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Cardiovascular Research 1998 39(3):665-673; doi:10.1016/S0008-6363(98)00152-7
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Endothelin B receptor-mediated vasoconstriction induced by endothelin A receptor antagonist

Yi Zhanga, John R. Oliverb and John D. Horowitza,*

aCardiology Unit, The Queen Elizabeth Hospital, University of Adelaide, 28 Woodville Road, Woodville South, SA 5011, Australia
bEndocrinology Unit, Flinders Medical Centre, Bedford Park, SA 5042, Australia

* Corresponding author. Tel.: +61-8-8222-6725; Fax: +61-8-8222-6030.

Received 14 November 1997; accepted 3 March 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The vasoconstrictor effect of endothelins (ET) is mediated by endothelin A (ETA) and endothelin B (ETB) receptors. Furthermore, ETB receptor stimulation results in release of vasodilators. Hence, ETA receptor antagonists should attenuate ET-mediated vasoconstriction. The aim of the present study was to evaluate and compare the effects of BQ-123, an ETA receptor antagonist, and bosentan, an ETA and ETB receptor antagonist, on coronary vasomotor tone, left ventricular systolic function and ET-1 efflux in the presence or absence of myocardial ischaemia/reperfusion. Methods: Isolated rat hearts were perfused using a Langendorff preparation. Global ischaemia was induced on average by 68±2% (±standard error of the mean) reduction of a baseline perfusion flow-rate 10 min after introduction of ET antagonists. Thirty minutes of ischaemia was followed by 30 min reperfusion. ET-1 efflux in coronary perfusate was measured by radioimmunoassay. Results: In non-ischaemic hearts (n=7), BQ-123 (10–6 M) perfusion induced a progressive decrease in coronary flow-rate compared with control group. This flow reduction persisted after wash-out of BQ-123. In contrast, bosentan (10–5 M, n=7) induced no change in perfusion rate. In the absence of ET antagonists (n=16), there was a 22±6% post-ischaemic increase in perfusion flow-rate. BQ-123 (n=5) but not bosentan (n=12) abolished this post-ischaemic increase in flow-rate. Neither BQ-123 nor bosentan induced significant variation in force of contraction. In ischaemic hearts, ischaemia per se induced a transient decrease in force of contraction. Bosentan significantly (P<0.05) accentuated and BQ-123 tended to accentuate (P=0.06) this decrease in force of contraction during ischaemia. Bosentan but not BQ-123 significantly impaired the recovery of systolic function during reperfusion (P<0.05). Both BQ-123 and bosentan perfusion increased ET-1 efflux rate to 730±188% and 315±81% respectively. This effect was abolished during ischaemia for BQ-123, but not for bosentan. Conclusions: In isolated perfused rat hearts, both BQ-123 and bosentan increased ET-1 efflux, but only BQ-123 exerted vasoconstrictor effects. These results thus generated the hypothesis that: (1) ET-1 release within the coronary vascular bed may be physiologically subject to negative feedback regulation mediated via ETA receptors; (2) ETA receptor antagonists increase ET-1 efflux, which may lead to net vasoconstriction via unopposed ETB stimulation. Furthermore, the negative inotropic effects observed during ischaemia suggest that ET is critical to the maintenance of systolic function during ischaemia.

KEYWORDS Isolated rat heart; Ischaemia; Endothelin; Endothelin receptor antagonists; Cardiac force of contraction


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Endothelins (ET) are a family of vasoactive hormones secreted mainly from endothelium. Their vasoconstrictor and vasodilator effects are mediated by two types of receptors, ETA and ETB [1]. The ETA receptor is classified by having greater affinity for ET-1 than ET-3 whereas the ETB receptor is non-isopeptide-selective. ETA receptor stimulation has generally been found to mediate vasoconstriction. The ETB receptor subtype located on smooth muscle also mediates vasoconstriction. It appears to be functionally distinct from the ETB subtype located on the vascular endothelium which mediates vasorelaxation via the release of NO [2, 3]. ETB receptors play a role in clearance of ET from the circulation by endothelial cells [4].

ET's potent vasoactive effects may have pathogenic relevance during myocardial ischaemia. Increased plasma ET concentrations are found in patients with unstable angina, myocardial infarction and immediately following percutaneous transluminal coronary angioplasty [5, 6]. Endogenous ET is also released into the coronary perfusate of isolated perfused rat hearts during ischaemia/reperfusion [7]. In cultured human endothelium, hypoxia induces ET gene expression and secretion [8]. On the other hand, ischaemia caused an increase in ET-1 binding site density [9, 10]. Furthermore, hypoxia increased the maximal responses of ET-1 and ET-3 in the rat isolated perfused mesenteric arterial bed [11].

Currently available methodologies for investigating the putative pathophysiological role of endogenous ET include the use of endothelin converting enzyme inhibitors, anti-endothelin antibodies and endothelin receptor antagonists. A specific ETA receptor antagonist, BQ-123, has been shown to inhibit ET-induced vasoconstriction in porcine coronary artery [3], to attenuate the hypertensive response to ET-1 and big ET in rats [12], and to reduce infarct size in a canine model [13]. However, BQ-123 failed to inhibit ET-induced coronary vasoconstriction in a Langendorff perfused rat heart model [14]. Seo et al. using bosentan (Ro 47-0203), an ETA/ETB receptor antagonist, demonstrated that both ETA and ETB receptors mediate contraction to ET-1 in isolated human blood vessels [15]. In isolated rat hearts, bosentan (10–5 M) increased coronary flow but did not influence contractility [16]. However, to date there are only limited data on the effect of ETA and ETA/B antagonists on the relationship between endothelin production and coronary vasomotor tone during ischaemia/reperfusion.

Exogenous ET-1 has also been shown to exert a positive inotropic effect on isolated cardiac myocytes and papillary muscles [17–21]. However, in vivo studies could not demonstrate the positive inotropic effect of exogenous ET-1, almost certainly because of concomitant induction of myocardial ischaemia via coronary vasoconstriction [22–27]. The development of ET receptor antagonists also offers a means of obtaining inferential information concerning the inotropic effects of ET in vivo. To date, studies with intravenous administration of bosentan in patients with severe chronic heart failure have demonstrated both an increase in cardiac output and a decrease in peripheral vascular resistance [28–31]; these findings suggest little fluctuation in underlying inotropic state. However, it is known that ET-1 is released during acute myocardial ischaemia [32]. Hence the inotropic effects of ET may fluctuate during ischaemia, with consequent variability in effects of ET antagonists in modifying contractile state during severe myocardial ischaemia.

The aim of the present study was to examine, in the perfused rat heart, the effects of BQ-123 and bosentan on coronary flow, release of ET-1 into coronary effluent and contractile state during, and following global myocardial ischaemia.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Isolated heart preparation
The protocol was approved by the institutional Animal Ethics Committee. Male Porton rats weighing 406±9 g (n=70) were anaesthetised with an intraperitoneal injection of sodium pentobarbitone (50 mg/kg). The chest of the animal was opened, the heart rapidly excised and arrested in ice-cold saline. The aorta was cannulated, and the heart transferred to a modified non-recirculating Langendorff apparatus perfused with modified Eagle's minimum essential medium (contained NaCl 116 mM, KCl 5.4 mM, NaHCO3 24 mM, MgSO4•7H2O 0.8 mM, NaH2PO4•2H2O 1.0 mM, CaCl2•2H2O 2.5 mM, pyruvic acid 5.0 mM, glucose 11 mM, bovine serum albumin 0.1%, insulin 10 U/l, pH 7.4) at a constant pressure of 70 cm H2O. Medium was equilibrated with O2–CO2 (95:5 v/v) at 37°C [33].

2.2 Protocol
Hearts were paced by placing electrodes against the right atrium in order to minimise rate-related effects on myocardial oxygen demand and coronary vasomotor tone. Pacing was performed at a rate 9±8% above the spontaneous heart rate (264±42 beats per min) with square wave pulses of 1 ms at a voltage 100% above threshold; the major purpose of achievement of constant heart rate was to avoid the emergence of contractile changes during ischaemia partially mediated via the Treppe effect.

After 40 min equilibration, baseline samples of coronary effluent were collected for 10 min. BQ-123 (Auspep, Australia) was dissolved directly in Eagle's minimum essential medium. Bosentan (Roche, Switzerland) was dissolved in 1 ml of distilled water at 37°C before addition to Eagle's minimum essential medium. Hearts were perfused with BQ-123 (10–6 M) or bosentan (10–5 M) for 40 min followed by 30 min with normal medium, minus ET receptor antagonist. In some hearts (n=5 for BQ-123; n=12 for bosentan), global ischaemia (perfusion flow reduced by 72.8±2.4% for BQ-123; 59.4±1.9% for bosentan) was induced 10 min after ET receptor antagonist perfusion had commenced. The basis for the disparate reductions in coronary perfusion rate with the two ET receptor antagonists was the development of bradyarrhythmias when bosentan-pretreated hearts were subjected to >67% flow reduction. While the basis for this phenomenon was not elucidated, the reductions in perfusion rate were maximal for maintenance of constant heart rate for both agents tested.

Volume of perfusate was measured by calibrated pipette as an index of coronary flow. Force of contraction and heart rate were monitored on a Grass SD 9 polygraph and dF/dt derived using MacLab v 3.5.2. via a force-displacement transducer. Coronary effluent was collected, snap frozen on dry ice and stored at –80°C. ET-1 concentration in the perfusate was measured by radioimmunoassay.

2.3 ET-1 radioimmunoassay
Perfusate was collected in tubes containing 0.1% trifluoroacetic acid, snap frozen and stored at –80°C until assayed. ET-1 was extracted from perfusate samples using Sep-Pak C18 columns (Waters, Milford, MA, USA). Recovery of ET-1 was 98±6% (n=40). Samples or ET-1 standard (Auspep) were incubated with rabbit anti-ET-1 serum (Peninsula, Belmont, CA, USA) for 48 h then with 125I ET-1 (Amersham, Little Chalfont, UK) for 16–24 h. Bound and free endothelin were separated using 17.6% polyethylene glycol 6000 and human {gamma} globulin to precipitate the bound fraction. Supernatant was discarded and the pellet counted in a gamma counter. Intra- and inter-assay coefficients of variation were 12.0% (n=5) at 0.802 fmol and 17.0% (n=11) at 1.433 fmol, respectively. Neither BQ-123 nor bosentan interfered with the measurement of ET-1 in the radioimmunoassay.

2.4 Analysis of data
Results are expressed as mean±standard error of the mean. ET-1 release rate was expressed as fg/min/unit wet weight of heart. Changes in perfusion flow, increase of ET-1 release and decrease of force of contraction during ischaemia were expressed as area under curves. Comparison of area under curves was performed using unpaired Student's t-test. All other comparisons were made utilising two factor analysis of variance (ANOVA), with Dunnett's test where appropriate for multiple comparisons. A P-value of <0.05 was considered statistically significant.

All studies conform with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication. No.85-23, revised 1985).


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effect of BQ-123 and bosentan on coronary perfusion flow-rate
3.1.1 Non-ischaemic hearts (Fig. 1)
Basal coronary perfusion rate was 7.9±0.3 ml/min/g wet heart weight. In control hearts (n=6), there was no significant fluctuation in perfusion rate over the duration of the experiment. During perfusion with BQ-123 (10–6 M) for 40 min, there was a progressive (P<0.0001) decrease in perfusion rate. The resultant difference between perfusion rates for BQ-123 and control hearts persisted (P=0.01) after wash-out of BQ-123. In contrast, bosentan (10–5 M) induced no significant changes in perfusion rate, either during, or subsequent to it.


Figure 1
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  		Fig. 1 Effects in non-ischaemic hearts of BQ-123 (10–6 M) ({square}, n=7) and bosentan (10–5 M) ({circ}, n=7) on perfusion flow-rate (PFR) compared with control (bullet, n=6).

 
3.1.2 Ischaemic hearts
In the absence of BQ-123 or bosentan, the induced decrease in flow-rate was followed by a phase of post-reperfusion increase in perfusion flow-rate (P<0.0001) (Fig. 2a and b). After perfusion with BQ-123 (Fig. 2a), post-ischaemic flow-rate was significantly (P<0.05) lower than for control hearts. Although bosentan tended to cause a mild increase in perfusion flow-rate during the pre-ischaemic phase, the effect was not significant. Bosentan did not significantly affect the post-ischaemic flow-rates (Fig. 2b).


Figure 2
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  		Fig. 2 Effect of BQ-123 (10–6 M) ({square}, n=5) and bosentan (10–5 M) ({circ}, n=12) on perfusion flow-rate (PFR) in ischaemic hearts compared with the same degree of ischaemia in absence of ET receptor antagonists ({blacktriangleup}, n=16,17).

 
3.2 Effect of BQ-123 and bosentan on ET-1 release
3.2.1 Non-ischaemic hearts (Fig. 3)
Basal ET-1 release rate was 4.70±0.024 fmol/s/g wet heart weight. In the absence of ischaemia and/or ET receptor antagonists, this basal rate did not fluctuate significantly. Almost immediately after BQ-123 perfusion had commenced, ET-1 efflux rate increased, the increase within 5 min being 730±188% (P<0.05). This increase in ET-1 efflux was sustained throughout the BQ-123 perfusion period. Ten minutes post BQ-123 wash-out, the ET-1 efflux rate had returned to baseline. Bosentan caused a gradual but sustained increase (P<0.0001) in ET-1 efflux rate with a peak increase of 315±81% after 30 min. The increase was significantly less than the increase in ET-1 efflux induced by BQ-123 (P<0.05). Following wash-out of either BQ-123 or bosentan, the ET-1 efflux rate returned towards baseline levels.


Figure 3
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  		Fig. 3 Effect of BQ-123 (10–6 M) ({square}, n=7) and bosentan (10–5 M) ({circ}, n=7) compared with control (bullet, n=6) on endothelin-1 (ET-1) efflux rate in non-ischaemic hearts.

 
3.2.2 Ischaemic hearts
In the absence of BQ-123, ischaemia (75.3±0.9% perfusion flow reduction) did not alter ET-1 efflux, but there was a transient increase in ET-1 efflux during reperfusion. The increases in efflux induced by BQ-123 was less marked during ischaemia than in its absence (P<0.05) (Fig. 4a). In the absence of bosentan, moderate ischaemia (58.2±0.9% flow reduction) did not affect ET-1 efflux. However, with the same level of ischaemia, ET-1 efflux in the bosentan-treated group was significantly increased (P<0.001) (Fig. 4b), there was a secondary increase (P<0.0001) in ET-1 efflux following reversal of ischaemia.


Figure 4
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  		Fig. 4 Effects in ischaemic–reperfused hearts of BQ-123 (10–6 M) ({square}, n=5) and bosentan (10–5 M) ({circ}, n=12) on endothelin-1 (ET-1) efflux rate compared with control ischaemia/reperfusion ({blacktriangleup}, n=16,17).

 
3.3 Effect of BQ-123 and bosentan on force of contraction
3.3.1 Non-ischaemic hearts
In the absence of ET-1 receptor antagonists, there was no significant change in force of contraction during the experimental period. BQ-123 or bosentan perfusion, started 10 min after the first baseline measurement, had no detectable effect on cardiac force of contraction. However, bosentan induced a significant change in the force–time interaction relative to that seen in control hearts (ANOVA; F=2.977, P=0.008). This implies a trend towards progressive reduction in force of contraction during bosentan infusion (Fig. 5).


Figure 5
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  		Fig. 5 Effects in non-ischaemic hearts of BQ-123 (10–6 M) ({square}, n=7) and bosentan (10–5 M) ({circ}, n=7) on force of contraction (Force) compared with control (bullet, n=6).

 
3.3.2 Ischaemic hearts
Severe ischaemia (n=16) per se induced a transient decrease in force of contraction (P<0.005). BQ-123 perfusion for 10 min prior to ischaemia had no effect on cardiac force of contraction. However, during global ischaemia, BQ-123 induced a trend (P=0.06, n=5) towards accentuation of ischaemia-induced decrease in cardiac force of contraction (Fig. 6a). Fig. 6b illustrates the effect of bosentan on cardiac force of contraction in ischaemic hearts. In the absence of bosentan, coronary flow reduction induced a transient decrease in force of contraction (P<0.05 vs. control, n=17). Ten minute pre-ischaemic bosentan perfusion did not alter the cardiac force of contraction. However, bosentan induced a further decrease in force of contraction (P<0.05, n=12) during the ischaemic phase. During reperfusion, the recovery of cardiac systolic function was impaired in bosentan-treated groups (P<0.05).


Figure 6
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  		Fig. 6 Effects in ischaemic–reperfused hearts of BQ-123 (10–6 M) ({square}, n=5) and bosentan (10–5 M) ({circ}, n=12) on cardiac force of contraction compared with control ischaemia/reperfusion ({blacktriangleup}, n=16,17).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The results of the current study indicate that the effects of the selective ETA receptor antagonist BQ-123 on coronary vasomotion and ET-1 efflux differ markedly from those of the ETA/ETB antagonist bosentan in the Langendorff-perfused rat heart. These differences are evident both in the presence and absence of transient myocardial ischaemia. Major differences were: (i) coronary vasoconstriction occurred during and after perfusion with BQ-123 but not bosentan; (ii) post-ischaemic perfusion flow-rate was significantly lower with BQ-123 than during control perfusion, while there were no significant differences between bosentan and control during this phase; and (iii) bosentan but not BQ-123 increased ET-1 efflux during ischaemia. Furthermore, while neither agent markedly affected contractile performance behaviour in the absence of ischaemia, bosentan significantly reduced force of cardiac contraction during myocardial ischaemia; BQ-123 tended to exert a similar effect (P=0.06). These findings have both physiological and pharmacological implications.

This is the first study demonstrating coronary vasoconstrictor effects of an ETA antagonist. Previous studies with BQ-123 have primarily addressed its inhibition of exogenous ET-induced vasoconstriction which is usually evident in the 0.1–10 µM range [3]. The concentration used in the present study (1 µM) is therefore assumed to be an effective concentration for the ETA receptors. Despite findings that BQ-123 reduces infarct size and inhibits anoxia-induced coronary vasoconstriction in canine models [13, 34], specific investigations of the interaction between endogenous ET and ETA receptor antagonists on vasomotor tone are relatively few. One investigation in isolated isovolumetric rat hearts by Han et al. [35]failed to demonstrate any effects of BQ-123 (estimated as 1 µM) on coronary vasomotor tone. However, in their study, effects of BQ-123 infusion on coronary flow were examined only after 5 min of infusion: in the current experiment no vasoconstriction was detectable at that stage. Furthermore, they did not examine either recovery of coronary flow after reperfusion, or effects of BQ-123 on ET-1 efflux from the heart. Information related to coronary vasomotor effects of bosentan is also somewhat limited. However, Dagassan et al. demonstrated a coronary vasodilator effect (coronary flow increased by 14%) of bosentan in pre-ischaemic rat heart, which did not persist during the immediate post-ischaemic phase [16]; these results are similar to those of the current study.

In this study, both BQ-123 and bosentan induced an increase in ET-1 efflux. The effect of BQ-123 accords with a previous finding in cultured human umbilical vein smooth-muscle cells where FR139 317, an ETA receptor antagonist, caused an immediate, transient but marked increase in ET levels, with a decrease in levels of big ET-1 in the supernatant [36]. With the recent identification of ETA receptors on endothelial cells [37, 38], it is possible that ET antagonists might inhibit a negative feedback mechanism involving ET-1 regulating its own release. There are no previous studies which have specifically investigated this possibility. Nevertheless, the implication that endogenously released ET might in some way exert homeostatic control on tissue ET concentrations, presumably via interaction with cardiac ETA receptors, is of considerable physiological interest.

In the specific case of bosentan, an additional mechanism might theoretically contribute to increased ET-1 efflux. There is considerable information showing that ETB receptors mediate ET clearance from the circulation, especially into pulmonary endothelium [4, 28, 38–42]. Studies of clearance of ET-1 from plasma into pulmonary tissue during acute ET-1 administration in intact dogs [39]and rats [4]and also in isolated perfused rat lungs [40]demonstrate specific inhibition of the clearance process by the selective ETB receptor antagonist BQ-788. These findings are also consistent with elevation of plasma ET-1 concentrations induced by bosentan [28, 41]. As regards possible specific effects of ETB receptor stimulation on ET-1 kinetics within the heart, Brunner and Doherty have demonstrated that BQ-788, but not the ETA antagonist PD 155 080, increased ET-1 release using a perfused rat heart model [42]: these results are at variance with the findings in our current experiments, which suggest primary or sole modulation of ET-1 efflux via a ETA receptor-dependent mechanism. While the basis for this discrepancy is not clear, there are a number of significant differences in experimental conditions. The utilization in Brunner and Doherty's experiments of (i) colloid-free Krebs solution (vs. albumin-containing medium), (ii) exclusion of potential effects of ventricular endocardial endothelium on ET-1 kinetics [42, 43](vs. ventricular perfusion utilizing current experimental design) and (iii) constant flow (vs. constant pressure) perfusion may contribute to this discrepancy. However, in their experiments, significant increases in ET-1 efflux with BQ-788 appeared progressively only after 75 min of perfusion, while the current experiments involved shorter duration of ET antagonist exposure. Irrespective of the reason, it must be emphasized that we have found no evidence of an ETB-selective mechanism for increased ET-1 efflux in the current experiments: the observation that stimulated ET-1 efflux is more pronounced with BQ-123 than with bosentan implies that, at least over the first 40 min of ET receptor antagonist perfusion, this phenomenon is ETA receptor-mediated.

The finding of coronary vasoconstriction during and after ETA receptor antagonist perfusion appears paradoxical since it implies activation of vasoconstrictor mechanisms both in the absence and presence of transient coronary flow reduction. It is conceivable that the increase in ET-1 efflux induced by BQ-123 mediated the observed vasoconstriction and that the total absence of vasoconstriction despite ET-1 release with bosentan is an indication that the increased ET-1 exerted its effects via the ETB receptors alone. Although the selectivity can be attributed to blockade of the ETA receptors mediating vasoconstriction, an additional factor may be the selectivity of the ETB receptor for ET-1 in low concentrations [14, 44]; which would render ETA antagonists to be relatively ineffective.

After BQ-123 wash-out, in the non-ischaemic heart, despite return of the ET-1 efflux to baseline, the vasoconstrictor effect of BQ-123 persisted. This effect is likely to reflect persistent stimulation of ETB receptors by ET-1, given the extremely slow dissociation rate of ET-1 from ETB receptor [45]. This hypothesis is also consistent with the lack of "rebound" vasoconstriction with the ETA/ETB antagonist bosentan, since this agent, although accelerating ET release, would have prevented binding of ET with ETB receptors. We are not aware of previous reports of "rebound" vasoconstriction after wash-out of an ET receptor antagonist. A model which takes into account the current observations is shown in Fig. 7. It is emphasised that this model is speculative and more detailed experiments exploring the source of the ET-1 will be required to assess its validity. Similarly, during post-ischaemic reperfusion, there was a significantly lower coronary flow for BQ-123 than control, while there was no such effect for bosentan (Fig. 2). The experimental design did not permit us to determine whether this difference reflected progressive differential effects of ET receptor antagonists on coronary vasomotor tone (i.e. continuation of the slight pre-ischaemic vasoconstriction with BQ-123 and vasodilation with bosentan), or whether it might represent a specific interaction with post-ischaemic vasodilator physiology.


Figure 7
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  		Fig. 7 Schema of proposed mechanisms underlying incremental ET-1 efflux and resultant ETB receptor-mediated coronary smooth muscle contraction in the presence of selective ETA antagonist (BQ-123).

 
The results of the current study as regards contractile performance of left ventricle also have implications regarding both the receptor mechanisms underlying positive inotropic effects of ET and also the potential homeostatic effects of these mechanisms during severe myocardial ischaemia. Furthermore, the effects observed with BQ-123 and bosentan are relevant to the use of ET receptor antagonists in patients prone to episodic myocardial ischaemia. In the current experiments, neither BQ-123 nor bosentan affected force of contraction during normal perfusion. However, during a moderate reduction in coronary flow, bosentan (10–5 M) tended to accentuate the ischaemia-induced reduction in force of contraction. These changes were not mediated via ET-induced ischaemia, as coronary flow was externally adjusted in order to eliminate variability between groups. Furthermore, heart rate was kept constant. The results therefore strongly imply a differential effect of endogenous ET on cardiac systolic function, consistent with incremental ET release [21]and/or effect during the ischaemic period.

The inconclusive results as regards BQ-123 may imply type 2 error; supplies of this agent were limited. No experiments with selective ETB antagonists were performed in this series. Nevertheless, the magnitude of the observed changes with bosentan (10–5 M) was no greater than that seen with BQ-123. Therefore, the results remain consistent with previous observations in isolated ferret papillary muscle [8]that the positive inotropic effects of ET are entirely mediated by ETA receptor stimulation. Surprisingly, in rabbit papillary (or ventricular) muscle, myocardial ETA receptors appear to mediate inotropic responses to ET-3 [9, 10], but not ET-1 [9]. A previous study in rat isolated right ventricular strips revealed that the ETA receptor antagonist BQ-123 exerted a moderate negative inotropic effect in the absence of ischaemia [23]. This indicates that ETA receptors mediate at least a component of the inotropic effect of endogenous ET in the rat. However, in the current study, no significant negative inotropic effects of BQ-123 or bosentan were detected during non-ischaemic perfusion.

The implied existence of an accentuated positive inotropic effect of endogenous ET during acute global ischaemia and/or early reperfusion period is a novel finding of this study. Such an effect would tend to be homeostatic, and its disruption is an area of potential concern regarding the future clinical use of ET receptor antagonists. However, currently available studies with whole animal models of myocardial ischaemia/reperfusion do not reveal any deleterious effect of ET receptor antagonists [24, 25]; indeed BQ-123 reduced infarct size in a canine model. [26]These results are not necessarily paradoxical. First, ET receptor antagonists may exert beneficial effects on coronary perfusion (direct or via collaterals) and on cardiac work (via afterload reduction); neither of these circumstances could be examined with the current experimental design. Furthermore, no studies to date have included detailed examination of ET receptor antagonist effects on haemodynamic status during myocardial ischaemia.

One of the unexpected results of the current study was the abolition of ET-1 efflux during ischaemia by BQ-123, with minimal changes during the ischaemic period in the presence of bosentan (Fig. 4). The mechanism of this effect was not elucidated in the current study. As the experimental design resulted in somewhat greater flow reductions during BQ-123 than during bosentan perfusion experiments, as discussed previously, it is possible that this small differential degree of ischaemia may have contributed to the effects observed. However, an alternative explanation would be an acute reduction in extent of ETA receptor occupancy during ischaemia in the case of 10–6 M BQ-123 in particular. Previous investigations have demonstrated externalization of ET receptors in rat heart during ischaemia, with a consequent increase in Bmax [9]. Hence, if ET-1 release in the presence of ETA antagonists were modulated by an ETA receptor-mediated negative "feedback" mechanism, an increase in ETA receptor density would provide a basis for decreased extent of ET by low concentration of ETA antagonists. The observed effects of BQ-123 in particular on contractile performance during ischaemia may reflect these mechanisms.

A possible limitation of the current study is inherent in the use of a colloid-perfused, rather then blood-perfused preparation. It is probable, for example, that extent of post-ischaemic increases in coronary flow would have been somewhat greater with blood-perfused preparations.

In conclusion, in isolated perfused rat hearts, both BQ-123 and bosentan increased ET-1 efflux, but only BQ-123 exerted vasoconstrictor effects. Furthermore, during ischaemia the effects of BQ-123 on ET-1 efflux were abolished, and bosentan induced a significant negative inotropic effect. These results offer new insights into possible local cardiac mechanisms of ET-1 release, and also indicate that transient exposure to the ETA antagonist BQ-123 may induce coronary vasoconstriction which persists unchanged during a wash-out phase. The results also strongly suggest that the importance of ET-induced positive inotropic effects is markedly accentuated during ischaemia. Clinical implications are that selective ETA antagonists may be disadvantageous relative to ETA/ETB receptor antagonists in the presence of myocardial ischaemia, and that if such agents were withdrawn, a gradual rather than an abrupt cessation would be preferable. Furthermore, the negative inotropic effects of ET receptor antagonists (especially selective ETA receptor antagonists) during ischaemia may limit their safety in patients with combined ischaemia and impaired left ventricular function.

Time for primary review 32 days.


   Acknowledgements
 
Dr. Zhang was supported by a scholarship from the University of Adelaide and The Queen Elizabeth Hospital. This work was supported in part by a grant from the National Health and Medical Research Council of Australia. Bosentan was kindly supplied by Hoffmann–La Roche. We wish to thank Prof. I.S. de la Lande for helpful advice and criticism.


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

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