Endothelin ET Research Articles

Effect of YM598, a selective endothelin ET A receptor antagonist, on endothelin-1-induced bone formation

We investigated the effect of endothelin-1 on bone formation in vitro and in vivo, and the effect of YM598, a novel selective endothelin ET A receptor antagonist, on endothelin-1-induced responses. In in vitro studies, the effect of endothelin-1 on cellular responses was investigated by measuring intracellular Ca 2+ levels, cell growth and alkaline phosphatase activity in the mouse osteoblast-like cell line MC3T3-E1. In in vivo studies, the effect of endothelin-1 on bone morphogenetic protein-2-induced ectopic bone formation in rats was investigated. A carrier containing bone morphogenetic protein-2 with or without endothelin-1 was subcutaneously implanted over the thorax, and the tissue (carrier) calcium content 3 weeks after implantation was evaluated. The inhibitory effect of YM598 on these responses was also investigated. In the in vitro studies, endothelin-1 (10 − 13 to 10 − 6 M) significantly increased intracellular Ca 2+ concentration, DNA synthesis and cell number in a concentration-dependent manner, while significantly decreasing alkaline phosphatase activity. YM598 (10 − 12 to 10 − 4 M) significantly inhibited these increases, as well as the decrease in alkaline phosphatase activity, in a concentration-dependent manner. In the in vivo studies, the tissue calcium content 3 weeks after carrier implantation was significantly higher in the group that received both bone morphogenetic protein-2 and endothelin-1 than in the group receiving bone morphogenetic protein-2 alone. Chronically administered YM598 (1 mg/kg/day) marginally inhibited this endothelin-1-potentiated ectopic bone formation. These results suggest that endothelin-1 may induce bone formation via endothelin ET A receptors in vitro and in vivo.

Endothelin ET A receptors and endothelium partially mediate the positive inotropic and lusitropic effects of angiotensin II

We analyzed the influence of endothelin-1 and endocardial endothelium on the myocardial effects of angiotensin-II. Angiotensin-II (10 − 9 –10 − 5 M) was tested in rabbit right papillary muscles in absence (Protocol-A) or presence of PD-145065 (10 − 7 M; Protocol-B), BQ-123 (10 − 7 M; Protocol-C) or losartan (10 − 6 M; Protocol-E), as well as, after removing the endocardial endothelium with Triton X-100 0.5% (Protocol-D). In Protocol-F increasing concentrations of endothelin-1 (10 − 10 –10 − 8 M) were added in presence of angiotensin-II (10 − 7 M) after selective removal of the endocardial endothelium. In Protocol-A, angiotensin-II had dose-dependent positive inotropic and lusitropic effects, maximal at 10 − 6 M increasing 122 ± 13% active tension, 117 ± 16% d T/d t max and 86 ± 9% d T/d t min. In Protocols B, C and D the inotropic and lusitropic effects of angiotensin-II were significantly attenuated. The same concentration (10 − 6 M) of angiotensin-II increased respectively 48 ± 11%, 59 ± 27% and 72 ± 16% active tension; 54 ± 14%, 54 ± 20% and 32 ± 9% d T/d t max; and 39 ± 8%, 48 ± 19% and 59 ± 11% d T/d t min; and 40 ± 10%. EC 50 for active tension significantly increased from − 7.8 ± 0.1 logM in Protocol A to − 7.1 ± 0.3, − 6.7 ± 0.4 and − 6.8 ± 0.3 logM in Protocols B, C and D respectively, while E max decreased from 106 ± 14% in Protocol A to 50 ± 14 and 51 ± 19% in Protocols B and C respectively, but did not significantly change in Protocol D (114 ± 25%). Losartan completely blocked the inotropic and lusitropic effects of angiotensin-II, while the attenuation of these effects after the selective removal of the endocardial endothelium was blunted by concomitant administration of endothelin-1 (Protocol F). In conclusion, angiotensin-II has a dose-dependent positive inotropic effect that depends, to a great extent, on endothelin ET A receptor activation and intact endocardial endothelium.

COX-2, Another Important Player in the Nitric Oxide–Endothelin Cross-Talk

See related article, pages 1439–1445 Traditionally, the role of the endothelium was thought to be primarily that of a selective barrier to the diffusion of macromolecules from the vessel lumen to the interstitial space. During the past 20 years, numerous additional roles for the endothelium have been defined such as regulation of vascular tone, modulation of inflammation, promotion as well as inhibition of vascular growth, and modulation of platelet aggregation and coagulation. Endothelial dysfunction is a characteristic feature of patients with cardiovascular risk factors such as hypercholesterolemia, hypertension, diabetes mellitus, and chronic smoking. More recent studies indicate that it may predict long-term atherosclerotic disease progression as well as cardiovascular event rate.1 There is a growing body of evidence that decreased endothelial bioavailability of nitric oxide (NO·) in particular attributable to increased production of reactive oxygen species, such as superoxide (O2·−), leads to an activation of the renin–angiotensin system,2,3 increased formation of cyclooxygenase (COX)-dependent vasoconstrictors,4 but also to increased expression of the most potent endogenous vasoconstrictor endothelin-1 (ET-1).5–9 Of the 4 active endothelins (ET-1 to ET-4) ET-1 is the predominant isoform in the cardiovascular system. ET-1 exerts its major cardiovascular effects through activation of 2 distinct G protein–coupled receptors, the ETA and ETB receptors. ETA receptors are found exclusively in smooth muscle cells. Endothelin-1 promotes vasoconstriction, mitogenesis, and thrombosis predominantly via binding to the ETA receptor. ETB receptors are localized to some extent in smooth muscle cells, but also in endothelial cells. Activation of ETB receptors has been demonstrated to cause the release of NO· and prostacyclin (PGI2).10,11 In normal states with p reserved vascular (endothelial) NO …

Endothelin ET A receptor blockade potentiates morphine analgesia but does not affect gastrointestinal transit in mice

Development of analgesic tolerance and constipation remain a major clinical concern during long-term administration of morphine in pain management. Central endothelin mechanisms are involved in morphine analgesia and tolerance. The present study was conducted to investigate the effect of intracerebroventricular (i.c.v.) and peripheral administration of endothelin ET A receptor antagonist, BMS182874, and endothelin ET B receptor agonist, IRL1620, on morphine analgesia and changes in gastrointestinal transit in male Swiss Webster mice. Results indicate that morphine (6 mg/kg, s.c.) produced a significant increase in tail flick latency compared to control group. Pretreatment with BMS182874 (50 μg, i.c.v.) significantly enhanced morphine-induced analgesia, while IRL1620 (30 μg, i.c.v.) pretreatment did not affect tail-flick latency values. Changes in gastrointestinal transit were measured by percent of distance traveled by charcoal in the small intestine of gastrointestinal tract. Percent distance traveled in morphine (6 mg/kg, s.c.) treated mice (48.45 ± 5.65%) was significantly lower ( P < 0.05) compared to control group (85.07 ± 1.82%). Administration of BMS182874 centrally (50 μg, i.c.v.) or peripherally (10 mg/kg, i.p.) did not affect morphine-induced inhibition of gastrointestinal transit. Pretreatment with IRL1620 (30 μg, i.c.v., or 10 mg/kg, i.v.) also did not affect morphine-induced inhibition of gastrointestinal transit. This study demonstrates that endothelin ET A receptor antagonists delivered to the CNS enhance morphine analgesia without affecting gastrointestinal transit.

Down-regulated VEGF expression in the diabetic heart is normalized by an endothelin ET A receptor antagonist

Recently, down-regulation of vascular endothelial growth factor (VEGF) in the heart was suggested as a potential molecular explanation for the increased risk of cardiovascular morbidity and mortality in patients with diabetes. Increased endothelin-1 production is reported in diabetes. Here, we report that down-regulated VEGF expression in the diabetic rat heart is normalized by an endothelin ET A receptor antagonist, suggesting that endothelin antagonism may be a novel therapeutic strategy for diabetic heart.

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 [(125)I]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.

Upregulation of endothelin ET B receptor-mediated vasoconstriction in rat coronary artery after organ culture

The aim of this study was to examine if endothelin ET B receptor-mediated contraction occurred in isolated segments of rat coronary arteries during organ culture. Presence of contractile endothelin ET B receptors was studied by measuring the change in isometric tension in rings of left anterior descending coronary arteries isolated from hearts of rats as response to application of the selective endothelin ET B receptor agonist, Sarafotoxin 6c and endothelin-1. In segments cultured 1 day in serum free Dulbecco's Modified Eagle's Medium, Sarafotoxin 6c induced a concentration dependent contraction with a pEC 50 value of 10.4 ± 0.21 and a maximal response of 3.9 ± 0.25 mN/mm ( n = 15). The maximal contraction was significantly larger than the responses measured in fresh tissue, where the mean value of the maximal contractions was 0.22 ± 0.03 mN/mm ( n = 17). The increased contraction to Sarafotoxin 6c after culturing could be eliminated with addition of the transcriptional blocker, actinomycin D, to the culture medium or be significantly attenuated by application of the translational inhibitor, cycloheximide. The vasoconstrictor effect of endothelin-1 or to depolarisation by high K +-solution was not modified after 1 day in culture medium. The experiments indicate that organ culture of rat coronary arteries upregulate endothelin ET B receptor-mediated contraction by inducing synthesis of new protein.

Vascular endothelin ET B receptor-mediated contraction requires phosphorylation of ERK1/2 proteins

In cardiovascular diseases, endothelin type B (ET B) receptors in arterial smooth muscle cells are upregulated. The present study revealed that organ culture of rat mesenteric artery segments enhanced endothelin ET B receptor-mediated contraction paralleled with increase in the receptor mRNA and protein expressions. The endothelin ET B receptor-mediated contraction was associated with increase in phosphorylation of extracellular regulation kinase 1 and 2 (ERK1/2) proteins and elevated levels of intracellular calcium. The elevation curve of intracellular calcium consisted of two phases: one rapid and one sustained. Inhibition of ERK1/2 phosphorylation by SB386023 or blockage of calcium channels by nifedipine significantly reduced the endothelin ET B receptor-mediated contraction ( P < 0.05) and decreased the sustained phase of intracellular calcium level, but not the rapid phase. Thus, phosphorylation of ERK1/2 proteins and elevation of intracellular calcium level are required for endothelin ET B receptor-mediated contraction in rat mesenteric artery.

Orofacial cold hyperalgesia due to infraorbital nerve constriction injury in rats: Reversal by endothelin receptor antagonists but not non-steroidal anti-inflammatory drugs

The susceptibility of changes in responsiveness to noxious cold stimulation of rats submitted to chronic constriction of the infraorbital nerve (CION) or carrageenan to drug inhibition was compared. Nocifensive responses were measured as total time rats engaged in bilateral facial grooming with both forepaws over the first 2 min following tetrafluoroethane spray application to the snout. Carrageenan (50 μg, s.c. into upper lip) caused short-lived ipsilateral cold hyperalgesia (peak at 3 h: vehicle 8.4 ± 1.3, carrageenan 21.2 ± 3.0 s) which was markedly suppressed by i.p. indomethacin (4 mg/kg), celecoxib (10 mg/kg) or s.c. dexamethasone (0.5 mg/kg), endothelin ET A or ET B receptor antagonists (BQ-123 and BQ-788, respectively; 10 nmol/lip). CION caused ipsilateral cold hyperalgesia between Days 2 and 12, which peaked on Days 4 (sham 15.3 ± 1.8, CION 32.4 ± 5.3 s) to 6. Established peak CION-induced cold hyperalgesia was unaffected by indomethacin and celecoxib, whereas dexamethasone, BQ-123, BQ-788, and i.v. injections of selective antagonists of ET A (atrasentan, 3–10 mg/kg) or ET B (A-192621, 5–20 mg/kg) receptors caused significant inhibitions lasting 1–2.5 h (peaks ∼65–90%). Bosentan (dual ET A/ET B receptor antagonist, 10 mg/kg, i.v.) abolished CION-induced cold hyperalgesia for up to 6 h. Thus, once established, CION-induced orofacial hyperalgesia to cold stimuli appears to lack an inflammatory component, but is alleviated by endothelin ET A and/or ET B receptor antagonists. If this CION injury model bears predictive value to trigeminal neuralgia (i.e., paroxysmal orofacial pain triggered by various stimuli), endothelin receptors might constitute new targets for treatment of this disorder.

Effects of antagonists for endothelin ET A and ET B receptors on coronary endothelial and myocardial function after ischemia-reperfusion in anesthetized goats

To compare the effects of antagonists for endothelin ET A and ET B receptors on the action of ischemia-reperfusion on endothelial and myocardial function, 30 min of partial or total occlusion followed by 60 min of reperfusion of the left circumflex coronary artery was induced in anesthetized goats treated with intracoronary administration of saline (vehicle), BQ-123 (endothelin ET A receptors antagonist) or BQ-788 (endothelin ET B receptors antagonist). During reperfusion after partial occlusion, coronary vascular conductance and left ventricle d P/d t were decreased after saline or BQ-788, and they normalized after BQ-123. In these three groups of animals, the coronary effects of acetylcholine (3–100 ng) and sodium nitroprusside (1–10 μg) during reperfusion were as under control. During reperfusion after total occlusion, coronary vascular conductance and left ventricle d P/d t were decreased after saline, and they normalized after BQ-123 or BQ-788. In these three groups of animals, the coronary effects of acetylcholine but not those of sodium nitroprusside during reperfusion were decreased after saline, and they reversed after BQ-123 or BQ-788. Therefore, selective antagonists of endothelin ET B and ET A receptors may produce similar protection of coronary vasculature and myocardium against reperfusion after severe ischemia. Selective antagonists of endothelin ET B receptors, contrarily to those of endothelin ET A receptors, may be ineffective to protect coronary vasculature and myocardium against reperfusion after mild ischemia.

Contribution of endothelin receptors and cyclooxygenase-derivatives to the altered response of the rabbit renal artery to endothelin-1 in diabetes

The influence of diabetes on regulatory mechanisms and specific receptors implicated in the response of isolated rabbit renal artery to endothelin-1 was examined. Endothelin-1 induced a concentration-dependent contraction that was less potent in arteries from diabetic rabbits than in arteries from control rabbits. Endothelium removal or N G-nitro- l-arginine (L-NOARG) enhanced contractions to endothelin-1 either in control and diabetic arteries. Indomethacin inhibited endothelin-1-induced response in control arteries, but enhanced it in diabetic arteries. In contrast to that observed in rubbed and in L-NOARG treated arteries, in the presence of indomethacin the contractile action of endothelin-1 was higher in diabetic arteries than in control arteries. Nimesulide enhanced endothelin-1 contractions both in control and diabetic arteries. Cyclo-( d-Asp-Pro- d-Val-Leu- d-Trp) (BQ-123, endothelin ET A receptor antagonist), attenuated endothelin-1 vasoconstriction in control rabbits, while vasoconstriction resulted increased in diabetic rabbits. 2,6-Dimethylpiperidinecarbonyl-γ-Methyl-Leu- N in-(Methoxycarbonyl)- d-Trp- d-Nle (BQ-788, endothelin ET B receptor antagonist), enhanced the contractile response in control rabbit arteries without modifying this response in diabetic rabbits. In summary, diabetes decreases the sensitivity of the rabbit renal artery to endothelin-1 by decreasing the ratio between vasoconstrictor and vasodilator prostanoids released after activation of endothelin ET A receptors.

Endothelin-1 as a central mediator of LPS-induced fever in rats

Fever induced by E. coli lipopolysaccharide (LPS) in rats is substantially reduced by blockade of central endothelin ET B receptors. This study explores the role of endothelin-1 as a central mediator of fever in rats, by investigating the effect of a pyrogenic dose of LPS on the levels of big endothelin-1 and endothelin-1 in the cerebrospinal fluid (CSF) and endothelin-1 in the plasma. We further assessed whether the increase in body temperature caused by central injection of endothelin-1 constitutes solely a hyperthermia or a true integrated febrile response. LPS (5 μg kg −1, i.v.) induced fever which peaked at 1.16 ± 0.24 °C within 2 h and remained stable up to 5 h. CSF levels of immunoreactive (ir) big endothelin-1 decreased to undetectable levels at 3 h after LPS, returning only partially at 5 h post-injection. CSF ir-endothelin-1 levels were undetectable in saline-treated animals, but reached 21.9 ± 5.2 fmol ml −1 at 3 h after LPS treatment. Plasma ir-endothelin-1 levels were unchanged after saline or LPS. Central injection of endothelin-1 (1 pmol, i.c.v.) caused long-lasting increases in body temperature (0.81 ± 0.17 °C, 3 h), but simultaneously decreased tail skin temperature (−1.10 ± 0.26 °C), indicating cutaneous vasoconstriction. Moreover, endothelin-1 induced fever (1.0 ± 0.3 °C, 3 h) when injected into the preoptic area of the anterior hypothalamus (100 fmol), but not i.v. (1 or 10 pmol). These data suggest that endothelin-1 is produced in the brain and acts centrally as a mediator of LPS-induced fever.

Vasoconstrictive Effect of Angiotensin IV in Isolated Rat Basilar Artery Independent of AT1 and AT2 Receptors

The effect of angiotensin IV (AngIV) was studied in freshly isolated rat basilar arteries (BAs) perfused at a constant rate. AngIV had no effect on basal BA perfusion pressure, but induced a marked concentration-dependent contraction in vessels precontracted by a 50-mM KCl solution (EC₅₀ = 44.5 ± 16 nM). This contraction was unaffected by the angiotensin AT₁ receptor antagonist candesartan or the angiotensin AT₂ receptor blocker PD123319, but was markedly inhibited by two different specific AT₄ receptor antagonists, Nle¹-Leu³¥(CH₂-NH₂)^3–4-AngIV and divalinal-AngIV. Removal of the endothelium abolished the contractile response to AngIV, and pretreatment of endothelium-intact arteries with the endothelin ET_A/ET_B receptors inhibitor PD142893 blocked the AngIV-induced contraction to the same extent. In BA pretreated with endothelin-1 (ET-1; 0.01 µM), AngIV-induced a concentration-dependent contraction, shifted to the left, compared with that observed with KCl precontraction, unaffected by candesartan but completely abolished by Nle¹-Leu³¥(CH₂-NH₂)^3–4-AngIV. The contractile effect was not affected by endothelium removal in the presence of exogenous ET-1, in contrast to KCl pretreated BA, suggesting that endothelium was mandatory to unmask the effect of AngIV as a source of endogenous ET-1 release. Taken together, these results indicate that low (nanomolar) concentrations of AngIV exert a constrictive effect mediated by its specific binding site AT₄ in the rat BA, and that this vasoactive effect is indirect and involves endogenous endothelin(s).

Effect of chymase on intraocular pressure in rabbits

Chymase is a chymotrypsin-like serine protease that is stored exclusively in the secretory granules of mast cells and converts big endothelins to endothelin-1 (1–31). The aim of this study was to evaluate the effect of chymase on intraocular pressure in rabbits. Chymase injection (3 and 10 mU) resulted in a trend toward increased intraocular pressure and a significant increase in intraocular pressure at a dose of 10 mU compared with the control. A specific chymase inhibitor, Suc-Val-Pro-Phe P(OPh) 2, attenuated the ocular hypertension induced by chymase. Endothelin-1 (1–31) also caused ocular hypertension, which was inhibited by a selective endothelin ET A receptor antagonist, cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123). Moreover, chymase-induced ocular hypertension was inhibited by BQ-123. These results suggest that chymase influences the regulation of intraocular pressure, and it is likely that the formation of endothelin-1 (1–31) and subsequent activation of endothelin ET A receptors are involved in the development of ocular hypertension induced by chymase.

Enhanced response of pig coronary arteries to endothelin-1 after ischemia–reperfusion. Role of endothelin receptors, nitric oxide and prostanoids

To analyse the coronary effects of endothelin-1 after ischemia–reperfusion, the left anterior descending coronary artery of anesthetized pigs was subjected to 30-min occlusion followed by 60-min reperfusion. Then, rings distal (ischemic arteries) and proximal (control arteries) to the occlusion were taken from this artery and prepared for isometric tension recording. The sensitivity of the contraction in response to endothelin-1 (3 × 10 − 10 –3 × 10 − 7 M) and the endothelin ET B receptor agonist IRL-1620 (3 × 10 − 10 –3 × 10 − 7 M) was greater in ischemic vessels. The endothelin ET A receptor antagonist BQ-123 (10 − 7 –3 × 10 − 6 M) decreased the sensitivity of the response to endothelin-1 similarly in ischemic and control arteries. The endothelin ET B receptor antagonist BQ-788 (10 − 6 M), endothelium removal or the inhibitor of nitric oxide synthesis N ω-nitro-L-arginine methyl ester (L-NAME 10 − 4 M) potentiated the response to endothelin-1 and IRL-1620 in control arteries only. The cyclooxygenase inhibitor meclofenamate (10 − 5 M) augmented the maximal response to endothelin-1 in control arteries, and reduced it in ischemic arteries. In precontracted arteries, IRL-1620 (3 × 10 − 11 –3 × 10 − 10 M) relaxed control but not ischemic arteries, and L-NAME or meclofenamate abolished this relaxation. Therefore, ischemia–reperfusion increases the coronary vasoconstriction in response to endothelin-1 probably due to impairment of endothelin ET B receptor-induced release of nitric oxide and prostacyclin, augmentation of the contractile response to activation of endothelin ET B receptors, and increased release of vasoconstrictor prostanoids.

Electrophysiological and haemodynamic effects of endothelin ETA and ETB receptors in normal and ischaemic working rabbit hearts

The aims of this study were to determine if endothelin-1 (ET-1) under normal and ischaemic conditions exhibits a direct arrhythmogenic effect that is independent of its ability to cause coronary vasoconstriction, and to determine the contribution of the ET(A) and ET(B) receptor subtype. ET(A/B) (with ET-1) and ET(A) (ET-1 in the presence of BQ-788) receptor activation resulted in a significant reduction in both epi- and endocardial monophasic action potential duration (MAPD(90)). ET(A) receptor activation reduced both epi- and endocardial effective refractory period (ERP). This MAPD(90) and ERP shortening were associated with a reduction in coronary flow, myocardial contractility and induction of ventricular fibrillation (VF) during ERP measurement. The ET(B) agonist sarafotoxin (S6c) had no marked, or concentration-dependent, effect on MAPD(90), ERP, myocardial contractility or induction of arrhythmias. Neither ET-1 nor S6c, given prior to coronary artery occlusion, significantly changed the ischaemia-induced dispersion of MAPD(90), ERP or the % incidence of VF. In conclusion, neither ET(A) nor ET(B) receptor stimulation has a direct arrhythmogenic effect in isolated rabbit hearts under normal or ischaemic conditions. The ET-1-induced arrhythmogenic effect observed in nonischaemic hearts is likely to be the result of the associated coronary vasoconstriction caused by ET(A) receptor stimulation resulting in myocardial ischaemia.

Glycosidation of an Endothelin ETA Receptor Antagonist and Diclofenac in Human Liver Microsomes: Aglycone-dependent UDP-sugar Selectivity

Following the finding that UGT2B7 catalyzes the transfer of the glycosyl group from both UDP-glucuronic acid (UDP-GlcA) and UDP-glucose (UDP-Glc) to an endothelin ET(A) receptor antagonist, 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], to form an acyl glucuronide and a glucoside (Tang et al., 2003), two additional nucleotide sugars [UDP-galactose (UDP-gal) and UDP-N-acetyl glucosamine (UDP-GlcNAc)] were examined as cosubstrates in human liver microsomes. It was found that UDP-gal, but not UDP-GlcNAc, also served as a sugar donor primarily through catalysis by UGT2B7, although at a significantly reduced catalytic rate. These three UDP-sugars showed pH-dependent kinetics and appeared to compete with each other, with IC50 values parallel to their respective apparent K(m) values. In contrast, only UDP-GlcA served as the sugar donor for the conjugation of diclofenac, a known UGT2B7 substrate, with an apparent K(m) for UDP-GlcA of 96 +/- 17 microM. UDP-Glc and UDP-gal, two futile sugar donors for diclofenac, were found to competitively inhibit the glucuronidation of this aglycone. Different from the case with Compound A, UDP-Glc and UDP-gal displayed K(i) values of 2054 +/- 108 microM and 1277 +/- 149 microM, >10-fold greater than the K(m) for UDP-GlcA, indicating that these two nucleotide sugars were also capable of binding to the enzyme but with a lower affinity. The findings of this study indicate that the selectivity of UGT2B7 toward UDP-sugars is aglycone-dependent. With Compound A as the acceptor substrate, human UGT2B7 becomes more accommodative in the transfer of the glycosyl group from UDP-sugars beyond UDP-GlcA. The mechanism may involve enzyme conformational changes associated with Compound A binding to the enzyme.

Effects of Long-Term Bosentan in Children With Pulmonary Arterial Hypertension

This study investigated the long-term outcome of children with pulmonary arterial hypertension (PAH) treated with bosentan therapy, with or without concomitant prostanoid therapy. Bosentan, an oral endothelin ET(A)/ET(B) receptor antagonist, improves hemodynamics and exercise capacity in adults with PAH; however, limited data are available on its long-term effects in children. In this retrospective study, 86 children with PAH (idiopathic, associated with congenital heart or connective tissue disease) started bosentan with or without concomitant intravenous epoprostenol or subcutaneous treprostinil therapy. Hemodynamics, World Health Organization (WHO) functional class, and safety data were collected. At the cutoff date, 68 patients (79%) were still treated with bosentan, 13 (15%) were discontinued, and 5 (6%) had died. Median exposure to bosentan was 14 months. In 90% of the patients (n = 78), WHO functional class improved (46%) or was unchanged (44%) with bosentan treatment. Mean pulmonary artery pressure and pulmonary vascular resistance decreased (64 +/- 3 mm Hg to 57 +/- 3 mm Hg, p = 0.005 and 20 +/- 2 U x m2 to 15 +/- 2 U x m2, p = 0.01, respectively; n = 49). Kaplan-Meier survival estimates at one and two years were 98% and 91%, respectively. The risk for worsening PAH was lower in patients in WHO functional class I/II at bosentan initiation than in patients in WHO class III/IV at bosentan initiation. These data suggest that bosentan, an oral endothelin ET(A)/ET(B) receptor antagonist, with or without concomitant prostanoid therapy, is safe and efficacious for the treatment of PAH in children.

Endothelin-1-induced translocation of RhoA is mediated by endothelin ET A receptors in rat bronchial smooth muscle

To clarify the receptor subtype(s) contributing to the RhoA activation by endothelin-1 in bronchial smooth muscle, the effects of BQ-123 [cycro(D-Asp-Pro-D-Val-Leu-D-Trp)], an endothelin ET A receptor antagonist, and/or BQ-788 [2,6-dimethylpiperidinecarbonyl-g-methyl-Leu-Nin-(Methoxycarbonyl)-D-Trp-D-Nle], an endothelin ET B receptor antagonist, on the endothelin-1-induced translocation of RhoA to plasma membrane were examined. Incubation of rat bronchial smooth muscle with endothelin-1 induced a distinct translocation of RhoA to plasma membrane, indicating an activation of RhoA by endothelin-1. The endothelin-1-induced translocation of RhoA was completely blocked by treatment with BQ-123, whereas BQ-788 had no effect. Thus, endothelin ET A but not ET B receptors might be involved in the endothelin-1-induced translocation of RhoA in rat bronchial smooth muscle.

The role of endothelin in FK506-induced vascular reactivity changes in rat perfused kidney

Tacrolimus (FK506) is an immunosuppressant agent that is widely used in transplanted patients. The aim of this study was to investigate the role of endothelin in the acute effects of FK506 on the vascular reactivity in perfused isolated rat renal and mesenteric vasculature. Left kidney/mesentery of male Wistar rats (230–300 g) were perfused by a constant flow and perfusion pressure was recorded. The responses to noradrenaline and sodium nitroprusside were obtained both in the absence and presence of FK506 (10 − 7 M) or polyoxyethylene hydrogenated castor oil 60 (HCO-60 and solvent of the drug at equivalent concentrations). FK506 significantly increased the noradrenaline-induced vasoconstrictor responses in renal, but not in mesenteric vascular beds. Bosentan (10 − 5 M), a nonselective endothelin ET-1 receptor antagonist given by perfusion, reversed the increase in noradrenaline responses in the kidney. Sodium nitroprusside-induced vasodilator responses in both renal and mesenteric vascular beds were significantly decreased by FK506. However, in renal vasculature, there was no significant difference between the inhibitory effects of FK506 and HCO-60, although the effect of the solvent was not significantly different from that of the control. While in the mesenteric bed, the solvent significantly inhibited nitroprusside-induced vasodilation, similar to that of FK506. The effect of FK506 on vasodilation in both vascular beds was not reversed by bosentan. Our results indicated that FK506 increased the reactivity of the renal vascular bed to noradrenaline through endothelin ET-1 receptor activation. The mechanism of impaired vasodilation due to FK506 appears to be due to its solvent action and is independent of endothelin release.