Mixed ET Research Articles

Potent anti-inflammatory and antinociceptive activity of the endothelin receptor antagonist bosentan in monoarthritic mice

IntroductionEndothelins are involved in tissue inflammation, pain, edema and cell migration. Our genome-wide microarray analysis revealed that endothelin-1 (ET-1) and endothelin-2 (ET-2) showed a marked up-regulation in dorsal root ganglia during the acute phase of arthritis. We therefore examined the effects of endothelin receptor antagonists on the development of arthritis and inflammatory pain in monoarthritic mice.MethodsGene expression was examined in lumbar dorsal root ganglia two days after induction of antigen-induced arthritis (AIA) using mRNA microarray analysis. Effects of drug treatment were determined by repeated assessment of joint swelling, pain-related behavior, and histopathological manifestations during AIA.ResultsDaily oral administration of the mixed ETA and ETB endothelin receptor antagonist bosentan significantly attenuated knee joint swelling and inflammation to an extent that was comparable to dexamethasone. In addition, bosentan reduced inflammatory mechanical hyperalgesia. Chronic bosentan administration also inhibited joint swelling and protected against inflammation and joint destruction during AIA flare-up reactions. In contrast, the ETA-selective antagonist ambrisentan failed to promote any detectable antiinflammatory or antinociceptive activity.ConclusionsThus, the present study reveals a pivotal role for the endothelin system in the development of arthritis and arthritic pain. We show that endothelin receptor antagonists can effectively control inflammation, pain and joint destruction during the course of arthritis. Our findings suggest that the antiinflammatory and antinociceptive effects of bosentan are predominantly mediated via the ETB receptor.

ENDOTOXIN CAUSES PULMONARY HYPERTENSION BY UPREGULATING SMOOTH MUSCLE ENDOTHELIN TYPE-B RECEPTORS

Endothelin-1 (ET-1), a potent vasoconstrictor and mitogen, is upregulated in pulmonary tissue during endotoxemia and contributes markedly to endotoxin-induced pulmonary hypertension. It is, however, unknown whether the ET receptors, ET(A) and ET(B), are differentially regulated in endotoxemic pulmonary vasculature and how this may impact on pulmonary vascular tone. To investigate this topic, we used isolated perfused lungs, pulmonary endothelial cells (ECs), and pulmonary vascular smooth muscle cells (SMCs) of the rat. During a 6-h endotoxin exposure, isolated perfused lungs developed significant pulmonary hypertension that was markedly attenuated by antagonizing ET(A) or ET(B) receptors using subtype-selective or a mixed ET(A/B) receptor antagonist. Peptide levels of big ET-1 and ET-1 and gene expression of prepro-ET-1 were increased after endotoxin challenge in all tissues. In endotoxemic isolated perfused lungs and ECs, the significant rise of mature ET-1 seen in controls after ET(B) receptor or mixed antagonism disappeared completely. However, this effect was preserved in endotoxemic SMCs. In ECs, endotoxin markedly downregulated maximum ET(B) receptor sites and ET(B) mRNA levels, whereas in SMCs, it generated substantial ET(B) receptor upregulation and moderate ET(A) receptor downregulation. The aldose reductase inhibitors sorbinil and zopolrestat mitigated endotoxin-induced pulmonary hypertension, ET-1 stimulation, and differential ET(B) receptor regulation. We conclude that endotoxin-induced pulmonary hypertension in the rat results from a loss of endothelial and concomitant gain of vascular smooth muscle ET(B) receptors. These changes are at least partly mediated by aldose reductase.

Role of IL-18 in overt pain-like behaviour in mice

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-γ, 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-γ −/− mice and by the treatment with bosentan (mixed endothelin ET A/ET B receptor antagonist), BQ 123 (cyclo[DTrp-DAsp-Pro-DVal-Leu], selective endothelin ET A receptor antagonist), BQ 788 ( N- cys-2,6 dimethylpiperidinocarbonyl- l-methylleucyl- d-1-methoxycarboyl- d-norleucine, selective endothelin ET B receptor antagonist) or indomethacin (cycloxigenase inhibitor). Thus, IL-18, IFN-γ, endothelin acting on endothelin ET A and ET B 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.

The endothelin system as a therapeutic target in cardiovascular disease: great expectations or bleak house?

There is considerable evidence that the potent vasoconstrictor endothelin-1 (ET-1) contributes to the pathogenesis of a variety of cardiovascular diseases. As such, pharmacological manipulation of the ET system might represent a promising therapeutic goal. Many clinical trials have assessed the potential of ET receptor antagonists in cardiovascular disease, the most positive of which have resulted in the licensing of the mixed ET receptor antagonist bosentan, and the selective ET(A) receptor antagonists, sitaxsentan and ambrisentan, for the treatment of pulmonary arterial hypertension (PAH). In contrast, despite encouraging data from in vitro and animal studies, outcomes in human heart failure have been disappointing, perhaps illustrating the risk of extrapolating preclinical work to man. Many further potential applications of these compounds, including resistant hypertension, chronic kidney disease, connective tissue disease and sub-arachnoid haemorrhage are currently being investigated in the clinic. Furthermore, experience from previous studies should enable improved trial design and scope remains for development of improved compounds and alternative therapeutic strategies. Although ET-converting enzyme inhibitors may represent one such alternative, there have been relatively few suitable compounds developed, and consequently, clinical experience with these agents remains extremely limited. Recent advances, together with an increased understanding of the biology of the ET system provided by improved experimental tools (including cell-specific transgenic deletion of ET receptors), should allow further targeting of clinical trials to diseases in which ET is involved and allow the therapeutic potential for targeting the ET system in cardiovascular disease to be fully realized.

Endothelin-1 and angiotensin II contribute to BNP but not c- fos gene expression response to elevated load in isolated mice hearts

The early events in the cardiac hypertrophic process induced by hemodynamic load include activation of B-type natriuretic peptide (BNP) and c- fos gene expression. However, it is unknown whether stretch acts directly or through local paracrine factors to trigger changes in cardiac gene expression. Herein we studied the involvement of endothelin-1 (ET-1) and angiotensin II (Ang II) in load-induced activation of left ventricular BNP and c- fos gene expression using an in vitro stretch model in isolated perfused adult mice hearts. Two-hour stretch induced by increasing coronary flow rate from 2 to 5 ml/min increased the expression of BNP and c- fos genes by 1.9- and 1.5-fold, respectively ( P < 0.001 and P < 0.05). A mixed ET A/B receptor antagonist bosentan attenuated the BNP gene expression response to load by 58% ( P < 0.005). A similar 53% inhibition was observed with the selective ET A receptor blocker BQ-123 ( P < 0.05). Type 1 Ang II receptor antagonist CV-11974 decreased the activation of BNP gene expression by 50% ( P < 0.05). In contrast, the activation of c- fos gene expression was not inhibited by antagonists of ET A/B and AT 1 receptors. Our results show that ET-1 and Ang II play a key role in the induction of BNP, but not c- fos gene expression in response to load in intact adult murine hearts.

Endothelin and the Ischaemic Heart

Soon after its identification as a powerful vasoconstrictor peptide, endothelin (ET-1) was implicated as a detrimental agent involved in determining the outcome of myocardial ischaemia and reperfusion. Early experimental studies demonstrated that ET(A) selective and mixed ET(A)/ET(B) receptor antagonists can reduce infarct size and prevent ischaemia-induced ventricular arrhythmias in models of ischaemia/reperfusion, implying that ET-1 acts through the ET(A) receptor to contribute to injury and arrhythmogenesis. However, as our understanding of the physiology of ET-1 has expanded, the role of ET-1 in the ischaemic heart appears ever more complex. Recent evidence suggests that ET-1 exerts actions on the heart that are not only detrimental (vasoconstriction, inhibition of NO production, activation of inflammatory cells), but which may also contribute to tissue repair, such as inhibition of cardiomyocyte apoptosis. In addition, ET-1-induced mast cell degranulation has been linked to a homeostatic mechanism that controls endogenous ET-1 levels, which may have important implications for the ischaemic heart. Furthermore the mechanism by which ET-1 promotes arrhythmogenesis remains controversial. Some studies imply a direct electrophysiological effect of ET-1, via ET(A) receptors, to increase monophasic action potential duration (MAPD) and induce early after-depolarisations (EADs), while other studies support the view that coronary constriction resulting in ischaemia is the basis for the generation of arrhythmias. Moreover, ET-1 can induce cardioprotection (precondition) against infarct size and ventricular arrhythmias, through as yet incompletely understood mechanisms. To enable us to identify the most appropriate means of targeting this system in a therapeutically meaningful way we need to continue to explore the physiology of ET-1, both in the normal and the ischaemic heart.

Endothelin upregulates the expression of vasopressin V 2 mRNA in the inner medullary collecting duct of the rat

Recent studies in our laboratory have demonstrated that bosentan, a mixed endothelin ET A/ET B receptor antagonist, prevented the upregulation of the arginine vasopressin (AVP) V 2 receptor in the inner medullary collecting duct (IMCD) of cardiomyopathic hamsters. These results suggested that endothelin-1 (ET-1) is involved in the upregulation of AVP V 2 receptors. Studies were performed to detect the effect of ET-1 on the expression of AVP V 2 receptors and the ET receptor mediating these effects within the IMCD of the rat. Rat IMCD tissue was isolated and incubated with the following: ET-1, or ET-1 in combination with ET A and ET B receptor antagonists BQ-123 and BQ-788, respectively, and sarafotoxin c (S6c), an ET B receptor-specific agonist. Tissue samples were then analyzed using quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blotting. ET-1 treatment resulted in increased V 2 mRNA from a control level of 186.8 ± 15.0 amol/μg total RNA to 430.7 ± 49.0 amol/μg total RNA ( P < .003). ET-1/ET A treatment resulted in no significant decrease in V 2 mRNA expression 335.0 ± 38.0 amol/μg total RNA. Whereas ET-1/ET B, and ET-1/ET B/ET A treatment resulted in V 2 mRNA approaching control 256.0 ± 15.0 amol/μg total RNA, and 215.6 ± 42.3 amol/μg total RNA. However, ET-3 treatment produced no significant changes in V 2 receptor mRNA expression. Sarafotoxin treatment corroborated both the ET-1 and ET receptor antagonist data, demonstrating striking significant increases in V 2 receptor mRNA and protein expression. S6c treatment increased V 2 mRNA expression from a control level of 199 ± 17.3 amol/μg total RNA to 284.3 ± 42.1 amol/μg total RNA ( P < 05). Western blotting revealed that changes in V 2 mRNA expression in the various treatment conditions were similar to changes in protein expression. Overall, these data indicate that in the IMCD ET-1 increases AVP V 2 receptor expression and these changes are mediated by the ET B receptor.

Endothelins contribute towards nociception induced by antigen in ovalbumin-sensitised mice.

1. The contribution of endogenous endothelins to nociceptive responses elicited by ovalbumin (OVA) in the hind-paw of mice sensitised to this antigen (50 microg OVA+5 mg Al(OH)(3), s.c., 14 days beforehand) was investigated. 2. Sensitised mice exhibited greater nocifensive responsiveness to intraplantar (i.pl.) OVA (total licking time over first 30 min: 85.2+/-14.6 s at 0.3 microg; 152.6+/-35.6 s at 1 microg) than nonsensitised animals (29.3+/-7.4 s at 1 microg). Nocifensive responses of sensitised mice to 0.3 microg OVA were inhibited by morphine (3 mg kg(-1), s.c.) or local depletion of mast cells (four daily i.pl. injections of compound 48/80). 3. Pretreatment with i.v. bosentan (mixed ET(A)/ET(B) receptor antagonist; 52 micromol kg(-1)) or A-122722.5 (selective ET(A) receptor antagonist; 6 micromol kg(-1)) reduced OVA-induced licking from 124.8+/-20.6 s to 45.7+/-13.0 s and 64.2+/-12.1 s, respectively, whereas A-192621.1 (selective ET(B) receptor antagonist; 25 micromol kg(-1)) enhanced them to 259.2+/-39.6 s. 4. Local i.pl. pretreatment with BQ-123 or BQ-788 (selective ET(A) or ET(B) receptor antagonists, respectively, each at 3 nmol) reduced OVA-induced licking (from 106.2+/-15.2 to 57.0+/-9.4 s and from 118.6+/-10.5 to 76.8+/-14.7 s, respectively). Sarafotoxin S6c (selective ETB receptor agonist, 30 pmol, i.pl., 30 min after OVA) induced nocifensive responses in OVA-sensitised, but not in nonsensitised, animals. 5. Compound 48/80 (0.3 microg, i.pl.) induced nocifensive responses per se and potentiated those induced by i.pl. capsaicin (0.1 microg). Treatment with BQ-123 (3 nmol, i.pl.) reduced only the hyperalgesic effect of compound 48/80, whereas BQ-788 (3 nmol) was ineffective. 6. Thus, immune-mediated Type I hypersensitivity reactions elicit mast cell- and endothelin-dependent nociception in the mouse hind-paw, which are mediated locally by both ET(A) and ET(B) receptors. The nocifensive response to antigen is amenable to blockade by systemic treatment with dual ET(A)/ET(B) or selective ET(A) receptor antagonists, but is sharply potentiated by systemic selective ET(B) receptor antagonist treatment. The apparently distinct roles played by ET(B) receptors in this phenomenon at local and other sites remain to be characterised.

Enrasentan, an antagonist of endothelin receptors.

Endothelins are powerful vasoconstrictor agents produced by endothelial cells and identified by Yanagisawa et al. in 1988. Two types of receptors for endothelins have been identified: ET(A) receptors are located on smooth muscle cells of the vascular wall and are responsible for endothelin-induced vasoconstriction while ET(B) receptors are located on endothelial cells and induce these cells to release NO and prostacyclin. Moreover, these peptides not only cause a potent and prolonged vasoconstriction but are also known to enhance cell proliferation and to stimulate extracellular matrix accumulation. High levels of plasma or tissue endothelins have been found in patients with heart failure, diabetes, stroke, primary pulmonary hypertension, liver cirrhosis and other diseases. Given these effects of endothelins, blocking their receptors might be a new way to reduce blood pressure and to treat other illnesses. Accordingly, many endothelin antagonists have been developed and evaluated in animals and humans. Enrasentan is a mixed ET(A) and ET(B) receptor antagonist with a higher affinity for ET(A) receptors, although it cannot be considered a selective antagonist. In an animal model of hypertension and cardiac hypertrophy the drug has reduced blood pressure, prevented cardiac hypertrophy and preserved myocardial function. In rats with hyperinsulinemia and hypertension enrasentan normalized blood pressure and prevented cardiac and renal damage. In rats with stroke the drug reduced the ischemic area in the brain. Enrasentan has been added to conventional treatment in patients with heart failure (NYHA Class 2-3) and no addictive effect of the drug has been observed. This is in contrast with results obtained in animal models and still has not been explained. In conclusion, many possible clinical applications can be suggested for this drug, but further studies are necessary to better evaluate its therapeutic efficacy.

Endothelin B receptors are expressed by astrocytes and regulate astrocyte hypertrophy in the normal and injured CNS.

The ability of mammalian central nervous system (CNS) neurons to survive and/or regenerate following injury is influenced by surrounding glial cells. To identify the factors that control glial cell function following CNS injury, we have focused on the endothelin B receptor (ET(B)R), which we show is expressed by the majority of astrocytes that are immunoreactive for glial acid fibrillary protein (GFAP) in both the normal and crushed rabbit optic nerve. Optic nerve crush induces a marked increase in ET(B)R and GFAP immunoreactivity (IR) without inducing a significant increase in the number of GFAP-IR astrocytes, suggesting that the crush-induced astrogliosis is due primarily to astrocyte hypertrophy. To define the role that endothelins play in driving this astrogliosis, artificial cerebrospinal fluid (CSF), ET-1 (an ET(A)R and ET(B)R agonist), or Bosentan (a mixed ET(A)R and ET(B)R antagonist) were infused via osmotic minipumps into noninjured and crushed optic nerves for 14 days. Infusion of ET-1 induced a hypertrophy of ET(B)R/GFAP-IR astrocytes in the normal optic nerve, with no additional hypertrophy in the crushed nerve, whereas infusion of Bosentan induced a significant decrease in the hypertrophy of ET(B)R/GFAP-IR astrocytes in the crushed but not in the normal optic nerve. These data suggest that pharmacological blockade of astrocyte ET(B)R receptors following CNS injury modulates glial scar formation and may provide a more permissive substrate for neuronal survival and regeneration.

Endothelin-3 induces both human and opossum gallbladder contraction mediated mainly by endothelin-B receptor subtype in vitro.

Endothelins are produced by gallbladder epithelial cells, suggesting a role in the regulation of gallbladder function. To characterize the effect of endothelin-3 (ET-3) on human and Australian possum gallbladder contractility and identify the receptor(s) involved. Human and possum gallbladder muscle strips were exposed to cumulative concentrations of ET-3 (10 pmol/L-100 nmol/L). Strips were pretreated with either tetrodotoxin (TTX) (1 micro mol/L), the selective ET receptor antagonists BQ-123 (ET(A)), BQ-788 (ET(B)), alone or together, or the mixed ET antagonist tezosentan (all 1 micro mol/L). Maximal changes in tone were measured and expressed as percentage of carbachol (100 micro mol/L)-induced tone. ANOVA was used for statistical analysis. Endothelin-3 induced a concentration-dependent increase in tone in both human and pos-sum strips (P < 0.05) and at 100 nmol/L represented 44.2 +/- 4.5% and 40.3 +/- 4.6% of carbachol-induced tone, respectively. The effect on human strips was TTX insensitive, whereas the possum concentration-response curve was shifted to the right. Individually, BQ-123 and BQ-788 shifted the human concentration-response curve to the right, but a greater inhibition by BQ-788 was achieved in the possum (P < 0.05). However, BQ-123 plus BQ-788 further reduced the ET-3 effect (P < 0.001) to a level comparable to that observed in the presence of tezosentan in both human and possum strips. Endothelin-3 produces potent gallbladder contraction in vitro, acting mainly via ET(B) receptors and also interacting with ET(A)receptors. The receptors are located on the smooth muscle, but in possum gallbladder, neural receptors may also be involved. These findings suggest that ET-3 may regulate motility of possum and human gallbladder.

Endothelin-1 and atherosclerosis: potential complications associated with endothelin-receptor blockade

Atherosclerotic vascular disease remains the single most prevalent cause of death and morbidity in the western world. Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that also possesses mitogenic activity on many cell types, including vascular smooth muscle cells. Raised plasma and tissue levels of ET-1 have been described in atherosclerosis in animal models and in man, suggesting that this peptide plays a pathophysiological role in this condition. Two main ET-1 receptors have been cloned (ET A and ET B). Mixed ET A/B and receptor subtype selective antagonists are now available. Since ET-1 is generally believed to be a ‘pathophysiological peptide’, we discuss the therapeutic potential of ET-1 antagonists in atherosclerosis and consider whether, at certain sites in this process, ET-1 may play a beneficial role. In such situations ET antagonism may be undesirable.

Differential effects of angiotensin II versus endothelin-1 inhibitions in hypertrophic left ventricular myocardium during transition to heart failure.

In view of their mutual crosstalk, the roles of angiotensin II (Ang II) and endothelin-1 (ET-1) in the myocardium are assumed to be synergistic and supplemental. In the phase of compensated left ventricular (LV) hypertrophy of Dahl salt-sensitive rats, Ang II peptide and the ACE mRNA in the LV were increased by 1.6- and 3.8-fold, respectively. In contrast, ET-1 peptide and the preproET-1 mRNA remained unchanged. In subsequent congestive heart failure (CHF), Ang II and ACE mRNA did not show further increases. But ET-1 and the mRNA were increased de novo by 5.3- and 4.1-fold, respectively. In ascending aorta-banded rats, the local activations of Ang II and ET-1 also showed a differential time course between LV hypertrophy and CHF. Long-term treatments of Dahl salt-sensitive rats with temocapril (an ACE inhibitor) and with bosentan (a mixed ET receptor blocker) equally improved long-term survival. Temocapril reduced the LV/body weight ratio and ameliorated LV fractional shortening. Conversely, although bosentan equally improved fractional shortening, it did not reduce the increase in LV mass. Combined treatment with these 2 drugs further ameliorated the animal's survival without additional decreases in systolic pressure. The pathophysiological roles in the myocardium during the transition to CHF differ qualitatively between Ang II and ET-1. Thus, long-term therapy with a combination of ACE inhibition and ET antagonism may provide a new approach for heart failure in humans.

Effect of an ET(B)-selective and a mixed ET(A/B) endothelin receptor antagonist on venomotor tone in deoxycorticosterone-salt hypertension.

Because the ET(B) receptor is important in venoconstriction, we examined the effects of a selective ET(B) receptor antagonist (A-1 92621) and a mixed ET(A/B) receptor antagonist (A-182086) on endogenous endothelin-1 (ET-1) contributions to elevated venomotor tone in deoxycorticosterone acetate-salt (DOCA-salt) hypertension. Changes in venomotor tone were assessed using repeated measurements of mean circulatory filling pressure (MCFP) in awake, uninephrectomized, DOCA-salt-treated rats and uninephrectomized sham rats following intravenous (i.v.) injections of the ET(B) antagonist (12 mg/kg i.v.) or the ET(A/B) antagonist (12 mg/kg i.v.) alone, or 1 h before ganglion blockade with hexamethonium (30 mg/kg i.v.). DOCA-salt rats were hypertensive and exhibited higher MCFP than sham normotensive rats. The ET(A/B) receptor antagonist lowered mean arterial blood pressure (MABP) in DOCA-salt and sham rats, but MCFP fell in DOCA-salt rats only. The ET(B) antagonist produced no changes in MCFP while MABP increased in both groups. Pre-treatment of DOCA-salt rats, but not sham rats, with either antagonist produced greater declines in MCFP following hexamethonium than after hexamethonium alone. The present study confirms previous findings of elevated MCFP in DOCA-salt hypertensive rats compared to normotensive rats, but is the first to show that venomotor tone is affected by the actions of endogenous ET-1 acting at ET(B) receptors to modulate sympathetic input to the veins, as well as direct actions of ET-1 on vascular smooth muscle (VSM) ET(A) receptors. We also showed that mixed ET(A/B) receptor antagonism was effective in lowering MCFP and MABP in DOCA-salt hypertensive rats.

Endotoxin treatment causes an upregulation of the endothelin system in the liver: amelioration of increased portal resistance by endothelin receptor antagonism.

Mechanisms underlying hepatic microcirculatory failure during endotoxemia are incompletely understood. Because endothelin-1 (ET-1) has been implicated in endotoxin-induced liver injury, we investigated the hepatic ET-1 system in endotoxin-treated rats. Rats were treated with endotoxin (Escherichia coli lipopolysaccharide; 3 mg/kg, i.p.), and various determinations were made 24 h later. Endotoxin treatment caused 11.2 +/- 1.6% weight loss, a decrease in mean arterial pressure (MAP; 96 +/- 5 mmHg vs 108 +/- 3 mmHg; P < 0.05) and an increase in portal pressure (11.6 +/- 1.3 mmHg vs 7.4 +/- 1 mmHg; P < 0.02). No significant changes in the serum levels of liver enzymes or hepatocellular necrosis were observed. Endotoxin caused increases in hepatic ET-1 (from 345 +/- 31 to 565 +/- 38 pg/g; P < 0.01), ET-1 receptor density (from 179 +/- 16 to 340 +/- 26 fmol/mg; P < 0.02), and mRNA expression of preproendothelin-1, and ET(A) and ET(B) receptors. While the serum nitric oxide (nitrite +/- nitrate) concentration was increased in endotoxin-treated rats, that of ET-1 remained unchanged. A mixed ET(A)/ET(B) receptor antagonist, TAK-044 (10 mg/kg, i.v.), reduced the weight loss from 11.2 +/- 1.6% to 5.9 +/- 2.9% (P < 0.05) and the portal pressure from 11.6 +/- 1.3 mmHg to 8.6 +/- 0.7 mmHg (P < 0.05) in endotoxin-treated rats. The mixed ET(A)/ET(B) receptor antagonist also caused an increase in serum ET-1 concentration, but did not affect serum nitric oxide and MAP in endotoxin-treated rats. These results suggest that the upregulated hepatic ET-1 system is an important mechanism of increased portal resistance and related complications of endotoxemia.

Role of endothelin in the control of peripheral vascular tone in human hypertension.

Endothelins are potent 21 amino acid vasoconstrictor isopeptides produced in different vascular tissues, including vascular endothelium. Endothelin-1 is the main endothelin generated by the endothelium and probably the most important in the cardiovascular system. Endothelin-1 acts through specific receptors termed ET(A), represented only on smooth muscle cells and having the function of growth promotion and mediating contractions, and ET(B), located both on smooth muscle cells, where they evoke contractions, and on endothelial cells, inducing relaxation by production of the endothelium-derived relaxing factor nitric oxide. In physiological conditions endothelin-1 administration causes vasodilation and vasoconstriction at low and high concentrations, respectively. However, administration of mixed ET(A)/(B) receptor antagonists causes slight or absent vasodilation, indicating that the direct vasoconstrictor effect of the peptide is probably masked by ET(B)-induced NO-dependent vasodilation. In essential hypertensive patients, the activity of exogenous endothelin-1 is either increased, similar or decreased as compared to normotensive subjects, depending on which vascular district or scheme of administration is considered. But although available evidence does not indicate increased endothelin-1 plasma levels in patients with essential hypertension, simultaneous antagonism of ET(A)/(B) receptors causes a greater degree of vasodilation in hypertensives than in normotensive subjects. Moreover administration of a selective ET(B) receptor antagonist causes vasoconstriction in normotensive subjects and vasodilation in essential hypertensive patients. Finally, the vasodilating effect of a mixed ET(A)/(B) receptor antagonist is inversely related to NO availability. Taken together these findings suggest that essential hypertension is characterized by increased endothelin-1 vasoconstrictor tone. This alteration seems to be dependent on decreased endothelial ET(B)-mediated NO production attributable to impaired NO availability. In such conditions endothelial ET(B)-induced vasodilation no longer compensates for the direct classical endothelin vasoconstrictor effect mediated by smooth muscle cell ET(A) and ET(B) receptors. Therefore endothelin-1 could potentially be involved in the pathogenesis of essential hypertension or of its complications, and blockade of this system is a fascinating new target for therapeutic intervention in this disease.

Heterozygous knock-Out of ET(B) receptors induces BQ-123-sensitive hypertension in the mouse.

Homozygous knock-out of ET(A) or ET(B) receptor genes results in lethal developmental phenotypes in the mouse. Such deleterious phenotypes do not occur in heterozygous littermates. However, it remains to be determined whether mice partially defective in ET(A) or ET(B) receptors display significant alterations in their responses to exogenous or endogenous endothelin-1 (ET-1). Furthermore, the anesthetized ET(B) (+/-) knock-out mice showed a significantly higher mean arterial blood pressure than the ET(A) (+/-) knock-out or their wild-type littermates. The pressor response to ET-1 but not to a selective ET(B) agonist, IRL-1620, was significantly reduced in the ET(A) (+/-) knock-out mice. In ET(B) (+/-) knock-out mice, the pressor effect of IRL-1620 was more markedly altered than those induced by ET-1. In wild-type mice, both ET(A) and ET(B) receptors were found to be involved in the pressor effect of ET-1, as confirmed by the significant and specific antagonism induced by either BQ-123 (ET(A) antagonist) or BQ-788 (ET(B) antagonist). Also, ET(A)-selective or mixed ET(A)/ET(B)- but not ET(B)-selective antagonists reversed the hypertensive state of the ET(B) (+/-) knock-out mice to the level of wild-type littermates. Finally, radiolabeled ET-1 plasmatic clearance was altered in ET(B) (+/-) but not ET(A) (+/-) knock-out mice when compared with wild-type animals. Thus, heterozygous knock-out of ET(B) receptors results in a hypertensive state, suggesting an important physiological role for that particular receptorial entity in opposing the endogenous ET-1-dependent pressor effects in the mouse.

Effect of endothelin antagonists, including the novel ET(A) receptor antagonist LBL 031, on endothelin-1 and lipopolysaccharide-induced microvascular leakage in rat airways.

1. The effect of the novel ET(A) receptor antagonist LBL 031 and other selective and mixed endothelin receptor antagonists on endothelin-1 (ET-1)-induced and lipopolysaccharide (LPS)-induced microvascular leakage was assessed in rat airways. 2. Intravenously administered ET-1 (1 nmole kg(-1)) or LPS (30 mg kg(-1)) caused a significant increase in microvascular leakage in rat airways when compared to vehicle treated animals. 3. Pre-treatment with the selective ET(A) receptor antagonists, LBL 031 or PD 156707, or the mixed ET(A/B) receptor antagonist, bosentan (each at 30 mg kg(-1)), reduced ET-1-induced leakage to baseline levels. ET-1-induced leakage was not reduced by pre-treatment with the ET(B) selective antagonist BQ 788 (3 mg kg(-1)). 4. Pre-treatment with the selective ET(A) receptor antagonist, LBL 031 (0.1 mg kg(-1)) or PD 156707 (10 mg kg(-1)), or the mixed ET(A/B) receptor antagonist, bosentan (30 mg kg(-1)), reduced LPS-induced leakage by 54, 48 and 59% respectively. LPS-induced leakage was not affected by pre-treatment with the ET(B) selective antagonist BQ 788 (3 mg kg(-1)). 5. The data suggests that ET-1-induced microvascular leakage in the rat airway is ET(A) receptor mediated and that part of the increase induced by LPS may be due to the actions of ET-1. Therefore, a potent ET(A) receptor selective antagonist, such as LBL 031, may provide a suitable treatment for inflammatory diseases of the airways, especially those involving LPS and having an exudative phase, such as the septic shock-induced adult respiratory distress syndrome.

Involvement of the Endothelin System in Experimental Critical Hind Limb Ischemia

Endothelin- (ET-1) is involved in the pathogenesis of several ischemic diseases. We investigated the hypotheses that ET-1 is involved in the pathogenesis of experimental critical hind limb ischemia and that ET-1 receptor antagonists have a protective effect. Critical hind limb ischemia was achieved by exclusion of the femoral artery and embolization of collateral vessels in rats. The induction of endothelin system components by ischemia was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) (mRNAs) and immunoassay (peptides) in the plasma and ischemic muscles 5 hr (H5), 5 days (D5) and 14 days (D14) after ischemia. Two groups of rats received 100 mg/kg/day of either Bosentan, a mixed ET(A/B) receptor antagonist (n = 12), or LU 135252, a selective ET(A) receptor antagonist (n = 9), and a control group without treatment (n = 12) served as control. Muscle blood flow and ischemia were monitored in the ischemic limb by laser Doppler and phosphorylase activity, respectively. The procedure induced an 80% decrease in muscle blood flow and complete suppression of phosphorylase activity without necrosis. At day 14, the tissue blood flow remained reduced by 70% and phosphorylase activity was suppressed completely. There was up-regulation of preproendothelin-1, preproET-3, endothelin converting enzyme-1, and ET(A). ET(B) receptor mRNAs in ischemic muscle at day 5 and day 14 was accompanied by an increase in muscle concentration of ET-1 at day 5, without significant changes in plasma endothelin. Treatment with Bosentan and LU 135252 increased tissue blood flow and reduced muscle ischemia at day 14. Tissue production of ET- 1 is up-regulated in experimental critical hind limb ischemia. Inhibition of the endothelin system by a mixed ET(A/B) receptor antagonist may protect, at least in part, against muscle injury.

Potent nonpeptide endothelin antagonists: synthesis and structure–activity relationships of pyrazole-5-carboxylic acids

We have previously reported the identification of pyrazole-5-carboxylic acids as a new class of endothelin antagonists from low affinity pyrazol-5-ol ligands, which were obtained by random screening assays. 1 We describe herein the synthesis and the structure–activity relationships (SARs) of these pyrazole-5-carboxylic acids with potent ET A selective, mixed ET A/ET B or moderately ET B selective antagonist activities.