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Cardiovascular Research 1998 39(3):609-616; doi:10.1016/S0008-6363(98)00172-2
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Reduced pulmonary metabolism of endothelin-1 in canine tachycardia-induced heart failure

Jocelyn Dupuisa,*, Gordon W Moeb and Peter Cernaceka

aDepartment of Medicine, Montreal Heart Institute, 5000 Bélanger Street East, Montreal, Quebec H1T 1C8, Canada
bSt. Michaels Hospital, Toronto, Ontario, Canada

* Corresponding author. Tel.: +1-514-376-3330 ext. 3542; Fax: +1-514-376-1355; E-mail: Dupuisj@icm.umontreal.ca

Received 4 February 1998; accepted 26 May 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusion
 References
 
Objectives: Plasma endothelin-1 (ET-1) increases in congestive heart failure (CHF). The pulmonary vascular bed could contribute to this increase through a reduced clearance. We evaluated the effect of tachycardia-induced CHF on pulmonary ET-1 kinetics. To discern between changes due to variations in pulmonary hemodynamics from true alterations of endothelial cell functions, we quantified ET-1 kinetics in isolated rat lungs under variable pressure and flow-rate conditions. Methods and Results: Indicator-dilution studies were performed in anesthetized dogs (n=14) before and 3 weeks after rapid ventricular pacing and in isolated lungs from healthy rats (n=4). In isolated lungs, graded increases in perfusion rate from 5–25 ml/min caused gradual reductions in ET-1 extraction from 60±1.5% to 17±4.9% (mean±S.D.). The capacity to clear ET-1 from the circulation, as computed from the permeability–surface area product (PS), however did not vary over this range of flows. CHF increased plasma ET-1 (11.2±11.4 vs. 5.2±1.6 fmol/ml, p<0.01), did not affect pulmonary ET-1 extraction (29.4±12.5% vs. 29.9±12.9%), but decreased the PS (8.3±5.4 cm3/s vs. 14.4±9.9 cm3/s, p=0.038). Contrary to the invariability of the PS in normal isolated rat lungs, CHF was associated with a positive relationship between the PS and pulmonary plasma flow (r=0.65, p<0.01). ET-1 binding studies in lung tissues showed no significant variations in ETA and ETB receptors densities but revealed a threefold decrease in binding affinity (p<0.01) that may explain the reduced clearance. Conclusion: CHF causes a reduction of pulmonary ET-1 clearance that likely contributes to the increased circulating ET-1 levels and reflects pulmonary metabolic dysfunction associated with this condition.

KEYWORDS Endothelin; Pulmonary circulation; Pacemaker; Endothelial function; Heart failure


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 5 Conclusion
 References
 
Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that contributes to the counterregulatory neurohumoral activation found in congestive heart failure (CHF) [1]. The increase in circulating ET-1 levels found in CHF correlates closely with the increase in pulmonary capillary wedge pressure (PCWP) as well as with pulmonary artery pressure [2]suggesting that the pulmonary circulation may contribute to the increase in ET-1 levels.

The pulmonary circulation plays an important role in the metabolism of ET-1 of various mammals. In rats, the majority of intravenously injected 125I-ET-1 is retained by the lungs, followed in importance by the liver and the heart [3]. Isolated rat lungs retain the ability to remove close to 90% of labeled ET-1 added to a recirculating perfusate [4]. More recently, we have confirmed these findings in vivo using the indicator-dilution technique in dogs and in humans and found, respectively, a 33±8% and 47±8% pulmonary extraction of ET-1 during a single pulmonary transit time [5, 6]. In the normal dog and human, the lung simultaneously releases a quantitatively similar amount of ET-1 explaining the absence of significant arterio–venous ET-1 gradient.

Studies in rats and dogs have shown that pulmonary ET-1 clearance is mediated through the endothelial ETB receptor [7, 8]; in dogs, the specific ETB antagonist BQ788 completely inhibits pulmonary ET-1 removal in vivo. Consequently, a downregulation or desensitization of this receptor could potentially contribute to a reduced clearance of this peptide and to the increase in circulating levels found in CHF. The tachycardia-induced model of CHF closely simulates human cardiomyopathy and is associated with the neurohumoral activation of CHF including an increase in plasma ET-1 levels [9]that correlates independently with PCWP and right atrial pressure. The aim of this study was therefore to determine if pulmonary ET-1 clearance is affected by tachycardia-induced CHF. In order to help in the interpretation of pulmonary ET-1 kinetics in heart failure, we also measured the effect of flow and pressure variations on ET-1 kinetics of isolated rat lungs.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 5 Conclusion
 References
 
2.1 Heart failure experiments
The investigations performed 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 1996). Fourteen mongrel dogs were studied. They were anesthetized with pentobarbital, intubated and mechanically ventilated using room air and continuously monitored using cutaneous electrocardiographic leads. A right femoral incision was done and the femoral artery and vein were isolated for catheter insertion. A multipurpose catheter was then inserted via the femoral artery and positioned about 2 cm above the aortic valve using fluoroscopic guidance: this catheter continuously monitored systemic pressure and was used as the collection catheter for the indicator-dilution experiment. A Swan–Ganz catheter was inserted in the vein and advanced to measure pressures of the right atrium, the pulmonary artery and the PCWP. After completion of these measurements, the Swan–Ganz catheter was withdrawn and replaced by a multipurpose catheter to be positioned in the right ventricular outflow tract and serve as the injection site for the indicator-dilution experiment. To realize the single bolus indicator-dilution experiment, a 2 ml bolus containing tracer 125I-ET-1 and Evans' blue dye labeled albumin was introduced into a 1.5 ml extension tubing connected to the catheter so that the whole of the bolus could be contained within the injection's line dead space. The bolus was then flushed with 10 ml of the dog's blood and blood collection was simultaneously begun from the collection line which had been connected to a Masterflex roller pump. The blood was pumped at 120 ml/min in a circular sample collector containing 50 heparinized tubes at a rate of 60 tubes/min. A permanent pacemaker was then installed as previously described [10]and, after 48 h of recovery, was programmed to pace at 200 beats/min. After a period of 3 weeks, the pacemaker was turned off and the animals were again anesthetized and instrumented for blood sampling, hemodynamic measurements and the indicator-dilution study.

2.2 Pulmonary ET-1 kinetics
The indicator-dilution technique was utilized to quantify the in vivo pulmonary metabolism of ET-1 as previously described [5]. Briefly, a bolus containing trace doses of 125I-ET-1 and a non-metabolizable vascular reference (Evans' blue dye bound to albumin) are injected in the pulmonary circulation and timed sequential outflow samples are collected. The quantity of both tracers in each of the collected samples is determined and normalized to the total amount of activity injected to obtain the fractional recovery of each tracer per ml of blood. The fractional recoveries can then be plotted as a function of time to construct the indicator-dilution curve. Cardiac output can then be computed as:


Formula 1

(1)
where q0 is the total amount of Evans' blue dye bound albumin injected and the denominator represents the area under the fractional recovery versus time curve for the same tracer. The recirculating tracer is mathematically removed by linear extrapolation of the exponential downslope on a semi-logarithmic plot. The mean transit time of tracer albumin through the lungs is computed as:


Formula 2

(2)

The central blood volume was computed as the product of cardiac output and albumin's mean transit time. This volume is composed mainly by the pulmonary blood volume (including the large pulmonary arteries) with a smaller contribution from the blood contained in the left atrium and ventricle. Tracer ET-1 extraction is calculated by the following equation:


Formula 3

(3)
where the right term represents the difference in the areas of the fractional recovery versus time curves for tracer albumin and ET. We have previously shown that during a single pulmonary transit time there is no apparent return into circulation of the tracer ET-1 that has been extracted by pulmonary tissue upto the time of recirculation [5]. ET-1 binds to the ETB receptor with high affinity [11]and autoradiographic studies have shown that ET-1 binding in lung tissue is resistant to dissociation [12]. These are compatible with the in vivo observation of the very short half-life of intravenously injected radioactive ET-1 in rats with most of the activity being retained by the lungs. Accordingly, we used the Renkin and Crone approximation for diffusible tracers which indicates that the fraction of the diffusible tracer (in this instance the extractable tracer ET-1) that reaches the outflow as a throughput would be exp(–PSc/Fc) times what would have reached the outflow if it had not crossed the capillary barrier (in this instance the non-diffusible tracer albumin). In this expression, PSc is the permeability–surface area product for the capillary and Fc is its plasma flow. This expression is usually utilized for the analysis of data obtained from the whole organ when it is applied to the upslope and peak of the dilution curve where all of the tracer is assumed to represent throughput without returning component. In the case of ET-1 where the form of the indicator dilution data and previous studies strongly suggest the absence of a returning component during a single pulmonary transit time, it becomes reasonable to apply the model to the whole of the dilution curve. Accordingly, the PS for ET-1 in the lungs was computed as:


Formula 4

(4)
where Fp is pulmonary plasma flow in ml/s and Ext is tracer ET-1 extraction.

2.3 Immunoreactive ET-1 measurements
Before each indicator-dilution study, 5 ml of blood was taken from the aortic catheter. Plasma ET-1 levels were determined by radioimmunoassay on extracted plasma using a commercial kit (Amersham, Arlington Heights, IL, USA). Rabbit anti-ET-1 serum was added to each sample and incubated for 16–24 h at 4°C. Competitive binding of the tracer 125I-ET-1 was achieved with an incubation period of 24 h. ET-1 was determined from the standard curve generated on a semi-logarithmic graph. The detection limit of the assay was 2.6 fmol/ml. Recovery was 73% and the intra-assay coefficient of variation was 8.7%. Cross reactivity of the assay with big ET-1 is of 37%.

2.4 ET metabolism in isolated rat lungs
Male Sprague–Dawley rats (n=4, Charles River, St. Constant, Quebec, Canada) were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) followed by 2000 U of i.p. heparin (Sigma, St. Louis, MO, USA). After stable anesthesia was obtained, the trachea was intubated and connected to a rodent ventilator (Harvard Apparatus, St. Laurent, Quebec, Canada) and ventilated using room air with a tidal volume of 1 ml and 2 cm H2O positive end-expiratory pressure.

A midline sternotomy was performed to expose the heart and lungs and the pulmonary artery was cannulated through an incision in the right ventricle to collect the effluent from the lungs. The lungs' perfusion was initiated by slowly infusing (2.0 ml/min) Krebs solution containing (mM): NaCl 120, NaHCO3 25, KCl 4.7, KH2PO4 1.18, MgSO4 1.17, CaCl2 2.5 and glucose 5.5. The Krebs was bubbled with 95% O2 and 5% CO2 to maintain a pH of 7.4. The lungs were then rapidly isolated and suspended in a warmed (37°C) water-jacketed chamber to be perfused in non-recirculating fashion with Krebs solution supplemented with 3% albumin at 37°C. The pulmonary flow was continuously measured with a transonic flow probe (Transonic, New York, NY, USA) connected to a flowmeter (model 208) and put on the circuit proximal to the pulmonary cannula. The perfusion pressure was also continuously recorded by a fluid filled pressure transducer connected to a Gould signal conditioner.

The lungs were then perfused under constant flow conditions for 20 min of stabilization using a Masterflex roller pump (Cole–Palmer, Niles, IL, USA) at a rate of 10 ml/min. Perfusion rate was then successively set at 5, 10, 15, 20, and 25 ml/min with an equilibration period of 5 min for each perfusion rate. A single bolus indicator-dilution experiment was carried at the end of each equilibration period by injecting a bolus in the perfusion cannula immediately proximal to the lungs and simultaneously collecting the lung effluent in a linear fraction collector containing 30 glass tubes. The collection times for the flow-rates of 5, 10, 15, 20, and 25 ml/min were 40, 28, 28, 15 and 13 s respectively.

The injection mixture was prepared by adding 0.5 µCi 125I-ET-1 (specific activity 2200 Ci/mmol; NEN, Boston, MA, USA) to 0.5 ml Evans' blue dye (5 mg/ml) and 1.5 ml 0.9% NaCl. Bovine serum albumin (Sigma) was then added at a concentration of 4%. A volume of 100 µl of the mixture was taken and injected at each of the above perfusion rates. Four 1:100 dilutions of the remaining bolus were done to serve as standards for the construction of the dilution curve. The tubes containing the lung effluent were processed by adding 2.0 ml 0.9% saline and vortexing. A 1 ml aliquot from each tube was pipetted into another tube and placed in a gamma counter to detect 125I activity and another 1 ml was placed in a spectrophotometer cuvette for measurement of Evans' blue dye absorbance (620–740 nm). The fractional recovery of each tracer in each sample was then determined and the indicator-dilution curves could be constructed as above. Pulmonary blood volume was computed as a product of perfusion flow-rate and labeled albumin mean transit time as described above. Tracer ET-1 extraction was computed using Eq. (3)with the exception that no extrapolation of the curves was necessary since no recirculation occurs in isolated lung preparation. The downslopes of the curves were therefore analyzed up to a point representing less than 5% of the peak.

2.5 ET receptor binding
Lung tissue from control (n=5) and heart failure dogs (n=6) were processed to prepare cell membranes. Approximately 100 mg of tissue from each lung was homogenized on ice in 2 ml of cold tris(hydroxymethyl)aminomethane (Tris) buffer (in mM: 25 Tris·HCl, 2 MgCl2, 250 sucrose, and 5 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid, pH 7.4) with a polytron (Brinkman, Rexdale, Ontario, Canada) at 13 000 rpm in six bursts of 15 s each. The homogenate was centrifuged at 1000 g for 10 min at 4°C, and the supernatant was collected and centrifuged at 35 000 g for 30 min at 4°C. The resulting pellet was resuspended in Tris buffer, aliquoted, and stored at –80°C. Amounts of protein were determined by the dye-binding method with bovine serum albumin as the standard. The type, density, and affinity of ET receptors in pulmonary vessels were assessed by competitive binding experiments. 125I-labeled ET-1 (20–25 pM) was added to each tube containing either 50 µl of Tris buffer or increasing concentrations of unlabeled ET-1 or BQ123. The binding reaction was initiated by adding 100 µl of membrane protein (1–4 µg/tube) to a final incubation volume of 200 µl. After 3 h at room temperature, the reaction was stopped by addition of 1 ml of cold phosphate-buffered saline containing 0.5% bovine serum albumin and rapid centrifugation at 12 000 g for 5 min. Radioactivity of the resulting pellet was determined in a gamma counter. Non-specific binding was measured in the presence of 2·10–7 M unlabeled ET-1. The dissociation rate constant (KD) and maximal binding (Bmax) for ET-1 were obtained using the receptor–fit competition program (London, Chagrin Falls, OH, USA).

2.6 Statistical analysis
Hemodynamic parameters, ET-1 levels and pulmonary ET-1 kinetic data measured at baseline and after the induction of heart failure were compared using two-tailed paired Student's t-test. The effect of perfusion flow-rate on the PS product, perfusion pressure and pulmonary vascular volume of isolated rat lungs was evaluated by a repeated measure analysis of variance (ANOVA) followed by multiple comparisons when a significant effect of flow-rate was found. Correlation of ET-1 levels and of the PS with hemodynamic parameters was done using a Pearson correlation matrix. Results from ET receptor binding studies were analyzed using Mann–Whitney tests. A p-value of less than 0.05 was considered significant. All values are reported as mean±S.D.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 5 Conclusion
 References
 
3.1 Effect of flow on ET-1 metabolism in isolated rat lungs
The graded increase in lung perfusion rate resulted in an almost linear decrease in pulmonary ET-1 extraction from 60±1.5% at 5 ml/min to 17±4.9% at 25 ml/min (Fig. 1). This was accompanied by the expected increase in pulmonary perfusion pressure. The PS for ET-1 removal by the isolated rat lungs was computed at each flow-rate (Fig. 2). There was no significant effect of flow-rate on the PS (p=0.094 by ANOVA), although there was a net tendency for the PS to increase in the transition from a very low flow-rate of 5–10 ml/min. At the highest flow of 25 ml/min, the mean PS tended to decrease but displayed a much wider standard deviation. Pulmonary vascular volume varied significantly with pulmonary perfusion flow-rate (Fig. 2): it increased in the transition form from 5 ml/min to 10 ml/min (from 1.06±0.10 ml to 1.51±0.08 ml) and remained unaffected with further increases in flow.


Figure 1
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  		Fig. 1 Effect of increasing perfusion flow-rate of isolated rat lungs (n=4) on the fractional single pass extraction of endothelin-1 (ET-1) and on the perfusion pressure. Percent ET-1 extraction decreased almost linearly as flow-rate and perfusion pressures were increased. **p<0.01 by repeated measures ANOVA.

 

Figure 2
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  		Fig. 2 Upper graph: effect of increasing perfusion flow-rate on the permeability–surface area product (PS) for endothelin-1 removal by isolated rat lungs. The PS tended to increase in the transition from 5–10 ml/min. There was no significant variation of the PS by repeated measures ANOVA. Lower graph: effect of increasing perfusion flow-rate on the pulmonary vascular volume of isolated rat lungs. **p<0.01 vs. 5 ml/min.

 
3.2 Pulmonary ET-1 metabolism in tachycardia-induced heart failure
Rapid ventricular pacing for a period of 3 weeks resulted in the development of CHF with a decrease in cardiac output and an increase in heart rate and left ventricular filling pressure (Table 1). There was also a significant increase in mean pulmonary artery pressure as well as pulmonary vascular resistance.


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  		Table 1 Hemodynamic parameters and endothelin levels

 
ET-1 levels (n=10) doubled with heart failure (p<0.05). There was a positive correlation between ET-1 levels and PCWP (r=0.54, p=0.008) as well as with mean pulmonary artery pressure (r=0.48, p=0.020) but no significant correlation with any of the other measured hemodynamic parameters.

Tracer ET-1 extraction was 29.9±12.9% at baseline and was not affected by the development of heart failure at 29.4±2.5% (Fig. 3). The PS for ET-1 removal by the lung however decreased from 14.4±9.9 cm3/s in controls to 8.3±5.4 cm3/s (p=0.038) in heart failure (Fig. 3). When the controls and CHF data were combined the PS did not correlate with ET-1 levels (r=–0.178, p=0.80) and any of the measured pressures but correlated directly with cardiac output (p<0.01) and pulmonary plasma flow (p<0.01, Fig. 4). The analysis remained similar when only the CHF data were analyzed. The central blood volume increased from 250±65 ml at baseline to 323±81 ml after the development of heart failure (p<0.01).


Figure 3
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  		Fig. 3 Fractional ET-1 extraction and permeability–surface area product (PS) in control conditions and after the development of congestive heart failure. **p<0.05 vs. control.

 

Figure 4
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  		Fig. 4 Variations of the PS product as a function of pulmonary plasma flow; (bullet) control, ({blacksquare}) heart failure.

 
Results from ET receptor binding experiments are assembled in Table 2. Heart failure was associated with a non-significant increase in both ETA and ETB Bmax values and a non-significant decrease in the percentage of ETB receptors from 65±15% to 55±15%. Binding affinity was greatly reduced by heart failure with a threefold increase in KD (p<0.01).


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  		Table 2 ET-1 receptor binding studies in lung tissues from controls and heart failure dogs

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
5 Conclusion
 References
 
This study was designed to determine if pulmonary clearance of circulating ET-1 is altered by tachycardia-induced CHF. We found that single pass pulmonary ET-1 extraction is unaffected by heart failure, but that plasma ET-1 clearance by the lungs, as measured by the PS for ET-1 removal, is reduced.

4.1 Pulmonary metabolism of ET-1 in the normal rat lung
In order to help in the interpretation of ET-1 kinetics in heart failure, we performed isolated rat lung studies under variable flow and pressure conditions. Since the PS is directly computed from pulmonary plasma flow and tracer ET-1 extraction, it became necessary to determine the effect of flow on these parameters in normal lungs. The PS will theoretically vary with the vascular endothelium surface area that can interact with a vascular substrate as well as with the activity of the specific removal process: in this instance the density and affinity of the endothelial ETB receptor. If those are kept constant, variations of flow will be accompanied by opposite variations in tracer extraction and the PS should remain constant.

As expected from the theoretical predictions of this model, we found an almost linear decrease in tracer ET-1 extraction as flow was increased from 5–25 ml/min. The PS did not significantly vary but demonstrated a mild tendency to increase in the transition from 5–10 ml/min and remained constant thereafter despite subsequent increases in perfusion rate up to 25 ml/min. This behavior of the PS must be analyzed with consideration of the effect of pressure on perfusion heterogeneity of the lung [13, 14]. In the transition from 5 to 10 ml/min the perfusion pressure increased from 3.17±0.2 mmHg to 6.8±0.5 mmHg. This very low perfusion pressure of 3.17±0.2 mmHg with a positive end expiratory pressure of 2 cm H2O (1.47 mmHg) represents a nonphysiologic situation equivalent to very low flow states such as cardiogenic shock. It is likely that the increase of perfusion pressure to 6.8±0.5 mmHg that accompanied the subsequent increase in flow resulted in recruitment of pulmonary capillaries with an increase in the vascular surface area accessible to tracer ET. The observed simultaneous increase of close to 50% in pulmonary blood volume from 1.06±0.11 ml to 1.51±0.81 ml is certainly compatible with this interpretation. Others have directly demonstrated the capacity to recruit pulmonary capillaries using in vivo video-microscopy during conditions that raise pulmonary blood flow and pressure [15]. Our data therefore demonstrate that in conditions of apparent maximal recruitment of the pulmonary vascular bed, the PS for ET-1 removal is constant over a wide range of flow and pressure conditions. However, under very low flow and perfusion pressure, derecruitment of pulmonary capillaries occurs thus reducing the PS. At the highest flow-rate of 25 ml/min, the PS for ET-1 removal showed a tendency to reduce despite maximal recruitment of the pulmonary circulation. We cannot exclude that at these high flow-rates, the high perfusion pressure (18.7±1.9 mmHg) may have caused mechanical disruption of the endothelial integrity with dysfunction of the endothelial ETB receptor explaining the mild non-significant reduction and the wide variability of the measured PS at this flow-rate.

Our data confirm the previously observed pulmonary extraction of ET-1 by rat lungs [4, 7]. In addition, we have characterized the kinetics of this extraction which varies inversely with pulmonary perfusion rate. The metabolic capacity of the normal rat lung to clear the perfusate of ET-1, the PS, is however remarkably constant over a wide range of normal blood flows. At very low flows, when the pulmonary circulation is not fully recruited, the PS tends to augment as the vascular surface area accessible to circulating ET-1 increases with higher perfusion pressures. CHF, which is associated with lower cardiac output (and lower pulmonary flow-rate) but higher pulmonary capillary pressures than in control conditions, thus represents a special condition in which the impact of these factors need to be considered for proper interpretation of ET-1 kinetics.

4.2 Pulmonary ET-1 kinetics in tachycardia-induced heart failure
As expected, dogs with CHF had lower cardiac outputs with higher mean pulmonary artery pressure and PCWP. Circulating ET-1 levels were increased by heart failure. The mean tracer ET-1 extraction of about 31% at baseline was unaffected by the development of heart failure but the capacity of the pulmonary circulation to clear the plasma of circulating ET-1, as computed from the PS, was reduced. To determine the meaning of this variation in the PS, we first need to consider the possible influence of the hemodynamic alterations of heart failure on this parameter. We have shown, in isolated rat lungs, that ET-1 extraction varies inversely with flow and that the PS will vary only if the perfused pulmonary vascular volume is increased. The pulmonary congestion of heart failure is associated with the so-called vascular redistribution since the less dependent portions of the lung become better perfused as pulmonary capillary pressures increase. Accordingly, we found an augmentation in the central blood volume with heart failure: even though part of this increase can be attributed to left ventricular dilatation, the order of magnitude that we found (a mean of 80 ml) supports a concomitant increase in the pulmonary blood volume. Such a rise would result in a greater perfused microvascular surface area that can interact with a vascular substrate. Our data however demonstrate a decrease in the PS despite an apparent increase (or at the minimum, no change) in the pulmonary vascular surface area. Furthermore, this decrease in the PS correlated with the severity of the reduction in cardiac output since tracer ET-1 extraction remained unchanged despite lower outputs. This reduction of tracer ET-1 clearance as pulmonary plasma flow is lowered is completely the opposite of what would be expected from theoretical predictions confirmed by the isolated rat lung data. Our data therefore strongly support the conclusion that pulmonary ET-1 clearance is reduced in this model of heart failure. This reduction occurs without any apparent decrease, and even an increase in the tracer accessible pulmonary vascular surface area so that intrinsic abnormalities of the extraction process must be responsible. We have recently demonstrated that isolated lungs from rats with myocardial infarction and mild secondary pulmonary hypertension also have a reduced pulmonary ET-1 clearance [16]and others have found a downregulation of the pulmonary ETB receptor in the same model [17]. Rats with monocrotaline-induced pulmonary hypertension also demonstrate a downregulation of the pulmonary ETB receptor [18]with a significant decrease in pulmonary ET-1 clearance [19]. The ETB receptor is uniquely responsible for pulmonary ET-1 removal in dogs which is completely abolished by in vivo administration of the specific ETB receptor antagonist BQ788 [8]. A downregulation or desensitization of this receptor may consequently occur as a result of the development of CHF. To evaluate this we performed ET-1 binding experiments in lung tissues and found a slight increase in densities of both ETA and ETB receptors, without achieving statistical significance. In contrast, the affinity of binding of ET-1 was markedly decreased by threefold in CHF dogs (p<0.01). Although the design of these experiments does not allow us to distinguish the changes in affinity of ETA vs. ETB receptors, it is most likely that the affinity of the ETB receptor to its ligand is substantially lower in this model of CHF, thus contributing to the decreased pulmonary clearance of ET-1. The exact mechanism by which CHF could lead to this abnormality and whether or not this is a specific alteration of endothelial cell function associated with heart failure or the manifestation of a more general dysfunction associated with pulmonary hypertension of any etiology will require additional studies.

The pathophysiological significance of this reduction in the metabolic capacity of the pulmonary circulation to clear ET-1 from the circulation is unclear. Although it must certainly contribute to the increase in circulating ET-1 levels found in this model of heart failure, our data also suggest that it is probably not the major contributor since the PS, although it correlated well with cardiac output, did not correlate with other parameters of the severity of heart failure (filling pressures) and more specifically, did not correlate with ET-1 levels. Like others, we however found significant correlation between ET-1 levels and pulmonary as well as left ventricular filling pressures [2, 9, 20]. This suggests that the systemic circulation must also substantially contribute to the increase in the levels of this peptide in relation with the severity of tachycardia-induced heart failure.

Stimulation of the endothelial ETB receptor by ET causes the release of nitric oxide [21]and ET-1 infusion causes transient pulmonary vasodilation [22]. In a dog model of pulmonary hypertension produced by injection of dehydromonocrotaline, the ETB receptor plays a protective role by attenuating basal pulmonary vascular tone [23]. In the rapid ventricular pacing model of heart failure, acute administration of specific ETB receptor antagonist RES-701-1 causes acute hemodynamic deterioration with a decrease in cardiac output, and an increase in PCWP and pulmonary vascular resistance [24]. Although highly speculative, these suggest that a reduced capacity of the endothelial ETB receptor to bind circulating ET-1 could adversely affect pulmonary hemodynamics by attenuating the potential protective role of the ETB receptor.

The pulmonary circulation is also an important site for ET-1 release into circulation: in normal dogs [5], humans [6], the lungs release into circulation an amount of ET-1 quantitatively similar to that that has been extracted, thus explaining the absence or minimal arterio–venous gradient. In the present study, we therefore cannot exclude that CHF may also cause an increased production and release of ET-1 by the lung that could also contribute to the observed augmentation in plasma ET-1.


   5 Conclusion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusion
References
 
Tachycardia-induced heart failure is associated with a reduced capacity of the pulmonary circulation to remove ET-1 from circulation. This may contribute to the observed increase in circulating ET-1 levels. Future studies are needed to determine if this newly described metabolic function of the pulmonary circulation is a simple marker of the pulmonary vascular endothelial dysfunction of CHF or if it bears a significant impact on the pathophysiology of this condition.

Time for primary review 30 days.


   Acknowledgements
 
The authors would like to thank Nathalie Ruel and Ana Albernaz for their expert technical assistance. This work was supported by the Fonds de la Recherche en Santé du Québec, the Heart and Stroke Foundation of Quebec, the Medical Research Council of Canada and the Fonds de Recherche de l'Institut de Cardiologie de Montréal.


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

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