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Vol. 280, Issue 2, 1102-1108, 1997
Clinical Pharmacology Unit, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 2QQ, United Kingdom
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Abstract |
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 Top
Abstract
Introduction
Methods
Results
Discussion
References
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We have determined the affinity and selectivity of a new nonpeptide antagonist PD156707 (sodium 2-benzo(1,3)dioxol-5-yl-4-(4-methoxy-phenyl)-4-oxo-3-(3,4,5-trimethoxybenzyl)-but-2-enoate) for human endothelin (ET)A and ETB receptors. In human coronary artery and saphenous vein the affinity of the ETA receptor for PD156707 was 0.15 ± 0.06 nM and 0.5 ± 0.13 nM, respectively. Competition experiments in human left ventricle and kidney revealed that PD156707 had 1,000- to 15,000-fold selectivity for the ETA receptor over the ETB receptor. This selectivity was confirmed autoradiographically. In human coronary artery, mammary artery and saphenous vein PD156707 (3-300 nM) potently antagonized the vasoconstrictor responses to ET-1. The pA2 values estimated from the Gaddum-Schild equation were 8.07 ± 0.09, 8.45 ± 0.11 and 8.70 ± 0.13, respectively. The concentration-response curves to ET-1 were shifted to the right in parallel fashion, without reduction of the maximum response. However, the regression lines fitted to the resulting Schild data deviated significantly from one. PD156707 appeared to be a more effective antagonist at lower concentrations than at the higher ones. It is possible that PD156707, a sodium salt, was reverting to a less soluble form which results in underestimation of its potency. These data show that PD156707 is a potent and selective antagonist at human ETA receptors and will be useful in clarifying the role of the endothelin peptides in human cardiovascular disease.
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Introduction |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The endothelins, a family of
three peptides designated ET-1, ET-2 and ET-3 (Masaki et
al., 1991
; Yanagisawa et al., 1988), are the most
potent constrictors of the human vasculature yet described (Davenport
et al., 1989). Both of the ET receptors, ETA and
ETB (Arai et al., 1990; Sakurai et
al., 1990), mediate vasoconstriction but the ETA
receptor predominates (>80%) in the medial layer of human blood
vessels (Davenport et al., 1995). The ETA
receptor is therefore responsible for the majority of the profound
constrictor response elicited by these peptides both in
vitro (Godfraind, 1993; Hay et al., 1993; Maguire
et al., 1994; Maguire and Davenport, 1995; Opgaard et
al., 1994; Riezebos et al., 1994) and in
vivo (Haynes et al., 1995). These data strongly imply a
therapeutic role for ETA-selective antagonists in
conditions of pathological vasospasm, e.g., subarachnoid
hemorrhage (Masaoka et al., 1989), in which plasma levels of
ET-1 are raised. Indeed ETA selective antagonists such as
BQ123 are effective in limiting tissue damage in some animal models of
vasospasm (e.g., Clozel and Watanabe, 1993).
Nonpeptide ET antagonists have been developed recently which exhibit a
range of receptor selectivities. We have previously reported that the
orally active, nonpeptide, butenolide PD155080 (Doherty et
al., 1995), had up to 1,000-fold selectivity for human ETA receptors compared with ETB receptors
(Maguire et al., 1995). With human cloned receptors a second
compound in this series, PD156707 (fig. 1) (Doherty
et al., 1995), has been shown to possess enhanced
selectivity for the ETA over the ETB receptor
and improved affinity for both receptors compared with PD155080. After
oral administration, PD156707 blocked the ETA pressor
response to infused ET-1 without affecting the ETB
depressor response, which indicated good bioavailability.
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As species differences in the ET receptors have been reported with
respect to their pharmacological profiles (Reynolds et al.,
1995a; Russell and Davenport, 1996), we have measured the affinity of
PD156707 for human ETA receptors in preparations of artery
and vein. The ETA subtype comprises more than 80% of the total ET receptor population on human vascular smooth muscle cells (Davenport et al., 1995). Competition data from coronary
artery and saphenous vein will therefore establish whether or not
PD156707 has high affinity for this receptor subtype. Selectivity for
one subtype over another is best demonstrated in tissues that express both ETA and ETB receptors. We have therefore
used sections of human left ventricular free wall and kidney. The
ratio of ETA:ETB in ventricle is
approximately 75%:25% (Molenaar et al., 1992), whereas this is reversed in kidney, with ETB receptors
predominating (30% ETA:70% ETB) (Karet
et al., 1993). The potency of PD156707 as an antagonist of
ET-1 constrictor responses was determined in human isolated coronary
artery, internal mammary artery and saphenous vein. Preliminary results
have been presented to the British Pharmacological Society (Maguire
et al., 1996).
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Methods |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Competition binding experiments. Human tissues were obtained with local ethical approval. The studies reported in this manuscript were carried out in accordance with the Declaration of Helsinki and/or with Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health.
For the binding experiments epicardial coronary arteries (n = 3, male, 21-50 years of age) and left ventricular free wall (n = 3, male, 54-58 years of age) were obtained from patients undergoing cardiac or cardiopulmonary transplantation for ischemic heart disease or cardiomyopathy. Saphenous veins were from patients receiving coronary artery bypass grafts (n = 43, 38 male and 5 female, 42-81 years of age). Sections of nondiseased kidney were obtained from patients (n = 3, male, 32-74 years of age) undergoing nephrectomy for nonobstructing tumors. All tissues were frozen in liquid nitrogen at the time of operation and stored at
70°C until
required.
Sections (10 µm) were cut from the media (smooth muscle layer) of
coronary arteries and from blocks of left ventricle and kidney (cortex
and medulla). Saphenous veins were dissected free of connective tissue,
ground at 70°C with a freezer mill (Glen Creston Ltd, Middlesex,
U.K.) and homogenized with ice-cold Tris-HCl buffer (50 mM Tris-HCl, 5 mM MgCl2, 5 mM ethylenediamine tetraacetic acid, 1 mM
ethyleneglycol-bis-(
-aminoethyl ether)-N,N,N
,N-tetraacetic acid,
100,000 IU/ml aprotinin, pH 7.4). The homogenate was centrifuged (1,000 × g, 1 min, 4°C), the pellet discarded and
the supernatant further purified by centrifugation at 40,000 × g for 30 min (4°C). The resulting pellet was resuspended
in Tris-HCl buffer and subjected to two additional centrifugation steps
(40,000 × g, 30 min, 4°C) with the final pellet
resuspended in HEPES buffer (50 mM HEPES, 5 mM MgCl2, 0.3%
bovine serum albumin, 4°C, pH 7.4). The concentration of protein was
determined (Biorad Laboratories Ltd., Hemelhempstead, Hertfordshire,
U.K.), the homogenate diluted to give a final concentration of 6 mg
protein/ml and stored at 70°C. When required the saphenous vein
homogenate was thawed, centrifuged (20,000 × g, 10 min, 4°C) and resuspended in HEPES buffer (23°C).
For competition binding experiments sections of coronary artery, left
ventricle and kidney and aliquots of saphenous vein homogenate (final
assay concentration, 2 mg/ml) were incubated for 2 h at 23°C
with 0.1 nM [125I]ET-1 (2000 Ci/mmol, Amersham
International plc, Buckinghamshire, U.K.) and increasing concentrations
(20 pM-100 µM) of PD156707. Nonspecific binding was determined by
the inclusion of 1 µM ET-1. Sections were then washed in ice-cold
Tris-HCl buffer (3 × 5 min) and the homogenates were centrifuged
(20,000 × g, 10 min, 4°C); the resulting pellets
were washed and recentrifuged in ice-cold Tris-HCl buffer (20,000 × g, 10 min). All tissues were then counted for
125I content. The amount of total binding for
[125I]ET-1 was typically 10,000 to 30,000 dpm per tissue
section or aliquot. In all instances specific binding was 85 to 95% of
the total. Data files from separate experiments generated by EBDA (McPherson, 1983) were coanalyzed by the nonlinear iterative
curve-fitting programme LIGAND (Munson and Rodbard, 1980) to calculate
values of equilibrium dissociation constant
(KD, expressed as nanomolar) and maximum
receptor density (Bmax, expressed as femtomoles
bound per milligram of protein). The presence of one, two or three
sites was determined by the F ratio test. Selectivity of
PD156707 for the ET receptor subtypes was estimated by the comparison
of the derived KD values.
Autoradiography.
To visually demonstrate the receptor
selectivity of PD156707 for ETA receptors additional
kidney sections were incubated for 2 h at 23°C, with 0.1 nM
[125I]ET-1 to label both ETA and
ETB receptors. Adjacent sections were incubated with 0.1 nM
[125I]ET-1 to which the selective agonist BQ3020
([Ala11,15]Ac-ET-1 (6-21), 200 nM) was added. This
concentration of BQ3020 was calculated from saturation data (Karet
et al., 1993) to block more than 99% of ETB
receptors, but less than 3% of ETA receptors, and
therefore reveal ETA receptor distribution. Similarly BQ123 was added at 100 nM, calculated to block more than 90% of
ETA receptors and less than 3% ETB receptors,
and so identify the latter. Finally, for comparison, the pattern of
receptor distribution was determined for [125I]ET-1 in
the presence of PD156707 (53 nM). This concentration of PD156707 was
calculated from the competition data to block more than 98% of
ETA receptors but less than 8% of ETB
receptors. The sections were washed in ice-cold Tris-HCl buffer (3 × 5 min), air-dried and apposed to radiation-sensitive Hyperfilm
-max (Amersham International plc, Buckinghamshire, UK) for 5 days.
In vitro pharmacological experiments. For in vitro pharmacological experiments blood vessels were collected in ice-cold Krebs' solution at the time of the operation and transported back to the laboratory. Coronary arteries were obtained from 12 patients (10 male, 2 female, 42-59 years of age) who were undergoing cardiac transplantation, and internal mammary arteries from 13 patients (11 male, 2 female, 46-71 years of age) and saphenous vein from 13 patients (10 male, 3 female, 47-74 years of age) were from patients receiving coronary artery bypass grafts.
Sections of coronary artery, mammary artery and saphenous vein were cut into 3- to 4-mm lengths, the endothelium was gently rubbed away with a blunt metal seeker (verified histologically), and the rings were suspended between two metal L-shaped hooks in 5-ml tissue baths containing oxygenated Krebs-Henseleit solution maintained at 37°C. Contractile responses were measured isometrically (F30 force transducers, Hugo Sachs Elektronik, March-Hugstetten, Germany) and recorded on a Graphtec chart recorder (Linton Instrumentation, Diss, Norfolk, UK). The vessels were allowed to equilibrate for 1 h, and then responses were elicited to 50 mM KCl at increasing levels of resting tension until no further increase in the magnitude of the KCl response was obtained. The preparations were then allowed to relax to their own resting tension before the effect of PD156707 was determined. PD156707 (3-300 nM) or vehicle (control) was added to the bathing medium for 30 min, then cumulative concentration-response curves were constructed to ET-1 (10
10-106 M). When
addition of a higher concentration of ET-1 elicited no further
contractile response, 50 mM KCl was added to determine the maximum
possible contractile response for each preparation. ET-1 responses were
subsequently expressed as a percent of this KCl response. Data for
Schild analysis (Arunlakshana and Schild, 1959) were derived from the
graphs of ET-1 concentration (log10) plotted against
response (percent KCl response) in the absence and presence of
increasing concentrations of PD156707. The slope of the resulting
Schild regressions, if not significantly different from one, were
constrained to one to determine the value of pKB for
PD156707 in each of the three blood vessels used.
Additional experiments were designed to show whether or not PD156707
(100 nM) could reverse a constrictor response to 30 nM ET-1. Rings of
saphenous vein (n = 3) were contracted with 30 nM ET-1
and the response allowed to develop fully over 30 to 40 min. Once the
maximum response was established, PD156707 (100 nM) was added to the
bath and the response was recorded. The effectiveness of PD156707 was
compared with that of BQ123 (3 µM).
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Results |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Affinity and selectivity of PD156707 for human
ETA and ETB
receptors.
PD156707 competed with subnanomolar affinity for the
majority of sites labeled by 0.1 nM [125I]ET-1 in
coronary artery (fig. 2a) and saphenous vein (fig. 2b). A small low-affinity component was detected only in the saphenous vein.
These data suggest that PD156707 has high affinity for human ETA receptors (table 1).
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) and microautoradiographical techniques (Karet et
al., 1993). ETA receptors are localized to the large
arcuate arteries and afferent veins at the corticomedullary junction
and the resistance arteries present particularly in the cortex (fig.
4B), whereas ETB receptors have a nonvascular distribution
(fig. 4C). The results also demonstrate that the pattern of receptor
distribution remaining after incubation with PD156707 (fig. 4D) is
identical with that achieved with the highly ETA-selective
antagonist BQ123 (fig. 4C) and different to that obtained with the
ETB agonist BQ3020 (fig. 4B).
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Antagonism of ET-1 constriction in human isolated blood vessels. ET-1 potently contracted rings of isolated coronary artery (n = 12), mammary artery (n = 13) and saphenous vein (n = 13) with EC50 values of 9.1 nM (5.6-15.0 nM), 4.3 nM (2.8-6.7 nM) and 1.7 nM (0.8-3.7 nM), respectively (geometric mean with 95% confidence intervals). The maximum response to ET-1 in each preparation was compared with that to 50 mM KCl added at the end of the experiment. For coronary artery, mammary artery and saphenous vein the mean KCl responses were 5.07 ± 0.87 g, 4.64 ± 1.10 g and 2.01 ± 0.43 g with maximum responses to ET-1 of 80.91 ± 4.32%, 79.22 ± 3.78% and 92.97 ± 1.91% respectively.
In each of the three preparations the response to ET-1 was antagonized by PD156707 which produced a rightward shift of the ET-1 concentration-response curve without significant diminution of the maximum response. This suggested that PD156707 was acting in a competitive manner; however, it was apparent that some of the lowest concentrations tested were as effective at displacing the control ET-1 concentration-response curve as some of the higher concentrations (fig. 5). From the Schild data it is clear that the resulting regression slopes deviated significantly from one (P < 0.05, Student's t test) and were 0.53 ± 0.17 in coronary artery, 0.37 ± 0.14 in mammary artery and 0.48 ± 0.17 in saphenous vein (fig. 6).
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Discussion |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Among the known vasoactive peptides ET possesses a unique
pharmacological profile. Not only is it the most potent vasoconstrictor yet described (Yanagisawa et al., 1988), but it also has an
unusually prolonged duration of action (Clarke, et al.,
1989; Ide et al., 1989; Weitzberg, et al., 1991).
In addition to its direct effects on vascular smooth muscle,
subthreshold concentrations of ET will enhance the response to other
vasospastic agents such as noradrenaline and 5-hydroxytryptamine
(Chester et al., 1992; Yang et al., 1990). Since
plasma ET levels are raised in a number of vascular disorders (see
Huggins et al., 1993, for review) it is possible that this peptide is responsible for, or exacerbates, the vasospasm which may
accompany conditions such as subarachnoid hemorrhage and ischemic heart
disease.
The clinical potential of ET antagonists has encouraged the design and
synthesis of orally active, nonpeptide antagonists such as the recently
described antagonist PD156707 (Doherty et al., 1995). In the
present study we have extended this pharmacological characterization by
evaluating the affinity and selectivity of PD156707 for native human ET
receptors and determining its potency as an antagonist of ET-1-mediated
vasoconstriction in human isolated blood vessel preparations.
In all of the human tissues investigated the ETA receptor
exhibited subnanomolar affinity for PD156707 whereas the
ETB receptor had only micromolar affinity. These data
confirm the selectivity of PD156707 for human ETA
receptors. We find a 1,000- to 15,000-fold higher affinity for
ETA compared with ETB receptors, which is comparable to that reported for PD156707 in animal tissue and cloned
human receptors (Reynolds et al., 1995b).
In functional experiments PD156707 potently inhibited the contractile
reponse to ET-1 observed in human isolated vascular preparations. The
concentration-response curves for ET-1 were shifted to the right in
parallel fashion without reduction of the maximum response which
indicates competitive antagonism. This concurred with observations by
Reynolds and colleagues in the rabbit femoral artery (Reynolds et
al., 1995b). However the ability of PD156707 to antagonize ET-1 in
these human blood vessel preparations did not appear to be completely
concentration-dependent. The Schild regressions deviated significantly
from one and it was apparent that the compound was more potent at the
lower concentrations tested compared with the higher concentrations
(fig. 5b). We do not have an explanation for the reduced potency of
PD156707 observed at the higher concentrations tested. One possibilty
that requires investigation is that PD156707 may be reverting to a less
soluble form (Doherty et al., 1995) leading to an
underestimation of the antagonist's potency.
It is noteworthy that the pA2 values estimated for PD156707
in human tissues at their most conservative (pA2 of
7.6-8.1 at 300 nM) are better than that reported for the antagonism of
ET-1 in rabbit femoral artery (Schild-derived pA2 = 7.5;
Reynolds et al., 1995b). We find that PD156707 is up to 50 times more potent as an antagonist of ET-1 contractions in human
vasculature in vitro (pA2~ = 9.2 at 3 nM) than
in the rabbit preparation. We are not able to give a reason for this
discrepancy as yet, although in binding experiments it appears that
members of this series of butenolide analogs have higher affinity for
human ETA receptors than rabbit ETA receptors,
whereas the converse is true for ETB receptors (Doherty
et al., 1995).
PD156707 has been shown to be effective in limiting tissue damage in
animal models of ischemia. After intravenous administration, PD156707
(3 µmol/kg bolus + 5 µmol/kg/h infusion) restored cerebral blood
flow to normal and reduced by 45% the volume of cerebral ischemic
damage in a cat model of focal ischemia (Patel et al., 1996). Crucially we were able to demonstrate the complete reversal of
the constrictor response to ET-1 by PD156707 in human saphenous vein
in vitro. This suggests that ETA-selective
antagonists such as PD156707 would be effective in conditions of
established vasospasm in man.
The high affinity and excellent selectivity of PD156707 demonstrated for human vascular ETA receptors suggests a potential therapeutic role for this and related compounds in conditions of pathological vasospasm in which the ET system is thought to be important.
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Acknowledgments |
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We thank Dr. Annette Doherty (Park-Davis Pharmaceutical Research) for supplying PD156707. We are grateful to the theatre and consultant staff at Papworth, Addenbrooke's and Hinchingbrooke's hospitals for permission to collect tissue specimens.
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Footnotes |
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Accepted for publication October 21, 1996.
Received for publication June 18, 1996.
1 Supported by grants from the British Heart Foundation, Royal Society and Isaac Newton Trust.
Send reprint requests to: Dr. Janet J. Maguire, Clinical Pharmacology Unit, University of Cambridge, Box 110, Addenbrooke's Hospital, Hills Rd., Cambridge, CB2 2QQ, U.K.
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Abbreviations |
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ET, endothelin;
PD156707, sodium
2-benzo(1,3)dioxol-5-yl-4-(4-methoxy-phenyl)-4-oxo-3-(3,4,5-trimethoxybenzyl)-but-2-enoate ;
PD155080, sodium 2-benzo(1,3)dioxol-5-
yl-3-benzyl-4-(4-methoxy-phenyl)-4-oxobut-2-enoate;
BQ123, cyclo(D-Asp-L-Pro-D-Val-L-Leu-D-Trp);
Tris, 2-amino-2-(hydroxymethyl)-1,3-propandiol;
HEPES, N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid].
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References |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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) and presence of 3 nM (
), 10 nM (
), 30 nM (
), 100 nM (
) or 300 nM (
) PD156707 determined in preparations of human
isolated coronary artery (a), mammary artery (b) and saphenous vein
(c). Data points are the mean ± S.E.M. from 3 to 13 individuals.
