Selective Endothelin ET Research Articles

Acyl glucuronidation and glucosidation of a new and selective endothelin ET(A) receptor antagonist in human liver microsomes.

Compound A [(+)-(5S,6R,7R)-2-isopropylamino-7-[4-methoxy-2-((2R)-3-methoxy-2-methylpropyl)-5-(3,4-methylenedioxyphenyl) cyclopenteno [1,2-b] pyridine 6-carboxylic acid] is a new and selective endothelin ET(A) receptor antagonist. It underwent significant acyl glucuronidation and acyl glucosidation in human liver microsomes supplemented with UDP-glucuronic acid (UDPGA) and UDP-glucose (UDPG). These two conjugations were observed in a panel of human liver microsomal samples (n = 16) that gave rise to varying activities but with no significant correlation with each other in the native and activator-treated microsomal preparations (r(2) <or= 0.4, p > 0.05). The lack of correlation may be explained by the involvement of multiple UDP-glucuronosyltransferases (UGTs; UGT1A1, 1A3, 1A9, 2B7 and 2B15) in the glucuronidation but essentially solely UGT2B7 in the glucosidation. Both reactions conformed to monophasic Michaelis-Menten kinetics in human liver microsomes. The glucuronidation reaction exhibited apparent K(m) values (mean +/- S.E.) for compound A and UDPGA of 8.4 +/- 0.6 and 605 +/- 35 microM, respectively, whereas the values for the glucosidation reaction were 10.2 +/- 1.5 and 670 +/- 120 microM, respectively. In both pooled human liver microsomes and expressed UGT2B7, UDPG and UDPGA competitively inhibited their counterpart conjugations with K(i) values close to their K(m) values, indicating a comparable affinity of the enzyme toward these two nucleotide sugars. We herein report a drug acyl glucoside formed in human liver microsomes at a considerable turnover rate and provide the evidence for a UGT isoform (UGT2B7) capable of transferring both glucuronic acid and glucose from UDPGA and UDPG to an aglycone.

Analysis of the time course for organ culture-induced endothelin ET B receptor upregulation in rat mesenteric arteries

In contrast to the constitutively expressed endothelin ET A receptor, the distribution of endothelin ET B receptors is more variable. The aim of the present study was to investigate the kinetics of organ culture-induced upregulation of contractile endothelin ET B receptors in rat mesenteric arteries at both mRNA and functional levels. Assessment of mRNA expression revealed low levels of endothelin ET B receptor mRNA relative to endothelin ET A receptor mRNA after 3 h of culture, which gradually increased to reach a plateau level after 24 h. Correspondingly, vessels cultured for 3 h showed a negligible contractile response the selective endothelin ET B receptor agonist sarafotoxin 6c. Subsequently, the contractile response to sarafotoxin 6c was successively increased during organ culture until 24 h and, thereafter, a further increase in potency was seen after 48 h. These results demonstrate a rapid induction of transcription within less than 7 h followed by an increase in the response to receptor stimulation.

Role of endothelin ET B receptors in the renal hemodynamic and excretory responses to big endothelin-1

We determined the role of endothelin ET B receptor in the renal hemodynamic and excretory responses to big endothelin-1, using A-192621, a selective endothelin ET B receptor antagonist and the spotting-lethal (sl) rat, which carries a naturally occurring deletion in the endothelin ET B receptor gene. An intravenous injection of big endothelin-1 produced a hypertensive effect, which is greater in wild-type (+/+) rats pretreated with A-192621 and in homozygous ( sl/ sl) rats. Big endothelin-1 markedly increased urine flow, urinary excretion of sodium and fractional excretion of sodium in wild-type rats treated with the vehicle. These excretory responses to big endothelin-1 were markedly reduced by pharmacological endothelin ET B receptor blockade. On the other hand, big endothelin-1 injection to the endothelin ET B receptor-deficient homozygous animals resulted in a small diuretic effect. When renal perfusion pressure was protected from big endothelin-1-induced hypertension by an aortic clamp, the excretory responses in vehicle-treated wild-type rats were markedly attenuated. In homozygous or A-192621-treated wild-type rats, there was a small but significant decreasing effect in urine flow. In addition, big endothelin-1 significantly elevated nitric oxide (NO) metabolite production in the kidney of wild-type rats but not in the homozygous rats. We suggest that the diuretic and natriuretic responses to big endothelin-1 consist of pressure-dependent and pressure-independent effects and that the increased NO production via the activation of endothelin ET B receptors in the kidney is closely related to the big endothelin-1-induced excretory responses.

Renal protective effect of YM598, a selective endothelin ET A receptor antagonist, against diabetic nephropathy in OLETF rats

We have investigated the effect of potassium ( E)- N-[6-methoxy-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl) pyrimidin-4-yl]-2-phenylenthenesulfonamidate (YM598), a selective endothelin ET A receptor antagonist, on renal function in Otsuka Long–Evans Tokushima Fatty (OLETF) rats, an animal model of type II diabetes. YM598 (0.1 or 1 mg kg −1), enalapril (5 mg kg −1), an angiotensin-converting enzyme inhibitor, or vehicle was administered once daily by gastric gavage to 22-week-old male Otsuka Long–Evans Tokushima Fatty rats for 32 weeks. Enalapril but not YM598 mildly lowered blood pressure in the diabetic rats. YM598 blunted the development of albuminuria in a dose-dependent manner. High dose of YM598 reduced albuminuria comparable to enalapril. Urinary endothelin-1 excretion was greater in the diabetic than in the control rats, and was not substantially influenced by the agents. These data suggest that endothelin is involved in the progression of diabetic nephropathy in Otsuka Long–Evans Tokushima Fatty rats, and an endothelin ET A receptor antagonist may be useful for the treatment of diabetic nephropathy.

Cyclooxygenase inhibitors attenuate endothelin ET B receptor-mediated contraction in human temporal artery

It is well documented that endothelin ET B receptor-mediated contraction develops in artery segments incubated in culture and that the reaction is augmented by proinflammatory cytokines, but little is known of the mechanisms involved. Segments of human temporal artery were incubated in organ culture for 2 days in the absence or presence of interleukin-1β (IL-1β), with or without nonsteroidal anti-inflammatory drugs, glucocorticoids or a nitric oxide synthase inhibitor. Thereafter, contractions were induced by the selective endothelin ET B receptor agonist, sarafotoxin S6c. Acetylsalicylic acid, indomethacin, nimesulide and rofecoxib were all effective in eliminating the increase in endothelin ET B receptor-mediated contraction induced by interleukin-1β, but only indomethacin and rofecoxib significantly reduced the spontaneous development of this reaction in cultured arteries. Dexamethasone and methylprednisolone augmented the reaction, and the nitric oxide synthase inhibitor had no effect. The results clearly indicate a role for cyclooxygenase, most likely cyclooxygenase-2, in endothelin ET B receptor-mediated contraction in this preparation.

Influence of indomethacin on effects of endothelin-1 on guinea pig isolated rings of common bile duct and sphincter of Oddi

The effects of endothelin-1 on motility of guinea pig extra-hepatic biliary tract portions were studied. Endothelin-1 (≤100 nM) failed to contract rings of hepatic, cystic, proximal or distal common bile ducts, or choledochal or papillary halves of sphincter of Oddi. At 100 nM, endothelin-1 or sarafotoxin S6c (selective endothelin ET B receptor agonist) inhibited contractions of choledochal (but not papillary) sphincter of Oddi to carbachol (1 μM) by 63±5 and 45±9%, respectively. In distal common bile duct, indomethacin (5.6 μM) unmasked potent contractile effects of endothelin-1 [EC 50 7.8 (5.5–11.1) nM; E MAX 80±6% of response to 80 mM KCl] and enhanced the contractile potency of carbachol (585-fold at EC 50 level), but not cholecystokinin C-terminal octapeptide. Inhibition of cholinergic responsiveness of the choledochal sphincter of Oddi by endothelin-1 was reduced by BQ-123 (1 μM; endothelin ET A receptor antagonist; cyclo[DTrp-DAsp-Pro-DVal-Leu]) and abolished by either BQ-123 plus BQ-788 (1 μM; endothelin ET B receptor antagonist; N- cis-2,6-dimethylpiperidinocarbonyl- l-γ-methylleucyl- d-1-methoxycarboyl- d-norleucine) or indomethacin. Thus, eicosanoids of the cyclo-oxygenase pathway (i.e. prostanoids) suppress endothelin-1-induced contractions of distal common bile duct and mediate endothelin ET A and ET B receptor-dependent inhibition of cholinergic responsiveness of the choledochal portion of the sphincter of Oddi.

Effects of the novel selective endothelin ET(A) receptor antagonist, SB 234551, on the cardiovascular responses to endotoxaemia in conscious rats.

1. In conscious, freely moving, male, Long Evans rats, regional haemodynamic responses to exogenous endothelin-1 (ET-1; 25, 50 and 250 pmol kg(-1) i.v.) were assessed in the presence of vehicle, or the selective ET(A)-receptor antagonist, SB 234551. On the following day, the effects of SB 234551 on the haemodynamic responses to lipopolysaccharide (LPS) infusion (150 microg kg(-1) h(-1), i.v.) were determined. 2. When SB 234551 was given i.v. by primed infusion at a dose of 0.3 mg kg(-1) bolus, 0.3 mg kg(-1) h(-1) infusion, it caused selective inhibition of the vasoconstrictor effects of exogenous endothelin-1, whereas at a dose of 1 mg kg(-1), 1 mg kg(-1) h(-1), SB 234551 also inhibited some of the vasodilator effects of endothelin-1. 3. Infusion of LPS, in the presence of vehicle, caused a short-lived (1 - 2 h) hypotension, tachycardia, and vasodilatation in renal, superior mesenteric and hindquarters vascular beds. Thereafter, blood pressure, heart rate and mesenteric vascular conductance returned to baseline values, but renal vasodilatation persisted, and there was vasoconstriction in the hindquarters. 4. In the presence of SB 234551 (0.3 mg kg(-1), 0.3 mg kg(-1) h(-1)), the early (1 - 2 h) cardiovascular responses to LPS infusion were unaffected, but the subsequent recovery of mean arterial blood pressure was impaired, due to developing vasodilatation in the mesenteric and, to a lesser extent, hindquarters, vascular beds. SB 234551 had no effect on the renal haemodynamic responses to LPS infusion. 5. The results confirm an important, regionally-selective, vasoconstrictor role for endogenous endothelin in this model of endotoxaemia.

Chelerythrine and genistein inhibit the endothelin-1-induced increase in myofilament Ca 2+ sensitivity in rabbit ventricular myocytes

We performed experiments to elucidate the cellular mechanism for the biphasic inotropic response to endothelin-1 of single rabbit ventricular myocytes loaded with a fluorescent dye, acetoxymethylester of indo-1. Endothelin-1 at 10 nM elicited a biphasic inotropic effect: a transient decrease in cell shortening and Ca 2+ transients followed by an increase in cell shortening without significant elevation of peak Ca 2+ transients. The selective endothelin ET A receptor antagonist FR139317 (2( R)-[2( R)-[2( S)-[(1-hexahydro-1 H-azepinyl)]carbonyl]amino-4-methylpentanoyl]amino-3-[3-(1-methyl-1 H-indolyl)propionyl]amino-3-(2-pyridyl)propionic acid) at 1 μM abolished the biphasic effect of endothelin-1 on cell shortening and Ca 2+ transients. The selective protein kinase C inhibitor chelerythrine at 1 μM and the tyrosine kinase inhibitor genistein at 5 μM inhibited the endothelin-1-induced increase in cell shortening without significantly affecting Ca 2+ transients and the transient decrease in cell shortening and Ca 2+ transients. The present results indicate that both protein kinase C and tyrosine kinase may contribute to the increase in myofilament Ca 2+ sensitivity induced by endothelin-1, whereas the decrease in Ca 2+ transients induced by endothelin-1 may be mediated by a signalling pathway different from that involved in the increase in cardiac contractility in rabbit ventricular myocytes.

Cytokine enhancement of endothelin ET B receptor-mediated contraction in human temporal artery

Segments of human temporal artery were incubated in organ culture for 2 days in the absence or presence of cytokines. Thereafter, contractions were induced by the selective endothelin ET B receptor agonist sarafotoxin S6c, a peptide that does not induce contraction in fresh human temporal artery. Interleukin-1β was most potent in increasing the sarafotoxin-induced contraction in cultured segments. Tumour necrosis factor (TNF)-α increased the magnitude of contraction to a similar degree, but at a higher dose. A significant increase was also induced by interferon-γ, but not by interleukin-6 at the concentrations used. The results suggest that endothelin ET B receptor-mediated contraction can be enhanced by pro-inflammatory cytokines in a concentration-dependent manner, and this may have relevance for pathophysiological conditions where inflammation and vasoactivity are important.

The contractile effects of endothelins on the smooth muscle of the rat prostate gland

Endothelin-1 elicited tonic contractions of rat prostatic smooth muscle that were unaffected by prazosin (0.5 μM), tetrodotoxin (1 μM) or guanethidine (10 μM). The rank order of potency of the endothelin isopeptides was endothelin-1>endothelin-2≥endothelin-3. Sarafotoxin S6B was approximately equipotent with endothelin-1 in eliciting tonic contractions, but neither of the selective endothelin ET B receptor-agonists, sarafotoxin S6C (0.1 nM–0.3 μM) and BQ3020 (Ac-[Ala 11,15]endothelin-1(6–21); 0.1 nM–0.3 μM), affected prostatic smooth muscle tone. The selective endothelin ET A receptor antagonist, BQ123 (cyclo( d-Asp- l-Pro- d-Val- l-Leu- d-Trp; 1 μM), attenuated responses to endothelin-1, -2 and -3, while the non-selective endothelin receptor antagonist bosentan (1 μM) and the selective endothelin ET B receptor antagonist BQ788, (Dmpc-γ-MeLeu 9- d-Trp(l-CO 2CH 3)- d-Nle-OH; 1 μM) attenuated responses to endothelin-3 only. Contractions induced by exogenous administration of noradrenaline were unaffected by preincubation of tissues in BQ123 (1 μM) indicating the selectivity of this antagonist. These data suggest that endothelins mediate contractions of the rat prostate by action at endothelin ET A receptors.

Endothelin receptor subtype antagonist activity of S-0139 in various isolated rabbit and canine arteries

Vascular responses to endothelin peptides have been proposed to be mainly mediated via subtypes of the endothelin receptor, endothelin ET A1, endothelin ET B1, and endothelin ET B2. The antagonist activity of 27- O-3-[2-(3-carboxy-acryloylamino)-5-hydroxyphenyl]acryloyloxy myricerone, sodium salt (S-0139) at these endothelin receptor subtypes was evaluated using isolated rabbit femoral, pulmonary, and mesenteric arteries. S-0139 competitively antagonized the endothelin-1-induced contraction mediated by the endothelin ET A1 receptor in endothelium-denuded rabbit femoral arteries with a p A 2 value of 8.6±0.1. Endothelin ET B2 receptor-mediated contraction induced by sarafotoxin S6c in endothelium-denuded rabbit pulmonary arteries was also inhibited by S-0139 with a p A 2 value of 5.6±0.1. The p A 2 value of S-0139 for the endothelin ET B1 receptor, evaluated from the endothelin-3-induced relaxant response in endothelium-intact rabbit mesenteric arteries, was 6.2±0.2. In isolated canine basilar, coronary, mesenteric and renal arteries, endothelin-1 caused concentration-dependent contractions with EC 50 values of 0.49±0.07, 0.61±0.25, 0.92±0.21 and 1.18±0.24 nM, respectively. S-0139 antagonized the endothelin-1-induced contraction in these arteries with p A 2 values of 8.0±0.1, 7.6±0.2, 7.6±0.2 and 7.6±0.1, respectively. These results suggest that S-0139 is a potent and selective endothelin ET A1 receptor antagonist, and that the contractions induced by endothelin-1 in canine basilar, coronary, mesenteric and renal arteries are mediated mainly via the endothelin ET A1 receptor subtype.

Negative inotropic effect of endothelin-1 in the mouse right ventricle

Effects of endothelin-1 on the contraction and cytosolic Ca 2+ concentrations ([Ca 2+] i) of the mouse right ventricle were investigated. Endothelin-1 (1–300 nM) elicited a negative inotropic effect in a concentration-dependent manner. The endothelin-1-induced negative inotropy was antagonized by a selective endothelin ET A receptor antagonist, BQ-123 (cyclo [Asp-Pro-Val-Leu-Trp-]; 3, 10 μM). Endothelin-1 reduced the peak amplitudes of both the [Ca 2+] i transient and contraction without changing inward Ca 2+ current. The relationship between peak amplitude of [Ca 2+] i and peak force generated by changing the extracellular Ca 2+ concentration ([Ca 2+] o) was not affected by endothelin-1. In addition, the trajectory of the [Ca 2+] i-contraction phase plane diagram obtained at 2 mM [Ca 2+] o in the absence of endothelin-1 was superimposable on that obtained at 4 mM [Ca 2+] o in the presence of endothelin-1 (300 nM). Endothelin-1 (300 nM) translocated protein kinase C from cytosol to membrane, suggesting activation of protein kinase C. Further, a selective protein kinase C inhibitor, bisindolylmaleimide I (10 μM), inhibited the endothelin-1-induced negative inotropy. These results suggest that endothelin-1 elicits negative inotropy by reducing the amplitude of the [Ca 2+] i transient without changing inward Ca 2+ current through the activation of the endothelin ET A receptor followed by protein kinase C activation in the mouse right ventricle.

Endothelin ET A receptor antagonism attenuates the pressor effects of nicotine in rats

The increased endothelin-1 levels observed after smoking may result from nicotine-stimulated endothelin-1 production by endothelial cells. In this study, we investigated the effects of selective endothelin ET A receptors antagonist Cycle d- a-aspartyl- l-prolyl- d-isoleucyl- d-tryptophyl (JKC 301) and of endothelin ET B receptors antagonist N- cis-2,6-dimethylpiperidino-carbonyl- l-gamma-methyl-leucyl- d- l- m ethoxycarbonyl-tryptophanyl-norleucine (BQ 788) on the changes in mean arterial pressure, heart rate, and plasma thromboxane B 2 (the stable product of thromboxane A 2) levels caused by increasing doses of nicotine (0.6, 2, 6, and 20 μmol/kg) in anesthetised rats. Nicotine (0.6, 2, and 6 μmol/kg) significantly increased the mean arterial pressure in control and BQ 788-pretreated rats, while only a nicotine dose of 2 μmol/kg) increased the mean arterial pressure in JKC 301-pretreated animals. There were no differences in the nicotine-induced changes in heart rate or in the increases in thromboxane B 2 levels among the groups treated with saline, JKC 301 and BQ 788. These results demonstrate that whereas the antagonism of endothelin ET A receptors attenuated the increase in blood pressure after nicotine injections, endothelin ET B receptor antagonism had no such effect. In addition, the antagonism of endothelin ET A or ET B receptors did not affect thromboxane A 2 production after nicotine administration. These findings suggest that endothelin-1 may have a role in the acute effects of nicotine.

Effects on haemodynamics by selective endothelin ET B receptor and combined endothelin ET A/ET B receptor antagonism during endotoxin shock

The endothelin system is highly activated during endotoxin and septic shock. To investigate this matter the selective non-peptide endothelin ET B receptor antagonist A-192621 ([2 R-(2α,3β,4α)]-4-(1,3-benzodioxol-5-yl)-1-[2-[2,6-diethylphenyl)amino]-2-oxoethyl]-2-(4-propoxy-phenyl)-3-pyrrolidinecarboxylic acid) was administered alone and in combination with the selective non-peptide endothelin ET A receptor antagonist PD 155080 (sodium 2-benzo[1,3]dioxol-5-yl-3-benzyl-4-(4-methoxy-phenyl)-4-oxobut-2-enoate) during established porcine endotoxin shock. Cardiopulmonary vascular function, metabolic parameters and plasma endothelin-1-like immunoreactivity levels were compared to a control group only receiving endotoxin. Administration of A-192621 alone resulted in cardiovascular collapse and death whereas combining A-192621 with PD 155080 abolished endotoxin induced pulmonary hypertension, enhanced cardiac performance and improved systemic oxygen delivery and acid–base balance. The beneficial effects of mixed endothelin ET A/ET B receptor antagonisms on the pulmonary and cardiovascular systems may result from blockage of constrictive endothelin receptors in and pulmonary circulation, reduced afterload and a direct inotropic effect. Possible mechanisms for the devastating effects by selective endothelin ET B receptor antagonism include increased endothelin ET A receptor-mediated vasoconstriction due to lack of endothelin ET B receptormediated vasodilation and decreased endothelin clearance from endothelin ET B receptor blockade. In conclusion, selective endothelin ET B receptor antagonism is deleterious whereas combined endothelin ET A and ET B receptor antagonism has favourable effects on haemodynamics, suggesting participation of the endothelin system in cardiopulmonary dysfunction during endotoxin shock.

Endothelin receptor blockade attenuates air embolization-induced pulmonary hypertension in sheep

We investigated the effects of two types of endothelin receptor antagonists on pulmonary hypertension induced by pulmonary air embolization in awake sheep. We prepared awake sheep with indwelling catheters inserted in blood vessels for continuous monitoring of pulmonary artery pressure, left atrial pressure and systemic arterial pressure. Cardiac output was measured every 30 min. The study consisted of two experiments, one with FR139317 (100 μg/kg/min; ( R)2-[( R)-2-[( S)-2-[1-(hexahydro-1 H-azepinyl)]-carbonyl]amino-4-methyl-pentanoyl]amino-3-[3-(1-methyl-1 H-indolyl)]propionyl)amino-3-(2-pyridyl)propionic acid), a selective endothelin ET A receptor antagonist, and the other with TAK-044 (100 μg/kg/h; cyclo[ d-α-aspartyl-3-[(4-phenylpiperazin-yl)carbonyl]- l-alanyl- l-α- aspartyl- d-2-(2-thienyl) glycyl- l-leucyl- d-tryptophyl] disodium salt), an endothelin ET A and ET B receptor antagonist. In the paired experiments, air was continuously (4.06 ml/min) infused into the main pulmonary artery for 3 h after the baseline pressures were stabilized. Sheep were treated or not treated with FR139317 or TAK-044. Pulmonary artery pressure was significantly higher than the baseline pressure after the start of air infusion. Both FR139317 and TAK-044 significantly attenuated the increase in pulmonary artery pressure during air embolization. Plasma endothelin -1 levels in both pulmonary and systemic arteries were equally and significantly increased after the start of air infusion. The results indicate that endothelin-1 release is attributable to the development of pulmonary hypertension during the course of air embolization in awake sheep.

Cytokines induce increased endothelin ET B receptor-mediated contraction

The effect of cytokines on the induction of contractile endothelin ET B receptors during organ culture was examined. Ring segments of rat superior mesenteric artery were used fresh or incubated for 24 h in Dulbecco's modified Eagle's medium alone, or with either interleukin-1β, tumor necrosis factor-α (TNF-α) or interleukin-2. In fresh arterial segments there was no endothelin ET B receptor-induced contraction. After incubation, the selective endothelin ET B receptor agonist sarafotoxin 6c evoked a contraction of 22±6% relative to that induced by 60 mM K +. The endothelin ET B receptor-induced contraction was further increased to 125±25% and 157±29% by interleukin-1β and TNF-α, respectively, while interleukin-2 did not alter the endothelin ET B receptor-induced contraction. The identity of the contractile receptor was confirmed as the endothelin ET B receptor by the use of an additional specific endothelin ET B receptor agonist, IRL 1620, and by antagonist experiments with FR 139317 and IRL 2500. The endothelin-1-induced contraction was not altered by either of the cytokines. Reverse transcriptase–polymerase chain reaction revealed increased levels of endothelin ET B mRNA, relative to endothelin ET A mRNA following organ culture, suggesting that contractile endothelin ET B receptors appear via de novo transcription. None of the cytokines changed the ratio of endothelin ET A and endothelin ET B receptor mRNA, indicating that the further increased sarafotoxin 6c-induced contraction is mediated through an enhancement of intracellular signalling mechanisms.

Effects of the endothelin ET A receptor antagonist, TA-0201, on pulmonary arteries isolated from hypoxic rats

To investigate the roles of endothelin-1 in the pathogenesis of hypoxic pulmonary hypertension, we studied the effects of a selective endothelin ET A receptor antagonist, TA-0201 ( N-(6-(2-(5-Bromopyrimidin-2-yloxy) ethoxy)-5-(4-methylphenyl) pyrimidin-4-yl)-4-(2-hydroxy-1,1-dimethylethyl) benzensulfonamide sodium salt sesquihydrate), on helical strips of pulmonary arteries isolated from hypoxia-induced pulmonary hypertensive rats as compared with those of normoxic rats. Endothelin-1-induced maximum contractions were significantly inhibited by exposure to hypoxia in the pulmonary arterial strips, but not in the mesenteric arterial strips. The hypoxia also induced right ventricular hypertrophy in rats. Addition of TA-0201 to the bath inhibited the endothelin-1-induced contraction of pulmonary arterial strips isolated from hypoxic rats more effectively than in those of normoxic rats. Oral administration of TA-0201 to hypoxic rats inhibited the hypoxia-induced right ventricular hypertrophy, and decreased the maximum contractile response to endothelin-1 in pulmonary arterial strips isolated from these rats. Those inhibitory effects induced by the oral administration of TA-0201 were not observed in the pulmonary arteries from normoxic rats or in the mesenteric arteries from both hypoxic and normoxic rats. These results suggest that endothelin-1 has important pathophysiological roles in hypoxia-induced pulmonary hypertension, and that TA-0201 may inhibit the endothelin-1-induced contraction through a change in the function of endothelin ET A receptor as well as competitive inhibition for endothelin ET A receptor.

Endothelin-1 affects capsaicin-evoked release of neuropeptides from rat vas deferens

Capsaicin-sensitive neurones release a number of neuropeptides, such as substance P, neurokinin A, somatostatin and calcitonin gene-related peptide (CGRP), which exert a number of effects on smooth muscle tissues. Endothelin-1 was thought to potentiate the capsaicin-evoked release of neuropeptides from sensory neurones of the rat. We have investigated the neuromodulatory effects of endothelin-1 on capsaicin-induced release of neurotransmitters from rat vas deferens. Capsaicin and human α calcitonin gene-related peptide (human αCGRP) reduced the rat vas deferens twitch responses induced by electrical field stimulation. Human β calcitonin gene-related peptide-(8–37) [human βCGRP-(8–37)] (1 μM), a selective αCGRP receptor antagonist, antagonized the inhibitory effects of both drugs. Endothelin-1 concentration dependently evoked an increase in basal tone of the musculature and potentiated the amplitude of the electrically stimulated responses, blocking inhibitory effects of capsaicin but not of human αCGRP. Moreover, endothelin-1 did not markedly change the inhibitory effects of papaverine (0.1–100 μM) or isoprenaline (1 nM–100 μM) on responses to electrical field stimulation. FR 139317 [( N, N-hexamethylene) carbamoyl-Leu- d-Trp( N-Me)- d-2-Pya], a selective endothelin ET A receptor antagonist, administered 30 min before endothelin-1 restored the capsaicin effects whereas BQ 788 [Dmpc-γ-MeLeu- d-Trp-(1-methoxycarbonyl)- d-Nle], a selective endothelin ET B receptor antagonist, was completely ineffective. The endothelin-1-induced block of the capsaicin effect was resistant to tetrodotoxin (1 μM) and 30-min pre-treatment with MEN 10.627 (cyclo[(Met-Asp-Trp-Phe-Dap-Leu) cyclo (2β–5β)]), a selective tachykinin NK 2 receptor antagonist, did not abolish the endothelin-1 effect on the inhibitory response to capsaicin. These results suggest that endothelin-1 selectively inhibits the capsaicin-induced release of neurotransmitters from rat vas deferens and these effects are mediated via endothelin ET A receptors but not by tachykinin release.

Endothelin ET A receptor antagonist reverses the inhibitory effect of platelet-derived growth factor on cytokine-induced nitric oxide production

Cytokines and cytokine-induced nitric oxide (NO) play important roles in inflammatory glomerular diseases, and both platelet-derived growth factor and transforming growth factor-β inhibit cytokine-induced NO production. In this study, we demonstrated that a selective endothelin ET A receptor antagonist, BQ-485 (Hexahydro-1 H-azepinylcarbonyl-Leu- d-Trp- d-Trp-OH), reversed the inhibitory effect of platelet-derived growth factor on cytokine-induced NO production, but not that of transforming growth factor-β. Our findings suggest a difference between the inhibitory mechanisms of platelet-derived growth factor and transforming growth factor-β on cytokine-induced NO production.

Essential role for endothelin ET(B) receptors in fever induced by LPS (E. coli) in rats.

1. The influence of endothelin receptor antagonists on febrile responses to E. coli lipopolysaccharide (LPS), interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and endothelin-1 (ET-1) was assessed in conscious rats. 2. Intravenous (i.v.) LPS (5.0 microg kg(-1)) markedly increased rectal temperature to a peak of 1.30 degrees C over baseline at 2.5 h. Pretreatment with the mixed endothelin ET(A)/ET(B) receptor antagonist bosentan (10 mg kg(-1), i.v.) or the selective endothelin ET(B) receptor antagonist BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D -1-methoxycarboyl-D-norleucine; 3 pmol, into a lateral cerebral ventricle-i.c.v.) reduced the peak response to LPS to 0.90 and 0.75 degrees C, respectively. The selective endothelin ET(A) receptor antagonist BQ-123 (cyclo[D-Trp-D-Asp-Pro-D-Val-Leu]; 3 pmol, i.c.v.) was ineffective. 3. Increases in temperature caused by IL-1beta (180 fmol, i.c.v.), TNF-alpha (14.4 pmol, i.c.v.) or IL-1beta (150 pmol kg(-1), i.v.) were unaffected by BQ-788 (3 pmol, i.c.v.). 4. Central injection of endothelin-1 (0.1 to 3 fmol, i.c.v.) caused slowly-developing and long-lasting increases in rectal temperature (starting 2 h after administration and peaking at 4-6 h between 0.90 and 1.15 degrees C) which were not clearly dose-dependent. The response to endothelin-1 (1 fmol, i.c.v.) was prevented by BQ-788, but not by BQ-123 (each at 3 pmol, i.c.v.). Intraperitoneal pretreatment with the cyclo-oxygenase inhibitor indomethacin (2 mg kg(-1)), which partially reduced LPS-induced fever, did not modify the hyperthermic response to endothelin-1 (3 fmol, i.c.v.). 5. Therefore, central endothelin(s) participates importantly in the development of LPS-induced fever, via activation of a prostanoid-independent endothelin ET(B) receptor-mediated mechanism possibly not situated downstream from IL-1beta or TNF-alpha in the fever cascade.