Inhibitory effects of endothelin-1 and endothelin-3 on prolactin release: possible involvement of endogenous endothelin isopeptides in the rat anterior pituitary.

Spontaneous prolactin release from the isolated rat anterior pituitary was inhibited by endothelin-1 in a dose-dependent manner (10(-8)-10(-6) M). Endothelin-3 also inhibited spontaneous prolactin release with an almost identical dose-response relationship as endothelin-1. These inhibitory effects were unaffected by application of a dopamine D2-receptor antagonist, YM-09151-2 (10(-7) M). Rat anterior and posterior pituitary glands were abundant in both endothelin-1 and endothelin-3, as compared with other regions of the brain. The present results suggest that endogenous endothelin-1 and endothelin-3 in the anterior and posterior pituitary are involved in the inhibitory regulation of prolactin secretion as autocrine or paracrine factors.     

Profibrotic effects of endothelin-1 via the ETA receptor in cultured human cardiac fibroblasts.

BACKGROUND/AIMS: Endothelin-1 (ET-1) has been implicated in pathologic remodelling and tissue repair processes in the heart. We investigated the effects of ET-1 on growth and collagen synthesis responses in cardiac fibroblasts isolated from human hearts. We also studied the receptor subtype(s) mediating such responses and the factors regulating their expression. METHODS: Fibroblasts were isolated from cardiac transplant recipient hearts and characterised by immunocytochemistry. Serum-starved cells were exposed to ET-1 and incorporation of [3H]proline and thymidine were measured as indexes of collagen and DNA synthesis respectively. Blocking experiments utilised the selective ETA receptor antagonist BQ123 and the ETB antagonist BQ788. RESULTS: ET-1 elicited a potent collagen synthesis response in cardiac fibroblasts, with a maximum 29+/-5% increase that was abolished by BQ123. Cardiac fibroblasts responded to ET-1 with a concentration-dependent decrease in DNA synthesis rate. The effects of ET-1 were similar to those of TGF-beta. Radioligand binding studies revealed the presence of high-affinity ET-1 binding sites on these cells, which were upregulated by treatment with the growth factors PDGF and EGF but downregulated by TGF-beta. CONCLUSIONS: These results therefore implicate ET-1 as a trophic agent in the human heart with the ability to influence the development of cardiac fibrosis.     

Endothelin stimulates glucose uptake and GLUT4 translocation via activation of endothelin ETA receptor in 3T3-L1 adipocytes

Endothelin-1 (ET-1) is a 21-amino acid peptide that binds to G-protein-coupled receptors to evoke biological responses. This report studies the effect of ET-1 on regulating glucose transport in 3T3-L1 adipocytes. ET-1, but not angiotensin II, stimulated glucose uptake in a dose-dependent manner with an EC50 value of 0.29 nM and a 2.47-fold stimulation at 100 nM. ET-1 stimulated glucose uptake in differentiated 3T3-L1 cells but had no effect in undifferentiated cells, although ET-1 stimulated phosphatidylinositol hydrolysis to a similar degree in both. The 3T3-L1 cells expressed approximately 560,000 sites/cell of ETA receptor, which was not altered during differentiation. Western blot analysis and immunofluorescence staining show that ET-1 stimulated the translocation of insulin-responsive aminopeptidase and GLUT4 to the plasma membrane. The effect of ET-1 on glucose uptake was blocked by A-216546, an antagonist selective for the ETA receptor. ET-1 treatment did not induce phosphorylation of insulin receptor beta-subunit, insulin receptor substrate-1, or Akt but stimulated the tyrosyl phosphorylation of a 75-kDa protein. Genistein (100 microM), an inhibitor of tyrosine kinases, inhibited ET-1-stimulated glucose uptake. Our results show that ET-1 stimulates GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes via activation of ETA receptor.     

Cloning and chromosomal localization of a human endothelin ETA receptor.

A cDNA clone encoding a human endothelin receptor was isolated from a placenta cDNA library. The deduced amino acid sequence of the clone is 94% identical to the bovine endothelin ETA receptor and represents the human homologue. The human endothelin ETA receptor gene was localized to chromosome 4 by analysis of its segregation pattern in rodent-human hybrids.     

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.     

Distribution of endothelin receptor subtypes ETA and ETB in the rat kidney.

The endothelin (ET) receptor system is markedly involved in the regulation of renal function under both physiological and pathophysiological conditions. The present study determined the detailed cellular localization of both ET receptor subtypes, ET(A) and ET(B), in the vascular and tubular system of the rat kidney by immunofluorescence microscopy. In the vascular system we observed both ET(A) and ET(B) receptors in the media of interlobular arteries and afferent and efferent arterioles. In interlobar and arcuate arteries, only ET(A) receptors were present on vascular smooth muscle cells. ET(B) receptor immunoreactivity was sparse on endothelial cells of renal arteries, whereas there was strong labeling of peritubular and glomerular capillaries as well as vasa recta endothelium. ET(A) receptors were evident on glomerular mesangial cells and pericytes of descending vasa recta bundles. In the renal tubular system, ET(B) receptors were located in epithelial cells of proximal tubules and inner medullary collecting ducts, whereas ET(A) receptors were found in distal tubules and cortical collecting ducts. Distribution of ET(A) and ET(B) receptors in the vascular and tubular system of the rat kidney reported in the present study supports the concept that both ET receptor subtypes cooperate in mediating renal cortical vasoconstriction but exert differential and partially antagonistic effects on renal medullary function.     

Pharmacological characteristics of endothelin receptors in the rabbit ventricular myocardium: The nonselective endothelin receptor antagonist PD 145065 antagonizes the positive inotropic effect of endothelin-3 but not of endothelin-1

Endothelin-3 (ET-3) elicited a concentration-dependent positive inotropic effect on rabbit papillary muscle, the maximal response being approximately 65% of the maximal response to isoproterenol. ET-1 induced a positive inotropic effect over the concentration range below 10^–9 M, at which ET-3 did not produce a positive inotropic effect, but the maximal response to ET-1 was equivalent to or slightly lower than that of ET-3. The nonselective ET receptor antagonist PD 145065 effectively antagonized the positive inotropic effect of ET-3 in a concentration-dependent manner and abolished it at 10^–5 M. PD 145065 decreased the positive inotropic effect induced by ET 1 at lower concentrations (< 10^–9 M) but it did not affect the main portion of the concentration-response curve for the positive inotropic effect, i.e., the effect induced by high concentrations (> 10^–9 M) of ET-1. PD 145065 antagonized also the positive inotropic effect of sarafotoxin S6c. PD 145065 inhibited the specific binding of [¹²⁵I]ET-1 and of [¹²⁵I]ET-3 with a high- and a low-affinity site for competition. ET_B selective ligands, RES-701-1 and sarafotoxin S6c, displaced [¹²⁵I^uc]ET-3 with high affinity but they scarcely affected the [1251]ET-1 binding. These findings indicate that different subtypes of the ET receptor are responsible for the induction of the positive inotropic effect of ET-3 and ET-1. ET receptors involved in the production of the positive inotropic effect in the rabbit ventricular myocardium have pharmacological characteristics that are different from those of conventional ET receptors originally classified based on the pharmacological findings in noncardiac tissues. The positive inotropic effect of ET-3 in the rabbit ventricular muscle may be mediated predominantly by ET_A1 receptors that are susceptible to PD 145065 as well as BQ-123 and FR139317, and partially mediated by ET_B receptors that are inhibitable with RES-701-1. ET_A2 receptors that are resistant to ET_A selective as well as nonselective antagonists may mainly be responsible for the positive inotropic effect of ET-1 in the rabbit ventricular muscle.     

Angiotensin II enhances endothelin-1-induced vasoconstriction through upregulating endothelin type A receptor

Endothelin-1 (ET-1) is the most potent vasoconstrictor by binding to endothelin receptors (ET_AR) in vascular smooth muscle cells (VSMCs). The complex of angiotensin II (Ang II) and Ang II type one receptor (AT₁R) acts as a transient constrictor of VSMCs. The synergistic effect of ET-1 and Ang II on blood pressure has been observed in rats; however, the underlying mechanism remains unclear. We hypothesize that Ang II leads to enhancing ET-1-mediated vasoconstriction through the activation of endothelin receptor in VSMCs. The ET-1-induced vasoconstriction, ET-1 binding, and endothelin receptor expression were explored in the isolated endothelium-denuded aortae and A-10 VSMCs. Ang II pretreatment enhanced ET-1-induced vasoconstriction and ET-1 binding to the aorta. Ang II enhanced ET_AR expression, but not ET_BR, in aorta and increased ET-1 binding, mainly to ET_AR in A-10 VSMCs. Moreover, Ang II-enhanced ET_AR expression was blunted and ET-1 binding was reduced by AT₁R antagonism or by inhibitors of PKC or ERK individually. In conclusion, Ang II enhances ET-1-induced vasoconstriction by upregulating ET_AR expression and ET-1/ET_AR binding, which may be because of the AngII/Ang II receptor pathways and the activation of PKC or ERK. These findings suggest the synergistic effect of Ang II and ET-1 on the pathogenic development of hypertension.     

Endogenous endothelin attenuates the pressor response to acute environmental stress via the ETA receptor

Clinical studies have documented an abrupt rise in plasma endothelin-1 (ET-1) coincident with an increase in mean arterial pressure (MAP) during the response to acute stress. We therefore examined the ET(A) and ET(B) receptor-dependent effects of ET-1 on the pressor response to acute environmental stress in ET-1-dependent hypertension. Stress was induced by administration of air jet pulses (3 min) in ET(B) receptor-deficient (ET(B) sl/sl) rats fed normal salt (NS; 0.8% NaCl), high salt (HS; 8% NaCl), and HS plus the ET(A) receptor antagonist ABT-627 (5 mg.kg(-1).day(-1)) on successive weeks. MAP was chronically monitored by telemetry. Total pressor response (area under the curve) was significantly reduced in ET(B) sl/sl rats maintained on a HS vs. NS diet [-6.8 mmHg (SD 18.7) vs. 29.3 mmHg (SD 8.1) x 3 min, P < 0.05]. Conversely, the total pressor response was augmented in both wild-type [34.2 mmHg (SD 29.2) x 3 min, P < 0.05 vs. NS] and ET(B) sl/sl rats [49.1 mmHg (SD 11.8) x 3 min, P < 0.05 vs. NS] by ABT-627. Blockade of ET(B) receptors in Sprague-Dawley rats caused an increase in basal MAP that was enhanced by HS and lowered by mixed ET(A)/ET(B) receptor antagonism; none of these treatments, however, had any effect on the pressor response. These data demonstrate that increasing endogenous ET-1 suppresses the pressor response to acute stress through ET(A) receptor activation in a genetic model of ET-1-dependent hypertension. These results are consistent with reports that ET-1 can attenuate sympathetically mediated responses.     

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.     

Evaluation of endothelin receptor populations using endothelin-1 biotinylated at lysine-9 sidechain.

Optimal conditions for biotinylation of endothelin-1 (Et-1) were determined using biotinylating reagents of variable linker arm length and mono- vs dual-biotinylated Et-1. Specific modification of lysine-9 sidechain with NHS-LC-biotin (Et-1[BtK9]) produced a derivative with maximal binding and retention of vascular smooth muscle contractile activity. The Et-1[BtK9] probe bound to Chinese hamster ovary cells transfected with EtA receptor cDNA (CHO[EtR]), but not untransfected cells. Binding to rat vascular smooth muscle cells (VSMC) was detectable at 0.01 nM with maximal binding at 1 nM. Displacement of 1 nM Et-1 [BtK9] binding by Et-1 indicated an IC50 value of 6 +/- 3 nM. Et-1 displaced Et-1[BtK9] binding to VSMC and CHO[EtR] to a greater extent than endothelin-3, indicating predominant expression of EtA receptor sub-type. Thus, biotinylation of Et-1 at the lysine-9 sidechain may be of general use for localization and typing of Et-receptor populations.     

Structural determinants for selective recognition of peptide ligands for endothelin receptor subtypes ETA and ETB

The molecular basis for recognition of peptide ligands endothelin‐1, ‐2 and ‐3 in endothelin receptors is poorly understood. Especially the origin of ligand selectivity for ET_A or ET_B is not clearly resolved. We derived sequence‐structure‐function relationships of peptides and receptors from mutational data and homology modeling. Our major findings are the dissection of peptide ligands into four epitopes and the delineation of four complementary structural portions on receptor side explaining ligand recognition in both endothelin receptor subtypes. In addition, structural determinants for ligand selectivity could be described. As a result, we could improve the selectivity of BQ3020 about 10‐fold by a single amino acid substitution, validating our hypothesis for ligand selectivity caused by different entrances to the receptors' transmembrane binding sites. A narrow tunnel shape in ET_A is restrictive for a selected group of peptide ligands' N‐termini, whereas a broad funnel‐shaped entrance in ET_B accepts a variety of different shapes and properties of ligands. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

A rabbit antiserum raised against the hexapeptide endothelin(16-21) shows different binding affinities for endothelin-1, -2 and -3.

A antiserum raised against the C-terminal hexapeptide ET16-21 common to ET-1, -2 and -3 was produced and characterized with respect to its binding properties for ET-1, -2, -3, ET16-21, the C-terminal octapeptide ET14-21, its derivative Phe21-ET14-21 and human big-ET-1. The antibody reacted with the peptides with decreasing binding affinities in the order: ET-1 greater than ET-2 greater than or equal to ET16-21 = ET 14-21 much greater than Phe21-ET14-21. It showed no crossreactivity with human big-ET-1. Similar results were obtained using [125I]ET-1, -2 or -3 as tracer. Substitution of Trp21 by Phe decreased the binding affinity of ET14-21 about 10 fold. Thus, the immunologically recognized sequence of the peptides is C-terminal and Trp21 seems to be important for high binding affinities. The significant differences in binding affinity observed for ET-1, -2, -3 and ET16-21 are consistent with an interaction of the C-terminal part of the endothelins with the bicyclic N-terminal part.     

Different stability of ligand-receptor complex formed with two endothelin receptor species, ETA and ETB.

There are at least two types of endothelin receptors, ETA and ETB, present in various tissues. We found that although biotinylated ET-1 could bind to both ETA and ETB receptors, the stability of the formed ligand-receptor complexes was different. When the preformed complexes of receptor (solubilized from canine brain and lung membranes) and biotinylated ET-1 were subjected to avidin agarose column chromatography, most of the ETA activity was recovered in the pass-through fraction and the remainder was recovered in the 0.5 M KSCN eluate as ligand-free forms. On the other hand, the ETB activity bound firmly to the avidin agarose column was eluted with 1.5 M KSCN. The detection of the complex of 125I-ET-1 and its receptor by SDS-PAGE run at a low temperature was only possible with the ETB fractions and the complex of 125I-ET-1 and ETA was unstable during the separation. These results suggest that the conformation of the ligand binding sites of canine ETA and ETB as well as the stability of their ligand-receptor complexes to SDS are significantly different. Similar observations were also obtained for human ETA and ETB receptors.     

Hybrid peptides constructed from RES-701-1, an endothelin B receptor antagonist, and endothelin; binding selectivity for endothelin receptors and their pharmacological activity

Hybrid peptides were constructed from endothelin B receptor (ET_B) selective antagonist RES-701-1 (1) and endothelin (ET-1). They have N-terminal 10 amino acids derived from 1 and C-terminal 10 amino acids derived from ET-1. RES-701-1(1-10)-[Ala15]ET-1(12-21) and its analogues substituted or truncated at the residues derived from RES-701-1 had proved to possess high receptor binding activity selective for ET_B as well as 1. Substitutions at the residues derived from ET-1 had produced some analogues that possessed high affinity not only for ET_B but for ET_A. Although all analogues had antagonistic effects on ET_A, some analogues had proved to function as agonist on ET_B confirmed by the changes in intracellular calcium concentrations of ET receptor-transfected COS-7 cells. We have found four types of ET receptor-binding peptides: (1) ET_B-selective agonist with weak ET_A antagonism (3, KT7421); (2) ET_B-selective antagonist with weak ET_A antagonism (29, KT7539); (3) ET_B agonist with potent ET_A antagonism (27, KT7538); and (4) non-selective ET_A/ET_B antagonist (26, KT7540).     

C-terminal modifications of an endothelin antagonist RES-701-1: Production of ETA/ETB dual selective analogs

Summary RES-701-1 is an endothelin B receptor (ET_B) selective peptidic antagonist, which has a novel cyclic structure of microbial origin. Modification at the C-terminal free carboxyl group of RES-701-1 by a methyl ester results in an ET_A/ET_B dual selective analog, which showed relatively high affinity for ET_A receptor subtype, while retaining the affinity for ET_B. The carboxyl-group-deleted analog with tryptamine as the C-terminal residue also showed relatively weak affinity for ET_A; however, benzyl ester or amide analogs did not show remarkable affinity for ET_A. It is suggested that the binding mode of RES-701-1 and its analogs is different from those of known ligands for ET receptors.