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Vol. 280, Issue 2, 959-965, 1997
Department of Pulmonary Pharmacology, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, Pennsylvania
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Abstract |
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 Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In this study the endothelin (ET) receptors mediating contractions produced by ET-1, ET-3 and the selective ETB ligands sarafotoxin 6c (S6c) and BQ-3020 in guinea pig bronchus were investigated using SB 209670, a nonpeptide, mixed ETA/ETB receptor antagonist, and the peptide ET receptor antagonists BQ-123 (ETA receptor-selective), BQ-788 (ETB receptor-selective) and RES-701 (ETB receptor-selective). SB 209670 (10 µM) antagonized concentrations induced by ET-1 (pKB = 6.1). In contrast, BQ-788 (10 µM) and BQ-123 (10 µM), either alone or in combination, were without significant effect on ET-1 concentration-response curves. SB 209670 (10 µM) and BQ-788 (10 µM) antagonized S6c concentration-response curves with pKB values of 6.6 and 5.5, respectively, whereas RES-701 (10 µM) and BQ-123 (10 µM) were without effect. SB 209670 (10 µM) was about a 10-fold less potent antagonist of contractions produced by ET-3 (pKB = 5.4) than of those elicited by S6c. BQ-788 (10 µM), RES-701 (10 µM) and BQ-123 (10 µM) were without effect on ET-3 concentration-response curves. BQ-788 (10 µM) had similar potencies for inhibition of contractions induced by S6c (pKB = 5.8) and BQ-3020 (pKB = 6.25). These data indicate that contractions induced by ET-1, ET-3, S6c and BQ-3020 in guinea pig bronchus appear to be mediated predominantly via stimulation of ETB receptors. However, these receptors are not very sensitive to the standard ETB receptor antagonists BQ-788 and RES-701, which suggests that responses produced by these ligands in this tissue involve activation not of the classical ETB receptor, but rather of an atypical ET receptor population. The results also provide additional evidence that the potencies of ET receptor antagonists depend upon the specific ET agonist.
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Introduction |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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ET-1, a member of a family of
21-amino acid peptides (Yanagisawa et al., 1988
; Inoue
et al., 1989; Masaki et al., 1992), exerts several effects in the lung, including contraction of airway smooth muscle and pulmonary vascular smooth muscle (Hay et al.,
1993a; Hay and Goldie, 1995). Furthermore, increased expression and
levels of ET-1 are detected in various lung diseases. On the basis of these observations, it has been proposed that ET-1 plays a significant role in the pathophysiology of pulmonary disorders, asthma in particular (Springall et al., 1991; Hay et al.,
1993a; Hay and Goldie, 1995).
In the search for therapeutics for pulmonary disease that are based on
antagonizing the effects of ET-1, it will be important to determine
what ET receptor subtypes are responsible for the diverse effects of
this mediator in the lung. Pharmacological, biochemical and molecular
biological studies indicate the presence of distinct receptor subtypes
(Yanagisawa and Masaki, 1989; Arai et al., 1990; Masaki
et al., 1992; Sakurai et al., 1992; Hay et al., 1993a; Hay and Goldie, 1995). Several years ago, two major ET
receptor subtypes were identified and characterized: an ETA receptor, which has a higher affinity for ET-1 or ET-2 than for ET-3,
and an ETB receptor, which has equal affinity for the three ET ligands (Arai et al., 1990; Sakurai et al.,
1990; Masaki et al., 1992). Recent research suggests the
presence of additional ET receptors, including an ETc
receptor (selective for ET-3) (Martin et al., 1990; Samson
et al., 1990; Masaki et al., 1992; Douglas et al., 1995) and subtypes of ETA (Yonemaya
et al., 1995) and ETB receptors (Sokolovsky
et al., 1992; Warner et al., 1993), although
considerable uncertainty exists in the area of ET receptor classification (Bax and Saxena, 1994).
In airways, both ETA and ETB receptors appear
to mediate contractions produced by ET-1 and other ET ligands, and both
species and regional differences are apparent in the relative
contribution of the two receptor subtypes to the responses (Hay
et al., 1993a, b; Henry, 1993; Battistini et al.,
1994a; Goldie et al., 1994; Hay and Goldie, 1995; Yonemaya
et al., 1995). In human bronchus, for example, responses to
ET-1 and S6c, an ETB receptor selective agonist (Williams
et al., 1991), appear to be mediated predominantly via stimulation of ETB receptors (Hay et
al., 1993b), although recent evidence suggests a contribution of
ETA receptors to the ET-1-induced contraction (Fukuroda
et al., 1996). Similarly, several studies in guinea pig
bronchus, based primarily on the activity of S6c and the effects of
BQ-123, a selective ETA receptor antagonist (Ihara et
al., 1992a), indicate that ET-induced responses in this tissue
involve mainly the activation of ETB receptors (Hay, 1992; Hay et al., 1993b; Battistini et al., 1994a,b;
Kizawa et al., 1994). Thus, in the context of ET
ligand-induced contractions, the guinea pig bronchus appears to be a
useful model system of human airways.
The major goal of the present study was to gain more information, using
nonpeptide and peptide receptor antagonists, on the characteristics of
the ETB receptors mediating contractions elicited by the ET
ligands ET-1, ET-3 and the ETB receptor selective agonists S6c (Williams et al., 1991) and BQ-3020 (Ihara et
al., 1992b) in isolated guinea pig bronchus. The compounds
examined were the nonpeptide, combined ETA and
ETB receptor antagonist SB 209670, which has high affinity
for the ETA receptor and lower but significant affinity for
the ETB receptor (Ohlstein et al., 1994a,b), the peptide ETB receptor selective antagonists BQ-788 (Ishikawa
et al., 1994) and RES-701 (Tanaka et al., 1994)
and the peptide ETA receptor selective antagonist BQ-123
(Ihara et al., 1992a).
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Materials and Methods |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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All experiments were performed in accordance with the guidelines of the Animal Care and Use Committee, SmithKline Beecham Pharmaceuticals.
Tissue preparation. Primary bronchi were removed from male Hartley guinea pigs (Hazelton Research Animals, Denver, PA; 450-650 g b.w.) and placed in modified Krebs-Henseleit solution. A 21-g syringe needle was inserted into the lumen of each bronchus, which was cleaned of adherent fat and connective tissue and cut into 4 to 5 rings of approximately 3 mm wide (o.d.). The epithelium was removed from each tissue by its rotation several times around the syringe needle. The individual rings from each bronchus were put randomly in the different treatment groups.
The tissues were then placed in 10-ml water-jacketed organ baths containing Krebs-Henseleit solution and connected via silk suture to Grass FT03C force-displacement transducers (Grass Instrument Co., Quincy, MA). Mechanical responses were recorded isometrically by MP100WS/Acknowledge data acquisition system (BIOPAC Systems, Goleta, CA) run on Macintosh computers. The composition of the Krebs-Henseleit solution, which was gassed with 95% O2/5% CO2 and maintained at 37°C, was as follows (mM):NaCl 113.0, KCl 4.8, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25.0 and glucose 5.5. Tissues were equilibrated under approximately 1.5 g resting load for at least 1 hr, and washed every 15 min with fresh Krebs-Henseleit solution, before the start of each experiment.Concentration-response curves.
After the equilibration
period, and before construction of agonist concentration-response
curves, tissues were exposed to 10 µM carbachol. After plateau of
this response, tissues were washed several times over 30 to 60 min
until the tension returned to base-line level. The preparations were
then left for at least 30 min before the start of the experiment. ET-1,
ET-3 and S6c concentration-response curves were obtained by their
cumulative addition to the organ bath in 3-fold increments according to
the technique of Van Rossum (1963). Each drug concentration was left in
contact with the preparation until the response reached a plateau before the subsequent agonist concentration was added. At the end of
the experiment, tissues were exposed again to 10 µM carbachol, which
yielded the reference contraction for data analysis. The carbachol-induced contraction was larger at the end than at the beginning of the experiment. The antagonists used in this study had no
effect on the ratio between the magnitudes of the carbachol-induced responses at the two different times, which indicates that they do not
have nonspecific effects on the contractile response. In experiments
examining the effects of antagonists, we added the compound under study
to the organ bath 30 min before addition of the contractile agonist.
Only one agonist concentration-response curve was generated per tissue.
Experiments were conducted in the presence of 1 µM sodium
meclofenamate, the cyclooxygenase inhibitor, which was added 45 min
before initiation of the curves.
Analysis of data.
All data are given as the mean ± S.E.M., and n represents the number of animals studied in a
particular group. Agonist-induced responses for each tissue were
expressed as a percentage of the reference contraction (10 µM
carbachol) obtained at the end of the experiment.
Concentration-response curves were analyzed using nonlinear
least-squares regression (Ohlstein et al., 1994a), and geometric mean EC50 values (pD2 values) were
calculated. Evidence indicates that the ET family of ligands may not
interact with their receptors in a classical manner that results in a
reversible competitive interaction among agonist, antagonist and
receptor (Marsault et al., 1991; Waggoner et al.,
1992; Ohlstein et al., 1995). However, for the purposes of
comparing the activities of compounds used in this study, as well as in
previous reports, we calculated antagonist potencies by assuming a
classical competitive interaction and expressed them as
pKB; pKB = 
log
[antagonist]/x 1, where x is the ratio
of agonist concentration required to elicit 50% of the maximal
contraction in the presence of the antagonist compared with that in its
absence (Arunlakshana and Schild, 1959). Results for control and
treated-tissues were analyzed for differences in both the
pD2 value (log EC50) and maximal contractile
response. Statistical analysis was conducted using ANOVA (Fisher's
protected least-squares difference) or two-tailed Student's
t test for paired samples, where appropriate, with P < .05 regarded as significant.
Drugs.
All drug solutions were made daily (from
stock solutions or powder) and stored on ice. The following drugs were
used: ET-1, ET-3, S6c, and BQ-3020 and BQ-123
(cyclo(D-Trp-D-Asp-Pro-D-Val-Leu) (American Peptide Co., Sunnyvale, CA) and carbachol and
dimethylsulphoxide (Sigma Chemical Co. St. Louis, MO). SB 209670 ((+)-(1S,2R,3S)-3-(2-carboxymethoxy-4-methoxyphenyl)-1-(3,4-methylenedioxy-phenyl)-5-(prop-1-yloxy)indane-2-carboxylic acid) and BQ-788
(N-cis-2,6-dimethylpiperidino-carbonyl-L-
-MeLeu-D-Try (1-CO2Me)-D-Nle) were synthesized by colleagues
in the Department of Medicinal Chemistry, SmithKline Beecham
Pharmaceuticals. RES-701 (cyclic(Gly1-Asn9)-(Try-His-Gly-Thr-Ala-Pro-Asp-Try-Phe-Phe-Asn-Tyr-Tyr-Trp)
and sodium meclofenamate were gifts from Warner Lambert (Ann Arbor, MI)
and Kyowa Hakko Kogyo Co., Ltd. (Tokyo, Japan), respectively.
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Results |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Contractile effects of ET-1, ET-3, S6c and BQ-3020.
ET-1,
ET-3, S6c and BQ-3020 produced concentration-dependent contractions of
guinea pig bronchus (fig. 1). S6c was about 10-fold more
potent than ET-1, ET-3 or BQ-3020 (P < .001); the latter three
agonists had similar potencies (table 1; fig. 1). ET-1 produced a greater maximal response than ET-3, S6c and BQ-3020, which
had equivalent efficacies (table 1; fig. 1). The effects of SB 209670 (ETA/ETB receptor antagonist), BQ-788
(ETB receptor antagonist), RES-701 (ETB
receptor antagonist) and BQ-123 (ETA receptor antagonist)
on contractions induced by ET-1, ET-3, S6c or BQ-3020 were examined.
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Effects of antagonists alone against ET-1-, ET-3-, S6c- and BQ-3020-induced contractions. SB 209670 (10 µM) antagonized concentrations induced by ET-1, a result reflected by a shift to the right in the agonist concentration-response curve, with a pKB value of 6.1 (table 2; fig. 2A). In contrast, BQ-788 (10 µM) or BQ-123 (10 µM) were without significant effect on ET-1 concentration-response curves (table 2; fig. 2B and C).
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; Kizawa et al., 1994; Yoneyama et al., 1995), we compared
the effects of BQ-788 on contractions induced by the selective
ETB agonists S6c and BQ-3020. BQ-788 (10 µM) antagonized
responses produced by the two ligands with similar potencies:
pKB = 5.8 vs. S6c and 6.25 vs.
BQ-3020 (n = 4) (fig. 5).
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Effects of combinations of antagonists against ET-1-, ET-3- and
S6c-induced contractions.
In view of the demonstration of both
ETA and ETB receptor populations in guinea pig
airways (Hay et al., 1993b; Battistini et al.,
1994a), we examined the effects of the combination of BQ-123 and BQ-788
or SB 209670 on ET-1-induced contractions.
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Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The present study, utilizing various ET ligands and peptide and nonpeptide ET receptor antagonists, indicates that contractions induced by ET-1, ET-3, S6c and BQ-3020 in guinea pig bronchus are mediated via activation of ETB rather than ETA receptors. However, the limited potencies of the ET receptor antagonists studied suggests that responses produced by the ET ligands in this tissue involve stimulation of an ETB receptor population that is not sensitive to the classical ETB receptor antagonists BQ-788 and RES-701 and thus may be mediated via activation of an atypical ET receptor population. In addition, the data provide further evidence that the potencies of ET receptor antagonists depend on the specific ET agonist under study.
Significant controversy exists in the area of ET receptor
classification. Only two mammalian ET receptor subtypes have been cloned to date: a receptor designated ETA, which has higher
affinity for ET-1 or ET-2 than for ET-3 or S6c, and another subtype,
named ETB, which does not discriminate among ET ligands
(Arai et al., 1990; Sakurai et al., 1990; Masaki
et al., 1992). Nevertheless, there are several proposals,
based on functional studies, for the existence of additional ET
receptor subtypes, including ETA1, ETA2,
ETB1 and ETB2 (Sokolovsky et al.,
1992; Warner et al., 1993; Bax and Saxena, 1994; Douglas
et al., 1995; Karaki et al., 1994a; Sudjarwo
et al., 1994; Yoneyama et al., 1995). A
complicating factor associated with ET receptor classification is that
some of the ET ligands do not appear to interact with the receptors in
a classical fashion that leads to a reversible, competitive interaction
among agonist, antagonist and receptor (Marsault et al.,
1991; Waggoner et al., 1992; Ohlstein et al.,
1995). Nevertheless, the utility of several structurally diverse ET
receptor antagonists, as well as further molecular biological studies,
should assist in clarifying the actual number, location and function of
the ET receptor subtypes.
Based on the present receptor classification, in this study the lack of
effect of BQ-123, a selective ETA receptor antagonist (Ihara et al., 1992a), and the potent contractile effects of
the selective ETB receptor agonists S6c (Williams et
al., 1991) and BQ-3020 (Ihara et al., 1992b),
concomitant with the increased potency of S6c vs. ET-1 and
ET-3, confirm previous observations and conclusions that responses
produced by ET ligands in guinea pig bronchus are mediated
predominantly, if not exclusively, via ETB
receptor activation (Hay, 1992; Hay et al., 1993b;
Battistini et al., 1994a; Kizawa et al., 1994).
Further characterization of the ETB receptors in this
tissue was explored using the nonpeptide, nonselective
receptor antagonist SB 209670, which has a high affinity for
ETA (Ki = 0.2 nM
vs. cloned human receptor) and also ETB
(Ki = 18 nM) receptors (Ohlstein
et al., 1994b), and the peptide, ETBreceptor
selective antagonists BQ-788 (IC50 = 12 nM for inhibition of [125I]-ET-1 binding to ETB receptors on
human Girardi heart cells, and IC50 > 1 µM for ET
receptors in human neuroblastoma cell line) (Ishikawa et
al., 1994) and RES-701 (IC50 = 10 nM and 20 nM for inhibition of [125I-ET-1 binding to rabbit lung
ETB and human ETB receptor, respectively) (Tanaka et al., 1994, 1995). Using these tool compounds
yielded two notable findings: 1) The antagonists were much less potent that anticipated on the basis of the results of previous binding and
functional studies (in many cases BQ-788 and RES-701 were without
effect on ET ligand-induced contraction). 2) The antagonist potency was
dependent on the specific ET ligand.
Regarding the limited antagonist potencies, given that SB 209670, BQ-788 and RES-701 have been reported to have affinities for human
ETB receptors in the 10 to 20 nM range (Ishikawa et al., 1994; Ohlstein et al., 1994b; Tanaka et
al., 1994), one might have anticipated reasonable functional
activity (manifest by marked shifts in ET ligand concentration-response
curves), especially with the high concentration tested, 10 µM. High
functional potency as ETB receptor antagonists has been
reported for SB 209670 (KB = 199 nM against ET-1-induced
contraction in rabbit pulmonary artery) (Ohlstein et al.,
1994b) and BQ-788 (pKB = 8.4 against BQ-3020-induced
contraction in rabbit pulmonary artery; and a 100-fold shift to the
right in the S6c concentration-response curve with 3 µM in rabbit
saphenous vein) (Ishikawa et al., 1994; Karaki et
al., 1994a). Nevertheless, in the present study, RES-701 was
without effect on ET-3- or S6c-induced contractions in guinea pig
bronchus, and BQ-788 had no effect on ET-1 or ET-3
concentration-response curves and had only a minor inhibitory effect on
contractions induced by S6c (pKB = 5.5). SB 209670 was the
most potent of the three compounds, inhibiting contractions induced by
ET-1, ET-3 and S6c, although its potency was limited (pKB = 5.4-6.6). The differences between radioligand binding and functional
potencies of the antagonists may be related to the pseudoirreversible
binding nature of the ET ligands (Marsault et al., 1991;
Waggoner et al., 1992; Ohlstein et al., 1995).
For example, the slow dissociation of endothelin from its receptor may
create nonequilibrium conditions that affect the estimation of receptor
dissociation constants. Alternatively, the lack of effect or limited
potency demonstrated in this study with BQ-788 and RES-701 suggests
that ETB-induced responses in this tissue are not so
sensitive to these prototypical ETB receptor antagonists as
reported previously in other tissues. Thus there may be differences in
the contractile ETB receptors, and responses elicited by
various ET ligands in guinea pig bronchus may be mediated by
stimulation of an atypical ET receptor population. Another possible
explanation for the observed findings is that the ET ligand-induced
contractions involve activation of multiple ET receptor subtypes, one
of which is not sensitive to the antagonists used in this study. The
lack of effect of the combination of BQ-788 and BQ-123 against
responses suggests that the receptor subtypes are not the classical
ETA and ETB receptors. Support of this
postulate requires molecular biological and pharmacological evidence
for additional ET receptor subtypes and determination of their
location in guinea pig bronchus.
The dependence of the potencies of the antagonists on the specific ET
ligand that was noted in the present study has been demonstrated
previously (Warner et al., 1993; Kizawa et al.,
1994; Ohlstein et al., 1994a; Yoneyama et al.,
1995). For example, in guinea pig bronchus, Ro 46-2005 had no effect on
responses induced by ET-1 and ET-3 but antagonized those elicited by
IRL 1620 (pKB = 6.35) (Kizawa et al., 1994). In
this study, SB 209670 was about 10-fold more potent at inhibiting
contractions induced by S6c than those produced by ET-3, and BQ-788
antagonized S6c- and BQ-3020-induced responses but not those elicited
by ET-1 or ET-3. The explanation for this phenomenon is unclear, but it
may indicate the presence of heterogeneous populations of ET receptors
for which different ET receptor antagonists have different affinities.
Another possibility is that a single population of ETB
receptors may exist in two different conformation states, as postulated
for the angiotensin II receptor (Robertson et al., 1994): an
active state coupled to contraction and an inactive state that is not
coupled to contraction. Alternatively, ET-1, S6c, ET-3 and BQ-3020 may
interact with different binding domains within a single population of
ETB receptors, and receptor antagonists may have
differential affinities for these domains (Hiley et al.,
1992). The findings of the present study may also reflect the
nonclassical interaction between some ET ligands and the ET receptors.
BQ-788 had no effect on ET-1 concentration-response curves in either
the absence or the presence of the ETA receptor antagonist BQ-123. This confirms that, unlike the guinea pig trachea, where both
ETA and ETB receptors are present (Hay et
al., 1993b; Battistini et al., 1994a), the guinea pig
bronchus has no functionally significant ETA receptors.
RES-701 antagonized the initial depressor response to ET-1
(ETB1-like mediated) in rats in vivo and has
been classified as an ETB receptor antagonist (Tanaka
et al., 1994; Karaki et al., 1994b). In this
study, RES-701 did not antagonize responses produced by S6c or ET-3 in
guinea pig bronchus. Similar findings have been observed in rabbit
trachea (Yonemaya et al., 1995). These data would support
the proposal that RES-701 does not antagonize the contractile
ETB receptor.
In conclusion, the present data from experiments utilizing various ET ligands and peptide and nonpeptide ET receptor antagonists indicate that contractions induced by ET-1, ET-3, S6c and BQ-3020 in guinea pig bronchus are mediated predominantly, if not exclusively, via activation of an ETB rather than an ETA receptor population. However, the limited potencies, and in some instances the lack of effects, of the ET receptor antagonists suggests that ET ligand-induced contractions in this tissue involve stimulation of an atypical ETB receptor population that is not sensitive to the classical ETB receptor antagonists, such as BQ-788 and RES-701. In addition, further evidence is provided that the potencies of the receptor antagonists depend on the ET agonist.
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Acknowledgments |
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The authors thank Kyowa Hakko Kogyo and Warner Lambert for their gifts of RES-701 and sodium meclofenamate, respectively, John D. Elliott, Amparo Lago and Aming Gao for synthesis of SB 209670 and BQ-788 and John Baugh for technical assistance.
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Footnotes |
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Accepted for publication October 21, 1996.
Received for publication June 14, 1996.
Send reprint requests to: Dr. Douglas W. P. Hay, Department of Pulmonary Pharmacology, UW2532, 709 Swedeland Road, King of Prussia, PA 19406.
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Abbreviations |
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ET, endothelin; ETB, endothelinB; ETA, endothelinA; S6c, sarafotoxin 6c.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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= ET-1
(n = 10); 
= S6c (n = 11); 
= ET-3 (n = 12); 
= BQ-3020 (n = 4).
Experiments were conducted in the presence of 1 µM meclofenamic
acid.
= +antagonist; 
= +BQ-123
and antagonist. In panel C, 
= + BQ-123. Experiments
were conducted in the presence of 1 µM meclofenamic acid.
