Endothelin receptor antagonists reduce the pressor effects of angiotensin II into the periaqueductal gray area of rats.

Injection of ANGII (0.01, 0.1 and 1 nmol/rat) into the periaqueductal gray (PAG) area significantly (P<0.01) increased, in a dose-dependent manner, the mean arterial blood pressure (MAP). The increases in blood pressure induced by ANGII (1 nmol; 37 +/- 4 mmHg, n=5) were greatly reduced (>85%) by pre-administration of the ET(A) receptor antagonist FR139317 (5 nmol/rat) to the PAG area, but were unaffected by the ET(B) receptor antagonist BQ-788 (5 nmol/rat). SB209670, non-selective ET(A)/ET(B) receptor antagonist, also reduced the effect induced by ANGII. These results suggest that endogenous endothelin-1, via an action on ET(A) receptors, may contribute to the pressor effects of ANGII within the PAG area of rats.     

Mechanism of hypoxic pulmonary vasoconstriction involves ET(A) receptor-mediated inhibition of K(ATP) channel

There is controversy on the role of endothelin (ET)-1 in the mechanism of hypoxic pulmonary vasoconstriction (HPV). Although HPV is inhibited by ET-1 subtype A (ET(A))-receptor antagonists in animals, it has been reported that ET(A)-receptor blockade does not affect HPV in isolated lungs. Thus we reassessed the role of ET-1 in HPV in both rats and isolated blood- and physiological salt solution (PSS)-perfused rat lungs. In rats, the ET(A)-receptor antagonist BQ-123 and the nonselective ET(A)- and ET(B)-receptor antagonist PD-145065, but not the ET(B)-receptor antagonist BQ-788, inhibited HPV. Similarly, BQ-123, but not BQ-788, attenuated HPV in blood-perfused lungs. In PSS-perfused lungs, either BQ-123, BQ-788, or the combination of both attenuated HPV equally. Inhibition of HPV by combined BQ-123 and BQ-788 in PSS-perfused lungs was prevented by costimulation with angiotensin II. The ATP-sensitive K(+) (K(ATP))-channel blocker glibenclamide also prevented inhibition of HPV by BQ-123 in both lungs and rats. These results suggest that ET-1 contributes to HPV in both isolated lungs and intact animals through ET(A) receptor-mediated suppression of K(ATP)-channel activity.     

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.     

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.     

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.     

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.     

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.     

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.     

A novel radioligand [125I]BQ-3020 selective for endothelin (ETB) receptors.

A linear endothelin (ET) analog, N-acetyl-LeuMetAspLysGluAlaValTyrPheAlaHisLeu-AspIleIleTrp (BQ-3020), is highly selective for ETB receptors. BQ-3020 displaces [125I]ET-1 binding to ETB receptors (nonselective to ET isopeptides) in porcine cerebellar membranes (IC50: 0.2nM) at a concentration 4,700 times lower than that to ETA receptors (selective to ET-1) on aortic vascular smooth muscle cells (VSMC) (IC50: 940nM). BQ-3020 as well as ET-1 and ET-3 elicits vasoconstriction in the rabbit pulmonary artery. The ETA antagonist BQ-123 failed to inhibit this BQ-3020-induced vasoconstriction. Furthermore, BQ-3020 elicits endothelium-dependent vasodilation. These data indicate that BQ-3020 has ETB agonistic activity. The radioligand [125I]BQ-3020 binds to cerebellar membranes at single high affinity sites (Kd = 34.4pM), whereas it scarcely binds to VSMC. [125I]BQ-3020 binding to the cerebellum was displaced by BQ-3020, ET-1 and ET-3 in a nonselective manner (IC50: 0.07-0.17nM). However, the binding of [125I]BQ-3020 was insensitive to the ETA antagonist BQ-123 and other bioactive peptides. Both [125I]ET-1 and [125I]BQ-3020 show slow onset and offset binding kinetics to ETB receptors. These data indicate that the radioligand [125I]BQ-3020 selectively labels ETB receptors and that the slow binding kinetics of ET-1 are dependent on the peptide sequence from Leu6 to Trp21, but not on the structure formed by its two disulfide bridges.     

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.     

Pronatriuretic peptide is a sensitive marker of the endocrine function of teleost heart

We recently characterized a novel heart-specific hormone from salmon (salmon cardiac peptide, sCP). We have now prepared a recombinant plasmid expressing the NH(2)-terminal fragment of pro-sCP (NT-pro-sCP) and used it to set up a specific RIA for the peptide. Because of the sensitivity of the assay and the high circulating levels, NT-pro-sCP can be measured from as little as 2 microl of serum. This enables repeated sampling from the same animal in different experimental setups. Mechanical load increased the release of NT-pro-sCP from isolated perfused salmon ventricle, in parallel with sCP. Bolus injection of human endothelin-1 (ET-1; 1 microg) in the dorsal aorta of salmon resulted in an extensive increase of serum NT-pro-sCP (from 0.99 +/- 0.11 to 4.6 +/-1.5 nmol/l). The response was abolished by pretreatment with a specific type A ET (ET(A)) receptor antagonist (BQ-123) but not with a type B ET receptor antagonist (BQ-788). The NT-pro-sCP levels had a good correlation with those of sCP (r(2) = 0.75). Our results demonstrate the practical usefulness of circulating NT-pro-sCP as a marker of the endocrine function of salmon heart. They also suggest that ET-1 has an important role in regulating sCP release from teleost heart by an ET(A) receptor-mediated mechanism.     

The endothelin receptor antagonist, BQ-123, inhibits angiotensin II-induced contractions in rabbit aorta.

The purpose of this study was to examine the specificity of the cyclic pentapeptide ET(A) receptor antagonist BQ-123. BQ-123 competitively antagonized endothelin-1-induced contractions in rabbit aorta, increases in inositol phosphates in cultured rat vascular smooth muscle A10 cells, and binding of [125I]endothelin-1 to the cloned ETA receptor cDNA expressed in Cos 7 cells. In contrast, BQ-123 was a weak antagonist of [125I]endothelin-3 binding to rat cerebellar membranes and to membranes from Cos 7 cells transfected with the cloned ETB receptor cDNA. BQ-123 shifted concentration-response curves in isolated rabbit aorta elicited by angiotensin II, but did not bind to angiotensin II receptors nor affect angiotensin II-induced increases in inositol phosphates. BQ-123 also did not affect contractions induced by KCl or norepinephrine. These data suggest that endothelin may play a role in angiotensin II-induced contractions of rabbit aorta.     

The blockade of endothelin A receptor protects astrocytes against hypoxic injury: common effects of BQ-123 anderythropoietin on the rejuvenation of the astrocyte population

In the present study the role of endothelin (ET) and its receptors (ETA-R and ETB-R) in cellular mechanisms underlying the resistance of astroglial cells to low oxygen level and development of hypoxia has been investigated. To define the influences of ET and its receptors on survival and on antigenic as well as morphologic differentiation of rat astroglial cells in normoxic (NC) and hypoxic culture (HC) the selective antagonists of ETA-R (BQ-123) and ETB-R (BQ-788) were used. Treatment of HC with BQ-123 caused an increase in cell number and inhibited the hypoxia-induced apoptosis by 37%. BQ-123 decreased the hypoxia-induced cytotoxicity in HC. These effects of BQ-123 were abolished in cultures simultaneously treated with BQ-123 and BQ-788. Administration of BQ-788 alone decreased the number of living cells in NC, but not in HC. The activity of caspase-3/-7 was not changed by exposure of NC and HC to BQ-788. The protection provided by BQ-123 to astroglial cells against cytotoxicity in NC and HC was similar to that of erythropoietin (EPO), a cytokine with established neuroprotective effects. The functional improvement of astroglial cells and slowing down of their differentiation under exposure to BQ-123, or EPO, or BQ-123 + EPO has been evidenced by an increased number of nestin+/glial fibrillary acidic protein-positive (GFAP+) astrocytes accompanied by decrease of nestin-/GFAP+ cells. The simultaneous treatment with BQ-123 and EPO additionally decreased the activities of caspase-3/-7 (64%) and release of LDH into the medium (94%). The benefits in the functional states of astrocytes obtained by combined treatment of HC with BQ-123 and EPO suggest a new therapeutic strategy in treatment of hypoxic brain injury.     

Endothelin-1 increases isoprenaline-enhanced cyclic AMP levels in cerebral cortex

We examined the effect of ET-1 on cyclic AMP levels in rat cerebral cortex. The peptide caused a concentration-dependent increase of [(3)H]cyclic AMP accumulation after 10 min of treatment. This effect was due to adenosine accumulation since it was inhibited by the treatment with adenosine deaminase. ET-1, apart from being able to increase cyclic AMP, also potentiated the cyclic AMP generated by isoprenaline in the presence of adenosine deaminase. Experiments performed in the presence of BQ-123 or BQ-788, specific ET(A) or ET(B) receptor antagonists respectively indicated that ET(B) was the receptor involved. This effect was dependent on extracellular and intracellular calcium concentration. These findings suggest that ET-1 plays a modulatory role in cyclic AMP generation systems in cerebral cortex.     

The renal and systemic hemodynamic effects of a nitric oxide-synthase inhibitor are reversed by a selective endothelin(a) receptor antagonist in men.

There is evidence for an interaction between nitric oxide (NO) and endothelin (ET) at the level of the renal vasculature. We hypothesized that acute renal effects of systemic NO synthase inhibition (NG-monomethyl-l-arginine, L-NMMA) may be blunted by coadministration of a specific ET(A) receptor antagonist (BQ-123) in healthy humans. Fifteen healthy young male subjects participated in this randomized, double-blind, placebo-controlled 3-way crossover study. These sodium-repleted volunteers received L-NMMA alone, or BQ-123 alone, or L-NMMA with a subsequent coinfusion of BQ-123. Renal plasma flow (RPF) and glomerular filtration rate (GFR) were determined with the PAH and inulin clearance method, respectively. Mean arterial pressure (MAP) and pulse rate were measured noninvasively at baseline and every 15 min after the start of the study period. L-NMMA alone reduced RPF (-22%, P < 0.001) and GFR (-8%, P < 0.009) and increased MAP (+10%, P < 0.001). BQ-123 alone did not affect these parameters. However, coinfusion of BQ-123 blunted the effects of L-NMMA on RPF (P < 0.001), GFR (P < 0.001), and MAP (P = 0.006). Peripheral and renal hemodynamic effects of acute systemic NO synthase inhibition are at least partially reversed by ET(A) receptor blockade with BQ-123. This indicates a functional antagonism between specific ET(A) receptor antagonist and NO synthase inhibitors at the level of the renal vasculature.     

Endothelin, PAF and thromboxane A2 in allergic pulmonary hyperreactivity in mice

The role of endothelin, PAF and thromboxane A2 in airway hyperreactivity (AHR) to carbachol induced by ovalbumin sensitization and challenge in Balb/c mice was investigated. Ovalbumin sensitization and challenge induced significant AHR to carbachol in actively sensitized and challenged mice. Treatment of these mice with the PAF antagonist CV-3988 (10 microg kg(-1), i.v.) completely abolished OVA-induced AHR to carbachol. Treatment of sensitized mice with the TxA2 antagonist L-654,664 (1 mg kg(-1), i.v.) partially blocked the induction of AHR in OVA-challenged mice. The intranasal administration of 50 pmol of the ET(A) receptor antagonist BQ-123 had no effect on the PIP but produced a significant reduction at the dose of 100 pmol. The intravenous administration of BQ-123 (100 pmol) reduced the PIP only at the highest doses of carbachol. The ET(B) receptor antagonist BQ-788 administered either via the intranasal or intravenous route had no effect on the PIP at the dose of 100 pmol. Na?ve mice treated with either U-44069 (25 or 100 microg kg(-1), i.v.), endothelin-1 (100 pmol, intranasally) or the ET(B) receptor agonist IRL-1620 (100 pmol, intranasally) showed a marked increase in airway reactivity to carbachol. These results suggest an important role for endothelin, PAF and thromboxane A2 in AHR in mice actively sensitized and challenged with ovalbumin.     

Two classes of endothelin receptors mediating contraction in esophageal muscularis mucosae

Endothelin (ET) causes contraction of the esophageal muscularis mucosae. To characterize the ET receptor subtypes involved in contraction, we measured contraction of isolated muscularis mucosae strips caused by ET-related peptides and binding of (125)I-ET-1 to cell membranes prepared from the guinea pig esophageal muscularis mucosae. Autoradiography demonstrated (125)I-ET-1 binding to the muscularis mucosae and muscularis propria. ET-1 caused tetrodotoxin and atropine-insensitive contraction of esophageal muscularis mucosae strips. The relative potencies for ET isopeptides to cause contraction were ET-1=ET-2>ET-3. FR-139317, an ET(A) receptor antagonist, or BQ-788, an ET(B) receptor antagonist, alone did not alter responses to ET-1. However, the combination of both antagonists almost abolished the ET-1-induced contraction, indicating synergistic inhibition. Desensitization to sarafotoxin S6c, an ET(B) receptor agonist, failed to abolish the response to ET-1, which was completely inhibited by FR-139317. These indicate the involvement of both ET(A) and ET(B) receptors in the contraction. Binding of (125)I-ET-1 to cell membranes of the muscularis mucosae was saturable and specific. Analysis of dose-inhibition curves demonstrated the presence of ET(A) and ET(B) receptors.This study demonstrates that the esophageal muscularis mucosae possesses both ET(A) and ET(B) receptors mediating muscle contraction. There is cooperation between these two subtypes of ET receptors in the esophagus mediating muscle contraction.     

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.     

Role of endothelin and endothelin A-type receptor in adaptation of the carotid body to chronic hypoxia

Chronic exposure in a low-PO(2) environment (i.e., chronic hypoxia, CH) elicits an elevated hypoxic ventilatory response and increased hypoxic chemosensitivity in arterial chemoreceptors in the carotid body. In the present study, we examine the hypothesis that changes in chemosensitivity are mediated by endothelin (ET), a 21-amino-acid peptide, and ET(A) receptors, both of which are normally expressed by O(2)-sensitive type I cells. Immunocytochemical staining showed incremental increases in ET and ET(A) expression in type I cells after 3, 7, and 14 days of CH (380 Torr). Peptide and receptor upregulation was confirmed in quantitative RT-PCR assays conducted after 14 days of CH. In vitro recordings of carotid sinus nerve activity after in vivo exposure to CH for 1-16 days demonstrated a time-dependent increase in chemoreceptor activity evoked by acute hypoxia. In normal carotid body, the specific ET(A) antagonist BQ-123 (5 microM) inhibited 11% of the nerve discharge elicited by hypoxia, and after 3 days of CH the drug diminished the hypoxia-evoked discharge by 20% (P < 0.01). This inhibitory effect progressed to 45% at day 9 of CH and to nearly 50% after 12, 14, and 16 days of CH. Furthermore, in the presence of BQ-123, the magnitude of the activity evoked by hypoxia did not differ in normal vs. CH preparations, indicating that the increased activity was the result of endogenous ET acting on an increasing number of ET(A). Collectively, our data suggest that ET and ET(A) autoreceptors on O(2)-sensitive type I cells play a critical role in CH-induced increased chemosensitivity in the rat carotid body.