Endothelin Receptor Binding Research Articles

Pharmacological and structural characterization of long-sarafotoxins, a new family of endothelin-like peptides: Role of the C-terminus extension

Long-sarafotoxins (l-SRTXs) have recently been identified in both the venom of Atractaspis microlepidota and that of Atractaspis irregularis. They are characterized by different C-terminus extensions that follow the invariant Trp21, which plays a crucial role in endothelin-receptor binding. We initially determined the toxicity and three-dimensional structures of two chemically synthesized l-SRTXs that have different C-terminus extensions, namely SRTX-m (24 aa, including extension “D-E-P”) and SRTX-i3 (25 aa, including extension “V-N-R-N”). Both peptides were shown to be highly toxic in mice and displayed the cysteine-stabilized α-helical motif that characterizes endothelins and short-SRTXs, to which a longer C-terminus with variable flexibility is added. To discern the functional and pharmacological consequences of the supplementary amino acids, different chimerical as well as truncated forms of SRTX were designed and synthesized. Thus, we either removed the extra-C-terminal residues of SRTX-m or i3, or grafted the latter onto the C-terminal extremity of a short-SRTX (s-SRTX) (ie. SRTX-b). Our competitive binding assays where SRTXs competed for iodinated endothelin-1 binding to cloned ETA and ETB receptor subtypes over-expressed in CHO cells, revealed the essential role of the C-terminus extensions for ET-receptor recognition. Indeed, l-SRTXs displayed an affinity three to four orders of magnitude lower as compared to SRTX-b for the two receptor subtypes. Moreover, grafting the C-terminus extension to SRTX-b induced a drastic decrease in affinity, while its removal (truncated l-SRTXs) yielded an affinity for ET-receptors similar to that of s-SRTXs. Furthermore, we established by intracellular Ca2+ measurements that l-SRTXs, as well as s-SRTXs, display agonistic activities. We thus confirmed in these functional assays the major difference in potency for these two SRTX families as well as the crucial role of the C-terminus extension in their various pharmacological profiles. Finally, one of the chimeric toxin synthesized in this study appears to be one of the most potent and selective ligand of the ETB receptor known to date.

EndothelinA-endothelinB receptor cross-talk and endothelin receptor binding

The magnitude of inhibition of an endothelin (ET)-1 response by selective blockade of the ET(A) or ET(B) receptors can be limited by apparent compensation mediated by the unblocked receptor. While the mechanism underlying this functionally defined interaction, or 'cross-talk', is not clear, binding studies suggest an interaction between the ET receptor subtypes. These binding studies are reviewed and suggest that, in general, they support the hypothesis that ET(A) and ET(B) receptor activation of intracellular signalling pathways influence ET-1 binding to these receptor subtypes. However, the relationship of these binding studies to functional effects and, thus, functional ET(A)-ET(B) receptor cross-talk, remains largely untested.

Positron emission tomography of [18F]‐big endothelin‐1 reveals renal excretion but tissue‐specific conversion to [18F]‐endothelin‐1 in lung and liver

Big endothelin-1 (ET-1) circulates in plasma but does not bind to ET receptors until converted to ET-1 by smooth muscle converting enzymes. We hypothesized that tissue-specific conversion of [(18)F]-big ET-1 to [(18)F]-ET-1 could be imaged dynamically in vivo within target organs as binding to ET receptors. [(18)F]-big ET-1 conversion imaged in vivo following infusion into rats using positron emission tomography (PET). [(18)F]-big ET-1 was rapidly cleared from the circulation (t(1/2)= 2.9 +/- 0.1 min). Whole body microPET images showed highest uptake of radioactivity in three major organs. In lungs and liver, time activity curves peaked within 2.5 min, then plateaued reaching equilibrium after 10 min, with no further decrease after 120 min. Phosphoramidon did not alter half life of [(18)F]-big ET-1 but uptake was reduced in lung (42%) and liver (45%) after 120 min, consistent with inhibition of enzyme conversion and reduction of ET-1 receptor binding. The ET(A) antagonist, FR139317 did not alter half-life of [(18)F]-big ET-1 (t(1/2)= 2.5 min) but radioactivity was reduced in all tissues except for kidney consistent with reduction in binding to ET(A) receptors. In kidney, however, the peak in radioactivity was higher but time to maximum accumulation was slower ( approximately 30 min), which was increased by phosphoramidon, reflecting renal excretion with low conversion and binding to ET receptors. A major site for conversion was within the vasculature of the lung and liver, whereas uptake in kidney was more complex, reflecting excretion of [(18)F]-big ET-1 without conversion to ET-1.

PET-compatible endothelin receptor radioligands: Synthesis and first in vitro and in vivo studies

The expression and function of endothelin (ET) receptors is abnormal in cardiovascular diseases, tumor progression, and tumor metastasis. In this study, we prepared two [ 18F]-fluorinated derivatives of the non-peptide ET A receptor antagonist PD 156707 and evaluated their ET receptor binding potencies. Ex vivo as well as in vivo biodistribution studies in mice were performed, as well as the metabolism of the radiotracer, which was examined by metabolite analysis in mice and rats. All tested derivatives of PD 156707 exhibited potent in vitro pharmacological characteristics with K i values comparable to that of the lead compound. The biodistribution studies showed a high accumulation of the tracer in bile and intestine. In vivo we were able to show that the visualization of the heart as a major target organ with high ET AR expression is possible.

Altered Endothelin Receptor Binding in Response to Nitric Oxide Synthase Inhibition in the Pregnant Rat

The authors evaluate the expression of endothelin-1 (ET-1) and its receptors in the uterus and placenta during maternal nitric oxide synthase (NOS) inhibition. Timed-pregnant rats received L-NAME (2.5 mg/kg/h) or saline from day 14 to 21 of gestation. Uterine and placental tissues collected on day 21 were assayed for preproET-1, ET( A), and ET(B) mRNA expression; localization and expression of ET-1 and receptor proteins; and receptor activity. NOS inhibition did not affect preproET-1 mRNA expression in the placenta or uterus. ET(A) expression decreased in the uterine free wall, but no other changes in receptor mRNA expression were observed in the uterus or placenta. ET-1 and receptor proteins were unchanged. Placental ET(A) and ET(B) receptor binding decreased. Uterine ET(A) receptor binding decreased in the placental bed. ET-1, a prominent mediator during NOS inhibition, is not of uterine or placental origin. Reduced receptor binding activity is the primary means by which these tissues regulate their response to ET-1 in the setting of NOS inhibition.

Synthesis and Endothelin Receptor Binding Affinity of a Novel Class of 2-Substituted-4-aryl-3-quinolinecarboxylic Acid Derivatives

The 21-amino acid peptide endothelin-1 (ET-1) is the predominant isoform of the endothelin peptide family, which includes ET-2, and ET-3. These peptides display a variety of physiological activities including vasoconstriction and the stimulation of cell proliferation in tissues both within and outside of the cardiovascular system. They exert their actions via activation of two distinct receptor subtypes, ET(A) and ET(B), belonging to the G protein-coupled receptor (GPCR) superfamily. Ligands of these receptors have received numerous citations in the recent pharmaceutical literature. In particular receptor antagonists, both ET(A)- and ET(B)-selective, as well as non-selective, have been described due to their wide therapeutic potential. As a part of our program toward the development of selective ET(A) ligands we have designed and we now report new molecules based on 2-substituted-4-aryl-3-quinolinecarboxylic acid moiety. Binding profile for some compounds (40, 44, 46, and 47) of this class showed a reasonable affinity and selectivity for ET(A) receptors.

Tanshinone IIA inhibits endothelin-1 production in TNF-?-induced brain microvascular endothelial cells through suppression of endothelin-converting enzyme-1 synthesis

To investigate the effects of tanshinone IIA (Tan IIA) on the regulation of the production of endothelin (ET)-1 (including large ET-1), mRNA levels of ET-1, endothelin-converting enzyme-1 (ECE-1), endothelin-A receptor (ETA) and endothelin-B receptor (ETB) induced by TNF-alpha in rat brain microvascular endothelial cells (BMVEC). The ET-1 release (including large ET-1) into the culture medium was determined by enzyme immunoassay. The levels of ET-1, ECE-1, ETA, and ETB mRNA were measured by RT-PCR. Endothelin receptor binding was also tested. The induction of ET-1 release by TNF-alpha from cultured BMVEC was dose-dependently reduced by Tan IIA, but large ET-1 levels progressively increased in response to Tan IIA; the mRNA expression of ET-1 was unaffected. Tan IIA also caused a decrease in ETA receptor mRNA and ECE-1 expression in a dose-dependent manner. Endothelin receptor binding was unaltered in BMVEC stimulated with TNF-alpha alone or a combination of TNF-alpha and Tan IIA. These findings suggest that Tan IIA may inhibit ET-1 production in TNF-alpha-induced BMVEC through the suppression of ECE-1 synthesis.

The effect of acute ischemia on ET-1 and its receptors in patients with underlying chronic ischemia of the lower limb.

Elevated plasma and tissue endothelin (ET)-1 levels in patients with critical limb ischemia (CLI) has been described. Here the effect of a period of acute ischemia and subsequent reperfusion on plasma ET-1 and tissue ET-1/ET receptors in skeletal muscle biopsies from CLI patients undergoing femoro-distal bypass surgery was studied. Peripheral and "local" blood and muscle biopsies were obtained from patients undergoing femoro-distal bypass surgery, at the start of the procedure (control), after a period of vascular clamping (ischemia), and after clamp release (reperfusion). Plasma ET-1 was determined by enzyme-linked immunosorbent assay. Tissue ET-1 was assessed by counting ET-1 immunostaining cells per unit area, and ET(A)/ET(B) receptors were identified on sections by in vitro autoradiography in which binding was quantitatively assessed by densitometry. There was no significant effect of ischemia or reperfusion on plasma ET-1 levels or on ET(A)/ET(B) receptor binding. However, tissue ET-1 increased during both acute ischemia and reperfusion (P < 0.05). A high proportion of positive ET-1 immunostaining was associated with microvessels and also exhibited a similar distribution to macrophages. Previously, it has been shown that both plasma ET-1 and tissue ET-1/ET receptors are increased in CLI patients compared with atherosclerotic controls. Also, increased muscle ET-1 levels have been described in acute ischemia caused by tourniquet application in nonischemic patients undergoing total knee replacement. In CLI patients, in whom ET-1 is already upregulated, this further increase may exacerbate existing pathologic processes and contribute to ischemia-reperfusion injury. ET-1 antagonists may therefore be useful adjuncts in CLI and other surgical procedures in which ischemia-reperfusion damage occurs.

4,4-Dinitro-5,6,7,8,9,10-hexahydro-4H-cyclonona[d]isoxazole

The title compound, C10H13N3O5, was designed as a potential endothelin receptor antagonist and as a probe of the structural requirements of the endothelin receptor binding site. The isoxazole ring is planar and the cyclo­nona­ne moiety lies approximately in the plane of the heterocycle. The mol­ecules are packed in the crystal structure by ionic and van der Waals inter­actions.

Anatomically distinct activation of endothelin-3 and the l-arginine/nitric oxide pathway in the kidney with advanced aging

Aging is associated with spontaneous degenerative changes of renal function and structure. The aim of this study was to determine changes of the endothelin (ET) system and NO tissue bioactivity during the physiological aging process. Renal protein expression of ET-1 and ET-3, ET A, and ET B receptor mRNA expression, ET receptor binding and distribution, and tissue NO metabolite content were determined in adult, middle-aged, and senescent normotensive female Wistar rats. In senescent animals, medullary ET-3 content increased 3.4-fold ( p < 0.05 vs. adult), whereas aging did not affect ET-3 levels in the cortex. Local NO bioavailability, determined by NO metabolite tissue measurements, decreased in the cortex only. ET receptor binding capacity—predominantly due to ET B receptor binding—was lower in medulla than in cortex. Aging had no effect on ET-1 binding capacity or ET receptor distribution, whereas with advanced age gene expression of both receptors decreased. In conclusion, aging causes distinctive expressional changes of the renal endothelin system in otherwise healthy rats. The pronounced increase of endothelin-3 in the renal medulla is associated with preservation of local NO metabolite levels, changes not observed in the cortex. These findings could be important for pathologies and possibly therapy associated with renal aging.

Altered Endothelin-1 Levels in Acute Lower Limb Ischemia and Reperfusion

Tourniquet-induced ischemia is often used in orthopedic and reconstructive procedures. This is associated with muscle damage and dysfunction, which limits tourniquet application time. Endothelin-1 (ET-1) is a potent vasoconstrictor, which has been implicated in ischemic conditions and ischemia-reperfusion injury. This study aimed to investigate the role of ET-1 in human skeletal muscle subjected to tourniquet-induced acute ischemia and reperfusion. Thirteen patients undergoing total knee replacement were studied. Plasma and muscle ET-1 concentrations were measured at the start of surgery, after an hour of acute ischemia, and 15 minutes following reperfusion. ET-1 receptor binding was also studied by use of autoradiography, and ET-1 mRNA expression investigated by use of real-time polymerase chain reaction (RT-PCR). Tissue ET-1 increased following the period of acute ischemia and persisted during reperfusion. ET-1 was associated with microvessels and macrophages in the muscle. No changes in circulating ET-1 levels, ET-1 mRNA expression, or ET-1 receptor binding were found. It is concluded that the ET-1 pathway is involved in acute ischemia and reperfusion and it may contribute to the muscle injury that occurs during surgical procedures.

Potential role of endothelin 1 in ischaemia-induced angiogenesis in critical leg ischaemia.

Ischaemia-induced angiogenesis occurs in critical leg ischaemia (CLI) and endothelin (ET) 1 may be involved in this process. The aim of this study was to quantify microvessels and study ET receptor expression and distribution in critically ischaemic leg muscle. Leg muscle biopsies were taken from 12 patients with CLI and 12 patients with no leg ischaemia. Microvessels were identified immunohistochemically on muscle sections, and the number of immunopositive cells was quantified. ETA and ETB receptor messenger RNA (mRNA) expression was studied using real-time quantitative reverse transcriptase-polymerase chain reaction, and receptor binding was localized and assessed by in vitro autoradiography. The number of microvessels in CLI muscle biopsies was 2.6 times higher than that in controls (P < 0.01). ETB receptor mRNA expression and binding were significantly increased in CLI tissue (P < 0.05), while ETA receptor levels were not significantly raised. High-resolution autoradiography showed that ET receptor binding was associated with microvessels. Angiogenesis occurs in CLI and raised ETB receptors within the muscle were associated with microvessels, suggesting that ET-1 may mediate angiogenesis via these receptors in critically ischaemic muscle.

Decreased endothelin binding and [Ca2+]i signaling in microvessels of DOCA-salt hypertensive rats.

The deoxycorticosterone acetate (DOCA)-salt model of hypertension is characterized by elevated vascular endothelin-1 (ET-1) and by reduced contraction to ET-1 in isolated mesenteric small arteries. The decreased contraction to ET-1 may be a compensatory mechanism caused by elevations in ET-1 and arterial pressure. The present study was designed to determine whether down-regulation of endothelin receptors or altered Ca2+ signaling contribute to the decreased contraction to ET-1. Contraction to ET-1 (10 to 10 mol/l) was significantly reduced in isolated mesenteric small arteries (87-286 microm intraluminal diameter) from DOCA-salt rats compared with placebo rats. Membrane protein was obtained for measurement of [125I]ET-1 receptor binding and ET receptor expression. Maximum binding was significantly reduced in vascular membranes from DOCA-salt rats (670 +/- 71 fmol/mg protein) compared with placebo rats (1165 +/- 75 fmol/mg protein), but binding affinity was unchanged. Conversely, ETA receptor protein was increased in DOCA-salt rat vessels. To assess Ca2+ signaling, freshly dissociated mesenteric small artery smooth muscle cells were loaded with fura-2 for measurement of the average myoplasmic free Ca2+ concentration ([Ca2+ ] ). The ET-1 (10 mol/l) induced increase in [Ca2+ ] was significantly less in cells from DOCA-salt rats compared with from placebo rats. This effect was not due to a loss of L-type Ca2+ channels since expression was increased in membrane protein from DOCA-salt rats compared with placebo rats, as measured by Western blot analysis. These findings indicate that decreases in receptor binding and Ca2+ signaling contribute to the impaired contraction to ET-1 in DOCA-salt hypertensive rats. However, these changes are not due to reduced expression of ETA receptors or L-type Ca2+ channels.

Effect of castration on endothelin receptors.

Endothelin (ET) plays a pivotal role in the pathogenesis of cell growth disorders such as cancer. Atrasentan (ABT-627), a selective antagonist for the ET receptor A (ET(A)), has shown benefit in controlling disease progression in men with hormone refractory prostate cancer who have undergone aggressive hormone ablation therapy. It is not known how hormone ablation affects ET-binding sites, although ET-1 and ET(A) expression are found to be elevated in prostate cancer patients. In this study, we examined the effect of castration on ET receptor binding in male beagle dogs. Three dogs were surgically castrated and another three sham-operated. The animals were sacrificed 1 week after operation and membranes were prepared from the prostate, heart, brain, kidney, liver and lung for ET-1, ET-3 and angiotensin II (A-II, as a control) binding studies. No significant difference in A-II binding was observed between castrated and sham-operated animals. However, ET-1 and ET-3 binding to prostate and brain membranes were altered significantly. From saturation binding studies using ET-1 in the prostate, the K(d) and B(max) values increased from 0.043 nM and 0.094 pmol/mg respectively in sham-operated dogs to 0.104 nM and 0.311 pmol/mg respectively in castrated animals. These results indicate that surgical castration in dogs produces a change in the ET receptor density in the prostate and brain, and may have implications for the effect of hormone ablation therapy on ET receptor expression in prostate cancer patients.

Role of ET-1 receptor binding and [Ca(2+)](i) in contraction of coronary arteries from DOCA-salt hypertensive rats.

Hypertension is associated with an increase in coronary artery disease, but little is known about the regulation of coronary vascular tone by endothelin-1 (ET-1) in hypertension. The present study evaluated the mechanisms mediating altered contraction to ET-1 in coronary small arteries from deoxycorticosterone acetate (DOCA)-salt hypertensive rats. DOCA-salt rats exhibited an increase in systolic blood pressure and plasma ET-1 levels compared with placebo rats. Contraction to ET-1 (1 x 10(-11) to 3 x 10(-8) M), measured in isolated coronary small arteries maintained at a constant intraluminal pressure of 40 mmHg, was largely reduced in vessels from DOCA-salt rats compared with placebo rats. To determine the role of endothelin receptor binding in the impaired contraction to ET-1, (125)I-labeled ET-1 receptor binding was measured in membranes isolated from coronary small arteries. Maximum binding (fmol/mg protein) and binding affinity were similar in coronary membranes from DOCA-salt rats compared with placebo rats. Changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) were measured in freshly dissociated coronary small artery smooth muscle cells loaded with fura 2. ET-1 (10(-9) M) produced a 30 +/- 9% increase in [Ca(2+)](i) in smooth muscle cells from placebo rats, but had no effect on cells from DOCA-salt rats (2 +/- 2%). In summary, the ET-1-induced coronary artery contraction and increase in [Ca(2+)](i) are impaired in DOCA-salt hypertensive rats, whereas endothelin receptor binding is not altered. These results suggest endothelin receptor uncoupling from signaling mechanisms and indicate that impaired [Ca(2+)](i) signaling contributes to the decrease in ET-1-induced contraction of coronary small arteries in DOCA-salt hypertensive rats.

Endothelin Receptor Remodeling Induces the Portal Venous Hyper-Response to Endothelin Following Endotoxin Pretreatment

The purpose of this study was to determine the changes in endothelin (ET) receptor subtype expression and their functional significance after endotoxin pretreatment. Rats were pretreated with lipopolysaccharide (LPS) or sterile saline (control). After 24 h, liver samples were homogenized and competitive receptor binding assays were performed. There was no significant difference in ET receptor binding affinity between the control and LPS groups. However, the receptor subtype density showed a significant increase in ET(B) receptors in LPS-treated rats, whereas the amount of ET(A) receptors was almost identical between the two groups. In control, almost all ET receptors (95%) were displaced by using combined ET(A) antagonist (BQ-610) and ET(B) agonist (IRL-1620) as competitors, whereas only 80% (P < 0.05 versus control) was displaced in LPS group, raising the possibility of novel type of ET receptor expression. An infusion of ET(B) agonist (Sarafotoxin 6c, S6c) through portal vein in isolated perfused livers produced the same pressure response in both LPS and control groups; however, the portal pressure increase in response to the ET-1, which binds all ET receptors, was significantly potentiated in LPS-treated rats compared to controls. We conclude that altered regulation of ET receptors, in particular, the appearance of ET binding capacity that is not displaced by ET(A) or ET(B) competitors, may explain the hyper-response of the portal venous system to ET-1 during endotoxemia.

ChemInform Abstract: Nonpeptide Endothelin Antagonists: From Lower Affinity Pyrazol-5-ols to Higher Affinity Pyrazole-5-carboxylic Acids.

Abstract Random screening of compounds in endothelin receptor (ET A and ET B ) binding assays led to the discovery of a new class of pyrazol-5-ol ligands. Characterization of structural features crucial for binding activities of these pyrazol-5-ols, by structure–activity-relationship (SAR) studies, allowed us to design a novel class of pyrazole-5-carboxylic acids as more potent ET antagonists.

Nonpeptide endothelin antagonists: from lower affinity pyrazol-5-ols to higher affinity pyrazole-5-carboxylic acids

Random screening of compounds in endothelin receptor (ET A and ET B) binding assays led to the discovery of a new class of pyrazol-5-ol ligands. Characterization of structural features crucial for binding activities of these pyrazol-5-ols, by structure–activity-relationship (SAR) studies, allowed us to design a novel class of pyrazole-5-carboxylic acids as more potent ET antagonists.

Synthesis and endothelin receptor binding activity of synthetic analogues of RES-1149-2

A direct synthesis of analogues 6 and 7 is described. The key transformations are the addition of dibromomethyl lithium to a ketone and the subsequent mild hydrolysis to a hemiacetal.

Upregulation of endothelin B receptors in kidneys of DOCA-salt hypertensive rats

Experiments were designed to elucidate the role of endothelin B receptors (ET(B)) on arterial pressure and renal function in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Male Sprague-Dawley rats underwent uninephrectomy and were treated with either DOCA and salt (0.9% NaCl to drink) or placebo. DOCA-salt rats given the ET(B)-selective antagonist, A-192621, for 1 wk (10 mg. kg(-1). day(-1) in the food) had significantly greater systolic arterial pressure compared with untreated DOCA-salt rats (208 +/- 7 vs. 182 +/- 4 mmHg) whereas pressure in placebo rats was unchanged. In DOCA-salt, but not placebo rats, A-192621 significantly decreased sodium and water excretion along with parallel decreases in food and water intake. To determine whether the response in DOCA-salt rats was due to increased expression of ET(B) receptors, endothelin receptor binding was performed by using membranes from renal medulla. Maximum binding (B(max)) of [(125)I]ET-1, [(125)I]ET-3, and [(125)I]IRL-1620 increased from 227 +/- 42, 146 +/- 28, and 21 +/- 1 fmol/mg protein, respectively, in placebo rats to 335 +/- 27, 300 +/- 38, and 61 +/- 6 fmol/mg protein, respectively, in DOCA-salt hypertensive rats. The fraction of receptors that are the ET(B) subtype was significantly increased in DOCA-salt (0.88 +/- 0.07) compared with placebo (0.64 +/- 0.01). The difference between [(125)I]ET-3 and [(125)I]IRL-1620 binding is consistent with possible ET(B) receptor subtypes in the kidney. These results indicate that ET(B) receptors in the renal medulla are up-regulated in the DOCA-salt hypertensive rat and may serve to maintain a lower arterial pressure by promoting salt and water excretion.