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Pulmonary hypertension (PH) is a severe chronic cardiopulmonary dysfunction characterized by impaired of pulmonary circulation. Current therapeutic drugs mainly act as vasodilators, leading to an unsatisfactory prognosis. The Rho/ROCK pathway plays an important role in the cardiovascular system. DL0805-1, a novel Rho kinase inhibitor, synthesized by our institute and showed a protective effect on lung tissues and reduced right ventricular systolic pressure in a hypertensive crisis rat model in our previous study. The present study aims to explore the efficacy of DL0805-1 on PH. The classical PH rat model induced by a single subcutaneous injection of monocrotaline was used to investigate the therapeutic effect of DL0805-1 on PH and the underlying mechanisms. The results showed that the high dose of DL0805-1 had a better effect on the survival rate and controlled right ventricular systolic pressure (RVSP) of PH rats than fasudil. DL0805-1 also exhibited a superior lung protective effect and significantly improved pulmonary vascular function compared with bosentan. Regarding molecular mechanisms, DL0805-1 inhibited the ROCK pathway in both pulmonary arteries and lung tissues. Taken together, DL0805-1 alleviated lung injury and vasculopathy in experimental PH rats. DL0805-1 has the potential to be developed as a candidate drug for the treatment of PH.

In the pulmonary vasculature there is clearance of endothelin-1 from the circulation mediated by endothelin ETB receptors. This study explored the haemodynamic effects of endothelin-1 and its clearance in the pulmonary and hindquarter vasculature in anaesthetised rats. Carotid and pulmonary artery pressures and pulmonary and hindquarter blood flows were measured. In each rat, a single endothelin-1 or sarafotoxin S6C cumulative dose-response curve was generated with or without antagonist pretreatment (i.v.). Endothelin-1 caused an acute fall in MAP and rise in hindquarter vascular conductance (HVC) followed by a marked increase in MAP at 5 min with falls in HVC and pulmonary vascular conductance (PVC). Bosentan (10, 20 & 30 mg/kg) pretreatment caused dose-dependent inhibition of the MAP increase as well as PVC and HVC decreases to endothelin-1. Similarly, macitentan (30 mg/kg) or ambrisentan (10 mg/kg) caused significant block of responses to endothelin-1. Sarafotoxin S6C caused acute falls in MAP and increases in HVC and then small falls in PVC and HVC, all prevented by pretreatment with ETB antagonist BQ788 (1 mg/kg). Pretreatment with BQ788 enhanced endothelin-1 potency by 2.5-fold in PVC and 2.4-fold in HVC. With BQ788 and bosentan, the fall in HVC response was completely blocked, but there were residual MAP rises and PVC falls at the highest endothelin-1 dose. Our work confirms the role of ETB receptors in the pulmonary vasculature that decrease the circulating levels of endothelin-1. This has important consequences in selecting an appropriate ETA and ETB dual receptor antagonist to effectively block endothelin-1-mediated pulmonary vasoconstriction.

The present study was designed to investigate the effect of late phase of whole body hypoxic preconditioning on endothelial-dependent vasorelaxation and cardioprotection from ischemia-reperfusion injury in spontaneously hypertensive rats (SHR). Hypoxic preconditioning was performed by subjecting rats to four episodes of alternate exposure to low O2 (8%) and normal air O2 of 10 min each. After 24 h, the mesenteric arteries and hearts were isolated to determine the vascular function and cardioprotection from ischemia-reperfusion (I/R) injury on the Langendorff apparatus. There was a significant impairment in acetylcholine-induced relaxation in norepinephrine precontracted arteries (endothelium-dependent function) and increase in I/R-induced myocardial injury in SHR in comparison to Wistar Kyoto rats (WKY). However, hypoxic preconditioning significantly restored endothelium-dependent relaxation in SHR and attenuated I/R injury in both SHR and WKY. Hypoxic preconditioning also led to an increase in the levels of endothelin-1 (not endothelin-2 or −3), vascular endothelial growth factor-A (VEGF-A) and HIF-1α levels. Pretreatment with bevacizumab (anti-VEGF-A) and bosentan (endothelin receptor blocker) significantly attenuated hypoxic preconditioning-induced restoration of endothelium-dependent relaxation and cardioprotection from I/R injury. These interventions also attenuated the levels of VEGF-A and HIF-1α without modulating the endothelin-1 levels. It may be concluded that an increase in the endothelin-1 levels with a subsequent increase in HIF-1α and VEGF expression may possibly contribute in improving endothelium-dependent vasorelaxation and protecting hearts from I/R injury in SHR during late phase of whole body hypoxic preconditioning.

Smoking-induced COPD is characterized by chronic airway inflammation, which becomes enhanced by bacterial infections resulting in accelerated disease progression called exacerbation. Alveolar macrophages (AM) release endothelin-1 (ET-1), IL-6, CCL-2 and MMP-9, all of which are linked to COPD pathogenesis and exacerbation. ET-1 signals via ETA- and ETB-receptors (ETAR, ETBR). This is blocked by endothelin receptor antagonists (ERAs), like bosentan, which targets both receptors, ETAR-selective ambrisentan and ETBR-specific BQ788. Therefore, ERAs could have anti-inflammatory potential, which might be useful in COPD and other inflammatory lung diseases. We hypothesized that ERAs suppress cytokine release from AM of smokers and COPD subjects induced by lipopolysaccharide (LPS), the most important immunogen of gram-negative bacteria. AM were isolated from the broncho-alveolar lavage (BAL) of n=29 subjects (11 non-smokers, 10 current smokers without COPD, 8 smokers with COPD), cultivated and stimulated with LPS in the presence or absence of ERAs. Cytokines were measured by ELISA. Endothelin receptor expression was investigated by RT-PCR and western blot. AM expressed ETAR and ETBR mRNA, but only ETBR protein was detected. LPS and ET-1 both induced IL-6, CCL-2 and MMP-9. LPS-induced IL-6 release was increased in COPD versus non-smokers and smokers. Bosentan, ambrisentan and BQ788 all partially reduced all cytokines without differences between cohorts. Specific ETBR inhibition was most effective. LPS induced ET-1, which was exclusively blocked by BQ788. In conclusion, LPS induces ET-1 release in AM, which in turn leads to CCL-2, IL-6 and MMP-9 expression rendering AM sensitive for ERAs. ERAs could have anti-inflammatory potential in smoking-induced COPD.

Pulmonary arterial hypertension (PAH), a condition that is defined by pulmonary vasculature constriction and remodeling, involves dysfunctional signaling of the serotonin (5-HT) receptors, 5-HT2A/2B/7. In a rat model of monocrotaline (MCT)-induced PAH, the effectiveness of RP5063 (RP), a dopamine and 5-HT receptor modulator, was evaluated as monotherapy and as an adjunct to standard PAH treatments. After a single 60 mg/kg dose of MCT, rats received vehicle (MCT+Veh; gavage twice-daily [b.i.d.]), RP (10 mg/kg; gavage b.i.d.), bosentan (B; 100 mg/kg; gavage BID), sildenafil (S; 50 mg/kg; gavage, BID), treprostinil (T; 100 ng/kg/min over 24 h intravenous), RP+B, RP+S, and RP+T for 28 days. Single-agent RP limited the functional and structural effects of PAH seen in the MCT+Veh group, with significant improvements in pulmonary hemodynamics, right ventricular (RV) hypertrophy, SO2, and pulmonary blood vessel structural changes. These effects appeared comparable with those associated with B, S, and T. Adjunctive RP treatment resulted in significantly lower mean pulmonary arterial pressures, RV systolic pressure. It also improved SO2 measurements, as compared with MCT+Veh (P

Pain reported by patients with head and neck cancer is characterized as persistent pain with mechanical allodynia. Pain management is inadequate for many patients, highlighting the need for improved therapies. We examined the hypothesis that the mixed endothelin ETA and ETB receptor antagonist, bosentan, reduces tumor-induced ongoing pain and evoked hypersensitivity in a rat model of facial cancer pain. Facial cancer was induced by inoculating a suspension of Walker-256 cells into the rat's right vibrissal pad. Tumor-bearing rats developed heat and tactile hypersensitivity along with increased spontaneous grooming behavior. Systemic morphine (2.5mg/kg, s.c.) blocked tumor-induced thermal and tactile hypersensitivity, with a lower dose (0.625mg/kg, s.c.) effective only against thermal hypersensitivity. Systemic bosentan blocked tumor-induced thermal hypersensitivity only at a high (300mg/kg, p.o.) dose, but failed to modify tactile hypersensitivity. Co-administration of the low doses of bosentan and morphine resulted in improved reduction of the tumor-induced heat and tactile hypersensitivity compared to either dose alone. Bosentan (100mg/kg, p.o.) reduced spontaneous grooming and induced conditioned place preference (CPP) selectively in tumor-bearing rats, suggesting that bosentan reduces tumor-induced ongoing pain at a lower dose than required to block tumor-induced hypersensitivity. This study provides evidence that endothelins may mediate tumor-induced ongoing pain and thermal hypersensitivity. In addition, bosentan enhanced morphine's effects on blocking tumor-induced heat and tactile hypersensitivity indicating that endothelin antagonists may be beneficial therapeutic targets that can be used to manage cancer-induced facial pain with opioid-sparing effects.

The present study aims to investigate the possibility that inhibiting the physiological function of endothelin-1 (ET-1) by blocking its receptors would significantly decrease the nephrotoxic effect of cisplatin. Therefore the study was designed to investigate the effect of treatment with BQ-123, the selective endothelin receptor-A (ET(A)) blocker, and bosentan, the non-selective endothelin receptor blocker, on the cisplatin-induced structural, functional, and biochemical alterations in the rat kidney. Rats were divided into four groups: control (given a single dose of normal saline, i.p.), cisplatin (given a single dose of cisplatin, 6mg/kg, i.p.), cisplatin+BQ-123 (1mg/kg, i.p.), and cisplatin+bosentan (30mg/kg, orally via gavage). Each of the two blockers was administered in two doses; 1h before and one day after the cisplatin dose. Acute cisplatin administration resulted in significant increases in blood urea nitrogen and serum creatinine concentrations at 96h following cisplatin injection. Increased concentrations of malondialdehyde, tumor necrosis factor-α (TNF-α) and caspase-3, decreased nitric oxide (NO) production and superoxide dismutase (SOD) activity in kidney homogenates were observed at 96h following cisplatin injection, in addition to a typical 'acute tubular necrosis' pattern. BQ-123 ameliorated the structural and functional injuries caused by cisplatin mainly via restoring SOD activity, in addition to other antioxidant parameters, NO, TNF-α and caspase-3 concentrations. This study further proves that ET(A) but not ETB receptors are involved in cisplatin-induced nephrotoxicity. The selective ET(A) antagonist BQ-123 ameliorated the cisplatin-induced deleterious effects and showed reno-protective effect against cisplatin-induced acute renal damage.

Paracetamol is one of the first rank drugs which cause hepatic damage during drug intoxications. Endothelin (ET) which is known as one of the most potent vasoactive agent has been shown to contribute in the pathophysiology of various diseases. We hypothesized that bosentan, which is a non-selective ET-1 receptor antagonist, can prevent liver damage. This study included 49 female rats. Groups; I: Healthy group, II: Paracetamol (2 g/kg orally). Groups 3, 4 and 5 received NAC 140 mg/kg (2 doses), BOS 45 mg/kg and BOS 90 mg/kg orally, respectively. 1 h after administration of pretreatment drugs, Groups 3, 4, 5 were given paracetamol. VI: received BOS 90 mg/kg. VII: received 140 mg/kg NAC (2 doses). According to biochemical results, TNF-α, ALT and AST levels were statistically increased in the paracetamol group, these parameters were improved in the bosentan groups. Paracetamol administration decreased SOD activity, GSH level and increased level of MDA in the liver, while bosentan administration significantly improved these parameters. In immunohistochemical staining ET-1 receptor expression was excessively increased in paracetamol group, but not in bosentan groups when compared to healthy control. All these results suggest that bosentan exerted protective effects against experimentally induced paracetamol toxicity in liver.

Background Endothelin receptor antagonists are approved for pulmonary arterial hypertension. Development of selective ETA-receptor antagonists over mixed or dual receptor antagonists has depended on a range of receptor binding assays, second messenger assays and functional blood vessel assays. This study compared the 3 clinically-approved endothelin receptor antagonists in assays of human isolated pulmonary and radial arteries in vitro. Methods Human isolated pulmonary (i.d. 5.5 mm) and human radial (i.d. 3.23 mm) artery ring segments were mounted in organ baths for isometric force measurement. Single concentration-contraction curves to endothelin-1 were constructed in the absence or presence of bosentan (1–10 µM), macitentan (0.03–0.3 µM) or ambrisentan (0.1–1 µM). Results All 3 endothelin antagonists caused competitive rightward shifts in the endothelin-1 concentration-response curves in both arteries. The Clark plot and analysis gave the following pKB values: bosentan, pulmonary artery 6.28±0.13 and radial artery 6.04±0.10; macitentan, pulmonary artery 8.02±0.13 and radial artery 7.49±0.08; and ambrisentan, pulmonary artery 7.38±0.13 and radial artery 6.96±0.10. Conclusions Noting the maximum plasma levels attained from recommended oral doses of each antagonist in volunteers, the pKB findings here show that there would be significant antagonism of endothelin-1 contraction in the pulmonary and radial arteries at therapeutic plasma levels. This functional assay confirms in human tissue that much higher plasma concentrations of endothelin-1 receptor antagonists are required to be effective than those predicted from binding or other biochemical assays.

The safety and effectiveness of drugs used to treat chronic diseases critically depend on their propensity to interact with co-administered drugs. Induction of enzymes and drug transporters involved in the clearance and distribution of drugs may critically reduce exposure with their substrates and thus lead to nonresponse. We therefore investigated the impact of the endothelin-1 receptor antagonists bosentan and ambrisentan on the expression of relevant human efflux and uptake transporters and on phase 1 and phase 2 enzymes. LS180 adenocarcinoma cells were treated for four days with bosentan or ambrisentan (1-50 μM), the positive control rifampicin, or medium only (negative control). For evaluation of bosentan also HuH-7 human hepatoma cells were used and treated similarly. Gene expression was quantified at the mRNA level by real-time reverse transcription polymerase chain reaction and for some genes also at the protein level by western blot analysis. Comparable to rifampicin, bosentan was a moderate to strong inductor for all cytochrome P450 isozymes and ATP-binding cassette transporters tested, and it also induced organic anion transporting polypeptides. 50 μM bosentan up-regulated e.g. CYP3A4 8.5-fold, ABCB1 5.1-fold, and ABCB11 1.9-fold at the mRNA level in LS180 cells. In HuH-7 cells induction was much less pronounced (e.g. CYP3A4 1.9-fold for bosentan). In contrast, ambrisentan only weakly induced some of the genes investigated in LS180 cells. These findings corroborate the in vivo finding that bosentan is much more prone to drug interactions than ambrisentan.

Nitric oxide (NO) induces vascular relaxation via cGMP in vascular smooth muscle (VSM) and is an important mediator of vascular tone during sepsis, as endothelial NO synthase (eNOS) may be upregulated during the early stages. Atrial natriuretic peptide (ANP) also stimulates cGMP via eNOS hence, this study aimed to investigate the role of NO in time-dependent altered vascular responses to ANP during the first 4h of exposure to bacterial lipopolysaccharide (LPS). We used male rat saphenous arteries [internal relaxed diameter 63-152 microm, n=48], mounted on a wire myograph and pre-constricted with phenylephrine. At 2h in the presence of LPS, there was increased relaxation to ANP in arteries exposed to LPS [16.3+/-2.4%, P0.05]. However the response to ANP was not altered by the NOS inhibitor Nomega-nitro-l-arginine methyl ester (L-NAME, 10(-4)M) and following denudation (vessels without endothelium). At 4h there was no longer increased relaxation to ANP in the presence of LPS. Moreover the vasodilator response to ANP was significantly reduced following L-NAME or denudation [4.4+/-1.0% and 4.3+/-1.1% respectively, P0.05]. However, the non-specific endothelin-1 (ET-1) receptor antagonist Bosentan [10(-5)M] increased dilatation in LPS exposed arteries at 1 and 2h, reaching significance at 4h [14.0+/-3.4%, P0.05]. In summary, an endothelial and NO dependent mechanism is responsible for increased relaxation to ANP following 2h exposure to LPS. However after 4h an endothelial and NO independent process involving ET-1 is responsible for decreased relaxation to ANP. The enhanced response to ANP may exacerbate early systemic vasodilatation during early sepsis.

There are evidences that targeting IL-18 might be beneficial to inhibit inflammatory symptoms, including hypernociception (decrease in nociceptive threshold). The mechanism of IL-18 mechanical hypernociception depends on endothelin in rats and mice. However, the role of IL-18 in overt pain-like behaviour remains undetermined. Therefore, we addressed the role of IL-18 in writhing response induced by intraperitoneal (i.p.) injection of phenyl-p-benzoquinone (PBQ) and acetic acid in mice. Firstly, it was detected that PBQ and acetic acid i.p. injection induced a dose-dependent number of writhes in Balb/c mice. Subsequently, it was observed that the PBQ - but not the acetic acid-induced writhes were diminished in IL-18 deficient ((-/-)) mice. Therefore, considering that IFN-gamma, endothelin and prostanoids mediate IL-18-induced mechanical hypernociception, we also investigated the role of these mediators in the same model of writhing response in which IL-18 participates. It was noticed that PBQ-induced writhes were diminished in IFN-gamma(-/-) mice and by the treatment with bosentan (mixed endothelin ETA/ETB receptor antagonist), BQ 123 (cyclo[DTrp-DAsp-Pro-DVal-Leu], selective endothelin ETA receptor antagonist), BQ 788 (N-cys-2,6 dimethylpiperidinocarbonyl-l-methylleucyl-d-1-methoxycarboyl-d-norleucine, selective endothelin ETB receptor antagonist) or indomethacin (cycloxigenase inhibitor). Thus, IL-18, IFN-gamma, endothelin acting on endothelin ETA and ETB receptors, and prostanoids mediate PBQ-induced writhing response in mice. To conclude, these results further advance the understanding of the physiopathology of overt pain-like behaviour, and suggest for the first time a role for IL-18 in writhing response in mice.

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.

Ventricular septal (150-200 microm) arteries were isolated from the hearts of six-week-old CD-1 mice and mounted on a pressure myograph. Equilibration of the vessels at 70 mm Hg for 60 min resulted in the development of spontaneous myogenic tone. Maximum tone observed in these vessels greatly exceeded that previously reported in septal arteries from rats. Inhibition of endothelin ET(A) and endothelin ET(B) receptors with bosentan (1 and 10 microM) reduced basal tone. Endothelin release required intact endothelial cells. The alpha(1)-adrenceptor selective agonists phenylephrine and methoxamine did not cause change in coronary tone, while the alpha(2)-adrenceptor selective agonists 6-allyl-2-amino5,6,7, 8-tetrahydro-4H-thiazolo-[4,5-d]azepin-dihydrochloride (BHT 920) and clonidine produced vasodilatation. Noradrenaline (1 nM-10 microM) induced a concentration-dependent vasodilatation, which was inhibited by concurrent treatment with yohimbine (10 microM) and propranolol (20 microM). Vasodilatation due to BHT 920 was abolished with vessel denudation, indicating the endothelial location of alpha(2)-adrenoceptors. Acetylcholine (1 nM-10 microM) caused an endothelium dependent vasodilatation; inhibition of nitric oxide synthase with N(omega)-nitro-L-arginine methyl ester attenuated this response. The endothelium-dependent vasodilators bradykinin and substance P produced no vasomotor effect in mouse coronary arteries. Differences between human and murine responses may impact on the relevance of the mouse coronary artery for use as a potential model of human coronary vessel diseases.

We have examined whether 4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-2,2′-bipyrimidin-4-yl]-benzenesulfonamide (bosentan; endothelin ETA/B receptor antagonist) and (R)2-[(R)-2-[(S)-2-[[1-(hexahydro-1H-azepinyl)]carbonyl] amino-4-methylpentanoil]amino-3-[3-(1-methyl-1H-indoyl)]propionyl]amino-3-(2-pyridyl) propionic acid (FR 139317; endothelin ETA receptor antagonist) inhibit contractions of human airway smooth muscle induced by endothelin-1 or Suc-[Glu9,Ala11,15]enthothelin-1-(8–21) (IRL 1620; endothelin ETB receptor agonist). Endothelin-1 and IRL 1620 were equipotent. Bosentan and FR 139317 (each 10 μM) produced a small shift in response curves to endothelin-1 (1.6- and 1.5-fold, respectively). However, bosentan was more potent against contractions elicited by IRL 1620 (10 μM, 11.2-fold shift) suggesting that these agonists exhibit different kinetic interactions with endothelin receptors or implying an interaction with a novel endothelin ETB receptor subtype in human airways.

The effects of the mixed endothelin ET(A)/endothelin ET(B) receptor antagonist Ro 47-0203 (bosentan, 4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-( 2-methoxy -phenoxy)-2,2'-bipyrimidin-4-yl] -benzenesulfonamide) and the selective endothelin ET(A) receptor antagonist PD155080 (sodium 2-benzo[1,3]dioxol-5-yl-3-benzyl-4-(4-me thoxy-phenyl)-4-oxobut+ ++-2-enoate) on plasma half-life and regional extraction of exogenous endothelin-1 as well as on the regional vascular effects of endothelin-1 were investigated in the pig in vivo. Bosentan but not PD155080 (5 mg/kg, i.v. bolus, both drugs) increased the arterial plasma levels of endothelin-1-like immunoreactivity. Neither of the drugs affected the plasma half-life of infused endothelin-1. In the spleen, both the extraction and vascular effects of exogenous endothelin-1 were attenuated by both bosentan and PD155080 whereas renal extraction and vascular effects in the kidney were unaffected by both drugs. In the lung, only bosentan decreased pulmonary extraction of endothelin-1. In conclusion, the bosentan-induced increase of circulating endothelin-1 seems to be related to blockade of endothelin-1 binding to endothelin ET(B) receptors. Blockade of these receptors does not influence the overall elimination of endothelin-1, however.

The effects of the mixed endothelin receptor antagonist bosentan on renal regional haemodynamics were investigated in rats with aorto-caval fistula, an experimental model of congestive heart failure. A matched group of normal rats served as control. Injection of bosentan (10 mg/kg i.v.) to the rats with decompensated congestive heart failure produced an increase in cortical (+ 20%) and medullary (+ 12%) blood flow, and a decrease in vascular resistance in the cortex (− 30%) and medulla (− 23%), while reducing mean arterial pressure by approximately 10 mm Hg. In rats with compensated congestive heart failure and in normal animals, infusion of bosentan did not affect blood pressure and cortical perfusion. These findings indicate that 1) endothelin receptor blockade produces beneficial effects on renal haemodynamics in rats with experimental congestive heart failure and 2) endothelin-1 may be involved in the pathogenesis of renal hypoperfusion only in decompensated congestive heart failure.

The involvement of endothelins in the cerebrovascular events which follow a focal ischemic insult in the rat was explored in the present study. Intravenous (i.v.) administration of bosentan (3, 15 and 30 mg/kg), an endothelin ETA and ETB receptor antagonist, prior to middle cerebral artery occlusion in the rat did not significantly alter cortical perfusion in these rats. A 62 +/- 3% reduction in laser doppler flow was observed 10 min after middle cerebral artery occlusion in the vehicle-treated group compared to a 49 +/- 5% reduction in laser doppler flow in the group receiving 15 mg/kg bosentan. Pre-treatment with intravenous bosentan (15 mg/kg) prior to middle cerebral artery occlusion in the rat also failed to elicit significant alterations in the reduction in regional cerebral blood flow (frontal cortex; 81 +/- 13 ml/100 g/min) and subsequent hemispheric volume of ischemic damage observed (94 +/- 9 mm3) compared to the vehicle treated animals (68 +/- 9 ml/100 g/min, 113 +/- 5 mm3, respectively). Minimal changes were also observed in these endpoints, when a 15 mg/kg dose of bosentan was administered following middle cerebral artery occlusion. In conclusion bosentan failed to expose a major role for endothelins in focal ischemic pathology in the rat.

Endothelin exerts a variety of biological effects in the lung which indicate that this peptide may have a role in the pathophysiology of a number of pulmonary diseases. In this study, the endothelin receptors on the human bronchus were compared with those on the guinea-pig trachea using the novel, non-peptide antagonist Ro 47-0203 (4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-2,2' -bipyrimidin-4-yl]-benzenesulphonamide, non-selective for endothelin ETA over endothelin ETB receptor) and the peptide antagonist BQ123 (cyclo(-D-Val-Leu-D-Trp-D-Asp-Pro), endothelin ETA receptor selective). On the human bronchus and guinea-pig trachea, the concentration-effect curve for endothelin-1 was shifted to the right by Ro 47-0203 (100 microM) with concentration ratios of 28.2 +/- 6.8 and 39.5 +/- 13.9, respectively but lower concentrations of the antagonist had no effect. Although the concentration-effect curve for sarafotoxin S6c on the human bronchus was shifted to the right by Ro 47-0203 (30 and 100 microM, concentration ratio: 6.88 +/- 1.72 and 69.7 +/- 17.2, respectively), equivalent degrees of inhibition could be obtained on guinea-pig trachea with lower concentrations of antagonist (10 and 30 microM, concentration ratio: 6.90 +/- 1.58 and 75.8 +/- 14.1 respectively). The lack of effect of BQ123 (10 microM) and the high concentrations of Ro 47-0203 needed to show antagonism indicate that endothelin receptors on both tissues are probably the endothelin ETB subtype. Although the antagonism by Ro 47-0203 is not classically competitive, the greater effect of Ro 47-0203 against sarafotoxin S6c on the guinea-pig trachea may reflect a difference between the endothelin ETB receptors on these tissues.

The effects of bosentan, a nonpeptide endothelin receptor antagonist, on endothelin-induced changes in coronary flow and myocardial ischaemic and reperfusion injury were investigated in the Langendorff perfused rat isolated heart. Endothelin-1 (0.012–0.4 nmol) evoked dose-dependent reduction in coronary flow, which was attenuated by bosentan (1.0–10 μM) in a concentration-related fashion. The inhibitory effect of bosentan lasted more than 30 min. The endothelin ETB receptor agonist Suc-[Glu9,Ala11,15]endothelin-1-(8–21) (IRL 1620) increased coronary flow in the absence but not in the presence of bosentan. In hearts subjected to 30 min of global ischaemia followed by 30 min of reperfusion, the recoveries of the left ventricular developed pressure, d Pdtmax, and coronary flow were significantly larger in a group given bosentan 10 μM at the start of ischaemia (92 ± 7%, 98 ± 8% and 83 ± 5%, respectively) than in a vehicle-treated group (70± 4%, 70 ± 6% and 42 ± 2%, respectively) at the end of the reperfusion period. During the reperfusion period, left ventricular end diastolic pressure was significantly lower in the bosentan group than in the vehicle group. The area of no-reflow in the bosentan group was 7 ± 3% of left ventricle compared to 21 ± 2% in the vehicle group (P < 0.01). Acetylcholine-induced endothelium-dependent vasodilatation was significantly reduced after ischaemia and reperfusion in the vehicle group but not in the bosentan group. It is concluded that bosentan attenuates the coronary vasoconstrictor effect elicited by endothelin and reduces ischaemia/reperfusion-induced myocardial and endothelial injury in the rat isolated heart. The results suggest that endogenous endothelin may be involved in the pathogenesis of myocardial ischaemic and reperfusion damage and a beneficial effect of bosentan in preventing myocardial and endothelial injury following ischaemia and reperfusion.

The effectiveness of intradermal (i.d.) BQ-123 (cyclo[D-Asp-Pro-D-Val-Leu-D-Trp]) and i.d. Ro 47-0203 (bosentan, 4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-2,2'-bipyr imidin-4 - yl]-benzene-sulfonamide) has been evaluated on local microvascular responses to endothelin-1 and endothelin-3, measured by a multiple site 133Xe clearance technique in rat skin in vivo. Intradermal injection of endothelin-1 (0.3 pmol/site) and endothelin-3 (10 pmol/site) induced a similar (approximately 50-60%) decrease in basal blood flow in rat skin. BQ-123 (3-1000 pmol/site), a selective endothelin ETA receptor antagonist, caused a significant dose-dependent decrease in the vasoconstriction induced by endothelin-1 (P < 0.05) but was less effective on vasoconstriction induced by endothelin-3. Bosentan (3-1000 pmol/site), a new non-peptide mixed antagonist of endothelin ETA and endothelin ETB receptors, significantly reduced the vasoconstriction induced by endothelin-1 but was less effective than BQ-123. BQ-123 and bosentan were similarly effective as antagonists of endothelin-3. BQ-123 and bosentan had no effect on basal blood flow and no inhibitory activity on vasoconstriction induced by vasopressin (0.03 pmol/site) or phenylephrine (300 pmol/site). These findings indicate that BQ-123 and bosentan are effective and selective inhibitors of the vasoconstriction induced by endothelins in the rat skin microvasculature.

The cerebrovascular actions of bosentan, a novel endothelin antagonist with effects at endothelin ETA and ETB receptors, have been examined in individual pial arterioles on the cortical surface of chloralose-anaesthetised cats. Subarachnoid perivascular microapplication of bosentan (0.3-300 microM) had minimal effect on pial arteriolar calibre. Subarachnoid perivascular microapplication of endothelin (10 nM) effected a marked reduction in pial arteriolar calibre (reduced by 39.2 +/- 2.7% from baseline). This vasomotor effect of topical endothelin could be attenuated either by co-administration of bosentan (IC50 approximately 1 microM) or by the intravenous administration of bosentan (17 mumol/kg). These investigations suggest that bosentan (applied topically or systemically) may be a valuable tool in the elucidation of the functional significance of endothelins in the cerebral circulation in vivo.

Endothelin peptides have been shown to increase cholinergic neurotransmission in the airway. Genetic differences in airway responsiveness to methacholine where reported in mice. The present study compared the airway reactivity to methacholine in C57Bl/6 and BALB/c mice, the involvement of endothelin on this reactivity and endothelin levels in lung homogenates. Whole airway reactivity was analyzed by means of an isolated lung preparation where lungs were perfused through the trachea with warm gassed Krebs solution at 5 ml/min, and changes in perfusion pressure triggered by methacholine at increasing bolus doses (0.1-100 microg) were recorded. We found that the maximal airway response to methacholine was much greater in C57Bl/6 than in BALB/c (Emax 34+/-2 vs 12+/-1 cmH(2)O, respectively). Bosentan (mixed endothelin A/B receptor antagonist; 10 mg/kg, i.p., 30 min before sacrifice) reduced lung responsiveness to methacholine in C57Bl/6 (58% at EC50 level) but had no effect in BALB/c mouse strain. This effect seems to be mediated by the endothelin ET(A) receptor since it was significantly reduced by the selective endothelin ET(A) receptor antagonist, BQ 123. Immunoreactive endothelin levels were higher in C57Bl/6 than in BALB/c lungs (43+/-5 vs 19+/-5 pg/g of tissue). In conclusion, airway reactivity to methacholine and lung endothelins content varies markedly between C57Bl/6 and BALB/c strains. Endothelins upregulate lung responsiveness to methacholine only in C57Bl/6, an effect achieved through the endothelin ET(A) receptor.

The effects of dual (ETA and ETB) endothelin receptor blockade on ventricular arrhythmogenesis during acute myocardial infarction are not well defined. We randomly allocated Wistar rats to bosentan (100 mg/kg daily, n=24), a dual endothelin receptor antagonist, or vehicle (n=23). After 7 days of treatment, myocardial infarction was induced by permanent coronary ligation. Ventricular tachyarrhythmias were evaluated for 24 h following ligation, using a miniature telemetry electrocardiogram recorder. Action potential duration was measured from monophasic epicardial recordings and sympathetic activation was assessed by heart rate variability and catecholamine serum level measurements. Compared to controls (1012+/-185 s), bosentan (59+/-24 s) markedly decreased (P

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.

The role of endothelin peptides was evaluated on survival and organ injury in a model of polymicrobial sepsis, induced by caecal ligation and puncture with particular emphasis on the timing of the administration of its blocker bosentan in Swiss albino mice (20-40 g). The cardiovascular response pattern in this experimental model was characterized by an early, "hyperdynamic" phase starting at 5 h, followed by a late but "hypodynamic" phase that commence after 20 h, provided that the animals are "resuscitated" by injecting 1 ml of saline i.p. at the end of the surgery. However, if saline resuscitation is omitted, then only hypodynamic pattern is observed starting at 5 h without any hyperdynamic phase. Thus, mice were first allocated into saline-resuscitated or unresuscitated groups and endothelin receptor antagonist bosentan (30 mg kg(-1), i.p., either 5 or 20 h after caecal ligation and puncture) was then administered. The control animals received the solvent of bosentan (i.e., saline: %0.9 NaCl, w/v). The survival rates in each group (n=14) were recorded over the following 144 h. In unresuscitated mice, the overall survival at 144 h was 14.3% in controls while bosentan treatment at 5 h (78.6%, P=0.0018) or 20 h (64.3%, P=0.0183) have both significantly improved the survival. However, in saline-resuscitated mice, bosentan administered at 20 h has significantly improved the survival (71.4%, P=0.0213) while its administration at 5 h has yielded exactly the same percent of survival (i.e., 21.4%) as observed in control animals. The beneficial effects of bosentan in preventing the tissue injury due to caecal ligation and puncture were also observed histopathologically in liver, spleen and kidney. Therefore, we concluded that the blockade of endothelin receptors by using bosentan during the later (hypodynamic) stages of septic shock is a promising therapeutic manoeuvre.

The binding affinities of (E)-N-[6-methoxy-5-(2-methoxyphenoxy)[2,2'-bipyrimidin]-4-yl]-2-phenylethenesulfonamide monopotassium salt (YM598) for native human endothelin ETA and ETB receptors expressed in human coronary artery smooth muscle cells (CASMC) and a human melanoma cell line, SK-Mel-28, respectively, were examined, and the results compared with those for the endothelin receptor antagonists atrasentan and bosentan. The in vivo endothelin ETA receptor inhibitory activities of YM598 and atrasentan were also compared through the suppression of the big endothelin-1-induced pressor response in pithed rats. Ki values of YM598, atrasentan, and bosentan for native human endothelin ETA receptors were 0.772, 0.0551, and 4.75 nM, while those for native human endothelin ETB receptors were 143, 4.80, and 40.9 nM, respectively. The calculated selectivity ratios of YM598, atrasentan, and bosentan for endothelin ETA versus ETB receptors were 185, 87 and 8.6, respectively. In pithed rats, YM598 and atrasentan inhibited the big endothelin-1 (1 nmol/kg)-induced pressor response in a dose-dependent manner on both intravenous and oral administration. The inhibitory effect of YM598 was less potent than that of atrasentan when these agents were intravenously administered, but closely similar on oral administration. These results suggest that YM598 has high selectivity for native human ETA against ETB receptors, and that YM598 is superior to atrasentan as an ETA receptor antagonist with regard to pharmacological bioavailability in rats.

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.

This study investigated the effects of bosentan, a dual endothelin ET(A) and ET(B) receptor antagonist, during hypoxia-reoxygenation of senescent aorta and during ischemia-reperfusion of senescent heart. Isolated aortic rings and isolated hearts from adult and senescent rats were submitted, respectively, to hypoxia/reoxygenation (20/30 min) and to low-flow ischemia/reperfusion (45/30 min), without or with bosentan (10(-5) M). In the aorta, bosentan treatment prevented the impairment of relaxation in response to acetylcholine after hypoxia-reoxygenation at both ages. In the heart, coronary flow recovery during reperfusion, which is lower in senescents than in adults (48% vs. 76% of baseline value, respectively; P0.05) was fully prevented by bosentan. Prevention of endothelial dysfunction during reoxygenation of hypoxic aorta and of coronary vasoconstriction during reperfusion of ischemic heart with a dual endothelin ET(A) and ET(B) receptor antagonist suggests a role of endothelin in the vulnerability of aorta to hypoxia-reoxygenation, and of coronary arteries to ischemia-reperfusion, especially during aging.

We examined the role of endothelin in ischaemia/reperfusion injury in skeletal muscle, using the endothelin receptor antagonist Bosentan. In the rat hindlimb tourniquet ischaemia model, one hindlimb was rendered ischaemic for 2 h at 36 degrees C, then blood flow was re-established for either 24 h to assess muscle survival or 1.5 h for a study of capillary perfusion. In the first set of rats, the gastrocnemius muscle was removed from the postischaemic limb and assessed for viability histochemically using the nitro blue tetrazolium stain. Tissue water content (a measure of oedema) and myeloperoxidase activity (a measure of neutrophil accumulation) were also assessed in the ischaemic muscle, the contralateral non-ischaemic muscle and the lungs. In the second set of rats, the hind limb was infused with India ink after 2-h ischaemia and 1.5-h reperfusion and the muscle was harvested, fixed and cleared. In control rats, muscle viability was 17+/-2% (S.E.M.). In rats treated with Bosentan (10 mg/kg, i.p.) 30 min before release of the tourniquet, muscle viability (48+/-7%) was significantly increased compared to the control group (P0.01). Bosentan treatment had no significant effect on tissue water content or myeloperoxidase activity in the ischaemic muscle, the contralateral non-ischaemic muscle or the lung. Immunoreactive endothelin levels in serum increased to a peak at 90 min of reperfusion and returned to control levels by 24-h reperfusion. India ink studies demonstrated a significantly increased functional capillary density in postischaemic Bosentan-treated muscles compared with postischaemic control muscles (P0.05). These results suggest that endothelin plays an important role in the necrosis which results from a period of ischaemia and reperfusion in skeletal muscle, by mediating a decrease in postischaemic microvascular perfusion.

Endothelin-1 elicited tonic contractions of rat prostatic smooth muscle that were unaffected by prazosin (0.5 microM), tetrodotoxin (1 microM) or guanethidine (10 microM). The rank order of potency of the endothelin isopeptides was endothelin-1>endothelin-2> or =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 microM) and BQ3020 (Ac-[Ala (11,15)]endothelin-1(6-21); 0.1 nM-0.3 microM), 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 microM), attenuated responses to endothelin-1, -2 and -3, while the non-selective endothelin receptor antagonist bosentan (1 microM) and the selective endothelin ET(B) receptor antagonist BQ788, (Dmpc-gamma-MeLeu(9)-D-Trp(l-CO(2)CH(3))-D-Nle-OH; 1 microM) attenuated responses to endothelin-3 only. Contractions induced by exogenous administration of noradrenaline were unaffected by preincubation of tissues in BQ123 (1 microM) indicating the selectivity of this antagonist. These data suggest that endothelins mediate contractions of the rat prostate by action at endothelin ET(A) receptors.

The modulatory effects of a non-selective endothelin receptor antagonist, bosentan, were investigated together with those of relatively selective inducible nitric oxide synthase inhibitors, aminoguanidine and L-canavanine, on mesenteric blood flow decrease, liver and spleen injury elicited by endotoxaemia. Swiss albino mice (20-40 g) were administered intraperitoneally bosentan (3, 10 or 30 mg kg(-1)), aminoguanidine (15 mg kg(-1)) or L-canavanine (20 or 100 mg kg(-1)) 10 min before they received saline or Escherichia coli endotoxin (10 mg kg(-1)). After 4 h, the mice were anaesthetized, mesenteric blood flow values were measured, spleen and liver weight/body weight ratios were determined and the organs were examined histopathologically. Endotoxin decreased mesenteric blood flow (ml min(-1), saline: 3.0 +/- 0.2; endotoxin: 2.2 +/- 0.2: n = 10, P < 0.05), increased the weight of liver (g per kg body weight, saline: 47.5 +/- 2.0; endotoxin: 60.8 +/- 1.9: n = 10, P < 0.05) and spleen (g per kg body weight, saline: 3.9 +/- 0.5; endotoxin: 8.6 +/- 0.9; n = 10, P < 0.01) while it inflicted significant histopathological injury to both organs. Bosentan was ineffective at 3 mg kg(-1) but at 10 and 30 mg kg(-1) doses, it abolished all the deleterious effects of endotoxin without exception. Aminoguanidine blocked most of the effects of endotoxin except those on spleen. In contrast, L-canavanine blocked only the endotoxin-induced increase in liver weight but itself increased spleen weight and failed to block any other effects of endotoxin. Thus, it can be speculated that the beneficial effects of aminoguanidine are produced largely by mechanisms other than selective inducible nitric oxide synthase inhibition since L-canavanine was not fully effective. The beneficial effects of endothelin inhibition by using bosentan in endotoxaemia can be further exploited for the understanding and the therapy of sepsis-related syndromes.

The influence of endothelin-1 on nociception induced by capsaicin was assessed in the mouse hindpaw. Local endothelin-1 injection (1 to 20 pmol/paw) 30 min prior to ipsilateral injection of capsaicin (0.1 microg/paw) increased, in a graded fashion, the time spent licking the injected paw. Maximal hyperalgesia was obtained with 10 pmol/paw of endothelin-1 (capsaicin-induced hindpaw licking time increased from 43 +/- 3 s to 114 +/- 7 s, n = 6), but no hyperalgesia was evident following 30 pmol/paw of endothelin-1. The selective endothelin ET(B) receptor agonists sarafotoxin S6c (< or = 30 pmol/paw) and IRL 1620 (i.e., Suc[Glu9,Ala11,15]endothelin-1-(10-21); < or = 100 pmol/paw) failed to induce hyperalgesia. Local treatment with BQ-123 (i.e., cyclo[DTrp-DAsp-Pro-DVal-Leu] 1 nmol/paw selective endothelin ET(A) receptor antagonist), 10 min before endothelin-1 (10 pmol/paw), fully blocked the hyperalgesic response, whereas similar treatment with the selective endothelin ET(B) receptor antagonist BQ-788 (i.e., N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D- 1-methoxy-carboyl-D-norleucine) was ineffective. Intravenous injection of bosentan (17 and 52 micromol/kg a non-peptidic mixed endothelin ET(A)/ET(B) receptor antagonist) or BMS 182874 (i.e., 5-[dimethylamino]-N-[3,4-dimethyl-5-isoxazolyl]-1-naphthalenesulph onamide; 10 and 30 micromol/kg; a non-peptidic selective endothelin ET(A) receptor antagonist), 1 h before endothelin-1, inhibited its hyperalgesic effect in a graded fashion and abolished the response at the higher doses. None of the antagonists modified nociception induced by capsaicin alone or the hyperalgesia induced by local injection of 5-hydroxytryptamine (5-HT; 2 nmol/paw, 30 min before capsaicin). Hyperalgesia induced by 5-HT was abolished by simultaneous injection of endothelin-1 or the endothelin ET(B) receptor agonist IRL 1620 (each at 30 pmol/paw). Therefore, local endothelin-1 exerts a dual influence in this model: at low doses it causes endothelin ET(A) receptor-mediated hyperalgesia (i.e., it potentiates capsaicin-induced nociception), whereas at higher doses it induces an anti-hyperalgesic effect against 5-HT which seems to be mediated via distinct endothelin ET (possibly ET(B)) receptors.

Changes in blood pressure, cardiac output, and total peripheral conductance evoked by intravenous infusions of [Arg 8 ]-vasopressin (vasopressin) were recorded before and after pretreatment with bosentan, a non-selective endothelin antagonist, in conscious unrestrained spontaneously hypertensive rats (SHR) and Wistar–Kyoto rats (WKY). The presser effects of vasopressin were exaggerated in SHR compared to WKY. Pretreatment with bosentan failed to change hemodynamic responses of WKY to vasopressin, but it blunted the increases in blood pressure and the decreases in conductance evoked by vasopressin in SHR. In contrast, bosentan failed to change cardiac output responses of SHR to vasopressin. Except at the highest dose of vasopressin, bosentan abolished the exaggerated pressor responsiveness of the SHR to vasopressin. The results suggest that endothelin contributes to the exaggerated pressor responsiveness of SHR to vasopressin, and that this effect is exerted at the level of the resistance vessels and not on factors that regulate cardiac output.

Activation of endothelial cells by vasoactive mediators, such as endothelins, may be an early, strategically important step in the initiation of inflammation in the intestine. In view of recent evidence that inflammatory bowel disease is associated with elevated intestinal concentrations of endothelins and upregulated expression of endothelin receptors on vascular endothelium in intestine, endothelins may become therapeutic targets in inflammatory bowel disease. The recent availability of an orally active, mixed endothelin receptor antagonist, bosentan, allowed us to examine the role of endothelins in a rat model of colitis. Colitis was induced by intra-rectal administration of trinitrobenzene sulphonic acid. In each treatment group, rats were treated with bosentan (10-60 mg/kg p.o.) 24 and 2 h prior to (pre-dose) or 1 h after the induction (post-induction) of colitis and all animals were treated every 24 h thereafter for 5 days. On day 6, stool consistency and the presence of adhesions in the peritoneal cavity were accessed. Colonic tissue samples were removed for determination of macroscopic and microscopic tissue injury, and myeloperoxidase activity. Colitis was typified by tissue ulceration in the distal colon and a corresponding 35-fold increase in myeloperoxidase activity compared to non-inflamed controls. Daily treatment with bosentan dose-dependently reduced colonic damage and myeloperoxidase activity when bosentan was given prior to induction of colitis. In the pre-dose group, the greatest beneficial effect of bosentan was observed at 60 mg/kg; colonic damage and granulocyte infiltration were attenuated by > 80%. A partial therapeutic effect of bosentan was also observed at 60 mg/kg when the pre-treatment regimen was excluded. These findings demonstrate that an orally active, mixed endothelin receptor antagonist has marked protective and therapeutic effects in an animal model of colitis.

This study analyzed if the paracrine liver endothelin system participates in the pathogenesis of CCl 4 -induced hepatotoxicity. Wistar Kyoto rats were divided into four groups: a bosentan (mixed endothelin ET A and ET B receptor antagonist) treated group with CCl 4 intoxication, a vehicle treated group with CCl 4 intoxication, a nontreated control group and a bosentan treated control group. Hepatotoxicity was assessed by determination of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) followed by histopathological examinations. Tissue endothelin-1 concentrations and expression of endothelin receptor subtypes were analyzed. The tissue levels of endothelin-1 in the liver of rats with CCl 4 intoxication were significantly higher than those in normal rats. Scatchard analysis revealed no differences in the density and binding constant of endothelin ET A and ET B receptor between rats with CCl 4 intoxication and controls. Bosentan treatment of rats undergoing CCl 4 inhalation resulted in a significant protection against elevation of ALT, AST, LDH and bilirubin. Histopathological examination of liver sections for necrotic, swollen and lipid-laden cells revealed findings that were in agreement with the serum enzyme data. In conclusion, this study showed that the paracrine endothelin system is involved in the pathogenesis of CCl 4 -induced hepatotoxicity and that the blockade of the stimulated liver endothelin system reduces CCl 4 -induced liver injury.