General

In mammals, the endothelin (ET) family comprises three endogenous isoforms, ET-1, ET-2 and ET-3 [20,43], and the receptors that mediate their effects have been classified as the endothelin ET_A and ET_B receptors.

Receptor structure

The two endothelin receptors have been isolated and cloned from mammalian tissues [1-2,6,20,24,28,32-33,43]. The structures of the mature receptors have been deduced from the nucleotide sequences of the cDNAs. The encoded proteins contain seven stretches of 20-27 hydrophobic amino acid (aa) residues in both receptors. This structure is consistent with a seven-transmembrane domain (7TM), G protein-coupled receptor belonging to the 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 sub-domains on each endothelin receptor. The extracellular loops, particularly between TM4-TM6, determine selectivity.

Receptor signalling

Endothelin is able to activate a number of signal transduction processes including phospholipase (PL) A₂, PLC and PLD, as well as cytosolic protein kinase activation. The receptors are able to couple to various types of G protein. Both ET_A and ET_B receptors expressed in COS7 cells were shown to couple to G_q, G₁₁, G_s and G_i2, suggesting that endothelin receptors may simultaneously stimulate multiple effectors via several types of G protein [37]. ET_A receptors expressed in CHO cells couple to G_q and G_s but not G_i. ET_B receptors couple to G_q and G_i. Coupling to G_s occurs through the second and third intracellular loops of the receptor. In order to couple with G_i through the third intracellular loop, palmitoylation of the C-terminal cysteine residues and C-terminus are necessary, whereas to couple with G_q only palmitoylation of the C-terminal domain is important [3,16].

Physiology

Endothelin receptors are widely expressed in all tissues, consistent with the physiological role of endothelins as ubiquitous endothelium-derived vasoactive peptides, contributing to the maintenance of vascular tone. Receptors are also localised to non-vascular structures such as epithelial cells as well as occurring in the central nervous system (CNS) on glia and neurones. Both ET_A and ET_B receptors are widely distributed, particularly in blood vessels. In human vessels, ET_A receptors are mainly located on vascular smooth muscle cells, with ET_B receptors being present on endothelial cells lining the vessel wall. ET_B receptors may play a role in the release of endothelium-derived relaxing factors such as nitric oxide (NO) and prostanoids from endothelial cells [40] where all three isoforms have a similar potency [11]. ET_A receptors present on smooth muscle cells are mainly responsible for contraction, but in animals this can vary depending on the species and vascular bed. In some blood vessels such as the rabbit saphenous vein, the rabbit jugular vein, rat renal vascular bed and porcine pulmonary vein, ET_B receptors mediate vasoconstriction. In other vessels, ET-1 is thought to mediate vasoconstriction by activating both receptors. Endothelin stimulates proliferation in a number of different cell types including smooth muscle cells (mainly via the ET_A receptor) or astrocytes (ET_B receptor). In most of these cells, endothelin is thought to be co-mitogenic, potentiating the actions of other growth factors such as PDGF.

Pharmacology

A selective ET_A receptor agonist has not been discovered to date. Sarafotoxin S6c [41] is a selective ET_B receptor agonist, as is [Ala^1,3,11,15]ET-1 [31], a linear analogue of ET-1 in which the disulphide bridges have been removed by substitution of Ala for Cys residues. ET_B receptors also bind the truncated alanine analogues BQ3020 ([Ala^11,15]Ac-ET-1_(6-21)) [18] and IRL1620 (Suc-(Glu⁹, Ala^11,15)-ET-1_(8-21)) [36]. Both compounds have been radiolabelled to give ligands that are highly selective and bind with sub-nanomolar affinity [27]. These peptides have the same or similar amino acid sequences in the C-terminus compared with ET-1. The compounds cause endothelium-dependent vasodilatation, in preparations such as porcine pulmonary artery, consistent with ET_B receptor-mediated release of relaxing factors from the endothelium.

The cyclic pentapeptide, BQ123 (cyclo-[D-Asp-L-Pro-D-Val-L-Leu-D-Trp-]) [17] is a highly selective ET_A receptor antagonist which has been radiolabelled [19]. This compound, and related cyclic peptides, was originally derived from BE18257 (cyclo-[D-Glu-L-Ala-allo-D-lle-L-Leu-D-Trp-]), a natural product of Streptomyces misakiensis. The modified linear peptide FR139317 (N-[(hexahydro-1-azepinyl)carbonyl]L-Leu(1-Me)D-Trp-3(2-pyridyl)-D-Ala [4] is also a selective ET_A receptor antagonist, and a structurally similar selective radioligand, [¹²⁵l]-PD151242 has been developed [10]. Non-peptide ET_A receptor-selective antagonists include PD155080 [25], PD156707 [12], SB234551 [29], L754142 [42], BMS182874 [35] and A127722 [30]. Unlike peptide antagonists, most of these compounds have good oral activity and some may cross the blood brain barrier. IRL2500 [5], RES7011 [38], BQ788 [21] (peptides) and Ro 46-8443 [7] (non-peptide) are selective ET_B receptor antagonists. Antagonists that block both ET_A and ET_B receptors include the peptide TAK044 [23] and the non-peptide compounds bosentan [9] and SB209670 [14].

Other receptors

The existence of alternative splice variants of the ET_B receptor has been reported. Cheng et al. [8] identified a novel cDNA from rat brain which produced a receptor protein with four amino acid substitutions. A variant ET_B receptor that results in a 10aa increase in the length of the second cytoplasmic domain has been described [34]. However, this did not result in any change in ligand affinities and the physiological importance is unclear. Elshourbagy et al. [15] discovered a splice variant from a human placental library; sequence analysis indicated the deduced polypeptide consisted of 436aa and had 91% sequence similarity to the known human ET_B receptor (442aa). However, although the variant displayed the same ligand binding properties of the wild-type, no functional response was detected. The cloning of an ET-3 specific receptor (ET-3 > ET-1) has been described from Xenopus laevis dermal melanophores [22]. The amino acid sequence had 47% sequence homology to the bovine ET_A receptor and 52% homology with the rat ET_B receptor, but no mammalian homologue has yet been identified.

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 ET_B receptors but not constrictor responses mediated by ET_B smooth muscle receptors [13,39]. However, in the ET_B receptor gene knock-out mouse, both the PD142893-sensitive vasodilator response and the PD142893-resistant contractile response to the ET_B agonist S6c were completely absent. These results indicate that the pharmacologically heterogeneous responses to S6c are mediated by ET_B receptors derived from the same gene [26].

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