ET-1-induced pulmonary vasoconstriction shifts from ET(A)- to ET(B)-receptor-mediated reaction after preconstriction.

Endothelin-1 (ET-1) has been reported to induce pulmonary vasoconstriction via either ET(A) or ET(B) receptors, and vasorelaxation after ET-1 injection has been observed. Our study investigated the effects of ET-1 in isolated rabbit lungs, which were studied at basal tone (part I) and after preconstriction (U-46619; part II). Pulmonary arterial pressure (PAP) and lung weight gain were monitored continuously. In part I, ET-1 (10(-8) M; n = 6; control) was injected after pretreatment with the ET(A)-receptor antagonist BQ-123 (10(-6) M; n = 6) or the ET(B)-receptor antagonist BQ-788 (10(-6) M; n = 6). The same protocol was carried out in part II after elevation of pulmonary vascular tone. ET-1 induced an immediate PAP increase (DeltaPAP 4.3 +/- 0.4 mmHg at 10 min) that was attenuated by pretreatment with BQ-123 (P < 0.05 at 10 min and P < 0.01 thereafter) and that was more pronounced after BQ-788 (P < 0.01 at 10 min and P < 0.001 thereafter). In part II, ET-1 induced an immediate rise in PAP with a maximum after 5 min (DeltaPAP 6.3 +/- 1.4 mmHg), leveling off at DeltaPAP 3.2 +/- 0.2 mmHg after 15 min. Pretreatment with BQ-123 failed to attenuate the increase. BQ-788 significantly reduced the peak pressure at 5 min (0.75 +/- 0.4 mmHg; P < 0.001) as well as the plateau pressure thereafter (P < 0.01). We conclude that ET-1 administration causes pulmonary vasoconstriction independent of basal vascular tone, and, at normal vascular tone, the vasoconstriction seems to be mediated via ET(A) receptors. BQ-788 treatment resulted in even more pronounced vasoconstriction. After pulmonary preconstriction, ET(A) antagonism exerted no effects on PAP, whereas ET(B) antagonism blocked the PAP increase. Therefore, ET-1-induced pulmonary vasoconstriction is shifted from an ET(A)-related to an ET(B)-mediated mechanism after pulmonary vascular preconstriction.     

Insulin sensitivity and big ET-1 conversion to ET-1 after ETA- or ETB-receptor blockade in humans.

Cardiovascular diseases are characterized by insulin resistance and elevated endothelin (ET)-1 levels. Furthermore, ET-1 induces insulin resistance. To elucidate this mechanism, six healthy subjects were studied during a hyperinsulinemic euglycemic clamp during infusion of (the ET-1 precursor) big ET-1 alone or after ET(A)- or ET(B)-receptor blockade. Insulin levels rose after big ET-1 with or without the ET(B) antagonist BQ-788 (P < 0.05) but were unchanged after the ET(A) antagonist BQ-123 + big ET-1. Infused glucose divided by insulin fell after big ET-1 with or without BQ-788 (P < 0.05). Insulin and infused glucose divided by insulin values were normalized by ET(A) blockade. Mean arterial blood pressure rose during big ET-1 with or without BQ-788 (P < 0.001) but was unchanged after BQ-123. Skeletal muscle, splanchnic, and renal blood flow responses to big ET-1 were abolished by BQ-123. ET-1 levels rose after big ET-1 (P < 0.01) in a similar way after BQ-123 or BQ-788, despite higher elimination capacity after ET(A) blockade. In conclusion, ET-1-induced reduction in insulin sensitivity and clearance as well as splanchnic and renal vasoconstriction are ET(A) mediated. ET(A)-receptor stimulation seems to inhibit the conversion of big ET-1 to ET-1.     

Hemodynamic and proinflammatory actions of endothelin-1 in guinea pig small intestine submucosal microcirculation

The hemodynamic and proinflammatory effects of endothelin-1 (ET-1) in proximal (1st/2nd order) and terminal (3rd/4th order) arterioles and venules were examined in small intestine submucosa of anesthetized guinea pigs. Vessel diameter (D), red blood cell velocity, and blood flow (Q) were determined in eight proximal and eight terminal microvessels before and at 20 min of ET-1 suffusion (10(-10), 10(-9), and 10(-8) M) and then with endothelin-A (ET(A))-receptor blockade with BQ-123 (10(-5) M). This protocol was repeated with platelet-activating factor (PAF) inhibition (WEB-2086, 1.0 mg/kg iv; n = 16). The ET-1-mediated microvascular responses were also examined with endothelin-B (ET(B))-receptor blockade using BQ-788 (10(-5) M; n = 11) alone or with ET(A+B)-receptor blockade with BQ-123 + BQ-788 (n = 10). Microvascular permeability was assessed by FITC-albumin (25 mg/kg iv) extravasation in seven series: 1) buffered modified Krebs solution suffusion (n = 6), 2) histamine suffusion (HIS; 10(-3) M, n = 5), 3) ET-1 suffusion (10(-8) M, n = 5), 4) BQ-123 (10(-5) M) plus ET-1 suffusion (n = 5), 5) PAF inhibition before ET-1 suffusion (n = 5), 6) histamine-1 (H1)-receptor blockade (diphenhydramine, 20 mg/kg iv) before ET-1 suffusion (n = 5), and 7) ET(B)-receptor blockade before (BQ-788 10(-5) M; n = 3) or with ET-1 suffusion (n = 3). D and Q decreased at 10(-8) M ET-1 and returned to control values with BQ-123 and BQ-123+BQ788 but not with BQ-788 in proximal microvessels. D did not change in terminal microvessels with ET-1 (10(-8) M) but decreased with BQ-788 and increased with BQ-123. PAF inhibition did not affect the D and Q responses of proximal microvessels to ET-1 but prevented the fall in Q in terminal microvessels with ET-1. ET-1 increased vascular permeability to approximately 1/3 of that with HIS; this response was prevented with BQ-123 and WEB-2086 but not with H1-receptor blockade. This is the first evidence that submucosal terminal microvessel flow is reduced with ET-1 independent of vessel diameter changes and that this response is associated with increased microvascular permeability mediated via ET(A)-receptor stimulation and PAF activation.     

Endothelin ET(A) but not ET(B) receptors mediate contraction of common bile duct

Endothelin (ET) causes contraction of the gallbladder. To investigate effects of ET in the common bile duct, we measured contraction of longitudinal muscle strips from guinea pig common bile ducts induced by ET-related peptides and binding of 125I-ET-1 to cell membranes prepared from the common bile duct. Visualization of 125I-ET-1 binding sites in tissue was performed by autoradiography. ET-1 caused tetrodotoxin and atropine-insensitive contraction. In terms of maximal tension of contraction, ET-1, ET-2 and ET-3 were equal in efficacy. However, sarafotoxin S6c, a selective ET(B) receptor agonist, caused only a negligible contraction. The relative potencies for ET isopeptides to cause contraction were ET-1=ET-2>ET-3. The ET-1-induced contraction was inhibited by BQ-123, an ET(A)-receptor-selective antagonist, but not by BQ-788, an ET(B)-receptor-selective antagonist. In addition, the combination of both antagonists, BQ-123 and BQ-788, inhibited ET-1 induced contraction but did not potentiate the inhibition caused by BQ-123 alone. These indicate that ET(A) but not ET(B) receptors mediate the contraction. Autoradiography localized 125I-ET-1 binding to the smooth muscle layer. Binding of 125I-ET-1 to the smooth muscle cell membranes was saturable and specific. Analysis of dose-inhibition curves indicated the presence of ET(A) and ET(B) receptors. These results demonstrate that ET causes contraction of longitudinal muscle of the common bile duct. Different from the gallbladder, which possesses both ET(A) and ET(B) receptors cooperating to mediate muscle contraction, the common bile duct possesses two classes of ET receptors, but only the ET(A) receptor mediates the contraction.     

Endothelin type A receptor antagonist normalizes blood pressure in rats exposed to eucapnic intermittent hypoxia

We have reported that eucapnic intermittent hypoxia (E-IH) causes systemic hypertension, elevates plasma endothelin 1 (ET-1) levels, and augments vascular reactivity to ET-1 and that a nonspecific ET-1 receptor antagonist acutely lowers blood pressure in E-IH-exposed rats. However, the effect of chronic ET-1 receptor inhibition has not been evaluated, and the ET receptor subtype mediating the vascular effects has not been established. We hypothesized that E-IH causes systemic hypertension through the increased ET-1 activation of vascular ET type A (ET(A)) receptors. We found that mean arterial pressure (MAP) increased after 14 days of 7 h/day E-IH exposure (109 +/- 2 to 137 +/- 4 mmHg; P < 0.005) but did not change in sham-exposed rats. The ET(A) receptor antagonist BQ-123 (10 to 1,000 nmol/kg iv) acutely decreased MAP dose dependently in conscious E-IH but not sham rats, and continuous infusion of BQ-123 (100 nmol.kg(-1).day(-1) sc for 14 days) prevented E-IH-induced increases in MAP. ET-1-induced constriction was augmented in small mesenteric arteries from rats exposed 14 days to E-IH compared with those from sham rats. Constriction was blocked by the ET(A) receptor antagonist BQ-123 (10 microM) but not by the ET type B (ET(B)) receptor antagonist BQ-788 (100 microM). ET(A) receptor mRNA content was greater in renal medulla and coronary arteries from E-IH rats. ET(B) receptor mRNA was not different in any tissues examined, whereas ET-1 mRNA was increased in the heart and in the renal medulla. Thus augmented ET-1-dependent vasoconstriction via vascular ET(A) receptors appears to elevate blood pressure in E-IH-exposed rats.     

The role of ET(A) and ET(B) receptor antagonists in acute and allergic inflammation in mice

In this study, we investigated the effects of the selective ET(A) (BQ-123) and ET(B) (BQ-788) receptor antagonists for endothelin-1 (ET-1) against several flogistic agent-induced paw edema formation and ovalbumin-induced allergic lung inflammation in mice. The intraplantar injection of BQ-123, but not BQ-788, significantly inhibited carrageenan-, PAF-, ET-1- and bradykinin-induced paw edema formation. The obtained inhibitions (1h after the inflammatory stimulus) were 79+/-5%, 55+/-4%, 55+/-6% and 74+/-4%, respectively. In carrageenan-induced paw edema, the mean ID(50) value for BQ-123 was 0.77 (0.27-2.23)nmol/paw. The neutrophil influx induced by carrageenan or PAF was reduced by BQ-123, with inhibitions of 55+/-2% and 72+/-4%, respectively. BQ-123 also inhibited the indirect macrophage influx induced by carrageenan (55+/-6%). However, BQ-788 failed to block the cell influx caused by either of these flogistic agents. When assessed in the bronchoalveolar lavage fluid in a murine model of asthma, both BQ-123 and BQ-788 significantly inhibited ovalbumin-induced eosinophil recruitment (78+/-6% and 71+/-8%), respectively. Neither neutrophil nor mononuclear cell counts were significantly affected by these drugs. Our findings indicate that ET(A), but not ET(B), selective ET-1 antagonists are capable of preventing the acute inflammatory responses induced by carrageenan, PAF, BK and ET-1. However, both ET(A) and ET(B) receptor antagonists were found to be effective in inhibiting the allergic response in a murine model of asthma.     

Central endothelin ET(B) receptors mediate IL-1-dependent fever induced by preformed pyrogenic factor and corticotropin-releasing factor in the rat

Blockade of central endothelin ET(B) receptors inhibits fever induced by LPS in conscious rats. The contribution of ET(B) receptor-mediated mechanisms to fever triggered by intracerebroventricular IL-6, PGE2, PGF(2alpha), corticotropin-releasing factor (CRF), and preformed pyrogenic factor derived from LPS-stimulated macrophages (PFPF) was examined. The influence of natural IL-1 receptor antagonist or soluble TNF receptor I on endothelin (ET)-1-induced fever was also assessed. The selective ET(B) receptor antagonist BQ-788 (3 pmol icv) abolished fever induced by intracerebroventricular ET-1 (1 pmol) or PFPF (200 ng) and reduced that caused by ICV CRF (1 nmol) but not by IL-6 (14.6 pmol), PGE2 (1.4 nmol), or PGF(2alpha) (2 nmol). CRF-induced fever was also attenuated by bosentan (dual ET(A)/ET(B) receptor antagonist; 10 mg/kg iv) but unaffected by BQ-123 (selective ET(A) receptor antagonist; 3 pmol icv). alpha-Helical CRF(9-41) (dual CRF1/CRF2 receptor antagonist; 6.5 nmol icv) attenuated fever induced by CRF but not by ET-1. Human IL-1 receptor antagonist (9.1 pmol) markedly reduced fever to IL-1beta (180 fmol) or ET-1 and attenuated that caused by PFPF or CRF. Murine soluble TNF receptor I (23.8 pmol) reduced fever to TNF-alpha (14.7 pmol) but not to ET-1. The results of the present study suggest that PFPF and CRF recruit the brain ET system to cause ET(B) receptor-mediated IL-1-dependent fever.     

Endothelin-1 and pancreatic islet vasculature: studies in vivo and on isolated, vascularly perfused pancreatic islets

Endothelin-1 (ET-1) is a potent endothelium-derived vasoconstrictor, which also stimulates insulin release. The aim of the present study was to evaluate whether exogenously administered ET-1 affected pancreatic islet blood flow in vivo in rats and the islet arteriolar reactivity in vitro in mice. Furthermore, we aimed to determine the ET-receptor subtype that was involved in such responses. When applying a microsphere technique for measurements of islet blood perfusion in vivo, we found that ET-1 (5 nmol/kg) consistently and markedly decreased total pancreatic and especially islet blood flow, despite having only minor effects on blood pressure. Neither endothelin A (ET(A)) receptor (BQ-123) nor endothelin-B (ET(B)) receptor (BQ-788) antagonists, alone or in combination, could prevent this reduction in blood flow. To avoid confounding interactions in vivo, we also examined the arteriolar vascular reactivity in isolated, perfused mouse islets. In the latter preparation, we demonstrated a dose-dependent constriction in response to ET-1. Administration of BQ-123 prevented this, whereas BQ-788 induced a right shift in the response. In conclusion, the pancreatic islet vasculature is highly sensitive to exogenous ET-1, which mediates its effect mainly through ET(A) receptors.     

Endothelium-derived reactive oxygen species and endothelin-1 attenuate NO-dependent pulmonary vasodilation following chronic hypoxia

Vasodilatory responses to exogenous nitric oxide (NO) are diminished following exposure to chronic hypoxia (CH) in isolated, perfused rat lungs. We hypothesized that both endothelium-derived reactive oxygen species (ROS) and endothelin-1 (ET-1) mediate this attenuated NO-dependent pulmonary vasodilation following CH. To test this hypothesis, we examined vasodilatory and vascular smooth muscle (VSM) Ca2+ responses to the NO donor spermine NONOate in UTP-constricted, isolated pressurized small pulmonary arteries from control and CH rats. Consistent with our previous findings in perfused lungs, we observed attenuated NO-dependent vasodilation following CH in endothelium-intact vessels. However, in endothelium-denuded vessels, responses to spermine NONOate were augmented in CH rats compared with controls, thus demonstrating an inhibitory influence of the endothelium on NO-dependent reactivity following CH. Whereas both the ROS scavenger tiron and the ETA receptor antagonist BQ-123 augmented NO-dependent reactivity in endothelium-intact vessels from CH rats, neither fully restored vasodilatory responses to those observed following endothelium denudation in vessels from CH rats. In contrast, the combination of tiron and BQ-123 or the nonselective ET receptor antagonist PD-145065 enhanced NO responsiveness in endothelium-intact vessels from CH rats similar to that observed following endothelium denudation. We conclude that both endothelium-derived ROS and ET-1 attenuate NO-dependent pulmonary vasodilation following CH. Furthermore, CH augments pulmonary VSM reactivity to NO.     

Endothelin-1 induces proliferation of human lung fibroblasts and IL-11 secretion through an ET(A) receptor-dependent activation of MAP kinases

Endothelin-1 (ET-1) is implicated in the fibrotic responses characterizing interstitial lung diseases, as well as in the airway remodeling process occurring in asthma. Within such a context, the aim of our study was to investigate, in primary cultures of normal human lung fibroblasts (NHLFs), the ET-1 receptor subtypes, and the intracellular signal transduction pathways involved in the proliferative effects of this peptide. Therefore, cells were exposed to ET-1 in the presence or absence of an overnight pre-treatment with either ET(A) or ET(B) selective receptor antagonists. After cell lysis, immunoblotting was performed using monoclonal antibodies against the phosphorylated, active forms of mitogen-activated protein kinases (MAPK). ET-1 induced a significant increase in MAPK phosphorylation pattern, and also stimulated fibroblast proliferation and IL-6/IL-11 release into cell culture supernatants. All these effects were inhibited by the selective ET(A) antagonist BQ-123, but not by the specific ET(B) antagonist BQ-788. The stimulatory influence of ET-1 on IL-11, but not on IL-6 secretion, was prevented by MAPK inhibitors. Therefore, such results suggest that in human lung fibroblasts ET-1 exerts a profibrogenic action via an ET(A) receptor-dependent, MAPK-mediated induction of IL-11 release and cell proliferation.     

ETA and ETB receptors are expressed in vascular adventitial fibroblasts

The adventitia has been recognized to play important roles in vascular oxidative stress, remodeling, and contraction. We recently demonstrated that adventitial fibroblasts are able to express endothelin (ET)-1 in response to ANG II. However, it is unclear whether ET-1 receptors are expressed in the adventitia. We therefore investigated the expression and roles of both ET(A) and ET(B) receptors in collagen synthesis and ET-1 clearance in adventitial fibroblasts. Adventitial fibroblasts were isolated and cultured from the mouse thoracic aorta by the explant method. Cultured cells were treated with ANG II (100 nmol/l) or ET-1 (10 pM) in the presence or absence of the ANG II type 1 receptor antagonist losartan (100 μM), the ET-1 receptor antagonists BQ-123 (ET(A) receptor, 1 μM) and BQ-788 (ET(B) receptor, 1 μM), and the ET(B) receptor agonist sarafotoxin 6C (100 nM). ET-1 peptide levels were determined by ELISA, whereas ET(A), ET(B), and collagen levels were determined by Western blot analysis. ANG II increased ET-1 peptide levels in a time-dependent manner. ANG II increased ET(A) and ET(B) receptor protein levels as well as collagen in a similar fashion. ANG II-induced collagen was reduced while in the presence of BQ-123, suggesting a role for the ET(A) receptor in the regulation of the extracellular matrix. ANG II treatment in the presence of BQ-788 significantly increased ET-1 peptide levels. Conversely, the ET(B) receptor agonist sarafotoxin 6C significantly decreased ET-1 peptide levels. These data implicate a role for the ET(B) receptor in the clearance of the ET-1 peptide. In conclusion, both ET(A) and ET(B) receptors are expressed in adventitial fibroblasts, which paves the ground for the biological significance of adventitial ET-1. The ET(A) receptor subtype mediates collagen I expression, whereas the ET(B) receptor subtype may play a protective role through increasing the clearance of the ET-1 peptide.     

Receptor subtype mediating the action of circulating endothelin on glucose metabolism and hemodynamics in perfused rat liver.

The subtype of endothelin receptor that mediates metabolic and hemodynamic effects of circulating endothelin was explored using perfused rat liver. Infusion of endothelin (ET)-1 or ET-3 into the portal vein at a concentration of 0.3 nM increased glucose and lactate output and decreased perfusion flow, although ET-3 was less effective than ET-1. The metabolic effects of ET-1 were observed even under costant-flow perfusion. Infusion of either sarafotoxin S6b or S6c, an ET(A)- or ET(B)-receptor agonist, mimicked the actions of ET-1 to an equal extent. The flow reduction and glucose production induced by ET-1 were partly attenuated by the ET(A)-receptor antagonist BQ485. By contrast, ET(B)-receptor antagonist BQ788 enhanced glucose production caused by ET-1 and ET-3 without affecting the hemodynamic change. The effects of ET-1 and ET-3 were almost totally inhibited by the combination of BQ485 and BQ788. These results suggest that both ET(A) and ET(B) receptors are involved in the metabolic and hemodynamic effects of circulating endothelin in rat liver, while the ET(A)-receptor-mediated action appears to be dominant.     

ET-1-induced bronchoconstriction is mediated via ETB receptor in mice

Nagase, Takahide, Tomoko Aoki, Teruaki Oka, YoshinosukeFukuchi, and Yasuyoshi Ouchi. ET-1-induced bronchoconstriction ismediated via ET_B receptor in mice.J. Appl. Physiol. 83(1): 46-51, 1997.Endothelin (ET)-1 is one of the most potent agonists of airwaysmooth muscle and can act via two different ET receptor subtypes, i.e.,ET_A andET_B. To determine the effects ofET-1 on in vivo pulmonary function and which ET receptors are involved in murine lungs, we investigated 1)the effects of ET and sarafotoxin S6c (S6c), a selectiveET_B agonist, on pulmonary functionand 2) the effects of BQ-123 andBQ-788, specific ET_A- andET_B-receptor antagonists, onET-1-induced bronchoconstriction. ICR mice were anesthetized and mechanically ventilated (frequency = 2.5 Hz, tidalvolume = 8 ml/kg, positive end-expiratory pressure = 3 cmH₂O). Intravenous ET-1, ET-2,and ET-3 increased lung resistance similarly and equipotently, whereasS6c elicited a greater degree of bronchoconstriction. Mice were thenpretreated with saline (Sal), BQ-123 (0.2, 1, and 5 mg/kg), or BQ-788(0.2, 1, and 5 mg/kg) before administration of ET-1(10⁷ mol/kg iv). No dose ofBQ-123 blocked ET-1-induced constriction, whereas pretreatment witheach dose of BQ-788 significantly inhibited ET-1-induced responses.There were significant differences in morphometrically assessed airwayconstriction between Sal and BQ-788 and between BQ-123 and BQ-788,whereas no significant difference was observed between Sal and BQ-123.There were no significant morphometric differences in the airway wallarea among the three groups. These observations suggest that theET_B- but notET_A-receptor subtype may mediatethe changes in murine pulmonary function in response to ET-1. Inaddition, the ET_B-receptorantagonist reduces ET-1-induced airway narrowing by affecting airwaysmooth muscle contraction in mice.

     

Endothelin causes contraction of human esophageal muscularis mucosae through interaction with both ETA and ETB receptors

Endothelin (ET) causes contraction of the muscularis mucosae in the guinea pig esophagus, but its role in the human esophagus remains unknown. To investigate effects of ET in the human esophagus, we measured contraction of isolated human esophageal muscularis mucosae strips caused by ET related peptides and binding of 125I-ET-1 to cell membranes prepared from the human esophageal muscularis mucosae. Autoradiography demonstrated specific binding of 125I-ET-1 to the muscularis mucosae and muscularis propria (muscularis externa) of the human esophagus. ET-1 caused tetrodotoxin and atropine-insensitive contraction of muscularis mucosae strips. In terms of the maximal tension of contraction, ET-1 and ET-2 were equal in efficacy. The relative potencies for ET related peptides to cause contraction were ET-1=ET-2>ET-3>sarafotoxin S6c (SX6c), an ETB receptor agonist. ET-1 caused contraction was mildly inhibited by BQ-123, an ETA receptor antagonist, and not by BQ-788, an ETB receptor antagonist. It was moderately inhibited by the combination of both antagonists, indicating synergistic inhibition. Furthermore, desensitization to SX6c with SX6c pretreatment failed to abolish the contractile response to ET-1, which was completely inhibited by BQ-123. These indicate the involvement of both ETA and ETB receptors in the contraction. Binding of 125I-ET-1 to cell membranes of the muscularis mucosae was saturable and specific. Analysis of dose-inhibition curves demonstrated the presence of ETA and ETB receptors. This study demonstrates that, the muscularis mucosae of the human esophagus, similar to that of the guinea pig esophagus, possesses both ETA and ETB receptors mediating muscle contraction.     

ET-1 induces cortical spreading depression via activation of the ETA receptor/phospholipase C pathway in vivo

Recently, it has been shown that brain topical superfusion of endothelin (ET)-1 at concentrations around 100 nM induces repetitive cortical spreading depressions (CSDs) in vivo. It has remained unclear whether this effect of ET-1 is related to a primary neuronal/astroglial effect, such as an increase in neuronal excitability or induction of interastroglial calcium waves, or a penumbra-like condition after vasoconstriction. In vitro, ET-1 regulates interastroglial communication via combined activation of ET(A) and ET(B) receptors, whereas it induces vasoconstriction via single activation of ET(A) receptors. We have determined the ET receptor profile and intracellular signaling pathway of ET-1-induced CSDs in vivo. In contrast to the ET(B) receptor antagonist BQ-788 and concentration dependently, the ET(A) receptor antagonist BQ-123 completely blocked the occurrence of ET-1-induced CSDs. The ET(B) receptor antagonist did not increase the efficacy of the ET(A) receptor antagonist. Direct stimulation of ET(B) receptors with the selective ET(B) agonist BQ-3020 did not trigger CSDs. The phospholipase C (PLC) antagonist U-73122 inhibited CSD occurrence in contrast to the protein kinase C inhibitor G?-6983. Our findings indicate that ET-1 induces CSDs through ET(A) receptor and PLC activation. We conclude that the induction of interastroglial calcium waves is unlikely the primary cause of ET-1-induced CSDs. On the basis of the receptor profile, likely primary targets of ET-1 mediating CSD are either neurons or vascular smooth muscle cells.     

Endothelin receptors in human and guinea-pig gallbladder muscle

We measured contraction of muscle strips caused by endothelin (ET) isopeptides and binding of (125)I-ET-1 to muscle cell membranes prepared from human and guinea-pig gallbladders. Visualization of (125)I-ET-1 binding sites in tissue was performed by autoradiography. Results in human were similar to those in guinea-pig. ET-1 caused tetrodotoxin and atropine-insensitive contraction. The relative potencies for ET isopeptides to cause contraction were ET-1=ET-2>ET-3. ET-1 caused contraction was only slightly inhibited by BQ-123 (potent ET(A) receptor antagonist) and not by BQ-788 (potent ET(B) receptor antagonist). It was inhibited by the combination of both. Autoradiography localized (125)I-ET-1 binding to the smooth muscle layer. Binding of (125)I-ET-1 to muscle cell membranes was saturable and specific. Analysis of dose-inhibition curves demonstrated the presence of two classes of receptors. One class (ET(A) receptor) had a high affinity for ET-1 and ET-2 but a low affinity for ET-3, and the other (ET(B) receptor) a high affinity for ET-1, ET-2 and ET-3. These results demonstrate that similar to guinea-pig, human gallbladder possesses both ET(A) and ET(B) receptors cooperating to mediate muscle contraction.     

Pulmonary vasoregulation by endothelin in conscious dogs after left lung transplantation.

We tested the hypothesis that regulation of the pulmonary circulation by endogenous endothelin (ET) during normoxia and hypoxia was altered in conscious dogs 1 mo after left lung autotransplantation (LLA). Sham-operated control and post-LLA dogs were chronically instrumented to measure the left pulmonary vascular pressure-flow (LP-Q) relationship. LP-Q plots were generated on separate days during normoxia and hypoxia (arterial PO(2) approximately 50 Torr) in the intact condition, after selective ET(A)-receptor inhibition (BQ-485), and after combined ET(A+B)-receptor inhibition (bosentan). Although LLA resulted in a chronic increase in pulmonary vascular resistance, the ET-receptor antagonists had no effect on the LP-Q relationship during normoxia in either group. The magnitude of hypoxic pulmonary vasoconstriction (HPV) was flow dependent in both groups, and the HPV response was potentiated post-LLA compared with control. ET(A)-receptor inhibition attenuated the HPV response to the same extent in both groups. ET(A+B)-receptor inhibition attenuated the HPV response to a greater extent than did ET(A)-receptor inhibition alone, and this effect was greater post-LLA compared with control. Plasma ET-1 concentration only increased during hypoxia in the LLA group. These results indicate that ET does not regulate the baseline LP-Q relationship in either group. Both ET(A)- and ET(B)-receptor activation mediate a component of HPV in conscious dogs, and the vasoconstrictor influence of ET(B)-receptor activation is enhanced post-LLA.     

The endothelin/sarafotoxin-induced increase of the proliferation of undifferentiated and DMSO-differentiated GEM-81 goldfish erythrophoroma cells is mediated by ETB receptors

Endothelins (ETs) and sarafotoxins (SRTXs) have been reported to exert ET(B)-mediated effects on vertebrate pigment cells. GEM-81 cell line, a red pigment cell-derived cutaneous tumor of the teleost Carassius auratus, expresses ET(B) receptors and can be differentiated with 1.5% DMSO treatment, thus constituting an useful model to investigate ET and SRTX effects on cultured fish pigment cells. Our aim was to characterize the pharmacology and biological effects mediated by ET receptors in DMSO-differentiated and undifferentiated cells. ET subtype receptors and their respective Ki values in both cell types were determined by competitive binding assays using (125)I ET-1 and BQ-485 (an ET(A) antagonist) or BQ-788 (an ET(B) antagonist). BQ-788, but not BQ-485, significantly reduced (125)I-ET-1 binding in both cell types, with similar low (Ki > nM) affinities. To determine the proliferation effects of ETs/SRTXs, cells were treated for 72 h with the hormones, and counted in a hemocytometer. The proliferation assays were repeated for SRTX S6c in the presence or absence of BQ-788. The results demonstrated that, with the exception of ET-1 (biphasic effect) and ET-3 (no significant effect) in undifferentiated GEM-81 cells, all the tested hormones induced increases in the proliferation of both types of cells. The hormones were equipotent in DMSO-differentiated cells, which exhibited increased sensitivity to ETs, but not to SRTXs, as compared with undifferentiated cells. The BQ-788 antagonistic effect was also exerted on the proliferation responses to SRTX S6c. These results corroborate the long and important evolutionary history of the ET/SRTX receptor system in vertebrate pigment cells.     

Dual role of endothelin-1 via ETA and ETB receptors in regulation of cardiac contractile function in mice

An increase in coronary perfusion pressure leads to increased cardiac contractility, a phenomenon known as the Gregg effect. Exogenous endothelin (ET)-1 exerts a positive inotropic effect; however, the role of endogenous ET-1 in the contractile response to elevated load is unknown. We characterized here the role of ETA and ETB receptors in regulation of contractility in isolated, perfused mouse hearts subjected to increased coronary flow. Elevation of coronary flow from 2 to 5 ml/min resulted in 80 +/- 10% increase in contractile force (P < 0.001). BQ-788 (ETB receptor antagonist) augmented the load-induced contractile response by 35% (P < 0.05), whereas bosentan (ETA/B receptor antagonist) and BQ-123 (ETA receptor antagonist) attenuated it by 34% and 56%, respectively (P < 0.05). CV-11974 (ANG II type 1 receptor antagonist) did not modify the increase in contractility. These results show that endogenous ET-1 is a key mediator of the Gregg effect in mouse hearts. Moreover, ET-1 has a dual role in the regulation of cardiac contractility: ETA receptor-mediated increase in contractile force is suppressed by ETB receptors.     

Endothelin-1 induces neutrophil recruitment in adaptive inflammation via TNFα and CXCL1/CXCR2 in mice

Endothelin mediates neutrophil recruitment during innate inflammation. Herein we address whether endothelin-1 (ET-1) is involved in neutrophil recruitment in adaptive inflammation in mice, and its mechanisms. Pharmacological treatments were used to determine the role of endothelin in neutrophil recruitment to the peritoneal cavity of mice challenged with antigen (ovalbumin) or ET-1. Levels of ET-1, tumour necrosis factor α (TNFα), and CXC chemokine ligand 1 (CXCL1) were determined by enzyme-linked immunosorbent assay. Neutrophil migration and flow cytometry analyses were performed 4 h after the intraperitoneal stimulus. ET-1 induced dose-dependent neutrophil recruitment to the peritoneal cavity. Treatment with the non-selective ET(A)/ET(B) receptor antagonist bosentan, and selective ET(A) or ET(B) receptor antagonists BQ-123 or BQ-788, respectively, inhibited ET-1- and ovalbumin-induced neutrophil migration to the peritoneal cavity. In agreement with the above, the antigen challenge significantly increased levels of ET-1 in peritoneal exudates. The ET-1- and ovalbumin-induced neutrophil recruitment were reduced in TNFR1 deficient mice, and by treatments targeting CXCL1 or CXC chemokine receptor 2 (CXCR2); further, treatment with bosentan, BQ-123, or BQ-788 inhibited ET-1- and antigen-induced production of TNFα and CXCL1. Furthermore, ET-1 and ovalbumin challenge induced an increase in the number of cells expressing the Gr1(+) markers in the granulocyte gate, CD11c(+) markers in the monocyte gate, and CD4(+) and CD45(+) (B220) markers in the lymphocyte gate in an ET(A)- and ET(B)-dependent manner, as determined by flow cytometry analysis, suggesting that ET-1 might be involved in the recruitment of neutrophils and other cells in adaptive inflammation. Therefore, the present study demonstrates that ET-1 is an important mediator for neutrophil recruitment in adaptive inflammation via TNFα and CXCL1/CXCR2-dependent mechanism.