Endothelin-1[1-31], acting as an ETA-receptor selective agonist, stimulates proliferation of cultured rat zona glomerulosa cells

Endothelin-1 (ET-1)[1-31] is a novel hypertensive peptide that mimics many of the vascular effects of the classic 21 amino acid peptide ET-1[1-21]. However, at variance with ET-1[1-21] that enhances aldosterone secretion from cultured rat zona glomerulosa (ZG) cells by acting via ETB receptors, ET-1[1-31] did not elicit such effect. Both ET-1[1-21] and ET-1[1-31] raised the proliferation rate of cultured ZG cells, the maximal effective concentration being 10(-8) M. This effect was blocked by the ETA-receptor antagonist BQ-123 and unaffected by the ETB-receptor antagonist BQ-788. Quantitative autoradiography showed that ET-1[1-21] displaced both [(125)I]PD-151242 binding to ETA receptors and [(125)I]BQ-3020 binding to ETB receptors in both rat ZG and adrenal medulla, while ET-1[1-31] displaced only [(125)I]BQ-3020 binding. The tyrosine kinase (TK) inhibitor tyrphostin-23 and the p42/p44 mitogen-activated protein kinase (MAPK) inhibitor PD-98059 abolished the proliferogenic effect of ET-1[1-31], while the protein kinase-C (PKC) inhibitor calphostin-C significantly reduced it. ET-1[1-31] (10(-8) M) stimulated TK and MAPK activity of dispersed ZG cells, an effect that was blocked by BQ-123. The stimulatory action of ET-1[1-31] on TK activity was annulled by tyrphostin-23, while that on MAPK activity was reduced by calphostin-C and abolished by either tyrphostin-23 and PD-98059. These data suggest that ET-1[1-31] is a selective agonist of the ETA-receptor subtype, and enhances proliferation of cultured rat ZG cells through the PKC- and TK-dependent activation of p42/p44 MAPK cascade.     

High affinity interaction of endothelin-3 with recombinant ETA receptors

Pharmacological evidence has suggested that endothelin-3 (ET-3) may act via a novel form of ET receptor that is shared by ETA receptor antagonists but not by ETB receptor selective agonists. This study analyses the properties of interaction of ET-3 with recombinant bovine ETA receptor. Apparent Kd(ET-3) values as low as 50 nM were defined from [125I]ET-1 binding experiments performed at low (5 microg/ml) protein concentrations in the assays. Larger (up to 1 microM) values were artefactually obtained in experiments performed at larger protein concentrations. The three monoiodo ET-3 derivatives were synthetized. ([125I]Y14)ET-3 did not recognize ETA receptors. ([125I]Y6)ET-3 labelled 18% of [125I]ET-1 binding sites with a Kd value of 320 pM. ([125I]Y13)ET-3 labelled 44% of [125I]ET-1 binding sites with a Kd value of 130 pM. High affinity ([125I]Y6)ET-3 and ([125I]Y13)ET-3 bindings were prevented by ET-1 (Kd = 5-7 pM), ET-3 (Kd = 70-250 pM), BQ-123 (Kd = 2 nM) and FR139317 (Kd = 2 nM) but not by low concentrations of 4-AlaET-1, sarafotoxin S6c or IRL1620. The three monoiodo ET-3 derivatives bound to recombinant rat ETB receptors with a pM affinity. The results suggest that ET-3, ([125I]Y6)ET-3 and ([125I]Y13)ET-3 should not be considered as ETB receptor specific ligands.     

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.     

Different distribution of endothelin receptor subtypes in pulmonary tissues revealed by the novel selective ligands BQ-123 and [Ala1,3,11,15]ET-1.

We have demonstrated the different distribution of two distinct endothelin (ET) receptor subtypes in porcine pulmonary tissues using a radioligand binding assay. The clear differentiation of the subtypes was made possible by the discovery of two compounds, BQ-123 and [Ala1,3,11,15]ET-1 (4AlaET-1), that are highly selective for ETA and ETB receptors, respectively. In the bronchus and lung parenchyma, BQ-123 inhibited 65% and 30% of [125I]ET-1 binding on the sensitive sites, while 4AlaET-1 displaced 25% and 60%, respectively. The combination of the two compounds completely inhibited ET-1 binding in both tissues. An autoradiographic study of [125I]ET-1 binding using BQ-123 and 4AlaET-1 also supported the different localization of two ET receptor subtypes in pulmonary tissues. In particular, the blood vessels and bronchi are rich in ETA, but the lung parenchyma is rich in ETB.     

Biological profiles of highly potent novel endothelin antagonists selective for the ETA receptor.

We describe novel potent endothelin (ET) antagonists that are highly potent and selective for the ETA receptor (selective to ET-1). Of the synthetic analogs based on ETA antagonist BE-18257A isolated from Streptomyces misakiensis (IC50 value for ETA receptor on porcine aortic smooth muscle cells (VSMCs); 1.4 microM), the compounds BQ-123 and BQ-153 greatly improved the binding affinity of [125I]ET-1 for ETA receptors on VSMCs (IC50; 7.3 and 8.6 nM, respectively), whereas they barely inhibited [125I]ET-1 binding to ETB receptors (nonselective with respect to isopeptides of ET family) in the cerebellar membranes (IC50; 18 and 54 microM, respectively). Associated with the increased affinity for ETA receptors, these peptides antagonized ET-1-induced constriction of isolated porcine coronary artery. However, there was a small amount of ET-1-induced vasoconstriction resistant to these antagonists, which paralleled the incomplete inhibition of [125I]ET-1 binding in the membrane of the aortic smooth muscle layer. These data suggest that the artery has both ETA and ETB receptors responsible for ET-1-induced vasoconstriction. The antagonists shifted the concentration-response curve to the right for ET-1 in the coronary artery, and increased the apparent dissociation constant in the Scatchard analysis of [125I]ET-1 binding on the VSMCs without affecting the binding capacity, indicative of the competitive antagonism for ETA receptor. In conscious rats, pretreatment with the antagonists markedly antagonized ET-1-induced sustained pressor responses in dose-dependent fashion without affecting ET-1-induced transient depressor action, suggesting that the pressor action is mediated by ETA receptors, while the depressor action is mediated by ETB receptors. In addition, pretreatment with the potent antagonists prevented ET-1-induced sudden death in mice. Thus, these potent ETA antagonists should provide a powerful tool for exploring the therapeutic uses of ETA antagonists in putative ET-1-related disorders.     

A novel ETA antagonist (BQ-123) inhibits endothelin-1-induced phosphoinositide breakdown and DNA synthesis in rat vascular smooth muscle cells.

The effects of a novel cyclic pentapeptide (BQ-123), an endothelin (ET) antagonist selective for the ETA receptor subtype, on phosphoinositide breakdown and DNA synthesis stimulated by ET-1 were studied in cultured rat vascular smooth muscle cells (VSMC). BQ-123 competitively inhibited the binding of [125I]ET-1 to VSMC with the apparent Ki of 4 x 10(-9) M. BQ-123 dose-dependently inhibited formation of inositol-1,4,5-trisphosphate and [3H]thymidine uptake stimulated by ET-1. These data suggest that the ET-1-induced DNA synthesis in VSMC is mainly mediated by ETA receptor subtype.     

PTHrP fragments 1-16 and 1-23 do not bind to either the ETA or the ETB endothelin receptors

Because of some isofunctional similarities with endothelin-1 (ET-1), it has been suggested that PTHrP(1-16) and PTHrP(1-23) could interact with osteoblast cells via ETA receptors. To document this interaction, we used the thoracic rat aorta and the guinea-pig lung parenchyma paradigms as ETA and ETB models, respectively. In addition, we also performed a series of competition experiments against [125I]ET-1, using transfected cells expressing the ETA or ETB receptor. So far, no agonistic nor antagonistic activities were observed in the ETA and ETB bioassays with the PTHrP fragments. Furthermore, both fragments were unable to displace [125I]ET-1 bound to cells expressing the ETA or ETB receptor.     

Nonpeptidic antagonists of ETA and ETB receptors reverse the ET-1-induced sustained increase of cytosolic and nuclear calcium in human aortic vascular smooth muscle cells

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.     

Upregulation of endothelin-1 binding in tissues of salt-loaded stroke-prone spontaneously hypertensive rats

Upon maintained on a 1% NaCl drinking solution beginning at 7 weeks of age, the stroke-prone spontaneously hypertensive rat (SHRsp) developed severe hypertension and stroke; most died by 16 weeks. The mechanism by which these diseases evolve remains unclear. Endothelin-1 (ET-1) is a potent, peptidic vasoconstrictor and is implicated in the pathogenesis of various cardiovascular, renal, and central nervous system diseases. The purpose of the present study was to compare the binding of [125I]ET-1 to the brain, heart, kidney, liver, and spleen membrane preparations of 16-week-old SHRsp and age-matched normotensive Wistar-Kyoto rats (WKY). The KD values for [125I]ET-1 binding to the corresponding tissues of the two strains were not significantly different, except in the brain (SHRsp: 17 +/- 1 pM; WKY: 24 +/- 1 pM). In contrast, the Bmax values measured in the brain, heart, kidney, and liver of SHRsp were 1.5- to 2.1-fold greater than those of their WKY counterparts. Competition of [125I]ET-1 binding to the membrane preparations by the specific ETA receptor antagonist BQ-123 or the specific ETB receptor agonist sarafotoxin S6c revealed a similar proportion of ETA and ETB receptor subtypes in the corresponding tissues of the two rat strains. These results indicate that ET-1 binding is upregulated in SHRsp and suggest that ET-1 may play a pathophysiological role in this animal model of genetic hypertension.     

ET-1 receptor gene expression and distribution in L1 and L2 cells from hypertensive sheep pulmonary artery

We examined gene and surface expression and activity of the endothelin (ET)-1 receptors (ETA and ETB) in subendothelial (L1) and inner medial (L2) cells from the main pulmonary artery of sheep with continuous air embolization (CAE)-induced chronic pulmonary hypertension (CPH). According to quantitative real-time RT-PCR, basal gene expression of both receptors was significantly higher in L2 than L1 cells, and hypertensive L2 cells showed significantly higher gene expression of ETB than controls. Expression of both genes in hypertensive L1 cells was similar to controls. Fluorescence-activated cell sorter analysis confirmed the increased distribution of ET(B) in hypertensive L2 cells. Although only the ETA receptors in control L2 cells showed significant binding of [125I]-labeled ET-1 at 1 h, both receptors bound ET-1 to hypertensive cells. Exposure to exogenous ET-1 for 18 h revealed that only the L2 cells internalized ET-1, and internalization by hypertensive L2 cells was significantly reduced when compared with controls. Treatment with ETA (BQ-610) and ETB (BQ-788) receptor antagonists demonstrated that both receptors contributed to internalization of ET-1 in control L2 cells, whereas in hypertensive cells only when both receptor antagonists were used in combination was significant suppression of ET-1 internalization found. We conclude that in sheep receiving CAE, alterations in ETB receptors in cells of the L2 layer may contribute to the maintenance of CPH via alterations in their expression, distribution, and activity.     

An endothelin B receptor-selective antagonist: IRL 1038, [Cys11-Cys15]-endothelin-1(11-21)

In the inhibition of specific binding of [125I]endothelins (ETs) to membrane from various tissues of rats, guinea pigs, pigs and humans, [Cys11-Cys15]-ET-1(11-21), IRL 1038, has a much higher affinity for ETB receptors (Ki = 6-11 nM) than for ETA receptors (Ki = 0.4-0.7 microM). In contraction assays, with ET-3 as a stimulant, 3 microM IRL 1038 antagonized the ETB receptor-mediated contraction of guinea pig ileal and tracheal smooth muscle without any significant agonistic activity, but did not effect the ETA receptor-mediated contraction of rat aortic smooth muscle. IRL 1038 is therefore, considered to be the first antagonist selective to the ETB receptor.     

Endothelin 1 and 3 enhance neuronal nitric oxide synthase activity through ETB receptors involving multiple signaling pathways in the rat anterior hypothalamus

We have previously reported that endothelin 1 and 3 (ET-1, ET-3) through the ETB receptor decrease norepinephrine release in the anterior hypothalamus and activate the nitric oxide (NO) pathway. In the present work we sought to establish the receptors and intracellular mechanisms underlying the increase in nitric oxide synthase (NOS) activity stimulated by ET-1 and ET-3 in the rat anterior hypothalamus. Results showed that ETs-stimulated NOS activity was inhibited by a selective ETB antagonist (BQ-788), but not by a selective ETA antagonist (BQ-610). In addition, NOS activity was not altered in the presence of an ETA agonist (sarafotoxin 6b), but it was enhanced in the presence of a ETB agonist (IRL-1620). Both Nomega-nitro-L-arginine methyl ester (NOS inhibitor), and 7-nitroindazole (neuronal NOS inhibitor) diminished ETs-stimulated NOS activity. The stimulatory effect of ETs on NOS activity was inhibited in the presence of PLC, PKC, PKA and CaMK-II inhibitors (U-73122, GF-109203X, H-89 and KN-62, respectively), and the IP3 receptor selective antagonist, 2-APB. Our results showed that both ET-1 and ET-3 modulate neuronal NOS activity through the ETB receptor in the rat anterior hypothalamus involving the participation of the PLC-PKC/IP3 pathway as well as PKA and CaMK-II.     

Dipeptide sulfonamides as endothelin ETA/ETB receptor antagonists

Endothelin-1 (ET-1) is a potent mitogen and modulator of vascular tone. It is synthesized and released from endothelial cells and acts upon two receptor subtypes designated as ETA and ETB. In this study, a series of potent dipeptide sulfonamide dual-endothelin ETA/ETB receptor antagonists were prepared to investigate their potential benefit in vascular diseases. CGS 31398 inhibited [125I]ET-1 binding to human ETA and ETB receptors expressed in Chinese hamster ovary (CHO) cells (ETA/CHO, ETB/CHO) with respective IC50 values of 0.26 and 0.12 nM. However, in anesthetized rats, this compound markedly potentiated ET-1-induced renal vascular resistance, a response normally observed with selective ETB receptor antagonists. To determine whether species differences account for these results, a direct comparison was made between binding to rat and rabbit aortic membranes versus functional antagonism in isolated rat aortic rings. It was found that CGS 31398 had potent affinity for the ETA receptor in rat and rabbit aorta with IC50 values of 0.87 and 0.79 nM, respectively. Inhibition of ET-1-induced contractions of rat aorta by the compound was considerably weaker than expected (pKB = 6.4), while that of sarafotoxin S6c induced contraction of dog saphenous vein (100% inhibition at 100 nM) was consistent with corresponding binding data. These results suggest that although CGS 31398 is a potent dual inhibitor of ETA/ETB receptor binding, it surprisingly displays potent ETB and weak ETA receptor antagonism in functional assays.     

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.     

A potent and specific agonist, Suc-[Glu9,Ala11,15]-endothelin-1(8-21), IRL 1620, for the ETB receptor.

A series of C-terminal linear peptides of endothelin (ET)-1 and their N alpha-succinyl (Suc) analogs were synthesized and their binding affinities for the two subtypes of ET receptor, ETA and ETB, in porcine lung membranes were examined. Among the synthetic analogs, Suc-[Glu9,Ala11,15]-ET-1(8-21), IRL 1620, was the most potent and specific ligand for the ETB receptor (KiETA/KiETB approximately equal to 120,000) as judged by the Ki values for ETA (1.9 microM) and ETB (16 pM) receptors. IRL 1620 was 60 times more selective for the ETB receptor than ET-3 (KiETA/KiETB approximately equal to 1,900). IRL 1620 (10(-9)-10(-7) M) induced contractions of the guinea pig trachea with a comparable potency to those of ET-1 or ET-3, suggesting that IRL 1620 is a potent ETB receptor agonist.     

Ala1,3,11,15]endothelin-1 analogs with ETB agonistic activity.

A linear peptide analog of endothelin (ET)-1, [Ala1,3,11,15]ET-1 (4AlaET-1), and its truncated peptide analogs were synthesized to study the structural requirements of ET-1 for the recognition of ETs-nonselective ETB receptors. ET-1 exhibited sub-nanomolar binding to two distinct ET receptor subtypes (ETA and ETB), but 4AlaET-1 bound to ETB with an affinity 1,700 times higher than that seen during binding to ETA. The truncated linear peptides 4AlaET-1(6-21), 4AlaET-1(8-21) and N-acetyl-4AlaET-1(10-21) still had high affinity for ETB, whereas 4AlaET-1(6-20) and 4AlaET-1(11-21) displayed remarkably reduced affinity for ETB. Therefore, ET-1 requires the Glu10-Trp21 sequence for ETB binding, but not the disulfide bridges. These ETB-binding peptides elicit endothelium-dependent vasorelaxation of porcine pulmonary arteries in parallel with the binding affinity for ETB, suggesting that they are ETB agonists.     

Venous smooth muscle contains vasoconstrictor ETB-like receptors.

Two endothelin (ET) receptor subtypes have been identified to date: the ETA receptor which preferentially binds ET-1 over ET-3, and the ETB receptor which is non-selective. This study characterized the ET receptor subtypes present in several vascular smooth muscle preparations using standard in vitro techniques. In all but one of the arteries tested, ET-3 was significantly less potent than ET-1. In contrast, the potency of ET-3 was very similar to that of ET-1 in all of the veins. The selective ETA receptor antagonist BQ-123 blunted the ET-1 contractions in rabbit carotid artery, but not in saphenous vein. The selective ETB receptor ligand sarafotoxin S6c contracted the rabbit saphenous vein, but not the carotid artery. These data suggest that vascular smooth muscle cells express ETA and ETB receptors. Stimulation of either receptor subtype can result in force development.     

Effects of human peptide endothelin-1 and two of its sterically unrestrained C-terminal fragments on coronaryvascular smooth muscle

Clearance of human peptide endothelin-1 (ET-1) has been proposed to follow a receptor pathway involving a cascade of ET-1 receptor endocytosis and lysosomal degradation by a family of proteinases expressed constitutively by most cells. Genetically distinct endopeptidases produce ET-1 and degrade mature peptide. The ET-1 degradation products were considered to be inactive, however, recent evidence suggests that ET-1 fragments sustain most of the homeostatic response produced by parent peptides. The purpose of this study was to establish whether the overall structure of human ET-1 or the structure of its C-terminus is responsible for the subtype-selectivity, down-regulation and clearance of endothelin, and whether D-aminoacid substitution in the moiety of synthetic peptide is involved in effective ET-1 antagonism in coronary vascular smooth muscle. To characterize specific mechanism(s) leading to subtype-selective ET-receptor down-regulation and/or to ET-1 antagonism, ligand binding studies were accomplished with radioactive human (1-21)ET-1 and with C-terminal ET-1 fragments, both peptide agonists and antagonists, in adult male porcine coronary artery vascular smooth muscle (CVSM). The subcellular membranes of CVSM were isolated by isopycnic gradient centrifugation. Exposure of porcine coronary artery to exogenous ET-1 induced endothelin-ETB selective down-regulation. ETA-mediated subtype-ETB down-regulation was observed with distribution of ligand-ETB receptor complexes in light, endosomal, membranes. The ETA selective PD151242 significantly attenuated [3H]-thymidine incorporation, and the ETB selective antagonist BQ788 blocked down-regulation observed in porcine vascular fibroblasts (PF). Preincubation of coronary arteries with ETB selective BQ3020 was accompanied with a more intense down-regulation. CONCLUSION: our data are indicative of short-term ETB selective down-regulation of endothelin receptors in coronary vascular smooth muscle after exposure to ET-1. The presence in the carboxy-terminus of (Ala11,15) substitution in peptide fragments IRL1620 and BQ3020 determined the differential specificity of ETB-receptor coupling and was important for subtype-ETB-receptor down-regulation. The activation of the dominating ETA-receptor by ET-1 facilitated mitogenic responses to ET-1 in porcine vascular fibroblasts.     

Production of nitric oxide from endothelial cells by 31-amino-acid-length endothelin-1, a novel vasoconstrictive product by human chymase

Human chymase selectively converts big endothelin (ET)-1 to 31-amino-acid-length ET-1 [ET-1(1-31)]. In this study we examined effect of ET-1(1-31) on endothelial function. ET-1(1-31) evoked contraction in a concentration-dependent manner at > 10(-8) M, which was about 10 times weaker than that of conventional ET-1 [ET-1(1-21)]. BQ485, an ETA receptor antagonist, completely abolished ET-1(1-31)-induced contraction, but BQ788, an ETB receptor antagonist, slightly enhanced it, suggesting that ET-1(1-31) relaxes artery via endothelium. On endothelial cells, ET-1(1-21) and ET-1(1-31) increased [Ca2+]i and produced NO, both of which were significantly inhibited by BQ788 and not by BQ485. These results indicate that ET-1(1-31) increased [Ca2+]i and produced NO in endothelial cells through ETB receptor similarly with ET-1(1-21), although slight difference in effect on smooth muscle cells.     

Modulatory effect of endothelin-1 and -3 on neuronal norepinephrine release in the rat posterior hypothalamus

Based upon the existence of high density of ET-receptors on catecholaminergic neurons of the hypothalamus, we studied the effects of endothelin-1 (ET-1) and endothelin-3 (ET-3) on neuronal norepinephrine (NE) release in the rat posterior hypothalamus. The intracellular pathways and receptors involved were also investigated. Neuronal NE release was enhanced by ET-1 and ET-3 (10 etaM). The selective antagonists of subtype A and B ET receptors (ETA, ETB) (100 etaM BQ-610 and 100 etaM BQ-788, respectively) abolished the increase induced by ET-1 but not by ET-3. The PLC inhibitor, U73122 (10 microM), abolished ET-1 and ET-3 response. GF-109203X (100 etaM) (PKC inhibitor) blocked the increase in NE release produced by ET-3 and partially blocked ET-1 response. The inositol 1,4,5-trisphosphate-induced calcium release inhibitor, 42 microM 2-APB, inhibited the stimulatory effect induced by ET-3 but not by ET-1. The PKA inhibitor, 500 etaM H-89, blocked the increase in neuronal NE release evoked by ET-1 but not by ET-3. Our results showed that ET-1 as well as ET-3 displayed an excitatory neuromodulatory effect on neuronal NE release in the rat posterior hypothalamus. ET-1 through an atypical ETA or ETB receptor activated the PLC/PKC signalling pathway as well as the cAMP pathway, whereas ET-3 through a non-ETA/non-ETB receptor activated the phosphoinositide pathway. Both ETs would enhance the sympathoexcitatory response elicited by the posterior hypothalamus and thus participate in cardiovascular regulation.