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Endothelin-1 (ET-1), an endogenous vasoactive peptide, has been found to play an important role in peripheral pain signaling. Acid-sensing ion channels (ASICs) are key sensors for extracellular protons and contribute to pain caused by tissue acidosis. It remains unclear whether an interaction exists between ET-1 and ASICs in primary sensory neurons. In this study, we reported that ET-1 enhanced the activity of ASICs in rat dorsal root ganglia (DRG) neurons. In whole-cell voltage-clamp recording, ASIC currents were evoked by brief local application of pH 6.0 external solution in the presence of TRPV1 channel blocker AMG9810. Pre-application with ET-1 (1−100 nM) dose-dependently increased the proton-evoked ASIC currents with an EC50 value of 7.42 ± 0.21 nM. Pre-application with ET-1 (30 nM) shifted the concentration–response curve of proton upwards with a maximal current response increase of 61.11% ± 4.33%. We showed that ET-1 enhanced ASIC currents through endothelin-A receptor (ETAR), but not endothelin-B receptor (ETBR) in both DRG neurons and CHO cells co-expressing ASIC3 and ETAR. ET-1 enhancement was inhibited by blockade of G-protein or protein kinase C signaling. In current-clamp recording, pre-application with ET-1 (30 nM) significantly increased acid-evoked firing in rat DRG neurons. Finally, we showed that pharmacological blockade of ASICs by amiloride or APETx2 significantly alleviated ET-1-induced flinching and mechanical hyperalgesia in rats. These results suggest that ET-1 sensitizes ASICs in primary sensory neurons via ETAR and PKC signaling pathway, which may contribute to peripheral ET-1-induced nociceptive behavior in rats.

Skeletal muscle insulin resistance is a hallmark of individuals with type 2 diabetes mellitus (T2D). In healthy individuals insulin stimulates vasodilation, which is markedly blunted in T2D; however, the mechanism(s) remain incompletely understood. Investigations in rodents indicate augmented endothelin-1 (ET-1) action as a major contributor. Human studies have been limited to young obese participants and focused exclusively on the ET-1 A (ETA) receptor. Herein, we have hypothesized that ETA receptor antagonism would improve insulin-stimulated vasodilation and glucose uptake in T2D, with further improvements observed during concurrent ETA + ET-1 B (ETB) antagonism. Arterial pressure (arterial line), leg blood flow (LBF; Doppler), and leg glucose uptake (LGU) were measured at rest, during hyperinsulinemia alone, and hyperinsulinemia with (1) femoral artery infusion of BQ-123, the selective ETA receptor antagonist (n = 10 control, n = 9 T2D) and then (2) addition of BQ-788 (selective ETB antagonist) for blockade of ETA and ETB receptors (n = 7 each). The LBF responses to hyperinsulinemia alone tended to be lower in T2D (controls: ∆161 ± 160 mL/minute; T2D: ∆58 ± 43 mL/minute, P = .08). BQ-123 during hyperinsulinemia augmented LBF to a greater extent in T2D (% change: controls: 14 ± 23%; T2D: 38 ± 21%, P = .029). LGU following BQ-123 increased similarly between groups (P = .85). Concurrent ETA + ETB antagonism did not further increase LBF or LGU in either group. Collectively, these findings suggest that during hyperinsulinemia ETA receptor activation restrains vasodilation more in T2D than controls while limiting glucose uptake similarly in both groups, with no further effect of ETB receptors (NCT04907838).

The present study investigated whether endothelin-1 acts via ETA or ETB receptors to mediate superoxide anion-induced pain and inflammation. Mice were treated with clazosentan (ETA receptor antagonist) or BQ-788 (ETB receptor antagonist) prior to stimulation with the superoxide anion donor, KO2. Intraplantar treatment with 30 nmol of clazosentan or BQ-788 reduced mechanical hyperalgesia (47% and 42%), thermal hyperalgesia (68% and 76%), oedema (50% and 30%); myeloperoxidase activity (64% and 32%), and overt-pain like behaviours, such as paw flinching (42% and 42%) and paw licking (38% and 62%), respectively. Similarly, intraperitoneal treatment with 30 nmol of clazosentan or BQ-788 reduced leukocyte recruitment to the peritoneal cavity (58% and 32%) and abdominal writhing (81% and 77%), respectively. Additionally, intraplantar treatment with clazosentan or BQ-788 decreased spinal (45% and 41%) and peripheral (47% and 47%) superoxide anion production as well as spinal (47% and 47%) and peripheral (...

The present study was aimed to explore the role of endothelins in remote preconditioning (RP)-induced myocardial protection in ischemia-reperfusion (IR) injury. RP stimulus was given by subjecting hind limb to four cycles of ischemia and reperfuion (5 minutes each) using blood pressure cuff in male rats. Following RP, hearts were isolated and subjected to 30 minutes of ischemia and 120 minutes of reperfusion on Langendorff apparatus. The extent of myocardial injury was determined by measuring the levels of LDH-1, CK-MB and cardiac troponin T (cTnT) in coronary effluent; caspase-3 activity and Bcl 2 expression in heart (apoptosis); infarct size by triphenyl tetrazolium chloride and contractility parameters including left ventricular developed pressure, dp/dtmax dp/dtmin and heart rate. RP reduced ischemia reperfusion-induced myocardial injury, increased the levels of endothelin 1 (in blood), Akt-P, GSK-3β-P and P-connexin 43 (in hearts). Pretreatment with ETA receptor antagonist, BQ 123 (1 and 2 mg/kg), ETB receptor antagonist, BQ 788 (1 and 3 mg/kg) and dual inhibitor of ETA and ETB receptor, bonsentan (25 and 50 mg/kg) abolished these effects of RP. However, the effects of bonsentan were more pronounced in comparison to BQ 123 and BQ 788. It is concluded that RP stimulus may release endothelin 1 in the blood, which may activate myocardial ETA and ETB receptors to trigger cardioprotection through connexin 43 and Akt/GSK-3β pathway.

This study assessed whether endothelin-1 (ET-1) helps mediate postischemic acute kidney injury (AKI) progression to chronic kidney disease (CKD). The impact(s) of potent ETA or ETB receptor-specific antagonists (Atrasentan and BQ-788, respectively) on disease progression were assessed 24 h or 2 weeks following 30 min of unilateral ischemia in CD-1 mice. Unilateral ischemia caused progressive renal ET-1 protein/mRNA increases with concomitant ETA, but not ETB, mRNA elevations. Extensive histone remodeling consistent with gene activation and increased RNA polymerase II (Pol II) binding occurred at the ET-1 gene. Unilateral ischemia produced progressive renal injury as indicated by severe histologic injury and a 40% loss of renal mass. Pre- and post-ischemia or just postischemic treatment with Atrasentan conferred dramatic protective effects such as decreased tubule/microvascular injury, normalized tissue lactate, and total preservation of renal mass. Nuclear KI-67 staining was not increased by Atrasentan, implying that increased tubule proliferation was not involved. Conversely, ETB blockade had no protective effect. Thus, our findings provide the first evidence that ET-1 operating through ETA can have a critical role in ischemic AKI progression to CKD. Blockade of ETA provided dramatic protection, indicating the functional significance of these results.

Recent evidence implicates endothelin in nociception, but it is unclear how endothelin activates trigeminal ganglion (TRG) neurons. In the present study, we investigated the expression of the endothelin receptors ETA and ETB and endothelin-induced responses in rat TRG neurons. Double-immunofluorescence studies demonstrated that ETA and ETB were expressed in TRG neurons and that 26% of ETA- or ETB-expressing neurons expressed both receptors. During whole-cell patch-clamp recording, endothelin-1 enhanced an induced current in response to capsaicin, a TRPV1 agonist, in approximately 20% of dissociated neurons. The enhancement was blocked by the PKC inhibitor chelerythrine and by the ETA antagonist BQ-123, but not by the ETB antagonist BQ-788. Ca2+-imaging showed that endothelin-1 increased the intracellular Ca2+ concentration in more than 20% of the dissociated neurons. Importantly, unlike the effect of endothelin-1 on capsaicin-induced current, the Ca2+ response was largely suppressed by BQ-788 but not by BQ-123. These results suggest that ETA-mediated TRPV1 hyperactivation via PKC activation and ETB-mediated Ca2+ mobilization occurs in different subsets of TRG neurons. These endothelin-induced responses may contribute to the induction of orofacial pain. The ETB-mediated function in TRG neurons is a special feature in the trigeminal system because of no ETB expression in dorsal root ganglion neurons.

Endothelin-1 induces contractions of human oviduct ampullar segment in follicular phase of menstrual cycle, acting on ET(A) receptors. The aim of our study was to investigate effects of endothelin-1, endothelin-2 and endothelin-3 on isolated ampullar segment of human oviducts, taken from the patients in luteal phase of menstrual cycle. Fallopian tubes were taken from 20 female patients (one tube from each patient) during abdominal hysterectomy with adnexectomy, due to extensive uterine fibroids. The oviduct ampulla was mounted in an organ bath longitudinally, and the tension of the isolated preparation was recorded with the isometric transducer. Endothelin-1 produced concentration-dependent tonic contraction of the isolated ampullar segment (EC(50)=6.80 +/- 1.2 x 10(-10)M), and concentration-dependent inhibition of its rhythmic contractions (EC(50)=7.86 +/- 2.3 x 10(-10)M). Endothelin-2 produced concentration-dependent tonic contraction of the isolated ampullar segment (EC(50)=4.56 +/- 0.3 x 10(-10)M), without affecting its rhythmic contractions. Endothelin-3 did not affect either tone or rhythmic contractions of the isolated preparations. Selective antagonist for ET(A) receptor subtype, BQ 123, produced inhibition of endothelin-1 effects on both tone (pA(2)=9.50) and spontaneous rhythmic contractions (pA(2)=10.73), while selective antagonist for ET(B) receptor subtype, BQ 788, produced only inhibition of endothelin-1 effects on tone (pA(2)=9.61), while the effect of endothelin-1 on spontaneous rhythmic contractions remained unaffected. The results of our study suggest that in the luteal phase both ET(A) and ET(B) receptors regulate tone, and only ET(A) receptors regulate rhythmic activity of human oviduct's ampullar segment.

Our previous work showed that ET-1 induced a concentration-dependent increase of cytosolic Ca2+ ([Ca]c) and nuclear Ca2+ ([Ca]n) in human aortic vascular smooth muscle cells (hVSMCs). In the present study, using hVSMCs and 3-dimensional confocal microscopy coupled to the Ca2+ fluorescent probe Fluo-3, we showed that peptidic antagonists of ETA and ETB receptors (BQ-123 (10−6 mol/L) and BQ-788 (10−7 mol/L), respectively) prevented, but did not reverse, ET-1-induced sustained increase of [Ca]c and [Ca]n. In contrast, nonpeptidic antagonists of ETA and ETB (respectively, BMS-182874 (10−8–10−6 mol/L) and A-192621 (10−7 mol/L)) both prevented and reversed ET-1-induced sustained increase of [Ca]c and [Ca]n. Furthermore, activation of the ETB receptor alone using the specific agonist IRL-1620 (10−9 mol/L) induced sustained increases of [Ca]c and [Ca]n, and subsequent administration of ET-1 (10−7 mol/L) further increased nuclear Ca2+. ET-1-induced increase of [Ca]c and [Ca]n was completely blocked by extracellular application of the Ca2+ chelator EGTA. Pretreatment with the G protein inhibitors pertussis toxin (PTX) and cholera toxin (CTX) also prevented the ET-1 response; however, strong membrane depolarization with KCl (30 mmol/L) subsequently induced sustained increase of [Ca]c and [Ca]n. Pretreatment of hVSMCs with either the PKC activator phorbol-12,13-dibutyrate or the PKC inhibitor bisindolylmaleimide did not affect ET-1-induced sustained increase of intracellular Ca2+. These results suggest that both ETA- and ETB-receptor activation contribute to ET-1-induced sustained increase of [Ca]c and [Ca]n in hVSMCs. Moreover, in contrast to the peptidic antagonists of ET-1 receptors, the nonpeptidic ETA-receptor antagonist BMS-182874 and the nonpeptidic ETB-receptor antagonist A-192621 were able to reverse the effect of ET-1. Nonpeptidic ETA- and ETB-receptor antagonists may therefore be better pharmacological tools for blocking ET-1-induced sustained increase of intracellular Ca2+ in hVSMCs. Our results also suggest that the ET-1-induced sustained increase of [Ca]c and [Ca]n is not mediated via activation of PKC, but via a PTX- and CTX-sensitive G protein calcium influx through the R-type Ca2+ channel.

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.

In rats, nitric oxide modulates renal autoregulation in steady-state experiments and the myogenic mechanism in dynamic studies. Interactive modulation of autoregulation by nitric oxide and endothelin-1, predominantly involving endothelin B receptors, has been reported although it remains unclear whether the interaction is synergistic or obligatory or whether it affects the myogenic component of autoregulation. Nonselective inhibition of nitric oxide synthase (Lω-nitro-l-arginine methyl-ester; l-NAME) with endothelin A and B selective receptor antagonists BQ-123 and BQ-788, all infused into the renal artery, plus time series analysis were used to test the interactive actions of nitric oxide and endothelin on renal vascular conductance and on autoregulation. Nonselective endothelin receptor antagonism blunted the constrictor response to subsequent l-NAME but had no effect on previously established l-NAME-induced vasoconstriction. BQ-123 did not affect conductance and caused only minor reduction in myogenic autoregulatory efficiency. Responses to BQ-123 and l-NAME were additive and not interactive. BQ-788 and l-NAME each caused strong vasoconstriction alone and in the presence of the other, indicating that coupling between nitric oxide- and endothelin B-mediated events is not obligatory. l-NAME augmented myogenic autoregulation, and subsequent BQ-788 did not alter this response. However, BQ-788 infused alone also enhanced myogenic autoregulation but resulted in significant impairment of myogenic autoregulation by subsequent l-NAME. Thus the interaction between nitric oxide and endothelin is clearly nonadditive and, because it is asymmetrical, cannot be explained simply by convergence on a common signal pathway. Instead one must postulate some degree of hierarchical organization and that nitric oxide acts downstream to endothelin B activation.

We describe characteristics of a selective endothelin (ET) ET(B) receptor antagonist, BQ-788 [N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D-1-methoxycarbonyltryptophanyl-D-norleucine], which is widely used to demonstrate the role of endogenous or exogenous ETs in vitro and in vivo. In vitro, BQ-788 potently and competitively inhibited (125)I-labeled ET-1 binding to ET(B) receptors in human Girrardi heart cells (hGH) with an IC(50) of 1.2 nM, but only poorly inhibited the binding to ET A receptors in human neuroblastoma cell line SK-N-MC cells (IC(50), 1300 nM). In isolated rabbit pulmonary arteries, BQ-788 showed no agonistic activity up to 10 microM and competitively inhibited the vasoconstriction induced by an ET(B)-selective agonist (pA(2), 8.4). BQ-788 also inhibited several bioactivities of ET-1, such as bronchoconstriction, cell proliferation, and clearance of perfused ET-1. Thus, it is confirmed that BQ-788 is a potent, selective ET(B) receptor antagonist. In vivo, in conscious rats, BQ-788, 3 mg/kg/h, i.v., completely inhibited a pharmacological dose of ET-1- or sarafotoxin6c (S6c) (0.5 nmol/kg, i.v.)-induced ET(B) receptor-mediated depressor, but not pressor responses. Furthermore, BQ-788 markedly increased the plasma concentration of ET-1, which is considered an index of potential ET(B) receptor blockade in vivo. In Dahl salt-sensitive hypertensive (DS) rats, BQ-788, 3 mg/kg/h, i.v., increased blood pressure by about 20 mm Hg. It is reported that BQ-788 also inhibited ET-1-induced bronchoconstriction, tumor growth and lipopolysaccharide-induced organ failure. These data suggest that BQ-788 is a good tool for demonstrating the role of ET-1 and ET(B) receptor subtypes in physiological and/or pathophysiological conditions.

We investigated the difference in the characteristics of endothelin-1 (ET-1)-induced contraction and the responses of intracellular Ca(2+) concentration ([Ca(2+)](i)) between rat small pulmonary artery and renal artery. ET-1 (30 nM) failed to elicit any contraction in renal arteries pretreated with 3 microM BQ-123, an ETA blocker. However, in the pulmonary artery a combination of BQ-123 and BQ-788, an ETB blocker (5 microM each), only partially inhibited the ET-1-induced contraction (by 25%). To focus on the ETA receptor, in the presence of 5 microM BQ-788, nitric oxide donors (sodium nitroprusside and (+/-)-S-nitroso-N-acetylpenicillamine) and forskolin reduced both the ET-1-induced contraction and increase in [Ca(2+)](i) in both pulmonary and renal arteries. However, the effects were stronger in the renal than in the pulmonary artery. ET-1-induced increase in [Ca(2+)](i) was only partially attenuated by 10 microM verapamil (to 81% of control) in pulmonary arteries but was reduced to 56.1% of control in renal arteries. Our results provide evidence that ET-1 may activate ET receptor(s) insensitive to both BQ-123 and BQ-788 in rat small pulmonary artery, at least under these conditions. Furthermore, the effects of relaxants such as L-type Ca(2+) channel blocker and nitric oxide donors on the ET-1-induced contraction were studied.

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 ETA and ETB receptor subtype. ETA/B (with ET-1) and ETA (ET-1 in the presence of BQ-788) receptor activation resulted in a significant reduction in both epi- and endocardial monophasic action potential duration (MAPD90). ETA receptor activation reduced both epi- and endocardial effective refractory period (ERP). This MAPD90 and ERP shortening were associated with a reduction in coronary flow, myocardial contractility and induction of ventricular fibrillation (VF) during ERP measurement. The ETB agonist sarafotoxin (S6c) had no marked, or concentration-dependent, effect on MAPD90, 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 MAPD90, ERP or the % incidence of VF. In conclusion, neither ETA nor ETB 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 ETA receptor stimulation resulting in myocardial ischaemia. Keywords: Endothelin-1, monophasic action potential duration, working heart, rabbit, sarafotoxin, BQ-788 Introduction Endothelin-1 (ET-1) levels have been shown to be increased during myocardial ischaemia in humans (Miyauchi et al., 1989; Yasuda et al., 1990), pigs (Wang et al., 1995) and rabbits (Vitola et al., 1996). Exogenous administration of ET-1 results in myocardial ischaemia in dogs through its potent coronary vasoconstrictor ability (Salvati et al., 1991) and causes ventricular arrhythmias (Merkely et al., 1998). Antagonists at ET-1 receptors protect against ischaemia-induced arrhythmias (Horkay et al., 2000; Kiss et al., 2000), supporting the view that ET-1 may cause myocardial ischaemia and play a role in the resultant arrhythmogenesis. ET-1 has also been shown to exhibit a direct arrhythmogenic effect, which is independent of its ability to cause myocardial ischaemia (Yorikane et al., 1991; Toth et al., 1995). However, the mechanism(s) that may underlie any such direct effect have not been clarified. Becker et al. (2000) showed that intracoronary administration of ET-1 induced ventricular arrhythmias that were not associated with changes in refractory periods or conduction delay, such changes usually being associated with myocardial ischaemia (Fozzard & Makielski, 1985). On the other hand, Szabo et al. (2000) reported that ET-1 induced arrhythmias and prolonged left ventricular (LV) monophasic action potential duration (MAPD) in dogs. It has been suggested that a mechanism, which may contribute to ET-1's direct arrhythmogenic effect, is regional heterogeneity in the electrophysiological effects of ET-1, increased dispersion of action potential duration and refractoriness and the generation of early after-depolarisations (EADs) (Geller et al., 1998; Duru et al., 2001). Thus, there exists controversy in the literature about the extent to which the arrhythmogenic effect of ET-1 is a consequence of the induction of myocardial ischaemia or a direct electrophysiological effect, and, if direct, what the mechanism is. To date, there have been no studies that have looked at the effect of ET-1 on dispersion of action potential duration and refractoriness; therefore, one of the aims of the current study is to determine if ET-1 modifies dispersion in such a way as to explain the arrhythmogenic effects of ET-1. ET-1 acts as an agonist at both ETA and ETB receptors. The arrhythmogenic action of exogenous ET-1, the MAPD prolongation and EAD formation has been shown to be blocked by the ETA selective antagonist, Lu 135.252, suggesting that these effects are mediated via ETA receptors (Kiss et al., 2000). In contrast, stimulation of ETB receptors with the selective agonist, sarafotoxin (S6c), exerted an antiarrhythmic action on ischaemia-induced arrhythmias in vivo (Crockett et al., 2000). Although ETB receptors are found within the heart predominantly in the conducting system and endocardium (Molenaar et al., 1993), no studies appear to have examined what electrophysiological effects they produce in whole hearts, if any such effects are more pronounced in the endo- vs the epicardium and if these could explain an antiarrhythmic action of ETB stimulation. The aims of this study were to use an isolated working rabbit heart model in which MAPD90, the effective refractory period (ERP) and conduction delay could be measured in both the endocardium and epicardium and correlated with haemodynamic variables to answer the following questions. Does ET-1, under normal and ischaemic conditions, have a direct arrhythmogenic effect that is independent of its ability to cause coronary vasoconstriction, and what is the contribution of ETA and ETB receptors to any observed effect? To address the latter question, studies were carried out using the selective ETB receptor agonist, S6c, and the mixed agonist, ET-1. Since there is no potent selective ETA agonist, the effects of ET-1 on ETA receptors were determined in the presence of the ETB antagonist, BQ-788 (Ishikawa et al., 1994). Regional myocardial ischaemia results in dispersion of action potential duration and refractoriness between the ischaemic and nonischaemic areas and it is thought that this dispersion contributes to the increased arrhythmogenicity. Therefore, in the current study, we used a regional model of ischaemia in order to determine if ET-1 or S6c modified this ischaemia-induced dispersion.

The participation of endothelins (ETs) in a model of neutrophil-dependent lung injury induced by intrabronchial instillation of rabbit antibodies to ovalbumin followed by i.v. injection of the antigens (Arthus reaction) was investigated. Hemorrhagic lesions were evaluated by measuring the extravasations of hemoglobin in lung parenchyma. From 5 min to 24 h after the Arthus reaction (AR), endothelin (ir-ET) levels in bronchoalveolar lavage fluid (BALF) and in plasma were measured by radioimmunoassay. BALF levels of ir-ET were not different between control and AR animals for the first 90 min after the antigen challenge but increased from 2 to 24 h after induction of AR. ET levels in the plasma did not change from the respective controls over the same 24 h period. Increased ir-ET in BALF was not affected by pretreatment with L-NAME (30 mg/kg, i.v.). A PAF antagonist (BN52021; 5 and 10 mg/kg, i.v.) increased ET content in BALF and decreased the intensity of the AR. Thiorphan (2 mg/kg, i.v.) inhibited the AR-induced hemorrhagic lesions in lungs. An ET(A) receptor antagonist, BQ-123 (1 mg/kg, i.v.) potentiated, whereas the ET(B) antagonist, BQ-788 (1 mg/kg, i.v.) inhibited the lung hemorrhage. It is concluded that ETs are released during and play a role in the lung AR.

The adrenal gland is highly vascularized with tightly regulated blood flow that is closely correlated with steroidogenesis. Mechanisms involved in the regulation of adrenal blood flow and vascular tone are largely unknown. The present study characterizes the contractile responses of isolated small cortical arteries from bovine adrenal glands. In endothelium-intact arteries, K(+), the thromboxane mimetic U46619, 5-hydroxytryptamine (5-HT), and endothelin-1 (ET-1) induced concentration-dependent contractions, whereas phenylephrine, norepinephrine, and ACTH were without effect. The EC(50)s for K(+), U46619, 5-HT, and ET-1 were 45 +/- 3 mm, 150 +/- 24 nm, 370 +/- 38 nm, and 2.8 +/- 0.8 nm, respectively. Contractions induced by U46619, 5-HT, and ET-1 were blocked by the thromboxane receptor antagonist SQ 29,548, the 5-HT(2A) receptor antagonist ketanserin, and the ET(A) receptor antagonist BQ 123, respectively. Removal of the endothelium caused a marked leftward shift of concentration responses to high K(+), U46619, 5-HT, and ET-1, and revealed contractile responses to phenylephrine and norepinephrine. In U46619-preconstricted arteries, BQ 123 converted ET-1-induced contractions to relaxations (maximal relaxation of 57 +/- 8%), which were subsequently blocked by the ET(B) receptor antagonist BQ 788. The ET(B)-mediated relaxations were endothelium dependent and inhibited by the nitric oxide synthase inhibitor N-nitro-l-arginine, the cytochrome P450 inhibitor SKF 525A, and high extracellular K(+), but not by the cyclooxygenase inhibitor indomethacin. These results demonstrate that small adrenal cortical arteries are highly responsive to various vasoconstrictor agents. The forceful contractile responses of these arterioles are consistent with their potential role in the regulation of adrenal blood flow.

Vasospasm in the vascular pedicle is a major cause of ischemic necrosis in autogenous skin transplantation (i.e., skin free flap surgery), and the pathophysiology is unclear. The clinical impression is that veins are more susceptible to vasospasm than arteries in the vascular pedicle of skin free flaps. The purpose of this study was to compare the vasoconstrictor response of the human radial artery (RA) and radial vein (RV) to endothelin (ET)-1 and to investigate the mechanism mediating ET-1-induced vasoconstriction. The isometric tension of RA and RV rings (4 mm) obtained from the vascular pedicle of human radial forearm skin free flaps were studied in organ chambers containing Krebs bicarbonate buffer. It was observed that ET-1 elicited concentration-dependent (5 x 10 (-11)to 2 x 10 (-8) ) contractions in RA and RV rings with similar contractile potency. However, the concentration-dependent contractile response to ET-1 was significantly (P < 0.05) higher in RV rings than in RA rings, with the maximum contractile response twice as high in RV rings than in RA rings. The contractile response to ET-1 in RA and RV rings was blocked by the ET receptor antagonist BQ 123 (10 (-5M)), but not by the ET receptor antagonist BQ 788 (5 x 10 (-6)). The ET(B) receptor agonist BQ 3020 (10 (-10) to 2 x 10(-8) ) had no significant contractile effect in RA and RV rings. Furthermore, the L-type Ca channel antagonist nifedipine (5 x 10 (-6)), the protein kinase C (PKC) inhibitor chelerythrine (10(-5M)), and the intracellular Ca chelator BAPTA-AM (10(-5M)) significantly reduced the contractile potency of ET-1 in RA rings and the maximum contractile response to ET-1 in RA and RV rings. It was concluded that the human RV is more responsive than RA to the contractile effect of ET-1. The contractile response to ET-1 in RA and RV is predominantly mediated by ET(A) receptors and the postreceptor mechanism involves L-type Ca (2+) channels, PKC, and intracellular Ca(2+).

The aim of the present study was to assess the role of endothelin (ET) in ischemia-reperfusion (I/R)-induced mucosal injury. Mucosal permeability (51Cr-EDTA clearance) and tissue myeloperoxidase (MPO) activity were significantly increased after 30 min of ischemia followed by 30 min of reperfusion. The I/R-induced increases in mucosal permeability and polymorphonuclear leukocyte (PMN) infiltration were significantly attenuated by pretreatments with ETA (BQ-485) and/or ETB (BQ-788) receptor antagonists. Monoclonal antibody (MAb) directed against intercellular adhesion molecule-1 (ICAM-1; MAb 1A29) and superoxide dismutase (SOD) pretreatments significantly attenuated the increased mucosal permeability and PMN infiltration in a similar manner as with ET receptor antagonists. Superior mesenteric artery blood flow was significantly reduced during the reperfusion period. Both ET receptor antagonists caused a significant rise in blood flow compared with an untreated I/R group. In conclusion, our data suggest that ETA and/or ETB receptors, ICAM-1, and superoxide play an important role in I/R-induced mucosal dysfunction and PMN infiltration. Furthermore, ET is involved in the pathogenesis of postreperfusion-induced damage and beneficial effects of ET receptor antagonism are related to an improvement of disturbed blood flow during the reperfusion period.

Autoregulation of renal blood flow (RBF) is an essential function of the renal microcirculation that has been previously shown to be blunted by excessive dietary salt. Endogenous endothelin 1 (ET-1) is increased following a high-salt (HS) diet and contributes to the control of RBF but the differential effects of ET-1 on renal microvessel autoregulation in response to HS remain to be established. We hypothesized that a HS diet increases endothelin receptor activation in normal Sprague-Dawley rats and blunts autoregulation of RBF. The role of ET-1 in the blunted autoregulation produced by a HS diet was assessed in vitro and in vivo using the blood-perfused juxtamedullary nephron preparation and anesthetized rats, respectively. Using highly selective antagonists, we observed that blockade of either ETAor ETBreceptors was sufficient to restore normal autoregulatory behavior in afferent arterioles from HS-fed rats. Additionally, normal autoregulatory behavior was restored in vivo in HS-fed rats by simultaneous ETAand ETBreceptor blockade, whereas blockade of ETBreceptors alone showed significant improvement of normal autoregulation of RBF. Consistent with this observation, autoregulation of RBF in ETBreceptor-deficient rats fed HS was similar to both ETB-deficient rats and transgenic control rats on normal-salt diets. These data support the hypothesis that endogenous ET-1, working through ETBand possibly ETAreceptors, contributes to the blunted renal autoregulatory behavior in rats fed a HS diet.

SUMMARY 1. Many diseases are associated with elevated endothelin (ET)-1 plasma concentrations. In order to understand the consequence of this elevation, in the present study the effects of exogenous ET-1 on the entire organsim were investigated, in particular with respect to the role of ETA and ETB receptors in the cardiovascular system. In open-chest rabbits, left ventricular (LV) pressure (LVPmax, LVPed), dP/dtmax and dP/dtmin were recorded in ejecting and isovolumically beating hearts to determine cardiac function. In addition, heart rate (HR), aortic pressure (AoP) and aortic flow (AoF) were measured. Total peripheral resistance (TPR) was calculated from mean AoP and AoF. 2. In the first series of experiments (n = 11), ET-1 (0.5 nmol/kg; bolus) produced a non-significant reduction in HR. Systolic function, in terms of AoF, LVPmax and dP/dtmax, was improved; for example, LVPmax was increased significantly (69 ± 10 vs 106 ± 20 mmHg for control and ET-1, respectively; P < 0.05). Similarly, early relaxation (dP/dtmin) was improved. In parallel, TPR rose significantly (0.25±0.07 vs 0.35±0.1 mmHg/min per mL for control and ET-1, respectively; P < 0.05). Isovolumic measurements showed corresponding responses. 3. In the second series of experiments (n = 7), animals were pretreated with an ETA receptor antagonist (330 nmol/min per kg FR 139317). After ETA receptor blockade, the administration of ET-1 had no significant effect on cardiac function or vasomotion. 4. In the third series of experiments (n = 6), animals were pretreated with an ETB receptor antagonist (10 nmol/min per kg BQ 788). In this series of experiments, the effects of ET-1 on cardiac function and vasomotion were the same as in the first series of experiments, except for the effect on HR, which decreased by 35% after ET-1. 5. In our experimental model, exogenous ET-1 exerted a clear-cut positive inotropic effect, together with the anticipated peripheral vasoconstriction via ETA receptors.

We sought to examine the effects of endothelin-1 on the intracellular free Ca(2+) concentration ([Ca(2+)](i)) and mitogenic response in the neuroblastoma cell line, B103 (B103 cells). The results obtained from an [125I] endothelin-1 binding assay demonstrated that B103 cells express the endothelin receptor. The B(max) and K(d) values for [125I]endothelin-1 binding were 70+/-36 fmol/mg protein and 52+/-13 pM, respectively. Endothelin-1 failed to stimulate cAMP formation, but it did inhibit forskolin-induced cAMP formation. Endothelin-1 also stimulated the accumulation of [3H]inositol phosphates. These results indicate that the endothelin receptor in B103 cells couples with G(i) and G(q) but not with G(s). Monitoring of [Ca(2+)](i) showed that endothelin-1 evoked a transient increase in [Ca(2+)](i); this remained even in the absence of extracellular Ca(2+). However, no sustained, endothelin-1-induced increase in [Ca(2+)](i) due to extracellular Ca(2+) influx was detected. The endothelin B receptor-selective antagonist, 2,6-Dimethylpiperidinecarbonyl-gamma-Methyl-Leu-N(in)-[Methoxycarbonyl]-D-Trp-D-Nle (BQ 788), abolished the endothelin-1-induced increase in [Ca(2+)](i), while the endothelin ET(A) receptor-selective antagonist, cyclo-D-Asp-Pro-D-Val-Leu-D-Trp (BQ 123), failed to inhibit it. These results indicate that B103 cells express endothelin ET(B) receptor or an endothelin ET(B)-like receptor predominantly and have no Ca(2+) channels activated by endothelin-1. Endothelin-1 activated mitogen-activated protein kinase in B103 cells. However, based on the data for 3-(4,5-dimethy-2-thiazolyl)-2,5-diphenyl tetrazolium bromide, [3H]thymidine incorporation, and apoptosis screening assays, endothelin-1 induces neither mitogenesis nor apoptosis. These results suggest that endothelin-1 has no role in the mitogenic response in B103 cells, and this is consistent with the notion that an endothelin-1-induced sustained increase in [Ca(2+)](i) plays a role in endothelin-1-induced cell proliferation.

Aims Endothelin-1 (ET-1) is a potent vasoconstrictor produced by the vascular endothelium. The interactions of ET with the mediators of the sympathetic nervous system and the renin-angiotensin-system in humans are unclear. Methods We studied the effects of the ETA-selective antagonist BQ-123 and the ETB-selective antagonist BQ-788 (both 10−10−10−8m) on ET-1 (10−16−10−10m), angiotensin II (AT, 10−16−10−10m) and noradrenaline (NA, 10−16−10−10m) induced vasoconstriction in the human skin microcirculation in vivo in 25 healthy male volunteers using laser Doppler flowmetry and double injection technique. Results BQ-123 caused a dose-dependent vasodilatation (maximum effect: + 949 ± 84 AUC-PU, P

The proliferative effects of endothelin-1 (ET-1), both alone and in combination with epidermal growth factor (EGF), and the effect of nitric oxide (NO) on the cell proliferation were investigated in cultured guinea pig bronchial smooth muscle cells. ET-1 (10-100 nM) alone augmented cell proliferation, and was additive to the effect of EGF (0.48 nM) in a concentration-dependent manner. An ET(A) antagonist, BQ-123 (10 microM), reduced the cell-proliferative effect of ET-1, whereas an ET(B) antagonist, BQ-788 (10 microM), did not influence the effect. A NO donor, SIN-1 (10 nM-1 microM), reduced the cell-proliferative effect of ET-1 in a concentration-dependent manner. The effect of SIN-1 (1 microM) was partly, but significantly, reversed by a soluble guanylyl cyclase inhibitor, ODQ (1 microM). These results suggest that ET-1 acts not only as a co-mitogen with EGF but also as a mitogen alone, and that its action is mediated through activation of ET(A) receptors. Therefore, ET-1 may contribute to airway remodeling, a pathophysiological hallmark of asthma. In addition, NO, which is produced mainly in the airway epithelium and is partly mediated through cGMP-dependent pathway, may reduce the phenomenon.

The effect of the novel ETA 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 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. Pre-treatment with the selective ETA receptor antagonists, LBL 031 or PD 156707, or the mixed ETA/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 ETB selective antagonist BQ 788 (3 mg kg−1). Pre-treatment with the selective ETA receptor antagonist, LBL 031 (0.1 mg kg−1) or PD 156707 (10 mg kg−1), or the mixed ETA/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 ETB selective antagonist BQ 788 (3 mg kg−1). The data suggests that ET-1-induced microvascular leakage in the rat airway is ETA receptor mediated and that part of the increase induced by LPS may be due to the actions of ET-1. Therefore, a potent ETA 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. British Journal of Pharmacology (2000) 131, 1129–1134; doi:10.1038/sj.bjp.0703691

The role of endothelin in the initial vasoconstrictor step of hyperacute xenogeneic rejection was investigated. Isolated rat livers were perfused in recirculation. Perfusion with human sera provided an ex vivo model of hyperacute rejection in a discordant combination. Perfusion of 10% xenogeneic serum induced a marked (70%) and sustained reduction of the liver flow and induced the release of endothelin into the perfusion medium. In contrast, perfusion of 10% allogeneic serum or of 10% decomplemented human serum induced a weak (25%) and transient reduction of the liver flow and induced the release of minimal amounts of endothelin. The simultaneous administration of BQ 123 and BQ 788, the respective antagonists of ETA and ETB endothelin receptors, or that of bosentan, a mixed ETA/ETB antagonist, antagonized the vasoconstrictor effect of 10% xenogeneic human serum, as well as that of 10−9 M endothelin-1. The vasoconstrictor effects of xenogeneic serum on liver circulation are, at least partly, mediated through the release of endothelin by the graft. British Journal of Pharmacology (2000) 130, 402–408; doi:10.1038/sj.bjp.0703295

We examined the participation of endothelin ET A and ET B receptors in modulation by endothelin-1 of adrenal catecholamine secretion during cholinergic activation in pentobarbital-anesthetized dogs. Drugs were infused intra-arterially into the adrenal gland. Splanchnic nerve stimulation (1 and 3 Hz) increased adrenal catecholamine output in a frequency-dependent manner. Endothelin-1 (0.2, 0.6, and 2 ng/kg/min) enhanced the catecholamine response induced by the 3-Hz nerve stimulation. Under pretreatment with an endothelin ET A receptor antagonist ( R )-2-[( R )-2-[( S )-2-[[1-(hexahydro-1 H -azepinyl)]carbonyl]amino-4-methylpentanoyl]amino-3-(2-pyridyl) propionic acid (FR139317) (1 μg/kg/min), endothelin-1 suppressed the 1- and 3- Hz nerve stimulation-induced catecholamine response in a dose-dependent manner. No inhibitory or facilitatory effect of endothelin-1 was observed under simultaneous pretreatment with FR139317 and an endothelin ET B receptor antagonist N - cis 2,6-dimethylpiperidinocarbonyl- l -γ-methylleucyl- d -1-methoxycarbonyltryptophanyl- d -norleucine (BQ-788) (1 μg/kg/min) or under pretreatment with BQ-788 alone. These results suggest that in the dog adrenal gland, endothelin-1 facilitates and inhibits adrenal catecholamine secretion during cholinergic activation by stimulating endothelin ET A and ET B receptors, respectively.