Yoshihiro Urade

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.

A novel subtype of endothelin B receptor mediating contraction in swine pulmonary vein.

Effects of agonists and antagonists of endothelin (ET) receptors were examined in swine pulmonary artery and vein and in rabbit saphenous vein. ET-1, but not ETB receptor agonists, sarafotoxin S6c (STXc) and IRL 1620, induced contraction in pulmonary artery. This effect was inhibited by the ETA receptor antagonists, BQ-123 and FR139317, but not by the ETB receptor antagonist, IRL 1038. Pulmonary artery precontracted by norepinephrine was relaxed by ET-3 in an endothelium-dependent manner. This relaxation was inhibited by IRL 1038 but not by BQ-123. In pulmonary vein, ET-1, ET-3, STXc and IRL 1620 induced contractions at a similar concentration range. ET-1 induced contraction also in saphenous vein. These contractions were not inhibited by BQ-123, FR139317 or IRL 1038. These results suggest that the isopeptide-selective ETA receptor mediates contraction in swine pulmonary artery whereas the isopeptide-nonselective ETB receptor mediates release of endothelium-derived relaxing factor. In contrast, contractions in the veins may be mediated by a novel subtype of isopeptide-nonselective ETB receptor which is not inhibited by IRL 1038.

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.

Induction of endothelium‐dependent relaxation in the rat aorta by IRL 1620, a novel and selective agonist at the endothelin ETB receptor

1 The effects of a novel and selective agonist at the endothelin ETB receptor, IRL 1620 (Suc‐[Glu9, Ala11,15] endothelin‐1 (8–21)), were examined in the isolated aorta of the rat. 2 IRL 1620 (1–300 nm) changed neither the resting tone nor the cytosolic Ca2+ level ([Ca2+]i) of the aorta without endothelium. In the presence of endothelium, however, IRL 1620 increased endothelial [Ca2+]i with little effect on the muscle tone. In the absence of external Ca2+, IRL 1620 still induced a transient increase in endothelial [Ca2+]i. 3 Noradrenaline (100 nm) increased both muscle [Ca2+]i and tension. IRL 1620 (1–300 nm) relaxed the muscle with an increase in endothelial [Ca2+]i only in the presence of endothelium. An inhibitor of nitric oxide synthase, 100 μm NG‐monomethyl‐l‐arginine, inhibited the relaxant effect of IRL 1620 but not the increase in endothelial [Ca2+]i. 4 In resting and noradrenaline‐stimulated aorta, the effects of IRL 1620 were inhibited by a selective antagonist of the ETB receptor, IRL 1038 (0.3–3 μm), although a selective antagonist of the ETA receptor, BQ‐123 (3 μm), was ineffective. Verapamil (10 μm) did not alter the effects of IRL 1620. 5 A muscarinic receptor agonist, carbachol (1 μm), also induced endothelium‐dependent relaxation with an increase in endothelial [Ca2+]i. However, the effects of carbachol were not inhibited by the ETB antagonist, IRL 1038 (3 μm). 6 These results suggest that IRL 1620 is a selective agonist at the ETB receptor which increases endothelial [Ca2+]i by releasing Ca2+ from storage sites and by opening non‐L type Ca2+ channels, activates nitric oxide synthase, releases nitric oxide, and relaxes vascular smooth muscle.

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.

ETB receptor antagonist, IRL 1038, selectively inhibits the endothelin-induced endothelium-dependent vascular relaxation

In isolated rat aorta, endothelin-1 induced contractions at lower concentrations than endothelin-3. The contractile effects were augmented by removing the endothelium. In contrast, endothelium-1 and endothelin-3 at similar concentrations induced endothelium-dependent relaxation in norepinephrine-stimulated aorta. IRL 1038 ([Cys11,Cys15]endothelin-1(11-21); 3 microM) augmented the contractile effects of endothelins only in the presence of the endothelium. IRL 1038 (0.3-3 microM) inhibited the endothelium-dependent relaxation induced by endothelins but not by carbachol. IRL 1038 itself did not change muscle tension. These results suggest that IRL 1038 is a novel antagonist of the ETB receptor responsible for the release of relaxing factor from the vascular endothelium.

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.

A reversible radioligand specific for the ETB receptor: [125I]Tyr13-Suc-[Glu9,Ala11,15]-endothelin-1(8- 21), [125I]IRL 1620.

Suc-[Glu9,Ala11,15]-endothelin(ET)-1(8-21), IRL 1620, is a linear ET-analog specific for the ET-isopeptide-nonselective ETB receptor. The radio-iodinated analog, [125I]IRL 1620, showed a single class of saturable binding to the ETB receptors in porcine lung membranes with a Kd of 18 pM and a Bmax of 930 fmol/mg protein, which are almost comparable to the values obtained with [125I]ET-3 (6 pM and 900 fmol/mg protein). In competitive binding assays with [125I]IRL 1620, unlabeled ET-1, ET-3, IRL 1620 and [monoiodo-Tyr13]-IRL 1620 showed almost identical displacement curves with Ki of 8 to 16 pM. However, [125I]IRL 1620 was dissociated from the binding sites by addition of an excess amount (100 nM) of any of these unlabeled peptides, each with the same t1/2 of 100 min. This was in marked contrast to [125I]ET-3 which was hardly dissociated from the binding sites.

Contraction of smooth muscle by activation of endothelin receptors on autonomic neurons

Endothelin receptors, predominantly of the ETB type, were localized to cell bodies, processes, and varicosities of cholinergic and adrenergic intramural autonomic neurons that were present in primary cultures of guinea pig tracheal smooth muscle. Stimulation of the neuronal ETB receptor produced a tetrodotoxin‐sensitive increase in the intracellular calcium concentration in neurons which was followed by contraction of the neighboring smooth muscle cells. These observations suggest that endothelins can induce smooth muscle contraction by means of a neuronally mediated mechanism, in addition to their direct actions on the smooth muscle.

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.