Pharmacological characterization of YM598, a selective endothelin-A receptor antagonist

The binding affinities of YM598, a novel endothelin-A (ETA) receptor antagonist, for native human ETA receptors expressed in human coronary artery smooth muscle cells and endothelin-B (ETB) subtypes in the human melanoma cell line SKMel- 28 were compared with those of atrasentan and bosentan. The in vivo ETA receptor antagonist activities of YM598 and atrasentan were also evaluated in pithed rats. The inhibitory dissociation constant values of YM598, atrasentan and bosentan were 0.772, 0.0551 and 4.75 nM, respectively, for native human ETA receptors, and 143, 4.80 and 40.9 nM, respectively, for native human ETB subtypes. The calculated selectivity ratios of YM598, atrasentan and bosentan for ETA versus ETB receptors were 222, 136 and 13.0, respectively. In pithed rats, YM598 and atrasentan inhibited the big endothelin-1 (1 nmol/kg)-induced pressor response in a dose-dependent manner, after both intravenous and oral administration. The inhibitory effect of YM598 was less potent than that of atrasentan when these agents were intravenously administered, but those of both agents were comparable when orally administered. These results suggest that YM598 has a high selectivity for native human ETA receptors against ETB receptors, and that YM598 is superior to atrasentan as an ETA receptor antagonist, with regard to pharmacological bioavailability in rats.     

Pharmacological characterization of YM598, an orally active and highly potent selective endothelin ET(A) receptor antagonist

We describe here the pharmacology of (E)-N-[6-methoxy-5-(2-methoxyphenoxy)[2,2'-bipyrimidin]-4-yl]-2-phenylethenesulfonamide monopotassium salt (YM598), a novel selective endothelin ET(A) receptor antagonist synthesized through the modification of the ET(A)/ET(B) non-selective antagonist, bosentan. YM598 inhibited [125I]endothelin-1 binding to cloned human endothelin ET(A) and ET(B) receptor, with K(i) of 0.697 and 569 nM, and inhibited endothelin-1-induced increases in intracellular Ca(2+) concentration in human and rat endothelin ET(A) receptor. YM598 also inhibited endothelin-1-induced vasoconstriction in isolated rat aorta with a pA(2) value of 7.6. In vivo, YM598 inhibited the pressor response to big endothelin-1, a precursor peptide of endothelin-1. DR(2) values of YM598 in pithed rats were 0.53 mg/kg, i.v. and 0.77 mg/kg, p.o., and its antagonism in conscious rats was maintained for more than 6.5 h at 1 mg/kg, p.o. In contrast, YM598 had no effect on the sarafotoxin S6c-induced depressor or pressor responses. YM598 showed not only superior antagonistic activity and higher-selectivity for endothelin ET(A) receptor in vitro, but at least a 30-fold higher potency in vivo than bosentan. In conclusion, YM598 is a potent and orally active selective endothelin ET(A) receptor antagonist.     

The increase in human plasma immunoreactive endothelin but not big endothelin-1 or its C-terminal fragment induced by systemic administration of the endothelin antagonist TAK-044.

1. We examined the effects of systemic infusion, in healthy human volunteers, of the endothelin antagonist TAK-044 on the plasma concentrations of mature endothelin, big endothelin-1 and the C-terminal fragment of big endothelin-1, by selective solid-phase extraction and specific radioimmunoassays. 2. Unlabelled TAK-044 competed with specific [125I]-endothelin-1 binding to human left ventricle tissue in a biphasic manner giving KD values of 0.11 nM and 26.8 nM at the ETA and ETB receptor subtypes, respectively, indicating a 244 fold selectivity for the ETA receptor subtype. 3. A 15 min intravenous infusion of placebo or 30 mg TAK-044 (giving a serum concentration of 2 nM, calculated to block > 95% of ETA but < 5% ETB receptors) had no effect on the immunoreactive plasma concentrations of the three peptides. 4. At the higher dose of 750 mg TAK-044 (giving a serum concentration of 80 nM, calculated to block > 99% of ETA and > 75% ETB receptors), the immunoreactive plasma endothelin concentrations were increased 3.3 fold over basal levels (P < 0.01). The concentrations of big endothelin-1 or C-terminal fragment of big endothelin-1 were unchanged. 5. At both doses of TAK-044, there were significant decreases in diastolic blood pressure, and peripheral vascular resistance, with corresponding increases in cardiac index and stroke index. There were no changes in systolic or mean arterial blood pressures or heart rate. 6. Since only the concentrations of the mature peptide were increased, we conclude that the most likely sources of endothelin contributing to the observed rise were displacement of receptor-bound peptide and reduction in plasma clearance rather than peptide synthesis.     

Upregulation of vascular ET(B) receptor gene expression after chronic ET(A) receptor blockade in prediabetic NOD mice

In the aorta of prediabetic non-obese diabetic mice, a model of human type 1 diabetes, we investigated gene expression of the endothelin receptors and contractility to big endothelin-1 and endothelin-1 at the ages of 10 and 16 weeks. A subgroup of 10- week-old animals was treated with the endothelin ETA receptor antagonist LU461314 (30 mg/kg per day for 6 weeks). Blood glucose levels were normal in all animals. Real-time polymerase chain reaction analysis revealed that vascular ETB receptor expression was higher in 10-week-old non-obese diabetic (NOD) mice compared with controls. In 16-week-old NOD mice, but not in control mice, ETB receptor mRNA was twofold lower (P < 0.05 vs 10-week-old NOD mice). In all groups ETA receptor expression was unaffected by age or treatment. Contractions to big endothelin-1 and endothelin-1 were lower in 10-week-old NOD mice compared with controls. Treatment with LU461314 increased ETB receptor expression in 16-week-old NOD mice, but had no effect on vascular contractility. These data indicate that dysregulation of ETB receptor expression and a decreased contractile response to big endothelin-1 and endothelin-1 are present in the prediabetic state of a model of human type 1 diabetes. These alterations occur independent of glucose levels. Furthermore, ETA receptor blockade is effective in increasing ETB receptor gene expression, suggesting a potential role for endothelin ETA antagonists in the treatment of type 1 diabetes.     

Renal protective effect of YM598, a selective endothelin type A receptor antagonist

We have investigated the protective effect of YM598, a selective endothelin type A receptor antagonist, against an endothelin-1-induced proliferation of rat mesangial cells and renal function in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an animal model of type II diabetes. YM598, but not K-8794, a selective endothelin type B receptor antagonist, inhibited the endothelin-1-induced proliferation of cultured mesangial cells derived from intact Wistar rats in a concentration-dependent manner. YM598 (0.1 or 1 mg/kg), enalapril (5 mg/kg), an angiotensin- converting enzyme inhibitor, or vehicle was administered once daily by gastric gavage to 22-week-old male OLETF rats for 32 weeks. YM598 blunted the development of albuminuria in a dose-dependent manner. A higher dose of YM598 reduced albuminuria comparable with enalapril. Urinary endothelin-1 excretion was greater in the diabetic rats than in the control rats, and was not substantially influenced by the agents. Enalapril, but not YM598, mildly lowered the blood pressure in the diabetic rats, indicating that blood pressure reduction is not involved in the major mechanism of the renoprotective effect of YM598 in OLETF rats. These data suggest that endothelin is involved in the progression of diabetic nephropathy in OLETF rats, and an endothelin type A antagonist is promising for the treatment of diabetic nephropathy.     

Mechanisms underlying diabetes enhancement of endothelin-1-induced contraction in rabbit basilar artery

The influence of alloxan-induced diabetes on the reactivity of rabbit basilar artery to endothelin-1 was examined. Endothelin-1 induced concentration-dependent contraction of basilar arteries that was higher in diabetic than in control rabbits. Endothelium removal produced a higher enhancement of the endothelin-1-induced contraction in control than in diabetic rabbits. N(G)-nitro-L-arginine (L-NOArg) enhanced the maximal contraction induced by endothelin-1 in control rabbits and potentiated this response in diabetic rabbits. Endothelin ETA receptor antagonist, cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123), inhibited endothelin-1-induced contraction in both rabbit groups. Endothelin ETB receptor antagonist, 2,6-Dimethylpiperidinecarbonyl-gamma-Methyl-Leu-Nin-(Methoxycarbonyl)-D-Trp-D-Nle (BQ-788), enhanced endothelin-1-induced contraction in control rabbits and decreased the potency of endothelin-1 in diabetic rabbits. Sodium nitroprusside-induced relaxation of basilar arteries was lower in diabetic than in control rabbits. These results suggest that mechanisms underlying rabbit basilar artery hyperreactivity to endothelin-1 include decreased endothelial modulation of endothelin-1-induced contraction, with impaired endothelial endothelin ETB receptor activity; decreased sensitivity to nitric oxide (NO) in vascular smooth muscle; and enhanced participation of muscular endothelin ETA and ETB receptors.     

Endothelin-1 (ET-1)-induced contraction in rat isolated trachea: involvement of ETA and ETB receptors and multiple signal transduction systems.

1. Quantitative autoradiographic, biochemical and functional studies were performed to investigate the endothelin receptor subtypes and signal transduction systems that mediate endothelin-1 (ET-1)-induced contraction in rat isolated tracheal smooth muscle. 2. Specific binding of 0.5 nM [125I]-ET-1 to tracheal smooth muscle was inhibited by at least 40% in the presence of either the ETA receptor selective ligand BQ-123 (1 microM) or the ETB receptor-selective ligand sarafotoxin S6c (30 nM), indicating the presence of both ETA and ETB receptors in this tissue. 3. ET-1 and sarafotoxin S6c were both potent spasmogens of rat isolated tracheal smooth muscle preparations. Sarafotoxin S6c-induced contractions were unaffected in the presence of the ETA receptor antagonist BQ-123 (10 microM), but were markedly attenuated in tissue previously exposed to 100 nM sarafotoxin S6c to induce ETB receptor desensitization. ET-1-induced contractions were, at most, only partially attenuated either by blocking the ETA receptor-effector system (with 10 microM BQ-123) or by desensitizing the ETB receptor-effector system with sarafotoxin S6c. However, ET-1-induced contractions were markedly attenuated by blocking both receptor-effector systems simultaneously. These findings suggest that ET-1 could induce contraction by stimulating either ETA or ETB receptors. 4. ET-1 (10 microM) induced a 7 fold increase in intracellular [3H]-inositol phosphate accumulation over basal levels in rat isolated tracheal smooth muscle. In contrast, sarafotoxin S6c (2.5 microM) increased intracellular [3H]-inositol phosphate accumulation by only 2 fold. ET-1-induced accumulation of [3H]-inositol phosphates was abolished by 10 microM BQ-123.(ABSTRACT TRUNCATED AT 250 WORDS)     

Paracrine renal endothelin system in rats with liver cirrhosis.

1. Liver cirrhosis was induced in rats by CCl4 administration. We analysed the expression of endothelin receptor subtypes in the renal cortex and medulla using Scatchard analysis and receptor autoradiography, and measured plasma as well as renal-tissue endothelin-1 concentrations using a specific radioimmunoassay. Furthermore, we analysed the effects of the non-selective (A/B) endothelin receptor antagonist, bosentan (6 and 100 mg kg-1 day-1) on mean arterial blood pressure, water and sodium excretion and glomerular filtration rate. 2. Our study revealed an overexpression of the endothelin B receptor (ETB) in the renal medulla of rats with liver cirrhosis (Cir: 2775 +/- 299 fmol mg-1; Con: 1695 +/- 255 fmol mg-1; n = 8; means +/- s.d., P < 0.01), whereas the density of ETB in the cortex and the endothelin A receptor (ETA) in the cortex and medulla were similar in both cirrhotic and control rats. Receptor autoradiography showed that the upregulation of medullary ETB in cirrhotic rats was due to an upregulation of ETB in the inner medullary collecting duct cells. 3. The tissue endothelin-1 concentrations were increased in the renal medulla of cirrhotic rats (Cir: 271 +/- 68 pg g-1wet wt.; Con: 153 +/- 36 pg g-1 wet wt., n = 8; means +/- s.d., P < 0.01). 4. The glomerular filtration rate was slightly decreased in cirrhotic rats but not altered after bosentan treatment in either cirrhotic or control rats. Bosentan decreased sodium excretion to a similar extent in both cirrhotic and control rats, whereas water excretion was significantly reduced by both dosages of bosentan in cirrhotic rats only (Cir + vehicle: 12.5 +/- 0.62 m day-1, Cir + 6 mg kg-1 day-1 bosentan: 8.6 +/- 1.0 ml day-1; Cir + 100 mg kg-1 day-1 bosentan: 7.4 +/- 0.6 ml day-1; n = 10; means +/- s.e.mean). 5. We therefore suggest that the upregulation of the medullary ETB in cirrhotic rats is involved in the regulation of water excretion in rats with CCl4-induced liver cirrhosis.     

Characterization of the binding of endothelin ETB selective ligands in human and rat heart.

1. We determined competition binding characteristics of endothelin ETB receptor selective ligands in human left ventricle and compared these values to those obtained with rat left ventricle. Sarafotoxin S6c, ET-3, BQ788 and IRL2500 competed against [125I]-PD151242 (ETA selective radioligand) with low affinity in human left ventricle, confirming the ETB selectivity of these compounds. 2. ET-3 competed with moderate selectivity for ETB over ETA receptors in human left ventricle and with slightly higher selectivity in rat left ventricle (460 and 1,400 fold, respectively). There was a small difference in the affinity of ETA receptors for ET-3 (KD ETA in human left ventricle = 0.07 +/- 0.02 microM; KD ETA in rat left ventricle = 0.27 +/- 0.08 microM; P = 0.05) but no difference in the affinity of ETB receptors for this ligand (KD ETB in human left ventricle = 0.15 +/- 0.06 nM; KD ETB in rat left ventricle = 0.19 +/- 0.03 nM). 3. The selectivity of sarafotoxin S6c for ETB over ETA receptors in human left ventricle was 5,900 fold compared with 59,400 fold in rat left ventricle. The affinity of ETA receptors for sarafotoxin S6c was higher in human than in rat left ventricle (KD ETA = 2.00 +/- 0.20 microM and 3.50 +/- 0.26 microM, respectively; P = 0.03), while the affinity of ETB receptors for this ligand was higher in rat left ventricle (KD ETB = 0.06 +/- 0.02 nM) than in human left ventricle (KD ETB = 0.34 +/- 0.13 nM) (P = 0.02). The affinity of ETB receptors for sarafotoxin S6c in rat left ventricle determined in the absence or presence of GTP was the same indicating that differing affinity states of ETB receptors in human and rat left ventricle do not account for the variation observed between species. 4. There was no difference in the affinity of ETA receptors for BQ788 (KD ETA = 1.01 +/- 0.20 microM and KD ETA = 1.39 +/- 0.35 microM) or for the novel ETB selective antagonist. IRL2500 (KD ETA = 30.0 +/- 20.8 microM and KD ETA = 55.6 +/- 9.93 microM) in human and rat left ventricle, respectively. ETB receptors had a significantly higher affinity for BQ788 (KD ETB = 9.8 +/- 1.3 nM and KD ETB = 31.0 +/- 5.4 nM; P = 0.02) and IRL2500 (KD ETB = 78.2 +/- 9.7 nM and KD ETB = 300.0 +/- 75.1 nM; P = 0.03) in human and rat left ventricle, respectively. The synthetically synthesized ETB selective antagonist RES-701-1 (0.1 -3 microM) failed to inhibit [125I]-ET-1 binding in either tissue. 5. In conclusion, we have compared equilibrium dissociation constants for a number of ETB selective compounds in human and rat heart. The affinity of ETB receptors for sarafotoxin S6c, BQ788 and IRL2500 differed in human and rat left ventricle. No difference in affinity was detected for ET-3 binding at ETB receptors. Sarafotoxin S6c binding was unaffected by GTP indicating that the different receptor affinities in human and rat heart cannot be explained by differing ETB receptor affinity states. This study highlights the need to consider differences in binding characteristics that may arise from the use of tissues obtained from different species.     

Effects of selective endothelin ET(A) receptor antagonists on endothelin-1-induced potentiation of cancer pain

In some diseases in which endothelin-1 production increases, e.g. prostate cancer, endothelin-1 is considered to be involved in the generation of pain. In the present study, we investigated the effects of a selective endothelin ET(A) receptor antagonist, (E)-N-[6-methoxy-5-(2-methoxyphenoxy)[2,2'-bipyrimidin]-4-yl]-2-phenylethenesulfonamide monopotassium salt (YM598), on the nociception potentiated by endothelin-1 in a cancer inoculation-induced pain model in mice, induced by inoculation of the androgen-independent human prostate cancer cell line PPC-1 into the hind paws of severe combined immunodeficiency (SCID) mice. No pain responses were observed in the sham-operated mice, whereas monophasic pain responses were observed in the PPC-1-inoculated mice. Endothelin-1 (1 to 10 pmol/paw) but not sarafotoxin S6c potentiated the pain response in prostate cancer-inoculated mice. Both YM598 and atrasentan (0.3 to 3 mg/kg, p.o.) significantly inhibited the endothelin-1 (10 pmol/paw)-induced potentiation of nociception in a dose-dependent manner. These results suggest that selective endothelin ET(A) receptor antagonists might relieve pain in patients with various diseases in which endothelin-1 production is increased, e.g. prostate cancer.     

Self-limiting enhancement by nitric oxide of oxygen free radical-induced endothelial cell injury: evidence against the dual action of NO as hydroxyl radical donor/scavenger.

1. In this study we used ligand binding techniques to determine the affinity and selectivity of endothelin receptor agonists and antagonists in human left ventricle which expresses both ETA and ETB receptors, and compared these results with cardiovascular tissues from rat and porcine hearts. 2. The linear tripeptide antagonist, FR139317 competed for [125I]-ET-1 binding to human left ventricle with over 200,000 fold selectivity for the ETA receptor (KD ETA = 1.20 +/- 0.28 nM, KDETB = 287 +/- 93 microM). The ETA-selective non-peptide antagonist, 50235, competed with lower affinity and selectivity (KDETA = 162 +/- 61 nM, KDETB = 171 +/- 42 microM) in this tissue. BQ123 and FR139317 also showed high selectivity (greater than 20,000 fold) and affinity in rat (BQ123: KDETA = 1.18 +/- 0.16 nM, KDETB = 1370 +/- 1150 microM; FR139317: KDETA = 2.28 +/- 0.30 nM, KDETB = 292 +/- 114 microM) and pig heart (BQ123: KDETA = 0.52 +/- 0.05 nM, KDETB = 70.4 +/- 4.0 microM; FR139317: KDETA = 2.17 +/- 0.51 nM, KDETB = 47.1 +/- 5.7 microM) (n > or = 3 individuals +/- s.e.mean). 3. Although BQ3020 competed with over 1000 fold selectivity for the ETB subtype in human heart (KDETB = 1.38 +/- 0.72 nM, KDETA = 2.04 +/- 0.21 microM) the peptide inhibited only the binding of [125I]-ET-1 at concentrations greater than 100 nM in rat and porcine heart. This is in contrast to the data from the ETA-selective antagonists which indicated the presence of ETB sites in these tissues from animal hearts. 4. The peptide antagonist, BQ788, had a low, micromolar affinity (KD = 1.98 +/- 0.13 microM) using human left ventricle and no significant selectivity for the human ETB-subtype in this tissue. 5. The non-peptide ET antagonists, Ro462005 (KD = 50.3 +/- 9.5 microM) and bosentan (Ro470203; KD = 77.9 +/- 7.9 nM) competed monophasically for [125I]-ET-1 binding sites in human left ventricle. 6. The results show that the ETA antagonists, BQ123 and FR139317, are highly selective for ETA receptors in all cardiac tissues tested, whereas BQ788 has a low affinity and no selectivity in this human tissue. Further we showed that there are species differences in the binding of BQ3020 to the ETB receptors in the hearts derived from human, rat and pig.     

Antihypertensive effect of an endothelin receptor antagonist in DOCA-salt spontaneously hypertensive rats.

1. Endothelin-1 gene expression is enhanced in aorta and mesenteric arteries, and possibly other vessels, of deoxycorticosterone acetate (DOCA)-salt hypertensive rats but is normal or reduced in spontaneously hypertensive rats (SHR). Bosentan, a mixed ETA/ETB endothelin receptor antagonist, blunts the development of elevated blood pressure of DOCA-salt hypertensive rats but not in SHR. In this study we investigated whether treatment of DOCA-salt SHR with bosentan would result in blunted rise in blood pressure. 2. SHR, aged 13 weeks, were implanted with silastic containing DOCA and offered 1% saline to drink. Systolic blood pressure was measured by the tail-cuff method. Endothelin-1 mRNA abundance in aorta and mesenteric arteries was measured by Northern blot analysis. Content of immunoreactive endothelin in blood vessels was measured by radioimmunoassay. 3. Systolic blood pressure rose less in bosentan-treated DOCA-salt SHR (to 223 +/- 2 mmHg) in comparison to the untreated rats (241 +/- 1), a small but significant difference (P < 0.001). However, blood pressure of bosentan-treated DOCA-salt SHR was still higher than in age-matched SHR. Endothelin-1 mRNA abundance and content of immunoreactive endothelin were increased in the aorta and the mesenteric arterial bed of DOCA-salt SHR, and were unaffected by treatment with bosentan. 4. These data support the hypothesis of a role of endothelin-1 in blood pressure elevation in this hypertensive model with malignant hypertension. They also support the hypothesis that an antihypertensive effect of the mixed ETA/ETB endothelin receptor antagonist, bosentan, is found when experimental hypertensive animals exhibit enhanced endothelin-1 gene expression in blood vessels.     

The orally active nonpeptide selective endothelin ETA receptor antagonist YM598 prevents and reverses the development of pulmonary hypertension in monocrotaline-treated rats

We investigated the preventive and therapeutic effects of the selective endothelin ETA receptor antagonist potassium(E)-N-[6-methoxy-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl)pyrimidin-4-yl]-2-phenylenthenesulfonamidate (YM598) on the development of pulmonary hypertension in monocrotaline-induced pulmonary hypertensive and hypoxemic rats. In the prevention study, oral administration of YM598 (0.1 and 1 mg/kg) or bosentan (30 mg/kg) for 4 weeks was started on the day following monocrotaline (60 mg/kg) injection. In the therapeutic study, oral administration of YM598 (0.1, 0.3 and 1 mg/kg) for 2 weeks was started 3 weeks after monocrotaline injection. In the prevention study, a marked increase in pulmonary arterial pressure and right ventricular hypertrophy, a decrease in right cardiac function and hypoxemia were observed. Histopathological examination indicated the presence of pulmonary remodeling, including medial wall thickening of the pulmonary microvasculature and alveolar disorders. YM598 suppressed the increase in pulmonary arterial pressure, right ventricular hypertrophy and systemic congestion, and improved the hypoxemia, but bosentan had only modest effects. Histopathological disorders were also ameliorated by YM598. In the therapeutic study, YM598 also ameliorated the pulmonary hypertension and hypoxemia in monocrotaline-treated rats. These results suggest that YM598 effectively prevented and reversed the development of pulmonary hypertension, and reduced the pulmonary vascular remodeling and parenchymal injury in monocrotaline-treated rats. YM598 also improved hypoxemia which accompanied with the severe pulmonary hypertension in these rats.     

Acute pro-inflammatory actions of endothelin-1 in the guinea-pig lung: involvement of ETA and ETB receptors.

1. Although recent observations suggest that endothelin-1 (ET-1) may play a role in the pathogenesis of asthma, to date little is known about the effects of ET-1 on parameters other than bronchoconstriction. The objectives of the present experiments were to study whether intravenously administered ET-1 could exert pro-inflammatory actions in the guinea-pig lung and to assess the involvement of endothelin ETA and ETB receptors in these events by using the ETA receptor-selective antagonist, FR 139317, the novel ETA/ETB receptor antagonist, bosentan and the ETB receptor-selective agonist, IRL 1620. 2. Bolus i.v. injection of ET-1 (0.1-1 nmol kg-1) to anaesthetized guinea-pigs evoked dose-dependent increases in mean arterial blood pressure which lasted for 6-12 min. This was accompanied by a dose-dependent haemoconcentration (8-15% plasma volume losses) and increases (up to 546%) in albumin extravasation in the trachea, upper and lower bronchi, but not in the pulmonary parenchyma. Qualitatively similar changes were observed following i.v. injection of the ETB receptor agonist, IRL 1620 (0.3 and 1 nmol kg-1), although IRL 1620 appeared to be about 3 times less potent than ET-1. The ETA receptor-selective antagonist, FR 139317 (2.5 mg kg-1) inhibited the ET-1 (1 nmol kg-1)-induced pressor response, haemoconcentration and albumin extravasation by 75, 77 and 60-70%, respectively, whereas it did not attenuate IRL 1620 (1 nmol kg-1)-induced changes. The ETA/ETB receptor antagonist, bosentan (10 mg kg-1) almost completely inhibited the pressor, haemoconcentration and permeability effects of both ET-1 and IRL 1620. 3. ET-1, but not IRL 1620 (0.1-1 nmol kg-1), produced a dose-dependent neutropenia with relative lymphocytosis and monocytosis, but did not induce influx of neutrophil granulocytes into pulmonary tissues or the bronchoalveolar space. ET-1 (1 nmol kg-1)-induced neutropenia was prevented by pretreatment of the animals with FR 139317 (2.5 mg kg-1), bosentan (10 mg kg-1) or adrenaline (90 nmol kg-1), indicating that ET-1 caused intravascular sequestration of neutrophil granulocytes. 4. ET-1 or IRL 1620 (10(-10)-10(-6) M) alone did not activate alveolar macrophages in vitro, whereas at a concentration of 10(-8) M, ET-1, but not IRL 1620, markedly potentiated superoxide production in response to f-Met-Leu-Phe (10(-9)-10(-7) M) and platelet-activating factor (PAF, 10(-9)-10(-7) M), but not to phorbol 12-myristate 13-acetate (10(-9) M).(ABSTRACT TRUNCATED AT 400 WORDS)     

Block of endothelin-1-induced release of thromboxane A2 from the guinea pig lung and nitric oxide from the rabbit kidney by a selective ETB receptor antagonist, BQ-788.

1. The present study characterizes the receptors responsible for endothelin-1-induced release of thromboxane A2 from the guinea pig lung and of endothelium-derived nitric oxide from the rabbit perfused kidney, by the use of the selective ETA receptor antagonist, BQ-123, and a novel selective ETB receptor antagonist, BQ-788. 2. In the guinea pig perfused lung, endothelin-1 (ET-1) (5 nM) induced a marked increase of thromboxane A2 which was reduced by 17 +/- 5.0, 70 +/- 1.0 and 93 +/- 1.2% by BQ-788 infused at concentrations of 1, 5 and 10 nM respectively. In contrast, BQ-123 (0.1 and 1.0 microM) had little or no effect on the ET-1-induced release of thromboxane A2. 3. In the same perfused model, the selective ETB agonist, IRL 1620 (50 nM), stimulated the release of thromboxane A2, but not prostacyclin. The eicosanoid-releasing properties of IRL 1620 were abolished by BQ-788 at 10 nM, yet were unaffected by BQ-123 (1 microM). 4. In the rabbit perfused kidney, BQ-788 (10 nM) potentiated the increase of perfusion pressure induced by endothelin-1 (1, 5 and 10 nM) by approximately 90%, but not that induced by angiotensin II (1 microM). Furthermore, the selective ETB receptor antagonist did not reduce the release of prostacyclin triggered by either peptide. 5. In another series of experiments, pretreatment of the perfused kidney with a nitric oxide synthase inhibitor, L-NAME (100 microM), potentiated the pressor responses to both endothelin-1 and angiotensin II. Under L-NAME treatment, BQ-788 did not further potentiate the pressor response to endothelin-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of the ETA/ETB receptor antagonist, bosentan on endothelin-1-induced myocardial ischaemia and oedema in the rat.

1. The purpose of this study were to assess the role of ETB receptors in mediating endothelin-1 (ET-1)-induced myocardial ischaemia and oedema in rats and to study the inhibitory action of the novel nonpeptide ETA/ETB receptor antagonist, bosentan on these actions of ET-1. 2. Intravenous bolus injection of ET-1 (1 nmol kg-1) into anaesthetized rats produced marked ST segment elevation of the electrocardiogram without causing arrhythmias. ST segment elevation developed within 30-50 s and persisted for at least 30 min following injection of the peptide. 3. Pretreatment of the animals with bosentan (10 mg kg-1, i.v.) inhibited on average by 96% the ST segment elevation elicited by ET-1 (1 nmol kg-1) compared to the 82% inhibition observed with the ETA receptor-selective antagonist, FR 139317 (2.5 mg kg-1, i.v.). 4. Bolus injection of ET-1 (1 nmol kg-1, i.v.) to conscious chronically catheterized rats evoked a transient depressor response followed by a prolonged pressor effect. Corresponding to changes in blood pressure, a transient tachycardia and a sustained bradycardia were observed. ET-1 (1 nmol kg-1) enhanced albumin extravasation by 119 and 93% in the left ventricle and right atrium, respectively, as measured by the local extravascualr accumulation of Evans blue dye. 5. Pretreatment of the animals with bosentan (10 mg kg-1) inhibited by 71 and 90% the depressor and pressor actions of ET-1 (1 nmol kg-1) and the accompanying tachycardia and bradycardia, respectively. FR 139317 (2.5 mg kg-1) attenuated the pressor response to ET-1 and accompanying bradycardia by 75%, without affecting the depressor action and accompanying tachycardia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacology of A-216546: a highly selective antagonist for endothelin ET(A) receptor

Endothelins, 21-amino acid peptides involved in the pathogenesis of various diseases, bind to endothelin ET(A) and ET(B) receptors to initiate their effects. Here, we characterize the pharmacology of A-216546 ([2S-(2,2-dimethylpentyl)-4S-(7-methoxy-1,3-benzodioxol-5-yl )-1-(N,N-di(n-butyl) aminocarbonylmethyl)-pyrrolidine-3R-carboxylic acid), a potent antagonist with > 25,000-fold selectivity for the endothelin ET(A) receptor. A-216546 inhibited [125I]endothelin-1 binding to cloned human endothelin ET(A) and ET(B) receptors competitively with Ki of 0.46 and 13,000 nM, and blocked endothelin-1-induced arachidonic acid release and phosphatidylinositol hydrolysis with IC50 of 0.59 and 3 nM, respectively. In isolated vessels, A-216546 inhibited endothelin ET(A) receptor-mediated endothelin-1-induced vasoconstriction, and endothelin ET(B) receptor-mediated sarafotoxin 6c-induced vasoconstriction with pA2 of 8.29 and 4.57, respectively. A-216546 was orally available in rat, dog and monkey. In vivo, A-216546 dose-dependently blocked endothelin-1-induced pressor response in conscious rats. Maximal inhibition remained constant for at least 8 h after dosing. In conclusion, A-216546 is a potent, highly endothelin ET(A) receptor-selective and orally available antagonist, and will be useful for treating endothelin-1-mediated diseases.     

In vitro and in vivo effects of endothelin-1 and YM598, a selective endothelin ET A receptor antagonist, on the lower urinary tract

We investigated the contractile response of the lower urinary tract to endothelin-1 in vitro (rabbits) and in vivo (dogs). We also assessed the effects of a selective endothelin ET_A receptor antagonist, (E)-N-[6-methoxy-5-(2-methoxyphenoxy)[2, 2′-bipyrimidin]-4-yl]-2-phenylethenesulfonamide monopotassium salt (YM598), on endothelin-1-induced contractile responses. In the in vitro study, endothelin-1 induced contractile responses in isolated rabbit bladder base, urethra, and prostate tissues. YM598 (10^− 7–10^− 5 M) antagonized these endothelin-1-induced contractile responses without affecting the maximal responses. In the in vivo study, endothelin-1 induced the elevation of non-prostatic urethral pressure as well as prostatic urethral pressure even in the presence of tamsulosin (10 μg/kg, i.v.) in anesthetized male dogs. YM598 (0.1–3 mg/kg, i.v.) inhibited these endothelin-1-induced contractile responses in a dose-dependent fashion. These results suggest that endothelin ET_A receptors play an important role in the lower urinary tract contraction, and that the selective endothelin ET_A receptor antagonist YM598 has ameliorating effects on various urinary dysfunctions, including benign prostatic hyperplasia.     

Participation of endogenous endothelin and ETA receptor in premicturition contractions in rats with bladder outlet obstruction

A relationship between endogenous endothelins and bladder overactivity has recently been suggested, but the related endothelin receptor subtype has not been identified. Here, to evaluate the involvement of endothelin-1 and its receptors in bladder overactivity, we investigated endothelin-1 levels and the expression of its receptors in the bladder of rats with bladder outlet obstruction (BOO), a model for bladder overactivity. We also investigated the effects of a selective endothelin ET_A receptor antagonist, (E)-N-[6-methoxy-5-(2-methoxyphenoxy)[2,2′-bipyrimidin]-4-yl]-2-phenylethenesulfonamide monopotassium salt (YM598), on bladder functions in conscious BOO rats. Partial obstruction of the urethra led to a progressive increase in bladder weight from weeks 1 to 6. Binding assays performed using plasma membranes prepared from these bladders to estimate endothelin receptor density from the maximum [¹²⁵I]endothelin-1 binding showed increased endothelin receptor density (about double) at 1, 2, and 6 weeks after the operation in the BOO bladder. The densities of endothelin ET_A receptors in the bladder of sham-operated and BOO rats at 2 weeks after operation were about 3.5 and 5 times those of endothelin ET_B receptors respectively. Furthermore, the endothelin-1 level was also increased in the BOO bladder. Two weeks after operation, BOO rats showed an increase in maximum bladder capacity and micturition volume and the generation of premicturition contractions. The frequency of premicturition contractions was dose-dependently reduced by YM598 (0.1–3 mg/kg, i.v.) without any effect on other voiding parameters in BOO rats. These data suggest that endothelin-1 and endothelin ET_A receptors might be involved in the generation of premicturition contractions in BOO rats, and that endothelin ET_A receptor antagonists such as YM598 may have ameliorating effects in patients with bladder overactivity associated with BOO.     

Synergistic inhibition by BQ-123 and BQ-788 of endothelin-1-induced contractions of the rabbit pulmonary artery.

In the rabbit isolated pulmonary artery, neither the ETA receptor antagonist, BQ-123 (10 microM), nor the ETB receptor antagonist, BQ-788 (10 microM), inhibited the contractions induced by 1 nM endothelin-1 (ET-1). However, the combination of BQ-123 and BQ-788 completely inhibited the ET-1-induced contraction. In contrast, the ETB-selective agonist, sarafotoxin S6c (1 nM)-induced contraction was completely inhibited by BQ-788 but not by BQ-123. In receptor binding assays, [125I]-ET-1 specific binding to pulmonary arterial membranes was inhibited by BQ-123 (1 microM) by approximately 20% and additive treatment with BQ-788 (1 microM) completely inhibited the BQ-123-resistant component of [125I]-ET-1 specific binding. The present study demonstrates synergistic inhibition by BQ-123 and BQ-788 of ET-1-induced contraction of the rabbit pulmonary artery and the coexistence of ETA and ETB receptors, suggesting that the activation of either only ETA or only ETB receptors may be sufficient to cause complete vasoconstriction. Therefore, blockade of both receptor subtypes would be necessary for the inhibition of some ETA/ETB composite types of responses.