Kyoko Oda

A potent and specific agonist, Suc-[Glu9, Ala11,15]-endothelin-1(8-21), IRL 1620, for the ETB receptor

A series of C-terminal linear peptides of endothelin (ET)-1 and their N alpha-succinyl (Suc) analogs were synthesized and their binding affinities for the two subtypes of ET receptor, ETA and ETB, in porcine lung membranes were examined. Among the synthetic analogs, Suc-[Glu9,Ala11,15]-ET-1(8-21), IRL 1620, was the most potent and specific ligand for the ETB receptor (KiETA/KiETB approximately equal to 120,000) as judged by the Ki values for ETA (1.9 microM) and ETB (16 pM) receptors. IRL 1620 was 60 times more selective for the ETB receptor than ET-3 (KiETA/KiETB approximately equal to 1,900). IRL 1620 (10(-9)-10(-7) M) induced contractions of the guinea pig trachea with a comparable potency to those of ET-1 or ET-3, suggesting that IRL 1620 is a potent ETB receptor agonist.

An endothelin B receptor-selective antagonist: IRL 1038, [Cys11-Cys15]-endothelin-1(11–21)

In the inhibition of specific binding or [125]endothelins (ETs) to membranes from various tissues of rats, guinea pigs, pigs and humans, [Cys11‐Cys15]‐ET‐1(11–21), IRL 1038, has a much higher affinity for ETB receptors (K i = 6–11 nM) than for ETA receptors (K i = 0.4–0.7 μM). In contraction assays, with ET‐3 as a stimulant, 3 μM IRL 1038 antagonized the ETB receptor‐mediated contraction of guinea pig ileal and tracheal smooth muscle without any significant agonistic activity, but did not effect the ETA receptor‐mediated contraction of rat aortic smooth muscle. IRL 1038 is, therefore, considered to be the first antagonist selective to the ETB receptor.

Autocrine receptors for endothelins in the primary culture of endothelial cells of human umbilical vein

Human umbilical vein endothelial cells (HUVECs) in primary culture produced and secreted endothelin I (ET‐1) actively. Specific binding of [125I]ET‐1 to these cells was not detectable because of the saturation of ET receptors with endogenously produced ET‐1. However, addition of phosphoramidon, an inhibitor of ET‐converting enzyme, to the medium reduced the production of ET‐1 and thus the receptors on HUVECs were made available for exogenously added [125I]ET‐1. Binding studies using phosphoramidon‐treated HUVECs indicated the existence of a non‐isopeptide‐selective type (ETB) or ET receptor with a K d of 17 pM. This receptor is thought to be involved in ET‐induced vasodilation in autocrine manner in vivo.

Endothelin stimulates both cAMP formation and phosphatidylinositol hydrolysis in cultured embryonic bovine tracheal cells

Embryonic bovine tracheal (EBTr) cells were found to possess receptors for endothelin (ET) of ET‐1‐selective (ETA)subtype with a K d for ET‐1 of 114 pM and a B max of 12.9 fmol/105 cells. Stimulation of EBTr cells with 100 pM to 100 nM ET‐1 increased the contents of both inositol phosphates and cAMP in a concentration‐dependent manner, indicating that the receptors are coupled to both phosphatidylinositol hydrolysis and cAMP formation in EBTr cells.

Both ETA and ETB receptors are involved in mitogen‐activated protein kinase activation and DNA synthesis of astrocytes: study using ETB receptor‐deficient rats (aganglionosis rats)

Endothelin (ET) is known to be a potent mitogen in astrocytes. However, the contribution and signalling pathway of ETA and/or ETB receptor to the proliferation of astrocytes remain unclear. We investigated ET‐induced DNA synthesis in astrocytes using ETB receptor‐deficient mutant rats (aganglionosis rats: sl/sl). Western blotting with anti‐ET receptor subtype‐specific antibodies and Scatchard analysis of binding revealed that ETB receptor expression in astrocytes depended on gene dosage (+/+: sl/+: sl/sl = 2: 1: 0), whereas ETA receptor expression was unchanged among the three genotypes. ET‐1 (10 nm) stimulated [3H]thymidine incorporation and mitogen‐activated protein kinase (MAP kinase) activity not only in +/+ via both ETA and ETB receptors, but also in sl/sl astrocytes via ETA receptor with about half the extent of those observed in +/+ astrocytes. Treatment with pertussis toxin (PTX) suppressed the ET‐1‐induced increases in the incorporation and MAP kinase activity in +/+, but not sl/sl astrocytes, indicating that the ETB receptor‐, but not the ETA receptor‐, mediated pathway to DNA synthesis involves PTX‐sensitive G proteins, e.g. Gi and/or Go (Gi/o). In +/+ astrocytes, ET‐1 (1 nm) stimulated cAMP accumulation, and the ETB receptor‐selective agonist IRL 1620 (1 nm) suppressed 10 μm forskolin‐induced cAMP accumulation, suggesting Gs coupling to the ETA receptor and Gi/o coupling to the ETB receptor. On the other hand, ET‐1 did not increase cAMP accumulation in sl/sl astrocytes, although ET‐1 (1 nm) suppressed the forskolin‐induced response, suggesting Gi/o coupling to the ETA receptor. Our results suggest the possibility that the selectivity of G protein for ETA receptor is changed from Gs to Gi/o in ETB receptor‐deficient astrocytes.

Two types of endothelin B receptors mediating relaxation in the guinea pig ileum

In guinea pig ileum, binding assays showed the existence of endothelin (ET) receptors of ETA (isopeptide-selective) and ETB (nonselective) subtypes. ETs induced relaxation followed by contraction. ET-1 induced greater contraction at lower concentrations than ET-3. An ETA antagonist, BQ-123, shifted the concentration-response curves for ETs to the right. An ETB antagonist, IRL 1038, shifted the concentration-response curve for ET-3 to the right and downwards with little effect on the curve for ET-1. In contrast, ET-1 and ET-3 induced relaxation at similar concentrations. The relaxation induced by ETs was composed of an initial transient relaxation followed by sustained relaxation. Only the transient phase was inhibited by IRL 1038 in a concentration-dependent manner. These results suggest that the ET-induced relaxation is mediated by two types of ETB receptor; transient and sustained relaxations are mediated respectively by IRL 1038-sensitive and IRL 1038-insensitive subtypes of ETB receptor. In contrast, the contractile effect seems to be mediated mainly by the ETA receptor and partially by an IRL 1038-sensitive subtype of ETB receptor.

Retraction concerning an endothelin B receptor-selective antagonist: Urade, Y., Fujitani, Y., Oda, K., Watakabe, T., Umemura, L., Takai, M., Okada, T., Sakata, K. and Karaki, H. (1992) FEBS Lett. 311, 12-16 : An endothelin B receptor-selective antagonist: IRL 1038, [Cys11-Cys15]-endothelin-1(11-21)

In the inhibition of specific binding of [125I]endothelins (ETs) to membrane from various tissues of rats, guinea pigs, pigs and humans, [Cys11-Cys15]-ET-1(11-21), IRL 1038, has a much higher affinity for ETB receptors (Ki = 6-11 nM) than for ETA receptors (Ki = 0.4-0.7 microM). In contraction assays, with ET-3 as a stimulant, 3 microM IRL 1038 antagonized the ETB receptor-mediated contraction of guinea pig ileal and tracheal smooth muscle without any significant agonistic activity, but did not effect the ETA receptor-mediated contraction of rat aortic smooth muscle. IRL 1038 is therefore, considered to be the first antagonist selective to the ETB receptor.The article describes the synthetic peptide IRL 1038 as having a much higher affinity to ETB receptors (Ki = 6 11 nM) than to ET, receptors (Ki = 4OC700 nM) in various tissue membranes. It was also shown that this peptide antagonized the ET,-mediated contraction of guinea pig ileal and tracheal smooth muscles. Since the publication of the data, we have synthesized more than 20 different batches of IRL 1038, some of which showed potencies and selectivities almost equivalent to the published data. However, the other batches, including batches synthesized by external suppliers, exhibited significantly higher Ki of 5&400 nM for the binding to ET, receptors in porcine lung membranes. We have very carefully investigated the physico-chemical properties of IRL 1038 in aqueous solutions at different pH (pH 7-10) and peptide concentrations, using fluorescence and proton NMR spectroscopy. At high pH (pH lo), the peptide does not aggregate and two kinds of non-aggregated states were observed by NMR spectroscopy. There was, however, no evident correlation between the two non-aggregated states and binding af-

Discovery of IRL 3461: a novel and potent endothelin antagonist with balanced ETA/ETB affinity

IRL 3461, N-butanesulfonyl-[N-(3,5-dimethylbenzoyl)-N-methyl-3-[4-(5-+ ++isoxazolyl) -phenyl]-alanyl]-(L)-valineamide, a potent and bifunctional (ETA + ETB) [Ki(ETA) = 1.8 nM, Ki(ETB) = 1.2 nM] antagonist was discovered by structural modification of IRL 2500, an ETB selective antagonist. IRL 3461 was found to be stable on incubation with human, rat, mouse, and guinea pig plasmas.

IRL 2500: A potent ETB selective endothelin antagonist

Abstract Combination of a glycine substitution scan on the C-terminal dodecapeptide analog of ET-1 and a substance P antagonist screen on the basis of a homology study of the rhodopsin superfamily of seven-transmembrane receptors yielded in the development of IRL 2500, a potent ETB selective endothelin antagonist.

Effect of a novel bifunctional endothelin receptor antagonist, IRL 3630A, on guinea pig respiratory mechanics.

This study characterized the in vitro pharmacological properties of a newly developed endothelin receptor antagonist, N-butanesulfonyl-[N-(3, 5-dimethylbenzoyl)-N-methyl-3-[4-(5-isoxazolyl)-phenyl]-(D)- alanyl]-( L)-valineamide sodium salt (IRL 3630A), and its in vivo effects on respiratory mechanics were determined. IRL 3630A showed highly balanced affinities to human endothelin ET(A) and ET(B) receptors, giving apparent K(i) values of 1.5 and 1.2 nM, respectively. This compound also potently antagonized the endothelin-1-induced intracellular Ca(2+) increases in both embryonic bovine tracheal (EBTr) cells expressing endothelin ET(A) receptors and human Girardi heart (hGH) cells expressing endothelin ET(B) receptors. In guinea pig isolated tracheas having both endothelin ET(A) and ET(B) receptors, IRL 3630A greatly inhibited endothelin-1-induced contraction (pA(2)=7.1), which was partially or scarcely suppressed by the endothelin ET(A) receptor antagonist cyclo[-(D)-Trp-(D)-Asp-(L)-Pro-(D)-Val-(L)-Leu-] (BQ-123) or the endothelin ET(B) receptor antagonist N-(3, 5-dimethylbenzoyl)-N-methyl-3-(4-phenyl)-(D)-phenylalanyl-(L)-t ryptop han (IRL 2500), respectively. Bolus i.v. injections of IRL 3630A administered into anaesthetized guinea pigs at 10 and 30 microg/kg inhibited endothelin-1 (1.3 microg/kg)-induced changes in respiratory resistance and compliance in a dose dependent manner, whereas both sodium 2-benzo[1, 3]dioxol-5-yl-4-(4-methoxy-phenyl)-4-oxo-3-(3,4, 5-trimethoxy-benzyl)-but-2-enoate (an endothelin ET(A) receptor antagonist: PD 156707) and IRL 2500 at doses of up to 30 microg/kg did not affect endothelin-1-induced changes in respiratory mechanics, reflecting the in vitro results. IRL 3630A is thus an effective bifunctional endothelin receptor antagonist, and will be useful in clarifying the role of endothelin in pulmonary diseases such as bronchial asthma.