Skip to main content
NCBI home page
The Journal of Physiology logoLink to The Journal of Physiology
. 1998 Sep 1;511(Pt 2):549–557. doi: 10.1111/j.1469-7793.1998.549bh.x

Role of endogenous endothelin in the regulation of basal coronary tone in the rat

Andrew T Goodwin 1, Mohamed Amrani 1, Caroline C Gray 1, Jay Jayakumar 1, Magdi H Yacoub 1
PMCID: PMC2231144  PMID: 9706030

Abstract

  1. Coronary vascular tone is a vital factor that regulates the delivery of oxygen to cardiac muscle. We tested the hypothesis that basal coronary tone may depend on the release of an endogenous vasoconstrictor peptide, endothelin (ET).

  2. Using an isolated, Krebs solution-perfused rat heart we measured the changes in coronary flow following the administration over a 30 min period of the ET antagonists Ro61-0612 (mixed ETA/ETB), PD155080 (ETA) and BQ788 (ETB).

  3. In a second series of experiments, hearts were randomly assigned to perfusion with plain Krebs solution, or with Krebs solution to which L-NAME and/or indomethacin had been added. The effect on coronary flow following the addition of Ro61-0612 was then measured.

  4. Perfusion with Ro61-0612 (10−4 M) alone increased coronary flow by 57.8 %vs. control (P = 0.00001). PD155080 (10−4 M) increased coronary flow by 28.9 % (P = 0.009), whereas BQ788 had no effect on coronary flow.

  5. In the second series of experiments, Ro61-0612 increased coronary flow by 6.6 ± 0.8 ml min−1 in hearts perfused with plain Krebs solution, by 3.8 ± 0.8 ml min−1 in hearts to which both L-NAME and indomethacin had been added, by 3.3 ± 0.7 ml min−1 in hearts to which L-NAME had been added, and by 6.9 ± 0.5 ml min−1 in hearts to which indomethacin had been added to the Krebs buffer.

  6. In hearts perfused with Krebs solution alone, nitric oxide (NO) release into the coronary sinus increased from 219.8 to 544.9 pmol min−1 g−1 following the addition of Ro61-0612 (P = 0.06). There was no detectable release of NO from hearts perfused with L-NAME alone or in combination with indomethacin either before or after the addition of Ro61-0612.

  7. We conclude that endogenous ET plays a role in coronary tone mediated via ETA receptors. This vasodilatation is partially due to an increase in endogenous NO release. However, a significant vasodilatation is still seen following the inhibition of NO synthesis. We propose that basal coronary tone depends on a balance between the endogenous release of vasodilators such as NO and vasoconstrictors such as ET.

Coronary vascular tone is a vital factor that regulates the delivery of oxygen to cardiac muscle. A number of factors are believed to be important in the regulation of basal coronary tone. These include a complex interaction between various circulating substances, neuronal control and vascular smooth muscle cells. Recently it has been observed that the vascular endothelium also plays a vital role through the secretion of various vasoactive factors that act locally on the vascular smooth muscle cells. This was first realized when it was shown that acetylcholine only produced a vasodilator response when applied to arterial ring segments in the presence of intact endothelial cells (Furchgott & Zawadzki, 1980). The importance of nitric oxide (NO) release in the regulation of coronary tone has since been demonstrated (Marin & Sanchez-Ferrer, 1990; Bassenge, 1991; Amrani et al. 1992; Smith et al. 1992). It has been suggested that coronary tone may depend on the balance between the secretion of various vasodilator and vasoconstrictor substances by the endothelium (Rubanyi, 1991; Stewart, 1991; Luscher & Tanner, 1993). One possible endothelium-derived vasoconstrictor is endothelin (ET).

The endothelins are a group of similar peptides (ET-1, ET-2 and ET-3). ET-1 is the most important of these and was first isolated from porcine aortic endothelial cells (Yanagisawa et al. 1988). There have been two ET receptors identified to date (ETA and ETB). The predominant receptor type is ETA, which mediates vasoconstriction of smooth muscle cells (Rubanyi & Polokoff, 1994). ETB receptors are present on both endothelial cells (where they mediate vasodilatation through the release of NO and prostacyclin) and smooth muscle cells (where they mediate vasoconstriction) (Hirata et al. 1993; Shetty et al. 1993). A large number of studies have shown that when ET is applied to human coronary arteries in vitro it causes a profound vasoconstriction (Chester et al. 1989, 1992). Similar results have been seen when ET is infused into animals (Clozel & Clozel, 1989; Kurihara et al. 1989; Hom et al. 1992). From these results it has been inferred that ET plays a role in resting vascular tone (Rubanyi, 1989; Luscher et al. 1990). However, all these studies have looked at the effects of adding exogenous endothelin to the coronary circulation. In addition, circulating plasma levels of ET are much lower than the doses of ET required to elicit a response in these studies. In view of this it is still unclear whether endogenous release of endothelin does play a role in the regulation of basal coronary tone.

In this study we have characterized the effects of endogenous ET on coronary tone using a number of recently synthesized ET antagonists. In addition, we have investigated the interaction between the endogenous vasodilators (NO and prostacyclin) and ET in the regulation of coronary tone.

METHODS

Animals

Male Sprague-Dawley rats weighing 300–330 g were used in all experiments. In all studies, animals received humane care in compliance with the ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research and the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 85–23, revised 1985).

Experimental preparation

The Langendorff isolated rat heart preparation was used in this study, as has already been described in detail elsewhere (Hearse et al. 1981; Goodwin et al. 1997). Briefly, the animals were killed by cervical dislocation. The femoral vein was immediately exposed and heparin (200 i.u.) was injected. The heart was then excised and immediately placed in ice-cold (4°C) Krebs solution. The aorta was rapidly cannulated (within approximately 30 s) and Langendorff perfusion was initiated. The hearts were perfused with Krebs-Henseleit bicarbonate buffer (pH 7.4) consisting of (mmol l−1): 118.5 NaCl, 25.0 NaHCO3, 4.8 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.25 CaCl2 and 11.0 glucose, which was continuously gassed with 95 % O2 and 5 % CO2, and maintained at 37°C. The Krebs buffer flowed from a reservoir 100 cm above the heart. The heart was suspended in a water-jacketed chamber maintained at 37°C.

Coronary flow was monitored by an electromagnetic flow probe (ECM2 20 ml, Scalar, Delft, Holland) mounted in the aortic flow-line, and connected to its compatible flowmeter (MDL 1401, Scalar, Delft, Holland). This provided an accurate (0.0-40.0 ml min−1) digital read-out of mean coronary flow, with a simultaneous hard copy recording through a connection with a chart recorder (RS3400, Gould Electronics, Hainault, Essex, UK). This allowed accurate monitoring of steady-state conditions (less than 0.1 ml min−1 change in coronary flow over 3 min).

Drugs and chemicals

The drugs used in this experiment were all made up to the required concentration in the Krebs buffer. The drugs used were Ro61-0612 (Hoffmann-La Roche Ltd, Basel, Switzerland), a non-peptide, non-selective ETA/ETB antagonist; PD155080 (Parke Davis, MI, USA), a non-peptide, selective ETA receptor antagonist; and BQ788 (Parke Davis), a peptide, selective ETB antagonist. In some of the experiments (see experimental time course below), 10−4 M L-NAME (Sigma), an inhibitor of NO synthase, and 10−6 M indomethacin (Sigma), an inhibitor of prostacyclin synthesis, were added to the Krebs buffer.

Experimental time courses

Role of endogenous ET in basal coronary tone

(Fig. 1A). The first series of experiments examined the role of endogenous ET in basal coronary tone by using the different ETA, ETB and ETA/ETB antagonists. Each heart was perfused in the Langendorff mode with plain Krebs solution until steady coronary flow was achieved. This takes approximately 15 min. Following this each heart was randomly assigned to receive plain Krebs solution (controls), or Krebs solution to which the ET antagonist under study had been added. Due to the length of the experimental protocol, only one concentration was used in each heart. The number of hearts studied at each concentration is shown in Table 1. The concentrations used were 10−6 to 10−4 M Ro61-0612; 10−6 to 10−4 M PD155080 and 10−7 to 10−5 M BQ788. Coronary flow was then recorded at 5 min intervals for a total of 30 min. A few hearts were perfused with the antagonists for 60 min. No additional effect was seen during this time (data not shown).

Figure 1. Experimental time courses.

Figure 1

Open in a new tab

A, protocol for experiments investigating the role of endogenous ET in basal coronary tone. B, protocol for experiment studying the role of endogenous ET-1 with and without blockade of NO and prostacyclin. Indo, indomethacin. Arrows mark sampling points for measurement of NO release in the coronary effluent.

Table 1.

Baseline characteristics in a series of experiments looking at the role of endogenous ET in basal coronary tone

Experimental group n Basal coronary flow (ml min−1) Significance vs. control
Control 5 12.7 ± 0.6
10−6m Ro 61–0612 3 11.1 ± 0.8 n.s.
10−5m Ro 61–0612 5 12.6 ± 0.2 n.s.
10−4m Ro 61–0612 4 11.9 ± 0.6 n.s.
Control 6 14.3 ± 0.3
10−6m PD155080 4 14.5 ± 0.2 n.s.
10−5m PD155080 5 15.2 ± 0.6 n.s.
10−4m PD155080 6 14.5 ± 0.5 n.s.
Control 4 13.1 ± 0.9
10−7m BQ788 4 14.2 ± 0.7 n.s.
10−6m BQ788 4 13.1 ± 0.6 n.s.
10−5m BQ788 4 11.7 ± 0.8 n.s.
Open in a new tab

n, number; n.s., not significant.

The role of endogenous ET in coronary tone with and without blockade of NO and prostacyclin

(Fig. 1B). The second series of experiments examined whether any coronary tone due to endogenous ET is modulated by the release of various endothelium-derived vasodilator substances, such as NO or prostacyclin. The hearts were divided into eight groups (Fig. 1B). In the first two groups the hearts were perfused with plain Krebs solution until steady coronary flow was achieved (approximately 15 min). Hearts were then perfused with plain Krebs solution (group K), or Krebs solution to which 10−4 M Ro61-0612 had been added (group K + Ro). Coronary flow was then recorded over a 10 min period. This time period was chosen as little additional effect was seen during longer perfusion for 30 min in the first series of experiments. In the remaining groups the hearts were initially perfused with Krebs solution to which L-NAME plus indomethacin (groups LI and LI + Ro), L-NAME alone (groups L and L + Ro), or indomethacin alone (groups I and I + Ro) had been added. In each case the hearts were allowed to stabilize until steady coronary flow was recorded. Following this hearts were allocated to either continue with Krebs solution (+L-NAME and/or indomethacin) (groups LI, L and I), or to Krebs (+L-NAME and/or indomethacin) to which 10−4 M Ro61-0612 had been added (groups LI + Ro, L + Ro and I + Ro). Coronary flow was recorded for a further 10 min. In hearts in groups K + Ro, LI + Ro, L + Ro and I + Ro, coronary sinus effluent samples (1 ml) were collected at the end of the initial stabilization period, and again following the 10 min of perfusion with Ro61-0612. These were immediately frozen and stored at −70°C for subsequent analysis of NO levels.

Measurement of nitric oxide release in the coronary sinus effluent

To determine total free NO production, the amount of its breakdown product (nitrite) was assayed with a chemiluminescence nitric oxide analyser (Sievers 270, CO, USA). Samples of the Krebs perfusate were injected into a purge vessel containing 1 % w/v potassium iodide in acetic acid; this allowed for the reduction of nitrite in the Krebs solution back to NO gas (Palmer et al. 1987). The gas given off by this reaction was then carried on a stream of nitrogen into a reaction chamber of the analyser, and a luminescent signal was then given off by the subsequent reaction of NO with ozone. Experimental samples were compared against a range of sodium nitrite standard concentrations. Release of NO was expressed in picomoles per minute per gram of dry weight. For this purpose, hearts were immediately freeze-clamped after each experiment, freeze-dried overnight, and then weighed.

Expression of results

In the first series of experiments, coronary flows are expressed as a percentage of the initial steady-state coronary flow. In the second series of experiments, coronary flow data are presented in millilitres per minute. Coronary flow data were compared by analysis of variance, followed by a Bonferroni test for multiple comparisons to indicate differences between groups. Pre- and post-NO release data were analysed using a Wilcoxon signed rank test. Values are given as means ± standard error of the mean (s.e.m.).

RESULTS

Role of endogenous ET in basal coronary tone

The baseline characteristics for each set of experiments is shown in Table 1. There were no significant differences between the groups at baseline. The effect of adding different concentrations of the non-selective ETA/ETB antagonist Ro61-0612 are shown in Fig. 2. This shows a dose-dependent increase in coronary flow when compared with perfusion with Krebs buffer alone. At the highest concentration (10−4 M), there was a 57.8 % increase in coronary flow (P = 0.00001 vs. control). In order to see whether this vasodilator response was due to ETA or ETB blockade, further experiments were performed with selective ET antagonists. The effect of adding different concentrations of the ETA-selective antagonist PD155080 are shown in Fig. 3. At a concentration of 10−4 M, there was a 28.9 % increase in coronary flow (P = 0.009 vs. control). Likewise, the effect of adding the selective ETB antagonist BQ788 is shown in Fig. 4. At the different concentrations studied there was no significant effect on coronary flow (P = n.s. vs. control).

Figure 2. Effect of perfusion with different concentrations (10−6 to 10−4 M) of the ETA/ETB antagonist Ro61-0612 on coronary flow.

Figure 2

Open in a new tab

Values are expressed as percentage changes from basal coronary flow. *P < 0.0001, control vs. 10−4 M Ro61-0612.

Figure 3. Effect of perfusion with different concentrations (10−6 to 10−4 M) of the ETA antagonist PD155080 on coronary flow.

Figure 3

Open in a new tab

Values are expressed as percentage changes from basal coronary flow. *P < 0.01, control vs. 10−4 M PD155080.

Figure 4. Effect of perfusion with different concentrations (10−7 to 10−5 M) of the ETB antagonist BQ788 on coronary flow.

Figure 4

Open in a new tab

Values are expressed as percentage changes from basal coronary flow.

The role of endogenous ET in coronary tone with and without blockade of NO and prostacyclin (Table 2 and Fig. 5)

Table 2.

Results of experiments looking at the effect of ETA/ETB antagonist (Ro61-0612) on coronary flow with and without NO/prostacyclin inhibition

Perfusion with plain Krebs solution
Group n Basal flow (ml min−1) Flow after 10 min perfusion (ml min−1) Change in flow (ml min−1)
K 5 12.7 (0.6) 12.8 (0.6) +0.1 (0.1)
K + Ro 4 11.9 (0.6) 18.5 (1.0) * +6.6 (0.8)
Perfusion with Krebs solution containing l-NAME and indomethacin
Group n Basal flow (ml min−1) Flow after 10 min perfusion (ml min−1) Change in flow (ml min−1)
LI 5 8.7 (0.5) 8.8 (0.4) +0.1 (0.1)
LI + Ro 6 9.3 (0.4) 13.1 (1.0)† +3.8 (0.8)
Perfusion with Krebs solution containing l-NAME
Group n Basal flow (ml min−1) Flow after 10 min perfusion (ml min−1) Change in flow (ml min−1)
L 5 8.5 (1.0) 8.4 (1.0) −0.1 (0.1)
L + Ro 5 8.4 (1.0) 11.7 (1.0)‡ +3.3 (0.7)
Perfusion with Krebs solution containing indomethacin
Group n Basal flow (ml min−1) Flow after 10 min perfusion (ml min−1) Change in flow (ml min−1)
I 5 12.0 (0.7) 12.2 (0.9) +0.2 (0.2)
I + Ro 5 12.3 (1.0) 19.2 (1.3)§ +6.9 (0.5)
Open in a new tab

For explanation of groups, see Fig. 1B. The table shows flow under basal conditions and following 10 min of perfusion with either ETA/ETB antagonist or controls (continuing perfusion with Krebs solution). n, number in each group. Significance: there was no significant difference in basal coronary flow between groups LI, L and I vs. group K. Likewise, there was no significant difference in basal coronary flow between groups LI + Ro, L + Ro and I + Ro vs. group K + Ro.

*

P < 0.01 vs. flow after 10 min, group K; P < 0.001 vs. basal flow, group K + Ro.

†

P < 0.05 vs. flow after 10 min, group LI; P = n.s. vs. basal flow, groupp LI + Ro.

‡

P = n.s. vs. flow after 10 min, group L; P = n.s. vs. basal flow, group L + Ro.

§

P < 0.001 vs. flow after 10 min, group I; P < 0.001 vs. basal flow, group I + Ro.

Figure 5. Increase in coronary flow following 10 min perfusion with Ro61-0612.

Figure 5

Open in a new tab

For explanation of groups, see Fig. 1B.

The results for the second series of experiments are shown in Table 2. Basal coronary flow was similar in the groups that were perfused with Krebs solution alone or Krebs solution containing indomethacin (K, K + Ro, I and I + Ro). However, in hearts perfused with L-NAME (groups LI, LI + Ro, L and L + Ro) there was a significant reduction in basal coronary flow by approximately 30 %. As would be expected, there was no effect on coronary flow following a further 10 min of perfusion with Krebs solution in groups K, LI, L and I. Perfusion with Ro61-0612, however, produced a vasodilator response in all four groups (K + Ro, LI + Ro, L + Ro, and I + Ro) (see Fig. 5). The vasodilatation seen was greatest in the groups perfused with either plain Krebs solution, or Krebs solution + indomethacin (groups K + Ro and I + Ro). The increase in coronary flow in these two groups was 6.6 and 6.9 ml min−1, respectively (K + Ro vs. I + Ro; P = n.s.). In the two groups to which L-NAME had been added, the increase in coronary flow was significantly less at 3.8 and 3.3 ml min−1, respectively (LI + Ro vs. L + Ro; P = n.s.).

NO release in the coronary effluent following administration of ETA/ETB antagonist

In the group of hearts perfused with plain Krebs solution (group K + Ro), the initial release of NO into the coronary sinus was 219.8 ± 151.8 pmol min−1 g−1. Following 10 min of perfusion with Ro61-0612, the NO release increased to 544.9 ± 227.6 pmol min−1 g−1 (P = 0.06 vs. baseline). In the hearts perfused with Krebs solution to which L-NAME alone or in combination with indomethacin had been added (groups LI + Ro and L + Ro), there was no detectable NO release at baseline, or following 10 min perfusion with Ro61-0612. In the hearts perfused with indomethacin alone (group I + Ro), basal NO release was 149.8 ± 112.9 pmol min−1 g−1 (P = n.s. vs. basal release group K + Ro). Following the addition of Ro61-0612, this increased to 355.8 ± 152.1 pmol min−1 g−1 (P = n.s. vs. basal release group I + Ro).

DISCUSSION

This study has shown for the first time that endogenous endothelin plays a role in basal coronary tone in this isolated Krebs solution-perfused rat model. This is mediated through ETA receptors. NO levels in the coronary sinus effluent are increased approximately twofold following the administration of the mixed ETA/ETB antagonist Ro61-0612. There was still a significant, but reduced, increase in coronary flow following blockade of endogenous ET in the presence of L-NAME. Inhibition of prostacyclin using indomethacin had no effect on basal coronary tone, or on the vasodilatation seen in response to ETA/ETB blockade. This suggests that the increase in coronary flow caused by perfusion with an ETA antagonist is due in part to the loss of basal vasoconstriction due to ET, and in part due to an increase in endogenous NO release.

A crystalloid-perfused, isolated heart, as used in this study, does not accurately represent the normal conditions experienced by the heart in vivo. However, the benefits of an isolated system are that it removes any confounding factors, such as circulating levels of catecholamines, neuronal control, and any effects due to neutrophil/endothelial cell interactions.

We have shown that Ro61-0612 (a non-selective ETA/ETB antagonist) and PD155080 (an ETA antagonist) both caused a significant increase in basal coronary flow, whereas BQ788 (an ETB antagonist) did not. This suggests that endogenous ET plays a role in basal coronary tone mediated through ETA receptors. These findings are important as they confirm that endogenous levels of ET can cause constriction of coronary arteries. Previous investigators have shown that administration of exogenous ET causes a profound vasoconstriction when applied to coronary arteries in vitro (Chester et al. 1989, 1992). There have been similar findings when ET has been injected into animals in vivo (Clozel & Clozel, 1989; Kurihara et al. 1989; Hom et al. 1992) and into healthy human volunteers (Pernow et al. 1996). The dose of ET required in order to elicit a response (1–10 nM) is much higher than circulating plasma concentrations of ET in healthy humans (1–5 pM) (Miyauchi et al. 1992; Haak et al. 1995; Kiowski et al. 1995). Although these studies confirm the presence of active ET receptors in coronary arteries, it is not possible to say what the role of endogenous ET would be. The reason for the large difference between the concentrations of endogenous and exogenous ET required may be because ET acts in a paracrine fashion, with ET released from endothelial cells acting locally on smooth muscle cells (Wagner et al. 1992). Locally released ET levels would therefore be many times higher than circulating plasma levels (Wagner et al. 1992). This may explain why ET blockade produced such a substantial effect in the present study.

At first glance our results suggest that use of a non-selective ETA/ETB antagonist causes a greater vasodilator effect (58 vs. 29 %) when compared with the ETA-selective antagonist. However, this is more likely to be due to the different potencies of the antagonists studied. To date there are no papers in the literature measuring the relative potency of Ro61-0612. Due to the lack of effect of the ETB antagonist, it is probable that the effects of the ETA antagonist would be similar to an equally potent non-selective ETA/ETB antagonist. Alternatively there may be different specificities/affinities of the different antagonists used.

ET has been shown to be a positive inotrope at low concentrations (Baydoun et al. 1989; Moravec et al. 1989). However, at higher doses coronary vasoconstriction occurs, resulting in reduced cardiac performance. One possible explanation of our results might be that there is an altered inotropic state of the heart during perfusion with an ET antagonist. The resulting change in oxygen requirement might then result in a compensatory change in coronary flow. However, this is unlikely to be the case, as in a previous study on isolated rat hearts using bosentan (an ETA/ETB antagonist), there was no effect on cardiac contractility in both paced and non-paced hearts (Dagassan et al. 1994).

A number of investigators have suggested that basal vascular tone may be due to a balance between the release of vasoconstrictor and vasodilator substances by the endothelium (Rubanyi, 1989, 1991; Stewart, 1991; Luscher & Tanner, 1993). In order to investigate this further, we studied the effect of ET receptor blockade in the presence or absence of NO and prostacyclin release. Perfusion with L-NAME alone (to inhibit NO) reduced basal coronary flow by approximately 30 %. This confirms previous findings that NO plays an important role in coronary tone (Amrani et al. 1992). The increase in coronary flow seen with ETA/ETB blockade is reduced following the inhibition of NO (6.6 vs. 3.3 ml min−1). This suggests that the increase in coronary flow seen in the presence of ET receptor blockade may be due, in part, to up-regulation of NO release. The measurement of NO in the coronary effluent confirmed that NO release was approximately doubled following 10 min of perfusion with the non-selective ETA/ETB antagonist.

In hearts perfused with indomethacin alone (to inhibit prostacyclin), there was no significant effect on basal coronary tone. Subsequent perfusion with Ro61-0612 resulted in a similar increase in coronary flow compared with controls (6.6 vs. 6.9 ml min−1). Prostacyclin release does not appear to play a significant role in basal coronary tone, or in the vasodilatation seen in response to ETA/ETB blockade, in this model.

There are a number of mechanisms by which release of NO and prostacyclin may be mediated. Stimulation of ETB receptors on endothelial cells has been shown to release NO and prostacyclin (Hirata et al. 1993; Shetty et al. 1993). However, inhibition of ETB receptors, as occurred in this study, still resulted in an increase in NO release. Alternatively, increased shear stress in the vessel wall, secondary to increased coronary flow, may stimulate NO release (Vequaud & Freslon, 1996; Wang & Diamond, 1997). It is possible that any flow-mediated release of NO may confound any results of our experiments. This is unlikely to be the case as experiments were performed both with and without L-NAME added to the perfusion buffer, in order to remove any secondary effects due to NO release.

In the series of experiments in which both NO and prostacyclin were inhibited, there was no detectable NO in the coronary effluent. Previous investigators have also shown that prostacyclin release is inhibited by the use of bosentan (an ETA/ETB antagonist) (Dagassan et al. 1994). Despite this, there was still a significant vasodilator response to the non-selective ETA/ETB antagonist in our experiments. This suggests that a significant proportion of the vasodilatation seen is indeed due to inhibition of endogenous ET. Alternatively, ET blockade results in the release of another, as yet undiscovered, vasodilator substance.

The vasodilator response in these experiments occurred maximally within 5–10 min of perfusion with the ETA/ETB antagonist. The rate of response in this study is quicker than that observed in vivo in human forearm vessels (Haynes & Webb, 1994). The maximal vasodilator response in human forearm was not seen until after approximately 1 h of perfusion with an ET antagonist. In some of our initial experiments, hearts were perfused for 60 min with the different antagonists (data not presented). However, no additional effect was seen during this time. There are a number of differences that may explain this. The studies are on different vascular beds, in different species and used different antagonists. In addition, our model is in isolation and removes the confounding factors of any counteracting influences such as circulating catecholamines and local neuronal control.

ET levels are increased in a number of pathological conditions, including hypertension, heart failure and following acute myocardial ischaemia (Watanabe et al. 1990; Cody, 1992; Maulik et al. 1992; Matheis et al. 1995; McGowan et al. 1995). It has been suggested that the raised ET levels may be responsible for an increase in coronary tone seen following myocardial infarction (Miyauchi et al. 1989; Salminen et al. 1989). There is experimental evidence accumulating, which suggests that elevated ET levels are responsible for the increase in peripheral vascular tone in patients with heart failure (Kiowski et al. 1995). The use of ET antagonists may therefore have a role to play in the treatment of some of these disorders, and in particular for the treatment of heart failure (Kaddoura et al. 1996; Sakai et al. 1996).

In summary, we conclude that endogenous ET plays a role in the maintenance of basal coronary tone in this isolated rat heart model. This is mediated through ETA receptors. These findings may have important future clinical implications in the treatment of a number of cardiovascular disorders.

Acknowledgments

This work was supported in part by the Harefield Hospital Heart Transplant Trust. We would also like to thank Parke Davis Pharmaceuticals and Hoffmann-La-Roche Ltd for their kind donation of the antagonists. Professor Yacoub is the British Heart Foundation Professor of Cardiac Surgery at Imperial College of Science Technology and Medicine, London.

References

  1. Amrani M, O'Shea J, Allen NJ, Harding SE, Jayakumar J, Pepper JR, Moncada S, Yacoub MH. Role of basal release of nitric oxide on coronary flow and mechanical performance of the isolated rat heart. The Journal of Physiology. 1992;456:681–687. doi: 10.1113/jphysiol.1992.sp019361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bassenge E. Endothelium-mediated regulation of coronary tone. Basic Research in Cardiology. 1991;86(suppl. 2):69–76. doi: 10.1007/978-3-642-72461-9_8. [DOI] [PubMed] [Google Scholar]
  3. Baydoun AR, Peers SH, Cirino G, Woodward B. Effects of endothelin-1 on the rat isolated heart. Journal of Cardiovascular Pharmacology. 1989;13(suppl. 5):S193–196. doi: 10.1097/00005344-198900135-00054. [DOI] [PubMed] [Google Scholar]
  4. Chester AH, Dashwood MR, Clarke JG, Larkin SW, Davies GJ, Tadjkarimi S, Maseri A, Yacoub MH. Influence of endothelin on human coronary arteries and localization of its binding sites. American Journal of Cardiology. 1989;63:1395–1398. doi: 10.1016/0002-9149(89)91055-2. [DOI] [PubMed] [Google Scholar]
  5. Chester AH, O'Neil GS, Allen SP, Luu TN, Tadjkarimi S, Yacoub MH. Effect of endothelin on normal and diseased human coronary arteries. European Journal of Clinical Investigation. 1992;22:210–213. doi: 10.1111/j.1365-2362.1992.tb01828.x. [DOI] [PubMed] [Google Scholar]
  6. Clozel JP, Clozel M. Effects of endothelin on the coronary vascular bed in open-chest dogs. Circulation Research. 1989;65:1193–1200. doi: 10.1161/01.res.65.5.1193. [DOI] [PubMed] [Google Scholar]
  7. Cody RJ. The potential role of endothelin as a vasoconstrictor substance in congestive heart failure. European Heart Journal. 1992;13:1573–1578. doi: 10.1093/oxfordjournals.eurheartj.a060103. [DOI] [PubMed] [Google Scholar]
  8. Dagassan PH, Breu V, Clozel M, Clozel JP. Role of endothelin during reperfusion after ischemia in isolated perfused rat heart. Journal of Cardiovascular Pharmacology. 1994;24:867–874. doi: 10.1097/00005344-199424060-00003. [DOI] [PubMed] [Google Scholar]
  9. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980;288:373–376. doi: 10.1038/288373a0. [DOI] [PubMed] [Google Scholar]
  10. Goodwin AT, Amrani M, Gray CC, Chester AH, Yacoub MH. Inhibition of endogenous endothelin during cardioplegia improves low coronary reflow following prolonged hypothermic arrest. European Journal of Cardiothoracic Surgery. 1997;11:981–987. doi: 10.1016/s1010-7940(96)01115-3. [DOI] [PubMed] [Google Scholar]
  11. Haak T, Matheis G, Kohleisen M, Ngo H, Beyersdorf F, Usadel KH. Endothelin during cardiovascular surgery: the effect of diltiazem and nitroglycerin. Journal of Cardiovascular Pharmacology. 1995;26(suppl. 3):S494–496. [PubMed] [Google Scholar]
  12. Haynes WG, Webb DJ. Contribution of endogenous generation of endothelin-1 to basal vascular tone. Lancet. 1994;344:852–854. doi: 10.1016/s0140-6736(94)92827-4. 10.1016/S0140-6736(94)92827-4. [DOI] [PubMed] [Google Scholar]
  13. Hearse DJ, Stewart DA, Braimbridge MV. Protection of the Ischemic Myocardium: Cardioplegia. New York: Raven Press; 1981. [Google Scholar]
  14. Hirata Y, Emori T, Eguchi S, Kanno K, Imai T, Ohta K, Marumo F. Endothelin receptor subtype B mediates synthesis of nitric oxide by cultured bovine endothelial cells. Journal of Clinical Investigation. 1993;91:1367–1373. doi: 10.1172/JCI116338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hom GJ, Touhey B, Rubanyi GM. Effects of intracoronary administration of endothelin in anesthetized dogs: comparison with Bay K 8644 and U 46619. Journal of Cardiovascular Pharmacology. 1992;19:194–200. doi: 10.1097/00005344-199202000-00007. [DOI] [PubMed] [Google Scholar]
  16. Kaddoura S, Firth JD, Boheler KR, Sugden PH, Poole Wilson PA. Endothelin-1 is involved in norepinephrine-induced ventricular hypertrophy in vivo. Acute effects of bosentan, an orally active, mixed endothelin ETA and ETB receptor antagonist. Circulation. 1996;93:2068–2079. doi: 10.1161/01.cir.93.11.2068. [DOI] [PubMed] [Google Scholar]
  17. Kiowski W, Sutsch G, Hunziker P, Muller P, Kim J, Oechslin E, Schmitt R, Jones R, Bertel O. Evidence for endothelin-1-mediated vasoconstriction in severe chronic heart failure. Lancet. 1995;346:732–736. doi: 10.1016/s0140-6736(95)91504-4. 10.1016/S0140-6736(95)91504-4. [DOI] [PubMed] [Google Scholar]
  18. Kurihara H, Yamaoki K, Nagai R, Yoshizumi M, Takaku F, Satoh H, Inui J, Yazaki Y. Endothelin: a potent vasoconstrictor associated with coronary vasospasm. Life Sciences. 1989;44:1937–1943. doi: 10.1016/0024-3205(89)90406-2. 10.1016/0024-3205(89)90406-2. [DOI] [PubMed] [Google Scholar]
  19. Luscher TF, Richard V, Tschudi M, Yang ZH, Boulanger C. Endothelial control of vascular tone in large and small coronary arteries. Journal of the American College of Cardiology. 1990;15:519–527. doi: 10.1016/0735-1097(90)90619-z. [DOI] [PubMed] [Google Scholar]
  20. Luscher TF, Tanner FC. Endothelial regulation of vascular tone and growth. American Journal of Hypertension. 1993;6:283–293. doi: 10.1093/ajh/6.7.283s. S. [DOI] [PubMed] [Google Scholar]
  21. Mcgowan FX, Jr, Davis PJ, Siewers RD, del Nido PJ. Coronary vasoconstriction mediated by endothelin-1 in neonates. Reversal by nitroglycerin. Journal of Thoracic and Cardiovascular Surgery. 1995;109:88–97. doi: 10.1016/S0022-5223(95)70424-8. [DOI] [PubMed] [Google Scholar]
  22. Marin J, Sanchez-Ferrer CF. Role of endothelium-formed nitric oxide on vascular responses. General Pharmacology. 1990;21:575–587. doi: 10.1016/0306-3623(90)91002-9. [DOI] [PubMed] [Google Scholar]
  23. Matheis G, Haak T, Beyersdorf F, Baretti R, Polywka C, Winkelmann BR. Circulating endothelin in patients undergoing coronary artery bypass grafting. European Journal of Cardiothoracic Surgery. 1995;9:269–274. doi: 10.1016/s1010-7940(05)80161-7. [DOI] [PubMed] [Google Scholar]
  24. Maulik N, Liu X, Subramanian R, Das DK. Release of endothelin during reperfusion of ischemic myocardium. ET-1 release from reperfused heart. American Journal of Cardiovascular Pathology. 1992;4:133–144. [PubMed] [Google Scholar]
  25. Miyauchi T, Yanagisawa M, Iida K, Ajisaka R, Suzuki N, Fujino M, Goto K, Masaki T, Sugishita Y. Age- and sex-related variation of plasma endothelin-1 concentration in normal and hypertensive subjects. American Heart Journal. 1992;123:1092–1093. doi: 10.1016/0002-8703(92)90734-d. 10.1016/0002-8703(92)90734-D. [DOI] [PubMed] [Google Scholar]
  26. Miyauchi T, Yanagisawa M, Tomizawa T, Sugishita Y, Suzuki N, Fujino M, Ajisaka R, Goto K, Masaki T. Increased plasma concentrations of endothelin-1 and big endothelin-1 in acute myocardial infarction. Lancet. 1989;2:53–54. doi: 10.1016/s0140-6736(89)90303-6. 10.1016/S0140-6736(89)90303-6. [DOI] [PubMed] [Google Scholar]
  27. Moravec CS, Reynolds EE, Stewart RW, Bond M. Endothelin is a positive inotropic agent in human and rat heart in vitro. Biochemical and Biophysical Research Communications. 1989;159:14–18. doi: 10.1016/0006-291x(89)92397-8. [DOI] [PubMed] [Google Scholar]
  28. Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 1987;327:524–526. doi: 10.1038/327524a0. 10.1038/327524a0. [DOI] [PubMed] [Google Scholar]
  29. Pernow J, Kaijser L, Lundberg JM, Ahlborg G. Comparable potent coronary constrictor effects of endothelin-1 and big endothelin-1 in humans. Circulation. 1996;94:2077–2082. doi: 10.1161/01.cir.94.9.2077. [DOI] [PubMed] [Google Scholar]
  30. Rubanyi GM. Maintenance of ‘basal’ vascular tone may represent a physiological role for endothelin. Journal of Vascular Medicine and Biology. 1989;1:315–316. [Google Scholar]
  31. Rubanyi GM. Endothelium-derived relaxing and contracting factors. Journal of Cellular Biochemistry. 1991;46:27–36. doi: 10.1002/jcb.240460106. [DOI] [PubMed] [Google Scholar]
  32. Rubanyi GM, Polokoff MA. Endothelins: molecular biology, biochemistry, pharmacology, physiology, and pathophysiology. Pharmacological Reviews. 1994;46:325–415. [PubMed] [Google Scholar]
  33. Sakai S, Miyauchi T, Kobayashi M, Yamaguchi I, Goto K, Sugishita Y. Inhibition of myocardial endothelin pathway improves long term survival in heart failure. Nature. 1996;384:353–355. doi: 10.1038/384353a0. 10.1038/384353a0. [DOI] [PubMed] [Google Scholar]
  34. Salminen K, Tikkanen I, Saijonmaa O, Nieminen M, Fyhrquist F, Frick MH. Modulation of coronary tone in acute myocardial infarction by endothelin. Lancet. 1989;2:747. doi: 10.1016/s0140-6736(89)90813-1. [DOI] [PubMed] [Google Scholar]
  35. Shetty SS, Okada T, Webb RL, Delgrande D, Lappe RW. Functionally distinct endothelin B receptors in vascular endothelium and smooth muscle. Biochemical and Biophysical Research Communications. 1993;191:459–464. doi: 10.1006/bbrc.1993.1240. 10.1006/bbrc.1993.1240. [DOI] [PubMed] [Google Scholar]
  36. Smith RE, Palmer RM, Bucknall CA, Moncada S. Role of nitric oxide synthesis in the regulation of coronary vascular tone in the isolated perfused rabbit heart. Cardiovascular Research. 1992;26:508–512. doi: 10.1093/cvr/26.5.508. [DOI] [PubMed] [Google Scholar]
  37. Stewart DJ. Role of EDRF and endothelin in coronary vasomotor control. Basic Research in Cardiology. 1991;86(suppl. 2):77–87. doi: 10.1007/978-3-642-72461-9_9. [DOI] [PubMed] [Google Scholar]
  38. Vequaud P, Freslon JL. Components of flow-induced dilation in rat perfused coronary artery. Cell Biology and Toxicology. 1996;12:227–232. doi: 10.1007/BF00438150. [DOI] [PubMed] [Google Scholar]
  39. Wagner OF, Christ G, Wojta J, Vierhapper H, Parzer S, Nowotny PJ, Schneider B, Waldhausl W, Binder BR. Polar secretion of endothelin-1 by cultured endothelial cells. Journal of Biological Chemistry. 1992;267:16066–16068. [PubMed] [Google Scholar]
  40. Wang W, Diamond SL. Does elevated nitric oxide production enhance the release of prostacyclin from shear stressed aortic endothelial cells? Biochemical and Biophysical Research Communications. 1997;233:748–751. doi: 10.1006/bbrc.1997.6548. 10.1006/bbrc.1997.6548. [DOI] [PubMed] [Google Scholar]
  41. Watanabe T, Suzuki N, Shimamoto N, Fujino M, Imada A. Endothelin in myocardial infarction. Nature. 1990;344:114. doi: 10.1038/344114a0. [DOI] [PubMed] [Google Scholar]
  42. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature. 1988;332:411–415. doi: 10.1038/332411a0. 10.1038/332411a0. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Physiology are provided here courtesy of The Physiological Society

RESOURCES