Pulmonary Big Endothelin Affects Coronary Tone and Leads to Enhanced, ETA-Mediated Coronary Constriction in Early Endothelial Dysfunction
Background Lung tissue produces a variety of mediators; however, little is known regarding how these mediators affect coronary regulation and myocardial contractility. In a novel rabbit lung-heart model, we investigated the possible influence exerted by pulmonary mediators on coronary tone both under normal conditions and in early endothelial dysfunction.
Methods and Results In our model, the effluent from the isolated lung is used to serially perfuse the coronary vessels of the isolated heart of the same animal. Compared with the hearts of control rabbits, isolated hearts of Watanabe rabbits revealed pharmacological evidence of endothelial dysfunction and a significant steeper decrease of coronary flow during serial perfusion of the coronary vessels with lung effluent (75±6% versus 89±3%). This decline in coronary flow was prevented by the nonselective endothelin (ET) antagonist PD-145065, the ETA antagonists BQ-123 and A-127722, and the endothelin-converting enzyme inhibitor phosphoramidon. The concentration of big ET in lung effluent ranged from 5.5 to 5.8 pmol/L in both control and Watanabe groups, with levels in corresponding coronary effluent falling to 0.9 to 1.1 pmol/L in controls and to 1.0 to 1.2 pmol/L in the Watanabe group. In either group, ET was not detected in lung effluent, but it rose significantly in coronary effluent during serial perfusion.
Conclusions Pulmonary big ET, locally converted into ET during coronary passage, causes an ETA-mediated elevation in coronary tone under basal conditions as well as an enhanced coronary constriction when early endothelial dysfunction is present.
It is well accepted that lung tissue in general and pulmonary endothelium in particular produce a variety of mediators, including various arachidonic acid metabolites,1 endothelium-derived relaxing factor,2 and several cytokines.3 4 5 However, little is known regarding how the lungs, through the release of these mediators into pulmonary circulation, affect coronary tone and cardiac contractility in specific physiological and pathophysiological states. The use of animal models to help clarify these processes has proved problematic, because it is difficult to decide whether cardiac effects are exclusively triggered by pulmonary mediators or caused by systemic effects, such as altered pulmonary gas exchange, compromised hemodynamics, and neuroregulatory activation.6 7
In response to this difficulty, we established a lung-heart model in which the coronary vessels of a Langendorff-perfused rabbit heart are serially perfused with blood-free effluent from the isolated lung of the same animal. This model allows coronary perfusion pressure and oxygen supply to be strictly controlled, irrespective of the experimental constraints imposed on the lung. In this way, focus can be directed to specific lung-mediated effects, with systemic interferences eliminated.
This study attempts to answer the question of whether coronary vascular tone is affected by mediators of pulmonary origin and, if so, to identify these substances. In addition, we compared findings in healthy New Zealand White rabbits with results obtained in atherosclerotic Watanabe rabbits to address the question of whether early systemic atherosclerosis alters lung-heart interactions in terms of coronary regulation.
Animals and Isolated Organ Preparation
Age-matched (14-week-old) male New Zealand White rabbits and male Watanabe rabbits (Charles River) weighing 2.0 to 2.3 kg were selected for this study. Watanabe rabbits constitute a highly appreciated atherosclerosis model: because of a heritable LDL receptor defect, the animals develop hypercholesterolemia from birth,8 with the ensuing atherosclerotic process closely resembling that observed in familial hypercholesterolemia in humans.9
For excision of heart and lung from the same animal, deep anesthesia with 40 to 80 mg thiopental sodium/kg body wt IP and anticoagulation with 1500 U heparin/kg body wt were performed. After tracheostomy and midsternal thoracotomy, the pulmonary artery was clamped; then a catheter was placed into the aorta, allowing the coronary vessels to be perfused in situ. The heart was rapidly removed and then perfused retrogradely through the aorta at a constant perfusion pressure of 60 mm Hg with a modified Krebs-Henseleit buffer (37.5°C) composed of the following constituents (in mmol/L): NaCl 127, KCl 3.7, CaCl 2.5, KH2PO4 1.2, MgSO4 1.1, NaHCO3 24.9, glucose 10, pyruvate 1.8, and HEPES 5.9. The pH was adjusted to 7.35 to 7.40, and the buffer was oxygenated with a mixture of 95% O2 and 5% CO2. LVP and RVP were measured with a polyethylene catheter, topped with a small latex balloon, inserted into the ventricular cavity. The balloons were slowly filled with saline solution until end-diastolic LVP and RVP stabilized between 1 and 3 mm Hg. Coronary flow rates were measured by an ultrasonic flow probe (Transonic Systems). Only those hearts were accepted that achieved a contractile performance of >70 mm Hg LVPmax and >1000 mm Hg/s maximum rate of pressure rise, dP/dtmax, and maximum rate of relaxation, dP/dtmin, respectively.
For lung excision, the pulmonary artery was cannulated and the lung immediately perfused in a nonrecirculatory mode with Krebs-Henseleit buffer (same composition, pH 7.35 to 7.40); pulsatile flow started at 10 to 20 mL/min. Lungs were ventilated (Servo 910, Siemens-Elema) through a tracheal cannula with 95% O2 and 5% CO2 (tidal volume, 10 to 12 mL/kg body wt, respiration rate 50 breaths per minute, positive end-expiratory pressure 1 mm Hg). Lungs were removed from the thorax and placed in a temperature-equilibrated organ chamber freely suspended from a force transducer for continuous monitoring of weight. The whole system, including perfusate reservoirs, tubing, and organ chamber, was gradually heated to 37.5°C. In parallel, pulmonary flow was gradually increased to 100 mL/min nonrecirculatory perfusion. Only those lungs were selected for the study that had a constant pulmonary arterial pressure (6 to 10 mm Hg), constant peak inflation pressures of 7 to 10 mm Hg, no weight gain (<0.5 g/h), and no signs of hemorrhage, edema, or atelectasis. Random light microscopic examination of the lungs revealed virtually no erythrocytes or platelets and only very few sticking leukocytes in the vascular bed. In addition, no evidence of interstitial or alveolar edema was found.
Endotoxin content of the perfusates ranged below 10 pg/mL (detection limit).
The apparatus used in our investigation (Fig 1⇓) was manufactured by Hugo Sachs Electronics. Modification of the device allows rapid switching from separate to serial organ perfusion; once the switch is made, the amount of lung effluent that equals coronary flow is rapidly conveyed (2 seconds) to the heart apparatus, where it is oxygenated before becoming perfused, at constant pressure, through the coronary vessels.
Serial perfusion groups. During an initial 45-minute period of separate organ perfusion, the lung was rinsed and controlled for pressure and weight invariance, and the heart was tested for endothelium-dependent and -independent coronary vasodilator response with the following substances. The endothelium-dependent vasodilator ACh was applied at a concentration of 2×10−6 mol/L, a level that produces the maximum vasodilator response in rabbit isolated hearts, according to results of our laboratory and others.10 To avoid coronary flow interference by ACh-induced bradycardia, hearts were electrically paced at 30 beats over basal heart rate during infusion. The endothelium-dependent vasodilator substance P was administered at a concentration of 2×10−10 mol/L. Nitroglycerin, acting independently of the endothelium, was infused at a concentration of 4×10−6 mol/L.
After the initial period of separate organ perfusion, the perfusion mode was immediately switched from separate to serial perfusion over 40 minutes, both in the control group (New Zealand White rabbits) and in the Watanabe group (n=7 for either group). Samples of pulmonary and coronary effluent were taken simultaneously at the end of separate perfusion and at 50, 500, and 1500 seconds during serial perfusion. After the admixture of 0.2 vol% BSA, the samples were rapidly frozen in liquid nitrogen and stored at −70°C for subsequent determination of ET and big ET levels (see below).
Cross-perfusion groups. To corroborate the data obtained in the serial perfusion experiments, we performed cross-perfusion of control hearts with the effluent of Watanabe lungs and, conversely, serially perfused Watanabe hearts with the effluent of control lungs (n=6, for either group). During the initial 45-minute period of separate organ perfusion, we tested the hearts for coronary vasodilator response with ACh and substance P as described. Subsequently, the endothelium-independent vasodilator SNP was infused, instead of nitroglycerin, at a concentration of 4×10−6 mol/L.
Mechanical flow reduction groups. Four isolated hearts of both control and Watanabe rabbits underwent mechanical flow reduction, ie, volume-controlled perfusion, to determine whether changes in mechanical performance (LVP, dP/dt) could be exclusively attributed to alterations in coronary flow. Mechanical flow reduction mimicked the average coronary flow response during serial perfusion.
Pharmacological intervention groups. Numerous reagents were applied before the switch from separate to serial perfusion, each to different subgroups of both the control and the Watanabe rabbits: DesArg9-[Leu]8-bradykinin (5×10−6 mol/L), a bradykinin BK1 receptor antagonist; Hoe-140 (1×10−6 mol/L), a bradykinin BK2 receptor antagonist; and saralasin (5×10−6 mol/L), a nonselective angiotensin II antagonist, were infused into the coronary circulation. Meclofenamic acid, a cyclooxygenase inhibitor (1×10−5 mol/L), was applied as pulmonary infusion (n=4 for each reagent).
In addition, the nonselective ET antagonist PD-145065 (1×10−5 mol/L); two different ETA receptor antagonists, BQ-123 (2×10−6 mol/L) and A-127722 (2×10−5 mol/L); the ETB antagonists IRL-1038 (4×10−6 mol/L) and IRL-1025 (5×10−6 mol/L); and the ECE inhibitor phosphoramidon (5×10−5 mol/L) were tested for their property of influencing the coronary effects observed during serial perfusion (n=6 for each reagent).
Exogenous vasoconstrictor groups. To compare the results obtained in the serial perfusion experiments with the effects of exogenous vasoconstrictors, isolated hearts of both control and Watanabe rabbits received a 10-minute coronary infusion of the thromboxane analogue U-46619 (5×10−9 mol/L), which was followed, after a 20-minute recovery period, by coronary infusion of human ET-1 (5 pmol/L) over a period of 10 minutes (n=3). Similarly, hearts of both control and Watanabe rabbits were treated with a coronary infusion of human big ET (5 pmol/L) subsequent to the initial administration of U-46619 (n=4).
Reagents. ACh, substance P, nitroglycerin, SNP, meclofenamic acid, saralasin, desArg9-[Leu8]-bradykinin, BQ-123, human ET-1, human big ET, and phosphoramidon were purchased from Sigma Chemical Co. PD-145065 and A-127722 were generously provided by Parke Davis and Abbott, respectively. IRL-1038 and IRL-1025 were gifts from CIBA International Research Laboratories. Hoe-140 was provided by Hoechst.
Measurement of ET and Big ET
ET and big ET levels in pulmonary and coronary effluent were determined with the use of two modified commercial ELISA kits (Immundiagnostik). The big ET kit is highly selective for all three isoforms of big ET(1-38), with cross-reactivity for big ET(22-38) and all ET isoforms measuring <1%. The ET kit displayed high selectivity for ET-1 and -2 (100% for both); with this kit, cross-reactivity for ET-3 (<5%) and big ET (<1%) is low.
Big ET was measured directly in perfusates, with calibration curves being constructed by the titration of exogenous human big ET(1-38), at 0.2 to 16 pmol/L, into blank perfusate. For ET measurement, the perfusates were first concentrated over C18 columns to lower the detection limit of the entire procedure to < 0.1 pmol/L. To this end, the perfusate was loaded onto preconditioned (3 mL methanol, 5 mL water) Sep-Pak C18 cartridges (Waters). ET was eluted with 2 mL 60% acetonitrile in 0.1% trifluoroacetic acid. The eluates were freeze-dried and the residues stored at −20°C. Redissolving the residues in 0.7 mL assay buffer (borate buffer [pH 8.5], 0.15% BSA) with the primary cartridge loading volume at 3.5 mL resulted in fivefold concentration of the effluent samples.
Calibration curves for the entire procedure relating to ET were constructed by titration of exogenous human ET, 0.02 to 3 pmol/L, into blank perfusate before C18 extraction.
Data are presented as mean±SD unless otherwise indicated. Differences between groups over time were analyzed with a nonparametric ANOVA for repeated measures.11 After global testing, a multiple-comparison procedure with Bonferroni-Holm adjustment of P was carried out.12
For comparison of the maximum vasodilator responses recorded in control and Watanabe hearts, we used the Mann-Whitney rank-sum test for unpaired data.
An error probability of P<.05 was regarded as significant. Lower levels of error probability are indicated, where used.
Coronary Vasodilator Response
Average coronary flow rates achieved in control and Watanabe hearts were nearly identical, with values recorded at 32.0±2.4 and 32.9±3.1 mL/min, respectively.
In control hearts, coronary infusion of ACh and substance P before serial perfusion or cross-perfusion evoked a flow increase measured at 46±4% and 53±5% of basal flow rate, respectively (Fig 2⇓). In contrast, infusion of ACh produced a drop in coronary flow (−11±2%) for Watanabe hearts. Moreover, the vasodilator effect of substance P was significantly attenuated in Watanabe hearts compared with control hearts (19±2% versus 53±5%). The vasodilator responses to the endothelium-independent reagent nitroglycerin did not differ significantly, with flow increases recorded at 29±3% and at 26±3% for control and Watanabe hearts, respectively. Similarly, the vasodilation induced by SNP in control hearts, 60±4%, was comparable to the effect observed in Watanabe hearts, 61±6%.
Serial Perfusion and Cross-Perfusion
Switching the perfusion mode from separate to serial resulted in significant coronary flow declines in both control and Watanabe serial perfusion groups. In controls, coronary flow rate dropped to 89±3% of basal flow rate (Fig 3⇓). The flow began to fall within 50 seconds of the switch, reaching a new steady state within 300 to 400 seconds and remaining stable during the entire 40-minute observation period. In the Watanabe group, the initiation of serial perfusion caused a significantly steeper drop in coronary flow compared with controls, with flow declining to 75±6% of basal flow rate.
In the cross-perfusion groups, perfusing control hearts with the effluent of Watanabe lungs led to a decline in coronary flow to 90±4% of basal flow rate, which was comparable to the effect measured during serial perfusion in controls (Fig 3⇑). In Watanabe hearts, the effluent of control lungs evoked a significantly steeper flow drop similar to that recorded in the Watanabe serial perfusion group, to 75±4% of basal flow rate.
In both control and Watanabe groups, coronary constriction was accompanied by decreases in mechanical performance, which corresponded to the degree of flow reduction (see Table 1⇓). These changes were not found to differ significantly from those after simple mechanical flow reduction.
ET and Big ET Measurement
In both the control and the Watanabe serial perfusion groups, ET levels in pulmonary effluent were measured at <0.1 pmol/L; these levels did not differ, at any time during the experiment, from nonspecific values of blank perfusate. At 50 seconds after the switch to serial perfusion, ET levels in coronary effluent continued to be <0.1 pmol/L in both groups. However, coronary effluent levels in both groups rose significantly at 500 seconds and at 1500 seconds after the switch (control, to 0.19±0.011 and 0.21±0.013 pmol/L; Watanabe, to 0.21±0.019 and 0.20±0.012 pmol/L, respectively) (Fig 4A⇓).
Big ET levels ranged from 5.5 to 5.8 pmol/L in pulmonary effluent, with values differing only slightly at various time intervals and between control and Watanabe groups. In coronary effluent, big ET could not be detected during separate perfusion, whereas during serial perfusion, coronary big ET was measured at a range of 0.9 to 1.2 pmol/L in both control and Watanabe groups. This range was significantly lower than that recorded in corresponding pulmonary effluent (Fig 4B⇑).
In both the control and Watanabe groups, coronary infusion of the following agents did not influence the decline in coronary flow during serial perfusion: saralasin (5×10−6 mol/L), a nonselective angiotensin II antagonist; desArg9-[Leu]8-bradykinin (5×10−6 mol/L), a bradykinin BK1 receptor antagonist; and Hoe-140 (1×10−6 mol/L), a bradykinin BK2 receptor antagonist. Similarly, pulmonary infusion of the cyclooxygenase inhibitor meclofenamic acid (1×10−5 mol/L) did not affect the decrease in coronary flow in either group (data not shown).
In contrast, flow reduction as well as the consecutive mechanical alteration were prevented in both groups by coronary infusion of the nonselective ET antagonist PD-145065 (1×10−5 mol/L) and the two ETA receptor antagonists, BQ-123 (2×10−6 mol/L) and A-127722 (2×10−5 mol/L) (Fig 5⇓, Table 2⇓). The effect was also hindered by coronary infusion of the ECE inhibitor phosphoramidon (5×10−5 mol/L). The ETA antagonists IRL-1038 (4×10−6 mol/L) and IRL-1025 (5×10−6 mol/L) had no effect on flow reduction and mechanical alteration.
Coronary administration of the thromboxane analogue U-46619 (5×10−9 mol/L) led to a decrement in coronary flow to 73±4% of the basal flow rate in control hearts. Coronary infusion of ET-1 at a concentration of 5 pmol/L produced a drop in coronary flow to 86±2%; an identical concentration of big ET caused the coronary flow to fall to 91±4% in control hearts. The corresponding values for Watanabe hearts, recorded at 45±5% for U-46619, 71±5% for ET-1, and 77±4% for big ET, were significantly lower than those observed in control hearts (Fig 6⇓). In both control and Watanabe hearts, ET induced a slightly but not significantly stronger constriction than big ET. Again, the alteration of the mechanical function closely corresponded to the degree of flow reduction (data not shown).
The present novel findings demonstrate that a basal luminal pulmonary release of big ET(1-38) occurs in isolated perfused rabbit lungs, unaccompanied by detectable amounts of the mature peptide ET(1-21). Because the lungs were perfused in a nonrecirculatory mode, artificial concentration of pulmonary mediators was avoided; therefore, the big ET levels recorded in the isolated organ approximate natural conditions as closely as possible. Approximating these conditions, in turn, is pivotal to a clean interpretation of the remote effect on coronary vascular bed.
Most likely, this release of big ET can be ascribed to the pulmonary vascular endothelium, which produces big ET-1 and ET-1 exclusively.13 Still, other potential sources, particularly vascular smooth muscle cells,14 cannot be completely ruled out. The question arises as to why only the precursor, big ET, is detectable in pulmonary outflow. To date, molecular and functional evidence has been gathered for two distinct isoforms of ECE, with ECE-1 functioning at neutral pH both inside the cell and on the cell surface and ECE-2 being active intracellularly at acidic pH.15 ECE activity has been documented not only for endothelial cells but also among vascular smooth muscle cells.16 Moreover, because conversion of exogenous big ET was shown to occur in isolated rabbit17 and rat lungs,18 we cannot assume that in our model, the lungs are completely unable to convert the precursor.
In principle, the following arguments may be considered. (1) For the nonrecirculatory mode of perfusion, conversion of the precursor may be prevented if the major site of big ET release is located downstream from major conversion. (2) Big ET is partially converted during the process of secretion, but the mature peptide so generated rapidly becomes trapped by its receptors because of its higher affinity; therefore, the mature peptide does not appear in pulmonary effluent.
Unfortunately, no data are available to show how ECE and sites of ET synthesis are precisely distributed along rabbit pulmonary vasculature, which is why the first hypothesis cannot be conclusively substantiated. But we favor the second argument, which is supported by the hypothesis of stoichiometric binding conditions for ET-1, as elaborated by Frelin and Guedin19 : because estimated ET receptor densities (10 to 100 nmol/L), both in isolated organs and in vivo, are considerably higher than the equilibrium dissociation constant Kd of the ligand receptor complex (≈1 pmol/L), nearly all ET molecules are rapidly bound to their receptors and thus do not enter vascular space. Thus, we interpret the documented levels of big ET in pulmonary effluent to be essentially a net result of precursor synthesis, followed by the immediate partial conversion to ET and the subsequent trapping of the mature peptide.
To date, most human and animal studies appear to have focused exclusively on plasma ET levels, aiming to determine whether net pulmonary clearance or net release of ET prevails in certain physiological and pathophysiological states.20 21 Compelling evidence exists that pulmonary circulation represents a major site of ET clearance,22 23 with net release shown to occur mainly in case of chronic pulmonary hypertension.24 25 To the best of our knowledge, comparable studies investigating arteriovenous pulmonary gradients of big ET have yet to be conducted.
In bolus injection experiments in humans26 and pigs27 and in histochemical studies,28 exogenous big ET was shown to be removed from plasma predominantly in the splanchnic and renal vascular beds rather than through the pulmonary circulation. In humans, a positive venoarterial gradient after big ET-1 infusion indicated significant conversion to ET only for the kidney,26 although ECE-1 is present in human lungs as well.29 Studies by Ishikawa et al17 and Hisaki et al18 did reveal a substantial conversion of exogenous big ET-1 taking place in isolated lungs, but the experimental conditions appear rather artificial in regard to the high concentrations of big ET applied (1 to 50 nmol/L), the recirculatory perfusion mode adopted, and the slow conversion kinetics observed (5 minutes to 1 hour). As a result, pulmonary release of big ET in the absence of significant pulmonary clearance of the peptide could result in considerable remote actions of this ET precursor, particularly in the adjacent coronary vascular bed.
In our investigation, serial lung-heart perfusion clearly demonstrated the remote effect on coronary function of big ET, causing coronary flow to fall to 89% of basal flow in the control group, New Zealand White rabbits. In previous experiments in rabbit isolated hearts, the lowest ET concentration found to increase coronary tone ranged between 1 and 10 pmol/L.30 We could reproduce this finding by administration of exogenous ET at a concentration of 5 pmol/L. We further demonstrated that exogenous big ET at a concentration of 5 pmol/L evokes a coronary constriction at a slightly lower level than the mature peptide, which is in agreement with findings revealing almost equipotency of ET and big ET with regard to their systemic pressor effect.31 32 These concentrations are similar to the levels of big ET we documented in pulmonary effluent. Because big ET displays an ≈140-fold lower affinity to ET receptors than ET does,33 the observed coronary constriction can be explained only by presuming that big ET converts at a high level into ET during coronary passage. This presumption is supported by our data showing that only 15% to 20% of the big ET concentration that had entered the coronary vascular bed was detected in coronary effluent. In addition, we observed a positive coronary gradient for ET during serial perfusion and a complete prevention of coronary flow reduction by the ECE inhibitor phosphoramidon.
Assuming that the amount of big ET extracted across the coronary circulation is completely converted into ET, which is, of course, only a simple approximation, the coronary gradient for big ET can be used to estimate the maximum coronary ET production caused by the inflow of pulmonary big ET. Moreover, a measure for the extraction of this ET fraction can be calculated (Table 3⇓). It should be emphasized that these calculations are very mechanistic, because possible alterations, initiated by the inflow of pulmonary big ET, in cardiac production and extraction of ETs are neglected. Nevertheless, our finding that >95% of the produced ET is trapped within the coronary circulation is in close agreement with previous experimental results revealing that the systemic pressor effect of big ET is attributed to local conversion of the precursor, with only a minor level of ET detectable in circulation.31 With regard to the time course, we documented a remarkable delay of the appearance of ET in coronary effluent compared with the vasoconstriction observed. This apparent discrepancy can be explained by the stoichiometric binding theory,19 which predicts that the vascular concentration of ET is effectively buffered by the high receptor concentration. As shown in Table 3⇓, nearly all ET molecules are trapped within the coronary vasculature at 50 seconds of serial perfusion. Thus, the concentration of ET detected in the coronary outflow poorly predicts the functional state of the tissue receptor system.
From our experiments, we cannot contend, of course, that the findings described are unique to the pulmonary circulation. Other vascular beds, for instance the renal circulation, might display the same activity; this item remains to be investigated. With the experimental setup described, reversing the circulation from the coronary outflow to the pulmonary artery was a logical consequence of the present results. However, a coronary impact on the pulmonary circulation could not be observed (unpublished data, K.S., T.D., M.L., K.A., G.B.), which can be explained by the low levels of ETs measured in coronary outflow. In addition, to approximate natural conditions, it was necessary to dilute the coronary effluent before it was perfused through the pulmonary bed, because coronary flow composes only a small fraction of pulmonary perfusion. Hence, coronary effects on the pulmonary vasculature are presumably blunted by physiological dilution.
The blockade of the vasoconstriction by nonselective (PD-145065) and ETA (BQ-123, A-127722) receptor antagonists, coupled with the failure of ETB antagonists (IRL-1038, IRL-1025), lends additional support to the assumption that coronary constriction in rabbits is predominantly ETA-mediated.34 However, in terms of mechanical performance (LVPmax, dP/dt), it was not possible to find either a significant difference between serial perfusion and sole mechanical flow reduction or a specific influence exerted by ET antagonists on the mechanical function of the isolated hearts to prove positive inotropic actions of the ETs observed in papillary muscle experiments.35 Other experiments, based on isolated rabbit hearts, have also failed to demonstrate these effects.36
Concerning the effect of the ETB antagonists, one could have expected a potentiation of the coronary constrictor response to pulmonary ETs, at least in control hearts, because there is a large body of evidence indicating ETB-mediated mitigation of the constriction induced by ET.37 38 Nevertheless, our results with IRL-1025 and IRL-1038 did not confirm these findings, which seems to be both a species-related problem and a consequence of big ET conversion. In contrast to the rat coronary vasculature,39 even the lowest effective concentration of ET does not evoke a transient coronary dilation in the rabbit isolated heart,30 suggesting that endothelium-dependent processes mediated by ETB receptors do not play a major role in the rabbit coronary system. Furthermore, even in species that normally display transient dilation to ET, the effect was reported to disappear if big ET, instead of the mature peptide, was used at an equipotent concentration.31 40 We would argue, therefore, against a substantial involvement of endothelial ETB receptors in the net vasoconstrictory effect caused by pulmonary big ET, which may account for the failure of the ETB antagonists.
In lung-heart preparations from Watanabe rabbits, we could show an ≈2.5-fold stronger coronary vasoconstrictor effect of pulmonary big ET compared with controls. This also applied to the administration of exogenous ET and big ET with regard to the magnitude and the time course of coronary constriction. In supplementary experiments using the thromboxane analogue U-46619, we confirmed that this coronary susceptibility is a general property, rather than an ET-specific phenomenon. These findings are unequivocally attributed to the compromised endothelial vasodilator capacity of Watanabe hearts. Endothelial dysfunction was evidenced by the selective defect of the endothelium-dependent vasodilation in response to ACh and substance P, with the endothelium-independent effect induced by nitroglycerin and SNP well preserved. Neither pulmonary release of big ET nor coronary capacity to convert the ET precursor seemed to be significantly altered compared with New Zealand White rabbits, as demonstrated by determination of pulmonary and coronary ET and big ET concentrations. In addition, in terms of coronary ET production initiated by the inflow of pulmonary big ET (Table 3⇑), we calculated nearly identical rates during the initial drop of coronary flow for control and Watanabe hearts at 50 seconds of serial perfusion. When the coronary flow had reached a new plateau, significantly lower rates were documented for Watanabe hearts. This was obviously due to the lower flow rate rather than the capacity to convert big ET.
It has recently been shown41 that Watanabe rabbits develop morphologically detectable atherosclerosis at 4 months of age, at the earliest, which supports previous results by Watanabe himself.8 We can therefore reason that the animals used in our investigation, 14 weeks old, suffered from only coronary endothelial dysfunction, rather than severe atherosclerotic damage. This dysfunction is well known to precede morphological alterations.42 43 Thus, together with the phenomenon of early endothelial dysfunction, the basal pulmonary vasoconstrictor impact is hypothesized to constitute a significant pathophysiological factor in ischemic heart disease, because it shifts the regulatory balance of the dysfunctional coronary endothelium further toward vasoconstriction, thereby enhancing coronary malperfusion. Cross-perfusion of Watanabe rabbit hearts with the effluent of control lungs and vice versa additionally excluded constrictor mechanisms inherent in Watanabe rabbit lungs that might have been nonspecifically blocked by the ET antagonists.
In conclusion, our findings suggest that pulmonary big ET may influence coronary vascular tone, the effect being enhanced in the case of early atherosclerosis with endothelial dysfunction. Further pathophysiological implications of these findings remain to be investigated.
Selected Abbreviations and Acronyms
|LVP||=||left ventricular pressure|
|RVP||=||right ventricular pressure|
Guest editor for this article was Thomas F. Lüscher, MD, University Hospital, Zürich, Switzerland.
- Received October 29, 1996.
- Revision received May 29, 1997.
- Accepted June 6, 1997.
- Copyright © 1997 by American Heart Association
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