Clinical Pharmacology Unit, University of Cambridge,
Centre for Clinical Investigation, Addenbrooke's Hospital,
Cambridge, United Kingdom
In mammals, the endothelin (ET) family comprises three endogenous
isoforms, ET-1, ET-2, and ET-3. ET-1 is the principal isoform in the
human cardiovascular system and remains the most potent and
long-lasting constrictor of human vessels discovered. In humans, endothelins mediate their actions via only two receptor types that have
been cloned and classified as the ETA and ETB
receptors in the first NC-IUPHAR (International Union of Pharmacology
Committee on Receptor Nomenclature and Drug Classification) report on
nomenclature in 1994. This report was compiled before the discovery of
the majority of endothelin receptor antagonists (particularly
nonpeptides) currently used in the characterization of receptors and
now updated in the present review. Endothelin receptors continue to be
classified according to their rank order of potency for the three
endogenous isoforms of endothelin. A selective ETA receptor
agonist has not been discovered, but highly selective antagonists
include peptides (BQ123,
cyclo-[D-Asp-L-Pro-D-Val-L-Leu-D-Trp-]; FR139317, N-
[(hexahydro-1-azepinyl)carbonyl]L-Leu(1-Me)D-Trp-3 (2-pyridyl)-D-Ala) and the generally more potent
nonpeptides, such as PD156707, SB234551, L754142, A127722, and
TBC11251. Sarafotoxin S6c, BQ3020
([Ala11,15]Ac-ET-1(6-21)), and IRL1620
[Suc-(Glu9,
Ala11,15)-ET-1(8-21)] are widely used
synthetic ETB receptor agonists. A limited number of
peptide (BQ788) and nonpeptide (A192621) ETB antagonists
have also been developed. They are generally less potent than
ETA antagonists and display lower selectivity (usually only
1 to 2 orders of magnitude) for the ETB receptor.
Radioligands highly selective for either ETA
(125I-PD151242, 125I-PD164333, and
3H-BQ123) or ETB receptors
(125I-BQ3020 and 125I-IRL1620) have further
consolidated classification into only these two types, with no strong
molecular or pharmacological evidence to support the existence of
further receptors in mammals.
 |
I. Introduction |
In mammals, the endothelin (ET1)
family comprises three endogenous isoforms, ET-1, ET-2, and ET-3
(Yanagisawa et al., 1988
; Inoue et al., 1989). These peptides mediate
their actions via two receptor types, classified as the
ETA and ETB receptors in the first NC-IUPHAR (International Union of Pharmacology Committee on
Receptor Nomenclature and Drug Classification) receptors report on
nomenclature by Masaki et al. (1994). This report was compiled before
the discovery of the majority of ET receptor antagonists (particularly
nonpeptides) currently used in the characterization of receptors and
now updated in the present review. The single proposed
ETB receptor antagonist included by Masaki et al.
(1994) was subsequently shown to lack efficacy and was withdrawn by the original discoverers (Urade et al., 1994). This review reflects both
peptide and nonpeptide ETB antagonists that are
the compounds of choice. A second major change is the inclusion of
radioligands that are highly selective for either
ETA (125I-PD151242,
125I-PD164333, and 3H-BQ123
(cyclo-[D-Asp-L-Pro-D-Val-L-Leu-D-Trp-])
or ETB receptors [125I-BQ3020
([Ala11,15]Ac-ET-1(6-21))
and 125I-IRL1620
(Suc-(Glu9,
Ala11,15)-ET-1(8-21))]
that have been crucial to consolidating the present classification.
Masaki et al. (1994) referred to a gene encoding a third receptor
(P32940) cloned from the amphibian Xenopus laevis dermal
melanophores, which was reported to be ET-3 specific (ET-3 > ET-1), the so-called ETC receptor. However, to
date, molecular and ligand binding techniques have failed to identify a
mammalian homolog. Since NC-IUPHAR is predominantly interested in human receptors, with an extension to mammalian receptors, this review follows the recommendation to exclude consideration of nonmammalian receptors in the classification.
ET-1 is the principal isoform in the human cardiovascular system and
remains the most ubiquitous, potent, and unusually long-lasting constrictor of human vessels discovered. ET-1 is unusual among the
mammalian bioactive peptides in being released from a dual secretory
pathway (Russell et al., 1998). The peptide is continuously released
from vascular endothelial cells by the constitutive pathway, producing
intense constriction of the underlying smooth muscle and contributing
to the maintenance of endogenous vascular tone (Haynes and Webb, 1994).
The peptide is also released from endothelial cell-specific storage
granules (Weibel-Palade bodies) in response to external physiological,
or perhaps pathophysiological, stimuli producing further
vasoconstriction (Russell et al., 1998). Thus, ET-1 functions as a
locally released, rather than circulating, hormone, and concentrations
are comparatively low in plasma and other tissues. ET-2 has been less
extensively studied than other ET peptides, but it is present in human
cardiovascular tissues and was as potent a vasoconstrictor as ET-1 in
human arteries and veins (Maguire and Davenport, 1995). Endothelial
cells do not synthesize ET-3, but the mature peptide is detectable in
plasma (Matsumoto et al., 1994) and other tissues, including heart and brain. ET-3 is unique in that it is the only endogenous isoform that
distinguishes between the two endothelin receptors. It has the same
affinity at the ETB receptor as ET-1 but, at
physiological concentrations, has little or no affinity for the
ETA.
The only endogenous peptides with a high degree of sequence similarity
to the ETs are the sarafotoxins (S6a, S6b, S6c, and S6d). This family
of 21-amino acid (aa) peptides was originally discovered in the venom
of a snake, Atractaspis engadensis (Takasaki et al., 1988).
| |
II. Cloned Endothelin Receptors |
Receptors can be identified by their amino acid structure and
provide unambiguous evidence for expression of a gene encoding a
particular type in specific cells or tissues. An increasing number of
mammalian species have been studied, but only two ET receptors have
been isolated and cloned (Table 1;
Arai et al., 1990; Sakurai et al., 1990; Adachi et al., 1991; Lin et
al., 1991; Nakamuta et al., 1991; Saito et al., 1991; Baynash et al.,
1994). The deduced amino acid sequences for the two human receptors
display only 59% similarity and are shown in Table
2. The amino acid sequences of
ETA receptors also differ between humans and
other species, for example by 9% between human and rat
ETA receptors and by 12% for the
ETB. These may contribute to differences in efficacy and potency of selective agonists and antagonists.
The structures of the mature receptors have been deduced from the
nucleotide sequences of the cDNAs. The encoded proteins contain seven
stretches of 20 to 27 hydrophobic aa residues in both receptors,
consistent with both subtypes belonging to the seven-transmembrane
(7TM) domain , G protein-coupled rhodopsin-type receptor superfamily.
Both receptors have an N-terminal signal sequence, which is rare among
heptahelical receptors, with a relatively long extracellular N-terminal
portion preceding the first transmembrane domain. There are two
separate ligand interaction subdomains on each endothelin receptor. The
extracellular loops, particularly between TM 4 to 6, determine selectivity.
At present, there is no justification for further types beyond the
current classification into ETA and
ETB in mammalian tissue. Functional studies have
suggested that PD142893 [Ac-(beta-phynyl) D-Phe-L-Leu-L-Asp-L-lle-L-lle-L-Trp],
a hexapeptide antagonist, can block the vasodilator actions of ET-1 at
endothelial ETB receptors but not constrictor
responses mediated by ETB smooth muscle receptors (Warner et al., 1993; Douglas et al., 1995). However, in the
ETB receptor gene knockout mouse, both the
PD142893-sensitive vasodilator response and the PD142893-resistant
contractile response to the ETB agonist
sarafotoxin S6c were completely absent. These results indicate that the
pharmacologically heterogeneous responses to S6c are mediated by
ETB receptors derived from the same gene
(Mizuguchi et al., 1997). In agreement, a very detailed binding study
(including PD142893) was unable to distinguish between
ETB receptors expressed by human isolated
endothelial cells compared with smooth muscle cells in culture (Flynn
et al., 1998). Furthermore, in human tissue, both
ETA- and ETB-selective
radiolabeled ligands bound with a single affinity and Hill slopes close
to unity (Molenaar et al., 1992; Davenport et al., 1994, 1998;
Davenport, 1997). Similarly, competition studies using unlabeled
ligands provided no evidence for further subtypes (Peter and Davenport,
1996; Russell and Davenport, 1996).
| |
III. Mammalian Splice Variants of EndothelinA and
EndothelinB Receptors |
Alternative splice variants of ET receptors have been reported but
to date these variants either show little or no change in binding
characteristics and their physiological or pathophysiological significance is unclear. The following is intended to be a guide only
because the field has not developed sufficiently with unequivocal quantitative evidence for significant expression and function in native
tissues rather than artificial cell lines, to make any firm
recommendation for classification.
The existence of alternative splice variants of the
ETB receptor in human and porcine tissue has been
reported. A variant human ETB receptor that
results in a 10-aa increase in the length of the second cytoplasmic
domain has been described (Shyamala et al., 1994). Messenger RNA
measured by reverse transcription-polymerase chain reaction in a
limited number of human tissues was found only in low abundance in
human brain (which expresses one of the highest densities of
ETB receptors) as well as the heart, lung, and
placenta but was not detected in other species tested (bovine, porcine,
and rat). The increase in amino acids did not result in any change in
either ligand affinities or signal transduction (cAMP and inositol
phosphate turnover), and the physiological importance of this variant
receptor is unclear.
Elshourbagy et al. (1996) discovered a second splice variant from a
human placental library. Analysis indicated that the deduced polypeptide was identical to the native ETB
sequence except that the 42 aa of the intracellular carboxy terminus of
the former was replaced with an alternative 36-aa sequence, bearing no
significant homology with other known proteins. Northern blot analysis
indicated an mRNA species of 2.7 kilobases, which was expressed in all
of a limited number of human tissues tested (lung, placenta, kidney, and skeletal muscle) in addition to mRNA encoding the native
ETB receptor. However, mRNA encoding the variant
was not particularly abundant. The relative ratio of each individual
variant mRNA was less than 10% of the total ETB
mRNA, with the intriguing exception of skeletal muscle where it
represented more than 40%. Two cell types were also examined,
endothelial and smooth muscle cells, but only mRNA encoding the native
receptor was detected. The cloned variant receptors expressed in COS
cells displayed similar binding properties for ET peptides compared
with expressed native receptors in the same cells, indicating
unsurprisingly that the splice variant had little or no effect on
ligand binding. However, functional studies showed that ET-stimulated
inositol phosphate accumulation in expressed native receptors was
abolished in cells transfected with the splice variant. These data
suggest the difference in the amino acid sequences between the two
receptors may alter functional coupling in the variant receptor.
Nambi et al. (2000) detected a novel cDNA from another species, porcine
cerebellum, that was predicted to encode an ETB
receptor also with alternate splicing of the carboxy terminus,
resulting in a deduced polypeptide of 429 aa, 14 residues shorter than
the wild-type receptor. The relative abundance of mRNA encoding the splice variant compared with the wild-type receptor was not reported, but mRNA was detected in ETB-rich tissues
including porcine lung, kidney, and cerebellum. However, the splice
variant did not alter the binding of radiolabeled ET-1 or functional
coupling when expressed in COS cells. The lack of effect on inositol
phosphate accumulation is in marked contrast to the human variant (see
above) previously described by this group. Combined with the lack of
sequence similarity between the human (38 aa) and porcine (29 aa)
carboxy terminal splice variants, it is not clear whether the porcine
variant is a homolog of the human or whether these are distinct splice variants.
Cheng et al. (1993) identified cDNA from rat brain, which they
described as producing a receptor protein with four amino acid substitutions that displayed equal affinity for the three ET isoforms. However, Cheng et al. (1993) probably described the correct rat ETB sequence, correcting a sequencing error in
the previously deduced sequence of Sakurai et al. (1990), for the
following reasons. The Cheng et al. (1993) sequence has 3 extra bases
in a 9-base span, which corrects a pair of adjacent frameshifts in the
Sakurai et al. sequence, making the DNA sequence identical to the mouse sequence (Hosoda et al., 1994) in the same region and matching 3 of 4 amino acids in the human sequence as opposed to 0 of 4 with the Sakurai
et al. sequence. Cheng et al. (1993) also report a different sequence
in the 5'-untranslated region, which could be an alternative first
exon, reflecting transcription initiating from an alternative promoter.
It is also possible, although less likely, that one of the
5'-untranslated region sequences is an artifact, such as a chimeric
cDNA or sequencing assembly error.
The human ETA receptor gene has been proposed to
give rise to at least three alternatively spliced
ETA receptor transcripts corresponding to
deletion of exon 3 (producing a protein with two membrane-spanning
domains), exon 4 (producing a protein with three membrane-spanning
domains), and exon 3 plus exon 4 (producing a protein lacking the third
and fourth domain) (Miyamoto et al., 1996; Bourgeois et al.,
1997). Although alternative transcripts were identified in human
tissues including lung, aorta, and atrium, the truncated receptors when
expressed in COS cell lines did not bind ET-1 (Miyamoto et al., 1996),
and a physiological role remains unclear. Intriguingly, mRNA encoding
the putative truncated receptor with the deletion of exon 3 plus 4 was
more abundant than the wild type in human melanoma cell lines and
melanoma tissue (Zhang et al., 1998).
| |
IV. Physiological Role of Receptors |
Endothelin receptors are widely expressed in all tissues, which is
consistent with the physiological role of endothelins as ubiquitous
endothelium-derived vasoactive peptides, contributing to the
maintenance of vascular tone. In humans, ETA
receptors predominate on the smooth muscle of blood vessels, and the
low density of ETB receptors (<15%) also
present on the smooth muscle contributes little to vasoconstriction in
either normal or diseased tissue (Maguire and Davenport, 1995).
ETB receptors are the principal type in the
kidney, localizing to nonvascular tissues. Evidence is emerging that
the ETB receptor functions as a "clearing
receptor" to remove ET from the circulation.
ETB receptors localized to the single layer of
endothelial cells that line all blood vessels, may play a role in the
release of endothelium-derived relaxing factors, such as nitric oxide
and prostanoids (Warner et al., 1989), where all three isoforms have a
similar potency (de Nucci et al., 1988). Although
ETA receptors present on smooth muscle cells are
mainly responsible for contraction throughout the human vasculature,
the situation in animals is more complex since the relative
contribution from activating constrictor ETB
receptors can vary, depending on the species and vascular bed. In some
blood vessels, such as the rabbit saphenous vein, rabbit jugular vein, rat renal vascular bed, and porcine pulmonary vein,
ETB receptors mediate vasoconstriction. In other
vessels, ET-1 is thought to mediate vasoconstriction by activating both receptors.
Receptors are also localized to nonvascular structures, such as
epithelial cells, as well as occurring in the central nervous system on
glia and neurones. Endothelin stimulates proliferation in a number of
different cell types, including smooth muscle cells (mainly via the
ETA subtype) or astrocytes
(ETB). In most of these cells, ET is thought to
be comitogenic, potentiating the actions of other growth factors such
as platelet-derived growth factor.
| |
V. Endogenous and Synthetic Agonists |
ET receptors continue to be classified (Table
3; Davenport, 2000) according to their
rank order of potency for the endogenous ET isoforms. A selective
ETA receptor agonist has not been discovered.
Sarafotoxin S6c is one of the most widely used
ETB-selective agonists, displaying over
200,000-fold selectivity in the rat (William et al., 1991), although
the peptide is much less selective in human tissues, perhaps reflecting
species differences in the receptors (Russell and Davenport, 1996).
[Ala1,3,11,15]ET-1 (Saeki et al., 1991), the
linear analog of ET-1 in which the disulfide bridges have been removed
by substitution of Ala for Cys residues, is
ETB-selective. The truncated linear synthetic analogs BQ3020 and IRL1620 are the most widely used selective synthetic
agonists to characterize ETB receptors. The
compounds cause endothelium-dependent vasodilatation in preparations
such as porcine pulmonary artery, which is consistent with
ETB receptor-mediated release of relaxing factors
from the endothelium.
| |
VI. Radiolabeled Agonists |
Most studies characterizing and localizing ET receptors use
125I-ET-1, directly labeled via the
Tyr13 (Table 3). This ligand binds with the same
affinity to both ETA and
ETB receptors and is stable under
nonphysiological binding conditions with little or no degradation of
labeled ET-1 being detected. 125I-ET-2,
125I-vasoactive intestinal contractor (the murine
isoform of ET-2), and 125I-sarafotoxin 6b have
been labeled and used in saturation assays where they also bind to both
receptors (Davenport and Morton, 1991; Maguire et al., 1996).
ET-3 can be labeled at Tyr6,
Tyr13, and Tyr14.
Tyr6 is generally used, as it is more difficult
to separate 125I-ET-3 labeled at the latter two
Tyr residues, although all three ET-3 ligands have similar affinities.
The selectivity of ET-3 for ETB versus
ETA receptors is often only about two orders and it is difficult to precisely delineate the two receptors using this
labeled peptide in saturation assays. ETB
receptors are usually characterized using
125I-BQ3020 (Ihara et al., 1992b; Molenaar et
al., 1992), which binds with subnanomolar affinity to the
ETB receptor, with at least 1500-fold selectivity
for this receptor over the ETA. Alternatively, the truncated analog 125I-IRL1620 can also be
used, particularly in animal tissues (Watakabe et al., 1992).
| |
VII. Antagonists |
Antagonists are currently classified as either
ETA--selective,
ETB-selective, or mixed antagonists that display
similar affinity for both receptors. The most highly selective peptide
antagonists (4 to 5 orders of selectivity) for the
ETA receptors are the cyclic pentapeptide BQ123
(Ihara et al., 1992a) and the modified linear peptide FR139317
(N-[(hexahydro-1-azepinyl)carbonyl]L-Leu(1-Me)D-Trp-3(2-pyridyl)-D-Ala; Aramori et al., 1993). Unlike peptide antagonists, many nonpeptide ETA receptor-selective antagonists have oral
bioavailability and some may cross the blood-brain barrier. The
majority are more potent, with pA2 values of up
to 10 compared with 7 or 8 for BQ123 or FR139317, but are less
selective, and plasma binding may also be significant in vivo.
| |
VIII. Radiolabeled EndothelinA Selective Antagonists |
125I-PD151242 is widely used to characterize
and localize ETA receptors. This linear
tetrapeptide analog of FR139317, binds with subnanomolar affinity to
the ETA receptor and has about 10,000-fold selectivity for this receptor in human and animal tissues. A nonpeptide ETA-selective ligand has also been developed,
125I-PD164333 (Davenport et al., 1998) with
comparable affinity as well as a tritiated ligand,
3H-BQ123 (Ihara et al., 1995). The above ligands
are available commercially either as catalog items or custom syntheses.
| |
IX. EndothelinB Selective Antagonists |
A more limited number of peptide (e.g., BQ788) and nonpeptide
(e.g., A192621) ETB antagonists have been
developed, reflecting the lack of clinical need for this type of
compound. They are less potent than ETA
antagonists and display lower selectivity (usually only 1 to 2 orders
of magnitude) for the ETB receptor (Table 3).
| |
X. EndothelinA/EndothelinB Antagonists |
The distinction between antagonists that are
ETA-selective and those that block both
ETA and ETB receptors is
not precise but generally the former display greater than 100-fold
selectivity for the ETA subtype, and the latter
less than 100-fold. These compounds are seldom reported as having equal
affinity for both receptors, and this should be taken into
consideration in experimental designs. Nonpeptide compounds included
bosentan (RO470203, Tracleer; Actelion, San Francisco, CA) (Clozel et
al., 1994), SB209670 (Elliott et al., 1994), SB217242
(enrasentan; Ohlstein et al., 1996), and RO610612 (tezosentan; Clozel
et al., 1999). Plasma binding may also be significant in vivo.
| |
XI. Conclusions |
In humans, ET peptides mediate their actions via only two receptor
types, classified as ETA and
ETB. There is no strong evidence to support the
existence of further receptors in mammals. Further research is required
to establish whether any of the potential splice variants in the
ETB receptor have a physiological or
pathophysiological role.
Supported by the British Heart Foundation. I
thank Tom Bonner for comments on ET splice variants and Michael
Spedding and Steve Watson for commenting on the manuscript.
Address correspondence to: Dr. Anthony P. Davenport, Clinical
Pharmacology Unit, University of Cambridge, Level 6, Centre for
Clinical Investigation, Box 110, Addenbrooke's Hospital, Cambridge, CB2 2QQ UK. E-mail: Apd10{at}medschl.cam.ac.uk
Composition of the endothelin receptor subcommittee of the
International Union of Pharmacology on Receptor Nomenclature and Drug
Classification: Anthony P. Davenport (Chairman), Clinical Pharmacology
Unit, University of Cambridge, Level 6, Centre for Clinical
Investigation, Box 100, Addenbrooke's Hospital, Cambridge C2 2QQ, UK;
Théophile Godfraind, Laboratoire de Pharmacologie, Université Catholique de Louvain, avenue Hippocrate 54, B-1200 Brussels, Belgium; Thomas F. Lüscher, University Hospital,
CH-8091 Zurich, Switzerland; Eliot Ohlstein, Pharmacological Sciences, Glaxo SmithKline Pharmaceuticals, 709 Swedeland Road, P.O. Box 1539, King of Prussia, PA 19406-0939; Gabor M. Rubanyi, Berlex Biosciences,
15049 San Pablo Avenue, Richmond, CA 94804; and Robert R. Ruffolo,
Wyeth Pharmaceuticals, 500 Arcola Road, Collegeville, PA 19426.
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-Phynyl)
D-Phe-L-Leu-L-Asp-L-lle-L-lle-L-Trp;
SB209670, (1RS,2SR,3RS)-3-(2-carboxymethoxy-4-methoxyphenyl)-5-(prop-1-yloxy)indane-2-carboxylic
acid.
