effective concentrations for NA (-logM) were as follows: LVP, 6.7 ± 0.1; +dP/dtmax, 6.5 ± 0.1; -dP/dtmax, 6.6 ± 0.1; and heart rate, 6.6 ± 0.3.

Figure 1. Reversible positive inotropic effects of NO donors.

Figure 1

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In the upper panel representative original recordings of the mean effect of DEA/NO and SNAP on left ventricular pressure of isolated constant-flow-perfused Langendorff hearts from Wistar rats are shown. The time point of subjection of the hearts to the drugs is indicated by an arrow. The lower panel shows the reversibility of the effect of 0.1 μm DEA/NO. The +dP/dtmax values are from 15 individual hearts before infusion of DEA/NO (equilibration) and after a washout period of 10 min (recovery). The direction of the individual changes are indicated by the connecting lines.

Effects of NO donors on myocardial contractility

All NO donors used in this study increased myocardial contractility as indicated by augmentation of left ventricular peak pressure, +dP/dtmax and -dP/dtmax. Overall, relaxation of the myocardium seems to be more affected than contraction (Figs 2–4). The mean changes of -dP/dtmax were always greater than the changes of +dP/dtmax and LVP; however, the respective concentration-response curves showed no significant differences (ANOVA). The organic nitrate GTN induced the smallest increase in myocardial contractility reaching a plateau at 1 μm, a concentration that had no effect on coronary perfusion pressure (Fig. 5). A further increase of its concentration had no additional effect (Fig. 2).

Figure 2. Effect of increasing concentrations of the organic nitrate glyceryl trinitrate (GTN) on left ventricular peak pressure (LVP), +dP/dtmax and -dP/dtmax of spontaneously beating constant-volume-perfused Langendorff hearts of normal Wistar rats (n = 32).

Figure 2

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Mean values of percentage changes related to the basal values before drug application are shown. Vertical bars indicate {"Single column legend" off}{"Single column legend" on}s.e.m. The maximal responses are significantly different from zero (P < 0.05, paired t test). Equilibration values for LVP, +dP/dtmax and -dP/dtmax of the hearts used for these experiments are 64.4 ± 4.1 mmHg, 2264 ± 243.3 mmHg s−1 and 1419 ± 96 mmHg s−1, respectively. The concentration-response curves are not significantly different from each other (ANOVA).

Figure 4. Effect of increasing concentrations of the spontaneous NO donor sodium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolat (DEA/NO) on left ventricular peak pressure (LVP), +dP/dtmax and -dP/dtmax of spontaneously beating constant-volume-perfused Langendorff hearts of normal Wistar rats (n = 23).

Figure 4

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Mean values of percentage changes related to the basal values before drug application are shown. Vertical bars indicate s.e.m. The maximal responses are significantly different from zero (P < 0.05, paired t test). Equilibration values for LVP, +dP/dtmax and -dP/dtmax of the hearts used for these experiments are 56.5 ± 3.9 mmHg, 1987 ± 91.21 mmHg s−1 and 1245 ± 83 mmHg s−1, respectively. The concentration- response curves are not significantly different from each other (ANOVA).

Figure 5. Effect of increasing concentrations of glyceryl trinitrate (GTN, n = 32), SNAP (n = 33) and DEA/NO (n = 23), on coronary perfusion pressure (CPP) of spontaneously beating constant-volume-perfused Langendorff hearts of normal Wistar rats.

Figure 5

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Mean values of percentage changes related to the basal values before drug application are shown. Vertical bars indicate s.e.m. The maximal responses are significantly different from zero (P < 0.05, paired t test). The concentration-response curves for DEA/NO and SNAP are not significantly different from each other (ANOVA).

The spontaneous NO donors SNAP and DEA/NO elicited a comparably stronger increase in myocardial contractility up to 0.1 μm of each drug. Contrary to the findings with GTN, a further increase of the concentration of SNAP and DEA/NO was associated with a decline of the positive effect on myocardial contraction resulting in a bell-shaped concentration-response curve (Fig 3 and Fig 4). Even the highest concentrations of SNAP (10 μm) and DEA/NO (100 μm), which correspond to NO concentrations of approximately 3 μm and 30 μm, respectively (see Methods), did not induce negative inotropic effects in terms of reduction of contractility below basal levels. The positive effects of the NO donors on myocardial contractility were fully reversible (Fig. 1). Even at the highest concentrations of the NO donors we observed no persistent effect. The contractility (+dP/dtmax) of the 8 hearts subjected to 10 μm DEA/NO was 2162 ± 118 mmHg s−1 before and 2173 ± 130.1 mmHg s−1 after infusion of the NO donor.

Figure 3. Effect of increasing concentrations of the spontaneous NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP), on left ventricular peak pressure (LVP), +dP/dtmax and -dP/dtmax of spontaneously beating constant-volume-perfused Langendorff hearts of normal Wistar rats (n = 33).

Figure 3

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Mean values of percentage changes related to the basal values before drug application are shown. Vertical bars indicate s.e.m. The maximal responses are significantly different from zero (P < 0.05, paired t test). Equilibration values for LVP, +dP/dtmax and -dP/dtmax of the hearts used for these experiments are 62.4 ± 5.9 mmHg, 2194 ± 181.0 mmHg s−1 and 1374 ± 101 mmHg s−1, respectively. The concentration- response curves are not significantly different from each other (ANOVA).

Effects of NO donors on coronary perfusion pressure

The basal coronary perfusion pressure progressively increased over the time of the experiment (Table 1) indicating an increase of vascular resistance in the coronary circulation. Infusion of GTN had little influence on CPP. Only high concentrations of GTN (10 μm, 100 μm) significantly reduced CPP (Fig. 5). By contrast, the spontaneous NO donors SNAP and DEA/NO induced a decrease of CPP in the range 0.1 nM to 10 μm indicating a vasorelaxation of coronary resistance vessels. At concentrations of SNAP and DEA/NO greater than 10 μm a progressive decline of this vasorelaxant action was observed. In fact, the highest concentration of DEA/NO (100 μm) did not significantly change control values of CPP (Fig. 5).

Effects of NO donors on heart rate

The basal spontaneous heart rate slightly decreased after 2 h of constant-flow perfusion (Table 1). Infusion of GTN had no effect on heart rate (Table 2). By contrast, infusion of SNAP or DEA/NO augmented heart rate in the concentration range 10 nM to 5 μm (Table 2). This slight tachycardic effect declined at 10 μm SNAP or DEA/NO. Interestingly, the concentration eliciting the maximal tachycardic effect of SNAP and DEA/NO was 50 times higher than the concentration causing a maximal inotropic effect.

Table 2.

Changes of heart rate induced by different NO donors

Concentration GTN n SNAP n DEA/NO n
100 pm — — −0.6 ± 1.0 7 −0.6 ± 0.4 15
1 nm −0.2 ± 0.4 15 0.6 ± 1.2 7 1.1 ± 0.7 15
10 nm 0.5 ± 0.7 15 2.0 ± 1.0 15 1.8 ± 0.7 15
100 nm 0.3 ± 0.4 15 2.2 ± 0.9* 15 3.4 ± 1.2* 15
1 μm 0.2 ± 1.6 11 4.2 ± 2.0* 11 0.9 ± 1.1 8
5 μm — — 7.3 ± 3.3* 11 5.3 ± 2.1* 8
10 μm 0.1 ± 0.8 11 2.9 ± 1.1* 11 2.2 ± 0.8* 8
100 μm 1.6 ± 1.0 6 — — 3.7 ± 3.3 8
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Values are expressed as mean ± s.e.m. percentage change of heart rate related to the baseline value before drug infusion as observed in n different hearts

*

(P < 0.05 vs. baseline, paired t test).

Isolated working heart preparation

Basal haemodynamic and flow parameters of the female working hearts are shown in Table 3. The baseline CF was identical to that of the Langendorff hearts used in this study. DEA/NO (0.1 μm) induced a pronounced vasodilatation in the working hearts as shown by a 58.0 ± 8.7 % increase in CF (Table 3). In parallel, DEA/NO induced small increases of CO, LVP, +dP/dtmax and -dP/dtmax (Fig. 6). As already indicated in Methods these inotropic effects cannot be related to an improved oxygen supply and therefore probably reflect direct effects of NO on myocardial contractility. The increase in -dP/dtmax was always greater than the corresponding increase in +dP/dtmax, which matches the effects of DEA/NO observed in constant-flow Langendorff preparations.

Table 3.

Changes of working heart functions after infusion of DEA/NO or adenosine in the absence and presence of NOS inhibition

CF (ml min−1 g−1) CO (ml min−1 g−1) LVP (mmHg) +dP/dtmax (mmHg s−1) −dP/dtmax (mmHg s−1) Heart rate (beats min−1)
Basal 9.9 ± 0.53 69.0 ± 5.5 83.8 ± 2.9 3089 ± 265 2989 ± 211 283 ± 15
DEA/NO 15.4 ± 0.62* 73.1 ± 4.7* 84.9 ± 2.6* 3282 ± 223* 3264 ± 247 295 ± 12
Basal 10.7 ± 0.45* 70.0 ± 4.8* 83.9 ± 2.1 3149 ± 213* 3060 ± 199 275 ± 12*
L-NA 7.0 ± 0.41* 56.8 ± 3.6* 80.4 ± 1.7* 2792 ± 158* 2470 ± 137* 260 ± 10
L-NA +adenosine 13.8 ± 1.06* 57.8 ± 4.8 81.1 ± 2.2 2938 ± 200 2510 ± 207 270 ± 13
L-NA 6.5 ± 0.38* 49.0 ± 4.6* 78.2 ± 2.2* 2578 ± 168* 2170 ± 133* 258 ± 11
L-NA + DEA/NO 13.1 ± 0.45* 70.1 ± 4.1* 84.2 ± 2.2* 3191 ± 192* 2808 ± 175* 282 ± 10*
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Values for coronary flow (CF), cardiac output (CO), left ventricular peak pressure (LVP), +dP/dtmax, −dP/dtmax and heart rate were measured at baseline and after infusion of 0.1 μm DEA/NO and 50 μm adenosine in the presence of the NOS inhibitor L-NA (100 μm) (for values of adenosine alone, see Methods). All values are expressed as means ± s.e.m. of 9 individual hearts

*

(P < 0.05 vs. previous condition (line above)).

Figure 6. Effect of 0.1 μm DEA/NO on +dP/dtmax, -dP/dtmax and cardiac output of spontaneously beating isolated working hearts of normal Wistar rats (n = 9).

Figure 6

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Mean values of percentage changes related to the basal values before drug application are shown (absolute values are given in Table 3). Vertical bars indicate s.e.m. Inhibition of NO synthase by L-NA strongly increases the positive inotropic effect of DEA/NO. Significant differences were calculated by two-tailed unpaired t test: †P < 0.05 vs. baseline, *P < 0.05 vs. DEA/NO.

Inhibition of NOS by 0.1 mM L-NA compromised cardiac function (Table 3). Coronary flow decreased by 34.0 ± 4.3 %, CO by 18.1 ± 3.3 %, LVP by 4.1 ± 0.9 %, +dP/dtmax by 10.8 ± 1.9 % and -dP/dtmax by 19.0 ± 2.0 %. It is most likely that the decrease in CF did not induce the reduction of myocardial contractility, since infusion of adenosine in the presence of L-NA, which increased CF over baseline values (Table 3), had no effect on myocardial contractility. In striking contrast, infusion of 0.1 μm DEA/NO, which increased CF to an extent identical to that with adenosine in the presence of L-NA (P > 0.5), strongly increased CO, +dP/dtmax and -dP/dtmax (Table 3, Fig. 6). This increase in myocardial contractility was significantly greater than the increase observed after infusion of DEA/NO before inhibition of NOS (Fig. 6). These results suggest that both the deterioration of heart function by L-NA and its re-establishment by DEA/NO are probably caused by depletion and subsequent replenishment of the cardiac NO levels, respectively.

To test the possibility that gender might have influenced the results in the working heart preparations we additionally investigated five hearts of male rats. In general, we observed no significant differences by comparing the results obtained in male and female hearts as indicated by the P values following the respective data given below. Basal CF was identical in hearts from male and female rats (P > 0.1). In hearts of male rats DEA/NO (0.1 μm) induced a pronounced increase in CF (38.0 ± 0.06 %; P > 0.15) and small increases of CO (2.6 ± 1.7 %; P > 0.16), +dP/dtmax (11.1 ± 1.3 %; P > 0.3) and -dP/dtmax (6.4 ± 0.6 %; P > 0.7) (see also Fig. 6). Similarly, inhibition of NOS by 0.1 mM L-NA in male hearts had identical depressing effects on cardiac function to those in female hearts. Coronary flow decreased by 29.7 ± 4.2 % (P > 0.56), CO by 16.6 ± 4.1 % (P > 0.8), LVP by 4.6 ± 0.9 % (P > 0.75), +dP/dtmax by 12.5 ± 2.0 % (P > 0.57) and -dP/dtmax by 17.3 ± 2.4 % (P > 0.65). Finally, infusion of 0.1 μm DEA/NO in the presence of 0.1 mM L-NA strongly increased CO (36.8 ± 9.9 %; P > 0.2), +dP/dtmax (21.5 ± 3.2 %; P > 0.6) and -dP/dtmax (20.0 ± 3.5 %; P > 0.3) (see also Fig. 6). These results indicate that there is no gender difference in the response of rat working hearts to DEA/NO.

As already observed in the Langendorff preparations, the effects of NO on heart rate were small. There was a slight increase in heart rate after infusion of DEA/NO, which reached statistical significance only in the presence of L-NA, which itself had a small negative chronotropic effect (Table 3).

DISCUSSION

In this study we investigated the effects of NO on myocardial function of the normal saline-perfused rat heart where shear stress-induced endothelial NO synthesis substantially contributes to total cardiac NO production. The new findings are that (1) an improvement of myocardial contractility occurred at final concentrations of exogenous NO up to approximately 1 μm in the coronary circulation and that (2) this effect was markedly increased after pharmacological inhibition of total endogenous NO synthesis. At concentrations of exogenous NO estimated to be within a range of 1–30 μm the positive inotropic effect declined and the increase in contractility disappeared. A direct depression of myocardial contraction was not observed. These results suggest that physiological and therapeutic NO concentrations support the development of myocardial contraction.

Effects on myocardial contractility

Pharmacological effects of GTN, SNAP and DEA/NO are probably caused by NO, which is liberated by different pathways from these drugs. Liberation of NO from SNAP occurs spontaneously, but is markedly enhanced in the presence of tissues, cell membranes or even free thiols. It is therefore difficult to estimate the concentration of NO in the coronary circulation after infusion of SNAP. The release of NO from organic nitrates requires an enzymatic bioactivation step (Ahlner et al. 1991) which also occurs in rat cardiac myocytes. Subjection of these cells to GTN or other typical organic nitrates results in increased cGMP levels (Kojda et al. 1996). Again, the actual concentration of NO in the coronary circulation or in the cardiomyocytes is difficult to estimate. In contrast, DEA/NO is stable in alkaline solutions and rapidly degrades to NO and the by-products diethylamine and N-nitrosodiethylamine at physiological pH (Keefer et al. 1996). The peak concentration of 3.384 ± 0.024 μm NO measured after degradation of 10 μm DEA/NO in Krebs-Henseleit buffer (see Methods) suggests that the maximal concentration of NO in the coronary circulation was approximately one-third of the infused concentration of DEA/NO. Accordingly, it is suggested that the maximal positive inotropic effect in rat isolated hearts occurs at a concentration range of 30–300 nM NO (Fig. 4).

The second new finding of our study is that the positive inotropic effect of NO was markedly enhanced after intramyocardial inhibition of total NOS activity by L-NA. NOS inhibition alone significantly reduced CO, contractility and CF. While readjustment of CF by adenosine had no influence on contractile parameters, a similar effect on CF induced by DEA/NO was accompanied by a 4-fold increase in CO. Therefore, the increase in CF induced by DEA/NO is not related to the observed increase of contractility. These results indicate that endogenous NO production in the rat heart significantly contributes to the physiology of myocardial contraction. This activity of NO in the heart seems to be of limited capacity, since activation of this pathway by exogenous NO in the presence of intact endogenous NO production has only minor inotropic effects.

The positive inotropic effect of NO in Langendorff and working rat heart preparations is consistent with previous findings. Positive inotropic effects of organic nitrates or sodium nitroprusside in different preparations of cardiac muscle from cat (Diamond et al. 1977), humans (Strauer, 1971), guinea-pigs (Korth, 1975) and rats (Kojda et al. 1996) have been reported. Likewise, GTN, SNAP and DEA/NO increase myocardial contractility in the dog heart in vivo (Preckel et al. 1997). A similar effect was shown in clinical studies long before it was known that the activity of therapeutic concentrations of GTN is dependent on the release of NO (Strauer & Scherpe, 1978; Hood et al. 1980). The positive effect of organic nitrates or NO on myocardial contraction is probably caused by cGMP (Mohan et al. 1996). According to previous reports the subsequent signalling steps probably include inhibition of cAMP hydrolysis by the cGMP-inhibited phosphodiesterase (PDE III) promoting increased Ca2+ current via L-type calcium channels (Ono & Trautwein, 1991; Méry et al. 1993; Kojda et al. 1996; Shirayama & Pappano, 1996). A recent study showed that NO might also stimulate cAMP synthesis independently of cGMP in cardiomyocytes (Vila-Petroff et al. 1999).

We cannot exclude enhanced degradation of NO leading to generation of peroxynitrite during perfusion with the NO donors. However, previous work has shown that increased degradation of NO at an oxygen tension of 700 mmHg versus 150 mmHg impacts only little in a perfused heart, where the passage time of the perfusate is smaller than a quarter (0.8 s) of the half-life of NO at 700 mmHg oxygen tension (4 s) (Kelm & Schrader, 1990). Furthermore, we have used a special infusion device to reduce the time between the direct contact of the stable non-oxygenised NO donor stock solution and the perfusion buffer to a minimum (see Methods). Even if small concentrations of peroxynitrite were generated due to the higher oxygen tension, its influence against the effects of NO is – according to published data – negligible (Schulz et al. 1997).

It is also noteworthy that the positive inotropic effect of 0.1 μm DEA/NO in Langendorff hearts of male rats was identical to that in working hearts of female rats indicating that gender is likely to alter the positive inotropic effect of NO in the rat. Another result of this study is that NO can also facilitate myocardial relaxation as indicated by the increase in -dP/dtmax (Figs 2–5, Table 3). This finding confirms previous reports (Paulus et al. 1994) and has been implicated in myocardial effects of factors released from the endocardium such as NO (Grocott-Mason et al. 1994; Shah, 1996).

In contrast to previous investigations, we did not observe direct negative inotropic effects of NO donors in the isolated heart in terms of a reduction of contractility below basal levels. In rabbit papillary muscle a very high concentration of authentic NO (100 μm) was found to reduce contractility (Ishibashi et al. 1993). Another investigation in guinea-pig cardiomyocytes has shown a negative inotropic effect of high concentrations of sodium nitroprusside (50 μm) in animals and man (Brady et al. 1993; Paulus et al. 1994). In isolated rat cardiomyocytes exposed to 100 μm of the NO donors SNAP or DEA/NO we found a depression of the contractile response to electrical stimulation (Kojda et al. 1996). The difference to the results presented here might be explained by different NO concentrations, different NO donors or different species and preparations. Furthermore, very high intramyocardial concentrations of nitric oxide can – depending on the respective rate of superoxide production – result in a considerable generation of peroxynitrite which has been shown to depress myocardial contractility at concentrations > 40 μm (Schulz et al. 1997).

The increase in myocardial contractility induced by NO was probably not mediated by indirect mechanisms such as a reduction in CPP (see below) or a release of endogenous NA. It has been shown previously that both exogenous and endogenous NO inhibits NA release in rat isolated hearts as measured by detection of NA in the coronary effluent (Schwarz et al. 1995). In addition, it is known that very low concentrations of NA (approximately 0.2 nM) appear in the coronary venous effluent of unstimulated rat hearts (Dart et al. 1984). This concentration of NA is not expected to change myocardial contractility as indicated by the dose dependency evaluated for NA (see Results).

Effects on the coronary perfusion

Infusion of SNAP and DEA/NO was associated with a substantial reduction of CPP (Fig. 5) that occurred in parallel with the inotropic effects. This decrease of CPP is consistent with the augmentation of CF in the rat working heart and with earlier studies demonstrating a potent vasodilator effect of NO in coronary microvessels (Harrison & Bates, 1993). The vasodilator effect of DEA/NO and SNAP might have contributed to the alterations of contractility of the isolated hearts. However, present evidence suggests that a reduction in CPP rather decreases myocardial contractility in constant-flow Langendorff preparations (Gregg, 1963). According to the ‘garden-hose’ hypothesis, this effect is related to a decrease of cardiac sarcomere lengths caused by the diminished distension of coronary vessels (Arnold et al. 1968; Poche et al. 1971). Thus, it is unlikely that coronary vasodilatation is the primary cause of the positive inotropic action of DEA/NO and SNAP in the Langendorff preparations. A similar conclusion can be drawn from the results of the experiments with the rat working heart. We have used adenosine to evaluate the effects of coronary vasodilatation on myocardial contractility. Adenosine is known to modulate cardiac contractility only in the presence of adrenergic stimulation (Schrader et al. 1977) but in the unstimulated spontaneously beating isolated rat heart the release of catecholamines is negligible (Dart et al. 1984). Adenosine strongly increased CF in the absence and presence of L-NA but had no effect on myocardial contractility (see Methods, Table 3), while infusion of DEA/NO, which increased CF to a similar extent, significantly enhanced myocardial contractility.

Effects on heart rate

The results of the present study show that exogenous NO is capable of increasing heart rate (Table 2). Although this effect was weak in the perfused rat heart, the result confirms previous investigations on chronotropic effects of NO showing that exogenous NO can stimulate the hyperpolarisation-activated inward current (Musialek et al. 1997). Other studies provided evidence for a positive chronotropic effect of endogenous NO. The NOS inhibitor L-NA and the respective methylester induce a negative chronotropic effect in the rat isolated heart (Pabla & Curtis, 1995; Kojda et al. 1997). In addition, disruption of the eNOS gene causes bradycardia in the mouse, which is aggravated by oral treatment with the NOS inhibitor NG-nitro-L-arginine-methylester (Shesely et al. 1996; Kojda et al. 1999). A contribution of the small positive chronotropic effect of DEA/NO and SNAP to the alterations of contractility of the isolated hearts is very unlikely. Previous investigations in adult rat ventricular cardiomyocytes have shown that stimulation rates between 4 and 6 Hz, which closely matches the heart rate range in our study, does not influence contractility (Borzak et al. 1991).

In summary, we have shown that submicromolar concentrations of NO induce a positive inotropic effect in Langendorff and working rat heart preparations where shear stress-induced endothelial NO synthesis substantially contributes to total cardiac NO production. This effect is markedly enhanced after inhibition of intramyocardial NOS activity. Our results indicate that the positive inotropic effect of NO appears to be almost maximally exploited by the endogenous NO production in the perfused heart.

Acknowledgments

This study was supported by a grant from the Deutsche Forschungsgemeinschaft, SFB 242 (Projekt A 11). The authors wish to thank Professor Larry Keefer, National Cancer Institute, Frederick, MD, USA, for kindly providing DEA/NO and Miss H. Winter, Institut für Physiologie, Leipzig, Germany, for technical assistance.

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