New drugs and emerging therapeutic targets in the endothelin signaling pathway and prospects for personalized precision medicine.

During the last thirty years since the discovery of endothelin-1, the therapeutic strategy that has evolved in the clinic, mainly in the treatment of pulmonary arterial hypertension, is to block the action of the peptide either at the ET(A) subtype or both receptors using orally active small molecule antagonists. Recently, there has been a rapid expansion in research targeting ET receptors using chemical entities other than small molecules, particularly monoclonal antibody antagonists and selective peptide agonists and antagonists. While usually sacrificing oral bio-availability, these compounds have other therapeutic advantages with the potential to considerably expand drug targets in the endothelin pathway and extend treatment to other pathophysiological conditions. Where the small molecule approach has been retained, a novel strategy to combine two vasoconstrictor targets, the angiotensin AT(1) receptor as well as the ET(A) receptor in the dual antagonist sparsentan has been developed. A second emerging strategy is to combine drugs that have two different targets, the ET(A) antagonist ambrisentan with the phosphodiesterase inhibitor tadalafil, to improve the treatment of pulmonary arterial hypertension. The solving of the crystal structure of the ET(B) receptor has the potential to identify allosteric binding sites for novel ligands. A further key advance is the experimental validation of a single nucleotide polymorphism that has genome wide significance in five vascular diseases and that significantly increases the amount of big endothelin-1 precursor in the plasma. This observation provides a rationale for testing this single nucleotide polymorphism to stratify patients for allocation to treatment with endothelin agents and highlights the potential to use personalized precision medicine in the endothelin field.

Endothelial cell-specific ETB receptor knockout: autoradiographic and histological characterisation and crucial role in the clearance of endothelin-1.

Inactivation of endothelin B receptors (ETB), either through selective pharmacological antagonism or genetic mutation, increases the circulating concentration of endothelin-1 (ET-1), suggesting ETB plays an important role in clearance of this peptide. However, the cellular site of ETB-mediated clearance has not yet been determined. We have used a novel mouse model of endothelial cell-specific knockout (KO) of ETB (EC ETB(-/-)) to evaluate the relative contribution of EC-ETB to the clearance of ET-1. Phenotypic evidence of EC-specific ETB KO was confirmed by immunocytochemistry and autoradiography. Binding of the radiolabelled selective ETB ligand BQ3020 was significantly and selectively decreased in EC-rich tissues of EC ETB(-/-) mice, including the lung, liver, and kidney. By contrast, ETA binding was unaltered. RT-PCR confirmed equal expression of ET-1 in tissue from EC ETB(-/-) mice and controls, despite increased concentration of plasma ET-1 in EC ETB(-/-). Clearance of an intravenous bolus of [(125)I]ET-1 was impaired in EC ETB(-/-) mice. Pretreatment with the selective ETB antagonist A192621 impaired [(125)I]ET-1 clearance in control animals to a similar extent, but did not further impair clearance in EC ETB(-/-) mice. These studies suggest that EC-ETB are largely responsible for the clearance of ET-1 from the circulation.

Kisspeptins: a multifunctional peptide system with a role in reproduction, cancer and the cardiovascular system.

Orphan G-protein-coupled receptors that have recently been paired with their cognate ligand are an often untapped resource for novel drug development. The KISS1 receptor (previously designated GPR54) has been paired with biologically active cleavage peptides of the KiSS-1 gene product, the kisspeptins (KP). The focus of this review is the emerging pharmacology and physiology of the KP. Genetic linkage analysis in humans revealed that mutations in KISS1 (GPR54, AXOR12 or hOT7T175) result in idiopathic hypogonadotrophic hypogonadism and knockout mouse studies confirmed this finding. Identification of KISS1 (GPR54) as a molecular switch for puberty subsequently led to the discovery that KP activate the GnRH cascade. Prior to the role of KISS1 (GPR54) in puberty being described, KP had been shown to be inhibitors of tumour metastasis across a range of cancers. Subsequently the mechanism of this inhibition has been suggested to be via altered cell motility and adhesiveness. PCR detected highest expression of KP and KISS1 (GPR54) in placenta, and changes in KP levels throughout pregnancy and expression in trophoblasts suggests a role in placentation. Placentation and metastasis are invasive processes that require angiogenesis. Investigation of KISS1 (GPR54) and KP in vasculature revealed discrete localisation of KISS1 (GPR54) to blood vessels prone to atherosclerosis and a potent vasoconstrictor action. A role for KP has also been shown in whole body homeostasis. KP are multifunctional peptides and further investigation is required to fully elucidate the complex pathways regulated by these peptides and how these pathways integrate in the whole body system.

Characterization of the snake venom ligand [125I]-DNP binding to natriuretic peptide receptor-A in human artery and potent DNP mediated vasodilatation.

BACKGROUND AND PURPOSE: The natriuretic peptides, ANP and BNP, modulate vascular smooth muscle tone in human conduit arteries. Surprisingly, the natriuretic peptide receptor-A (NPR-A) has not been visualized using radioligand binding in these vessels. A new member of this peptide family, Dendroaspis natriuretic peptide (DNP) identified from snake venom, has been proposed to be present in human plasma and endothelial cells. Also, recently a novel radioligand, [(125)I]-DNP, has been characterized as selective for NPR-A in human heart. EXPERIMENTAL APPROACH: Our aims were to investigate expression and function of NPR-A receptors in human mammary artery using [(125)I]-DNP to quantify receptor density, immunocytochemistry to delineate the cellular distribution of the receptor and in vitro pharmacology to compare DNP induced vasodilatation to that of ANP. KEY RESULTS: Saturable, sub-nanomolar affinity [(125)I]-DNP binding was detected to smooth muscle of mammary artery, with receptor density of approximately 2 fmol mg(-1) protein, comparable to that of other vasoactive peptides. NPR-A immunoreactivity was localised to vascular smooth muscle cells and this was confirmed with fluorescence dual labelling. NPR-A expression was not detected in the endothelium. Like ANP, DNP fully reversed the constrictor response to ET-1 in endothelium intact or denuded mammary artery, with comparable nanomolar potencies. CONCLUSIONS AND IMPLICATIONS: This is the first characterization of NPR-A in human mammary artery using [(125)I]-DNP and we provide evidence for the presence of receptor protein on vascular smooth muscle cells, but not endothelial cells. This implies that the observed vasodilatation is predominantly mediated via direct activation of smooth muscle NPR-A.

Maternal dexamethasone increases endothelin-1 sensitivity and endothelin a receptor expression in ovine foetal placental arteries.

Despite National Institutes of Health recommendations to administer antenatal steroids as a single course to women threatening preterm delivery, repeated treatments are often given. We investigated effects of repeated dexamethasone (DM) administered to the ewe on small maternal and foetal placental arteries. We hypothesized that DM would increase responsiveness to endothelin-1 (ET-1) and norepinephrine (NE) and that foetal arteries would react differently to ET-1 and NE compared to maternal arteries. Ewes received three treatments beginning at 103, 110, and 117 days of gestation (dGA). Each treatment consisted of four IM injections of 2mg DM or saline at 12-h intervals. At 119 dGA, in vitro functional studies were performed using Mulvany wire myography and endothelin receptor (ETR) expression was quantified using real-time RTPCR and receptor ligand autoradiography. Foetal placental arteries demonstrated greater maximal contractility to ET-1 and lesser maximal contractility to NE compared to maternal arteries. DM increased the maximal contraction elicited by ET-1 and NE in foetal but not maternal placental arteries. DM also increased the abundance of type-A ETR but not type-B ETR mRNA in foetal but not maternal placental arteries. However, within the whole placentome, DM increased the abundance of type-B ETR and decreased type-A ETR mRNA, which was confirmed by similar changes in ETR binding specifically within the labyrinth region. In summary, repeated DM treatment results in agonist and vascular bed specific responses within the placenta.

Discovery of recently adopted orphan receptors for apelin, urotensin II, and ghrelin identified using novel radioligands and functional role in the human cardiovascular system.

Using novel synthetic radioligands, we have discovered receptors for the recently paired apelin (APJ orphan receptor), ghrelin (GHS orphan receptor), and urotensin II (orphan GPR14) in the human cardiovascular system and determined their anatomical localisation. In addition, we have established functional vasoactive properties for these three peptides as potential vasoconstrictor/vasodilator mediators and provided evidence for alteration of receptor density in cardiovascular disease. We find that receptors for apelin, ghrelin, and urotensin II are widely distributed in human cardiovascular tissue, suggesting perhaps vasoactive roles for these peptides in human vascular physiology and a potential role in pathophysiology. Apelin and urotensin II are potent vasoconstrictors with low efficacy, consistent with their low receptor density. Ghrelin receptor density was increased (approximately three- to fourfold) with atherosclerosis of coronary artery disease and accelerated atherosclerosis of saphenous vein grafts, compared with normal vessels, highlighting a potentially beneficial role for this novel vasodilator peptide in human vascular disease. Our approach has demonstrated one successful strategy for translating genetic information encoding recently paired orphan receptor ligands into discovery of function. This study has the advantage of focussing on the actual disease processes, which allow the more precise identification of novel therapeutic targets.

Vasoconstrictor activity of novel endothelin peptide, ET-1(1 - 31), in human mammary and coronary arteries in vitro.

1. The ability of the putative chymase product of big endothelin-1 (big ET-1), ET-1(1 - 31), to constrict isolated endothelium-denuded preparations of human coronary and internal mammary artery was determined. 2. pD2 values in coronary and mammary artery respectively were 8.21+/-0.12 (n=14) and 8.55+/-0.11 (n=12) for ET-1, 6.74+/-0.11 (n=16) and 7.10+/-0.08 (n=16) for ET-1(1 - 31) and 6.92+/-0.10 (n=15) and 7.23+/-0.11 (n=12) for big ET-1. ET-1(1 - 31) was significantly less potent than ET-1 (P<0.001, Student's t-test) and equipotent with big ET-1. 3. Vasoconstrictor responses to 100 - 700 nM ET-1(1 - 31) were significantly (P<0.05, Student's paired t-test) attenuated by the ET(A) antagonist PD156707 (100 nM). 4. There was no effect of the ECE inhibitor PD159790 (30 microM), the ECE/NEP inhibitor phosphoramidon (100 microM) or the serine protease inhibitor chymostatin (100 microM) on ET-1(1 - 31) responses in either artery. 5. Radioimmunoassay detected significant levels of mature ET in the bathing medium of coronary (1.6+/-0.5 nM, n=14) and mammary (2.1+/-0.6 nM, n=14) arteries, suggesting that conversion of ET-1(1 - 31) to ET-1 contributed to the observed vasoconstriction. 6. ET-1(1 - 31) competed for specific [(125)I]-ET-1 binding to ET(A) and ET(B) receptors in human left ventricle with a pooled K(D) of 71.6+/-7.0 nM (n=3). 7. Therefore, in human arteries the novel peptide ET-1(1 - 31) mediated vasoconstriction via activation of the ET(A) receptor. The conversion of ET-1(1 - 31) to ET-1, by an as yet unidentified protease, must contribute wholly or partly to the observed constrictor response. Chymase generated ET-1(1 - 31) may therefore represent an alternative precursor for ET-1 production in the human vasculature.

Effect of in vivo fetal infusion of dexamethasone at 0.75 GA on fetal ovine resistance artery responses to ET-1.

At 110-111 days gestation, instrumented fetal sheep were administered saline or dexamethasone (2.2 microgram. kg(-1). h(-1) iv) for 48 h. Measurement of fetal blood pressure showed a greater increase in dexamethasone-treated (n = 6) compared with control (n = 5) fetuses (7.3 +/- 2.3 vs. 0.6 +/- 2.3 mmHg, P < 0.05). Fetuses were delivered by cesarean section, and the femoral muscle and brain were obtained under halothane anesthesia. Femoral and middle cerebral arteries (approximately 320-micrometer internal diameter) were evaluated using wire myography. Sensitivity to KCl (2.5-125 mM) and the magnitude of the maximal vasoconstriction to 125 mM K(+) were similar in femoral and middle cerebral arteries from dexamethasone-treated vs. control fetuses. Acetylcholine-induced vasorelaxation was similar in femoral arteries from control and dexamethasone-treated fetuses. Middle cerebral arteries did not relax to acetylcholine. Sensitivity to endothelin-1 (ET-1; 0.1 pM-0.1 microM) and magnitude of the ET-1-induced vasoconstriction were greater in femoral arteries from dexamethasone-treated vs. control fetuses (P < 0.05). Autoradiographical studies with receptor-specific ligands demonstrated increased ET(A)-receptor binding, the principal receptor subtype, in femoral muscle vessels (P < 0.001) but decreased ET(A)-receptor binding in middle cerebral arteries (P < 0.01) from dexamethasone-treated compared with control fetuses. Relatively little ET(B)-receptor binding was evident in all tissues examined. We conclude that hyperreactivity to ET-1, due to increased ET(A)-receptor binding, may be involved in the dexamethasone-induced increase in peripheral vascular resistance in fetal sheep in vivo.

Cellular expression of isoforms of endothelin-converting enzyme-1 (ECE-1c, ECE-1b and ECE-1a) and endothelin-converting enzyme-2.

Our aim was to compare the cellular expression of endothelin-converting enzyme-1 (ECE-1) isoforms and ECE-2 using immunocytochemistry in normal and diseased human tissue. Intense ECE-1b immunoreactivity was present within renal and pulmonary epithelial cells with lower levels of staining displayed by ECE-1c, ECE-1a and ECE-2 antisera. Staining was detected with all antisera (except ECE-1a) within the endothelium of renal and pulmonary vessels having a range of lumen diameters as well as pial arteries and intracerebral vessels penetrating brain. ECE-1b, ECE-1c and ECE-2 immunoreactivity was localized to perivascular astrocytes and neuronal processes in the cerebral cortex. In diseased vessels, ECE-1c, ECE-1b and ECE-2 antisera stained macrophages infiltrating atherosclerotic plaques within coronary arteries. These results suggest ECE-1b and ECE-2 may be widely expressed in normal tissue from humans and inhibition of ECE-1 isoforms and ECE-2 expressed by cells such as macrophages in pathophysiological tissue may be an additional therapeutic target in cardiovascular disease.

Expression and characterization of the human endothelin-A-receptor in Pichia pastoris: influence of N-terminal epitope tags.

Knowledge of the three-dimensional structure of the endothelin-A receptor (ET(A)) will provide important information for rational drug design of antagonists and agonists. In order to produce correctly folded and active receptor protein for physical characterization by such techniques as X-ray crystallography and circular dichroism spectroscopy, we have cloned and expressed the human ET(A)-receptor in Pichia pastoris. The expression constructs all contained signal sequences to direct the expressed protein to the membrane fraction. Fidelity of folding and the subtype specificity of the expressed receptor was demonstrated by ligand binding studies using 125I-labelled endothelin-1 (ET-1). Expression of receptor with two different N-terminal epitope 'tags' had little effect on the affinity of ET-1 for the receptor. The strain of P. pastoris employed did influence the quantity of receptor expressed.

Tissue-specific modulation of endothelin receptors in a rat model of hypertension.

Adenovirus gene transfer of endothelin-1 (ET-1) in rats causes a transient elevation of plasma ET-1 levels, leading to systemic hypertension. Our aim was to evaluate modulation of both ET receptor subtypes in this experimental model. Recombinant adenovirus encoding either the human preproendothelin-1 (Ad.CMV.ET-1) or beta-galactosidase (Ad.CMV.beta-gal) as control was injected systemically into rats. Elevated plasma ET-1 levels and systemic blood pressure were confirmed 96 h after viral administration. Competition binding studies were carried out using tissues from liver, heart, kidney and brain to measure affinities and receptor densities. In the liver, both ET receptor densities were significantly reduced in the Ad.CMV.ET-1 group. In the heart, only the endothelin-A- (ET(A)) receptor density was significantly reduced. In the kidney and brain, the density of the ET receptors did not differ from the control group. In all tissues studied, there was no change in affinities between the two groups. The tissue-specific modulation of ET receptors and the fine regulation of ET(A)-receptors in the heart support the suggested role of the ET system in the development of cardiovascular diseases.

Endothelin-A-receptors in human aorta and pulmonary arteries are downregulated in patients with cardiovascular disease: an adaptive response to increased levels of endothelin-1?

Circulating levels of endothelin-1 (ET-1) are elevated in a number of pathophysiological conditions. Our aim was to determine the effect of overexpression of the peptide on ET receptors in human blood vessels. Aorta and pulmonary arteries were removed from patients with dilated cardiomyopathy (CDM), ischaemic heart disease (IHD), and primary pulmonary hypertension (PPH) and compared with controls. Sections of the medial (smooth muscle) layer were incubated with [125I]ET-1 and increasing concentrations of FR139317, an endothelin-A- (ET(A)) selective antagonist. FR139317 competed for the binding of iodinated ET-1 monophasically, indicating that all vessels examined expressed ET(A)-receptors in the media. ET(B)-receptors could not be detected, either in the control vessels or in those from patients with disease. There was no change in affinity (K(D)) but there was a significant (*p < 0.05) reduction in ET(A)-receptor density (Bmax) by 20-50% in diseased tissues, compared with controls. These results suggest that ET(A)-receptors are downregulated as an adaptive response to increased levels of ET-1.

Orphan-receptor ligand human urotensin II: receptor localization in human tissues and comparison of vasoconstrictor responses with endothelin-1.

We have determined the distribution of receptors for human urotensin-II (U-II) in human and rat CNS and peripheral tissues. In rat, [(125)I]-U-II binding density was highest in the abducens nucleus of brainstem (139.6+/-14 amol mm(-2)). Moderate levels were detected in dorsal horn of spinal cord and lower levels in aorta (22. 5+/-6 amol mm(-2)). In human tissues density was highest in skeletal muscle and cerebral cortex ( approximately 30 amol mm(-2)), with lower levels (<15 amol mm(-2)) in kidney cortex and left ventricle. Little binding was identified in atria, conducting system of the heart and lung parenchyma. Receptor density was less in human coronary artery smooth muscle (14.6+/-3 amol mm(-2), n=10) than rat aorta with no significant difference between normal and atherosclerotic vessels. In human skeletal muscle [(125)I]-U-II bound to a single receptor population with K(D)=0.24+/-0.17 nM and B(max)=1.97+/-1.1 fmol mg(-1) protein (n=4). U-II contracted human coronary, mammary and radial arteries, saphenous and umbilical veins with sub-nanomolar EC(50) values. U-II was 50 times more potent in arteries and <10 times more potent in veins than endothelin-1 (ET-1). The maximum response to U-II ( approximately 20% of control KCl) was significantly less than to ET-1 ( approximately 80% KCl). In contrast, in rat aorta, U-II and ET-1 were equipotent with similar maximum responses. This is the first report of high affinity receptors for [(125)I]-U-II in human CNS and peripheral tissues. This peptide produces potent, low efficacy, vasoconstriction in human arteries and veins. These data suggest a potential role for U-II in human physiology.

Expression of endothelin(A) receptors in human gliomas and meningiomas, with high affinity for the selective antagonist PD156707.

OBJECTIVE: Endothelin (ET) immunoreactivity, ET production, and specific ET receptors have been identified in the brain. Changes in ET concentration or receptor expression have been implicated in the pathophysiological changes in vasospasm after subarachnoid hemorrhage and in cerebral neoplasia. In this study, we have characterized the ET(A) and ET(B) receptor subtypes present in human normal cerebral cortex (NCC) and two common central nervous system tumors, i.e., meningioma (MNG) and glioblastoma multiforme (GBM). A knowledge of the ET receptor subtypes present may provide a novel therapeutic target for newly developed ET antagonists. METHODS: Saturation, competition, and autoradiographic studies were performed with the subtype-specific radioligands 125I-PD151242 and 125I-BQ3020, to characterize the ET(A) and ET(B) receptors present in NCC, MNG, and GBM. RESULTS: NCC expresses high-affinity ET(A) receptors on pial and intraparenchymal vessels and high-affinity ET(B) receptors on glia and neurons. MNGs express mainly (85%) high-affinity ET(A) receptors in a diffuse pattern, whereas GBMs express high-affinity ET(A) receptors on the neovasculature and ET(B) receptors on the nonvascular elements. The ET antagonist PD156707 (Kd = 0.059 nmol/L) showed a higher affinity for the ET(A) receptor subtype than did bosentan (Kd = 1.1 nmol/L). CONCLUSION: ET(A) receptors are expressed in high concentrations in MNGs and in the vasculature of NCC and GBMs. The ET(A)-selective antagonist PD156707 may be of potential therapeutic value in vascular and neoplastic diseases of the central nervous system.

Endothelin-converting enzyme in the human vasculature: evidence for differential conversion of big endothelin-3 by endothelial and smooth-muscle cells.

Our aim was to localize endothelin-converting enzyme (ECE) in human saphenous vein grafts and to quantify enzymic activity in cultured human endothelial and smooth-muscle cells. Immunoreactive ECE localized to the endothelium and infiltrating macrophages in vein grafts, but little or no immunoreactivity was detected within the media or proliferated smooth muscle of the occlusive lesion. Cultures of human umbilical vein endothelial cells were incubated with big endothelin-1 (ET-1) (10 nM) to measure extracellular conversion. After 2 h the concentration of mature peptide in the medium was increased by 162.7 +/- 21.6 pM (n = 3 +/- SEM) above basal. Permeabilization of the cells increased conversion to 1077.9 +/- 52.8 pM, suggesting that about 85% of ECE activity was located intracellularly. In both cases, activity was inhibited by phosphoramidon but not by thiorphan. In contrast, conversion of big ET-3 (10 nM) under the same conditions was not detected in either intact or permeabilized cells after 2 h. Big ET-3 and big ET-1 were converted by a phosphoramidon-sensitive/thiorphan-insensitive enzyme on the surface of confluent cultures of human umbilical vein smooth-muscle cells, with concentrations of the corresponding mature peptides increasing by 99.5 +/- 14.5 pM and 222.2 +/- 11.6 pM, respectively. These results suggest that smooth-muscle cells could be responsible for the synthesis of ET-3 present in plasma and for additional processing of big ET-1 released by endothelial cells.

Characterization of site-directed antisera against endothelin-converting enzymes.

Site-directed antisera have been developed against the two endothelin-converting enzyme-1 (ECE-1) isoforms cloned to date in humans, ECE-1 alpha and ECE-1 beta. Antisera were raised in rabbits against synthetic peptides corresponding to the deduced amino acid sequences that differ between ECE-1 alpha and ECE-1 beta. Antisera were highly selective for their corresponding antigen (titer 1 x 10(4)) and did not detect ET-1 or big ET-1. Furthermore, no detectable crossreactivity was observed between the different site-specific antisera and the other immunizing peptides, suggesting that the antisera would be selective for ECE-1 alpha and ECE-1 beta. Standard displacement curves have been developed to determine the levels of immunoreactive ECE-1 alpha and ECE-1 beta in solubilized microsomal fractions of human tissue. In conclusion, we have described the first production and characterization of site-directed antisera raised against ECE-1 alpha and ECE-1 beta capable of discriminating between the two ECE-1 isoforms. Furthermore, using these antisera, we have found that ECE-1 alpha appears to be the predominant isoform in human tissue.

Characterization of [125I]-PD164333, an ETA selective non-peptide radiolabelled antagonist, in normal and diseased human tissues.

1 We have synthesized a new low molecular weight, non-peptide radioligand, [125I]-PD164333, an analogue of the orally active butenolide antagonists of the endothelin ETA receptor. 2 Analysis of saturation binding assays demonstrated that [125I]-PD164333 bound with high affinity to a single population of receptors (n > or = 3 individuals +/- s.e.mean) in human aorta (KD=0.26+/-0.08 nM; Bmax=8.8+/-3.95 fmol mg(-1) protein), left ventricle from the heart (KD=0.16+/-0.02 nM; Bmax=34.2+/-3.02 fmol mg(-1) protein) and kidney (KD=1.24+/-0.16 nM; Bmax=125.3+/-35.07 fmol mg(-1) protein). In each case Hill slopes were close to unity. 3 In kinetic experiments, the binding of [125I]-PD164333 to ETA receptors in sections of heart was time-dependent and rapid at 23 degrees C. The data were fitted to a one site model, with an association rate constant (K1 of 2.66+/-0.213x10(8) M(-1) min(-1), and a half-time for association of 11 min. The binding was reversible at 23 degrees C: analysis of the data indicated [125I]-PD164333 dissociated from a single site, with a dissociation rate constant of 0.0031+/-0.0004 min(-1), a half-time for dissociation of 216 min and a KD calculated from these kinetic data of 0.01 nM. 4 Unlabelled PD164333 inhibited the binding of [125I]-ET-1 to left ventricle (which expresses both subtypes) in a biphasic manner with a KDETA of 0.99+/-0.32 nM and KDETB of 2.41+/-0.22 microM, giving a selectivity of 2500 fold. ETA-selective ligands competed monophasically for [125I]-PD164333 binding in left ventricle, a one site fit was preferred to a two site model giving similar nanomolar affinities: BQ123, KD=3.93+/-0.18 nM; FR139317 KD=3.53+/-0.69 nM. In contrast, the ETB selective agonists, BQ3020 and sarafotoxin S6c (1 microM) did not inhibit binding. 5 In human isolated saphenous vein, unlabelled PD164333 was a functional antagonist, producing parallel rightward shifts of the endothelin-1 (ET-1) concentration-response curve (pA2=8.84) and a slope of unity. 6 In the human brain, autoradiography revealed high levels of [125I]-PD164333 binding to the pial arteries of the cerebral cortex and to the numerous smaller intercerebral vessels penetrating the underlying grey and white matter. Conduit and resistance vessels contributing to the control of blood pressure from the heart, kidney, lungs and adrenal also displayed high densities of binding. In diseased vessels, binding of [125I]-PD164333 was confined to the medial layer of both coronary arteries with advanced atherosclerotic lesions or occluded saphenous vein grafts. In contrast, little or no binding was detected in the proliferated smooth muscle of the intimal layer or occluded lesion. 7 These results show [125I]-PD164333 is a specific, high affinity, reversible non-peptide radioligand for human ETA receptors, which will facilitate the further characterization of this subtype, in vitro and in vivo.

Affinity and selectivity of PD156707, a novel nonpeptide endothelin antagonist, for human ET(A) and ET(B) receptors.

We have determined the affinity and selectivity of a new nonpeptide antagonist PD156707 (sodium 2-benzo(1,3ioxol-5-yl-4-(4-methoxy-pheny l)-4-oxo-3-(3,4,5-trime tho xybenzyl)-but-2-enoate) for human endothelin (ET)(A) and ET(B) receptors. In human coronary artery and saphenous vein the affinity of the ET(A) receptor for PD156707 was 0.15 +/- 0.06 nM and 0.5 +/- 0.13 nM, respectively. Competition experiments in human left ventricle and kidney revealed that PD156707 had 1,000- to 15,000-fold selectivity for the ET(A) receptor over the ET(B) receptor. This selectivity was confirmed autoradiographically. In human coronary artery, mammary artery and saphenous vein PD156707 (3-300 nM) potently antagonized the vasoconstrictor responses to ET-1. The pA2 values estimated from the Gaddum-Schild equation were 8.07 +/- 0.09, 8.45 +/- 0.11 and 8.70 +/- 0.13, respectively. The concentration-response curves to ET-1 were shifted to the right in parallel fashion, without reduction of the maximum response. However, the regression lines fitted to the resulting Schild data deviated significantly from one. PD156707 appeared to be a more effective antagonist at lower concentrations than at the higher ones. It is possible that PD156707, a sodium salt, was reverting to a less soluble form which results in underestimation of its potency. These data show that PD156707 is a potent and selective antagonist at human ET(A) receptors and will be useful in clarifying the role of the endothelin peptides in human cardiovascular disease.

Differential distribution of endothelin peptides and receptors in human adrenal gland.

Sub-type selective ligands revealed a differential distribution of endothelin (ET) receptors within human adrenal glands. High densities of ETA receptors were localized, using [125I]-PD151242, to the smooth muscle layer of the arteries, smaller vessels within the capsular plexus and to the secretory cells of zona glomerulosa (KD = 139.8 +/- 39.7, Bmax = 69.7 +/- 9.1 fmol mg-1 protein, mean of 3 individuals+/-sem). ETB receptors were present in the medulla (KD = 145.2 +/- 16.4, Bmax = 75.5 +/- 12.3), zona glomerulosa (KD = 100.6 +/- 35.1, Bmax = 63.1 +/- 10.0), fasiculata (KD 145.1 +/- 16.2, Bmax = 67.9 +/- 6.9) and reticularis (KD = 118.2 +/- 18.6, Bmax = 71.9 +/- 6.5). ETB receptors were not detected within the smooth muscle of the vasculature. Messenger RNA encoding both sub-types was present in adrenals. ET-like immunoreactivity was localized to the cytoplasm of the endothelial cells from arteries supplying the gland and resistance vessels within the capsular plexus. Staining was also detected in these cells using anti-big ET-1 and less intensely with anti-big ET-2 antisera but not within cells within the cortex or medulla. Big ET-3-like immunoreactivity was localized to secretory cells of the medulla. Staining was not found using antiserum that could detect ET-3, suggesting further processing of big ET-3 may occur within the plasma, and that the adrenals could be a source of ET-3. The presence of ET-1 was confirmed by high performance liquid chromatography and radioimmunoassay although ET-3 was not detected. The results suggest that ET-1 is the predominant mature isoform, which is localized mainly to adrenal vasculature, particularly the capsular plexus, and may contribute to blood flow regulation in the gland.

Failure of BQ123, a more potent antagonist of sarafotoxin 6b than of endothelin-1, to distinguish between these agonists in binding experiments.

1. In homogenates of human saphenous vein, [125I]-ET-1 and [125I]-S6b each labelled a single population of high affinity binding sites with K(D) values of 0.64 +/- 0.11 nM and 0.55 +/- 0.08 nM respectively. Hill slopes were close to one. However, the density of receptors labelled by [125I]-ET-1 was significantly greater than that by [125I]-S6b (187.6 +/- 23.0 compared to 91.7 +/- 23.6 fmol mg-1 protein, P < 0.02). 2. BQ123, an ET(A-)selective antagonist, inhibited specific [125I]-ET-1 and [125I]-S6b binding with equal affinity. BQ123 competed in a biphasic manner for both [125I]-ET-1 (0.1 nM) and [125I]-S6b (0.1 nM) with ET(A) K(D) values of 0.55 +/- 0.17 nM and 0.52 +/- 0.02 nM and ET(B) K(D) values of 14.4 +/- 2.60 microM and 11.2 +/- 0.31 microM respectively. S6b monophasically inhibited 0.1 nM [125I]-ET-1 (K(D) 1.16 +/- 0.9 nM) but competed for 0.25 nM [125I]-ET-1 in a biphasic manner (K(D) high affinity site 1.99 +/- 0.84 nM, K(D) low affinity site 0.68 +/- 0.63 microM, ratio 67% : 33%). 3. BQ123 antagonized the vasoconstrictor responses of ET-1 with a pK(B) value of 6.47 whereas BQ123 exhibited 50 fold higher affinity against S6b-mediated vasoconstriction with a pK(B) value of 8.18. Regression slopes were 0.80 +/- 0.13 and 1.08 +/- 0.11 respectively. 4. In desensitization experiments, S6b (300 nM) did not contract preparations which were no longer responsive to ET-1 whereas a small contraction to ET-1 (300 nM) was obtained in preparations rendered unresponsive to S6b. 5. Medial sections of non-diseased human aorta, which express only ET(A) receptors, were used to compare dissociation rates of the two agonists. The time course for the dissociation of [125I]-ET-1 and [125I]-S6b was similar with 20-30% of each ligand dissociating at 4 h. 6. These data suggest that whilst BQ123, in common with other endothelin antagonists, is a much more potent blocker of S6b contractile responses than of ET-1 contractile responses, this is not reflected by the equal affinity of BQ123 determined in competition binding experiments against both [125I]-ET-1 and [125I]-S6b. This discrepancy in antagonist potency is probably not due to a marked difference in the rate of dissociation of [125I]-ET-1 and [125I]-S6b from endothelin receptors. One possible explanation is that ET-1 is activating an additional population of receptors which may have lower affinity for BQ123. This is suggested by the discrepancy in receptor density identified by [125I]-ET-1 and [125I]-S6b.