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.

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.

Endothelin-1 mRNA is widely expressed in porcine and human tissues.

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide isolated from porcine endothelial cells. We have previously demonstrated widespread binding sites for ET-1 in blood vessels, heart, kidney, adrenal, lung, and brain in a distribution that paralleled that of endothelial cells. To determine whether these cells are capable of synthesizing ET-1 in close proximity to its binding sites, amplification of cDNA using the polymerase chain reaction and in situ hybridization were used to study the distribution of ET-1 mRNA. We have found widespread transcription of ET-1 mRNA in human and porcine tissues. The identity of the transcripts was confirmed by prediction of restriction fragment lengths or sequencing. In situ hybridization in the kidney showed that the regional expression of these transcripts is localized, probably to small blood vessels, but the failure to visualize ET-1 mRNA in the capillaries may reflect absence of expression or insufficient sensitivity of the technique. These results should permit investigation of the role of ET-1 as a local factor in vascular pathophysiology.

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.

Endothelin.

In humans, the endothelins (ETs) comprise a family of three 21-amino-acid peptides, ET-1, ET-2 and ET-3. ET-1 is synthesised from a biologically inactive precursor, Big ET-1, by an unusual hydrolysis of the Trp21 -Val22 bond by the endothelin converting enzyme (ECE-1). In humans, there are four isoforms (ECE-1a-d) derived from a single gene by the action of alternative promoters. Structurally, they differ only in the amino acid sequence of the extreme N-terminus. A second enzyme, ECE-2, also exists as four isoforms and differs from ECE-1 in requiring an acidic pH for optimal activity. Human chymase can also cleave Big ET-1 to ET-1, which is cleaved, in turn, to the mature peptide as an alternative pathway. ET-1 is the principal isoform in the human cardiovascular system and remains one of the most potent constrictors of human vessels discovered. ET-1 is unusual in being released from a dual secretory pathway. The peptide is continuously released from vascular endothelial cells by the constitutive pathway, producing intense constriction of the underlying smooth muscle and contributing to the maintenance of endogenous vascular tone. ET-1 is also released from endothelial cell-specific storage granules (Weibel-Palade bodies) in response to external stimuli. ETs mediate their action by activating two G protein-coupled receptor sub-types, ETA and ET(B). Two therapeutic strategies have emerged to oppose the actions of ET-1, namely inhibition of the synthetic enzyme by combined ECE/neutral endopeptidase inhibitors such as SLV306, and receptor antagonists such as bosentan. The ET system is up-regulated in atherosclerosis, and ET antagonists may be of benefit in reducing blood pressure in essential hypertension. Bosentan, the first ET antagonist approved for clinical use, represents a significant new therapeutic strategy in the treatment of pulmonary arterial hypertension (PAH).

Imaging atherosclerotic plaque inflammation with [18F]-fluorodeoxyglucose positron emission tomography.

BACKGROUND: Atherosclerotic plaque rupture is usually a consequence of inflammatory cell activity within the plaque. Current imaging techniques provide anatomic data but no indication of plaque inflammation. The glucose analogue [18F]-fluorodeoxyglucose (18FDG) can be used to image inflammatory cell activity non-invasively by PET. In this study we tested whether 18FDG-PET imaging can identify inflammation within carotid artery atherosclerotic plaques. METHODS AND RESULTS: Eight patients with symptomatic carotid atherosclerosis were imaged using 18FDG-PET and co-registered CT. Symptomatic carotid plaques were visible in 18FDG-PET images acquired 3 hours post-18FDG injection. The estimated net 18FDG accumulation rate (plaque/integral plasma) in symptomatic lesions was 27% higher than in contralateral asymptomatic lesions. There was no measurable 18FDG uptake into normal carotid arteries. Autoradiography of excised plaques confirmed accumulation of deoxyglucose in macrophage-rich areas of the plaque. CONCLUSIONS: This study demonstrates that atherosclerotic plaque inflammation can be imaged with 18FDG-PET, and that symptomatic, unstable plaques accumulate more 18FDG than asymptomatic lesions.

The endothelin system in human saphenous vein graft disease.

Saphenous vein graft stenosis is a significant clinical complication for coronary artery bypass patients. Endothelin-1, a peptide synthesised by vascular endothelial cells, is the most potent known vasoconstrictor and has mitogenic properties. Recent advances in our knowledge of endothelin-1 synthesis and endothelin receptor expression and function in normal and atherosclerotic human saphenous vein imply a role for the peptide in the progression of vein graft failure. Manipulation of the endothelin system, by selective receptor antagonism or inhibition of the specific endothelin-converting enzymes may, therefore, represent a novel therapeutic target for treating vein graft disease.

Endothelin receptor antagonism in patients with chronic heart failure.

OBJECTIVE: The relative importance of ETA and ETB receptors in mediating the constrictor effects of endogenous endothelin-1 in patients with chronic heart failure is not known. The primary purpose of this study was to compare the acute effects of selective ETA and ETB receptor antagonists in vivo in healthy subjects and patients with chronic heart failure. Our secondary aim was to examine more closely the effect of chronic heart failure on endothelin biosynthesis. METHODS: We studied the effects of BQ-123 (a selective ETA antagonist) and BQ-788 (a selective ETB antagonist) in ten healthy subjects and ten patients with chronic heart failure. Locally active doses of each antagonist were infused into the non-dominant brachial artery for 90 min on separate days at least 1 week apart. Changes in forearm blood flow were measured by venous occlusion plethysmography. Venous blood samples were obtained prior to antagonist infusion for assay of total endothelin, big endothelin-1 and C-terminal fragment immunoreactivity. RESULTS: BQ-123 (100 nmol/min) increased blood flow by 54+/-10% (P<0.001) and 30+/-5% (P<0.001) in controls and heart failure patients, respectively. BQ-788 (1 nmol/min) reduced blood flow by 15+/-5% (P=0. 036) and 9+/-4% (P=0.001) in controls and heart failure patients, respectively. Total endothelin immunoreactivity was non significantly greater in heart failure patients than controls (6. 8+/-1.4 vs. 4.6+/-0.5 pM; P=0.13). Big endothelin-1 (2.6+/-0.4 vs. 1. 7+/-0.1 pM; P=0.04) and C-terminal fragment immunoreactivity (2. 1+/-0.3 vs. 0.6+/-0.1 pM; P<0.0001) were each significantly greater in heart failure patients than controls. CONCLUSIONS: Selective ETA receptor antagonism caused vasodilatation in the peripheral circulation of healthy subjects and patients with chronic heart failure while selective ETB receptor antagonism caused vasoconstriction in each group. ETB receptor antagonism may therefore cause potentially deleterious vasoconstriction in chronic heart failure. Chronic heart failure is associated with a significant increase in plasma big endothelin-1 and C-terminal fragment immunoreactivity.

Presence of messenger ribonucleic acid for endothelin-1, endothelin-2, and endothelin-3 in human endometrium and a change in the ratio of ETA and ETB receptor subtype across the menstrual cycle.

The aim of the present study was to determine whether mRNA for the three endothelin peptides (endothelin-1, endothelin-2, and endothelin-3) and the two known receptor subtypes (ETA and ETB) was present in human endometrium at different stages of the menstrual cycle (menstrual, early and mid-proliferative, and early, mid-, and late secretory). Endometrium was obtained from women undergoing surgery for benign disease, and total RNA was extracted using a guanidinium isothiocyanate method. mRNA for endothelin peptide and receptor was detected using the reverse transcriptase-polymerase chain reaction with nested oligonucleotide primers. mRNA for endothelin-1, endothelin-2, and endothelin-3 was demonstrated throughout the menstrual cycle, and three splice variants of mRNA encoding endothelin-3 were found in all samples. The ratio of ETA to ETB receptor mRNA was found to change throughout the menstrual cycle. In the proliferative phase, amplified cDNA product was almost exclusively confined to the ETA receptor, whereas an increase in the amplified product of the ETB receptor cDNA was seen in the secretory and menstrual phases. These studies show that mRNA for endothelin-1, endothelin-2, and endothelin-3 is present in human endometrium at all stages of the menstrual cycle and suggest that different physiological actions of the endothelin peptides may be mediated through changes in the ratio of the ETA and ETB receptor subtypes.

The endothelin peptides ET-1, ET-2, ET-3 and sarafotoxin S6b are co-mitogenic with platelet-derived growth factor for vascular smooth muscle cells.

We have investigated whether any of the three isoforms of endothelin (ET) ET-1, ET-2 and ET-3 or the structurally similar peptide sarafotoxin S6b is mitogenic on its own for rat vascular smooth muscle cells in culture. DNA synthesis was determined by a peroxidase-linked double antibody technique to detect bromodeoxyuridine incorporation into the nucleus and stained nuclei were counted by image analysis. None of the ET peptides or sarafotoxin S6b (up to 100 nM) was capable of initiating DNA synthesis in the absence of platelet derived growth factor (PDGF) or fetal calf serum. All the peptides potentiated the mitogenic effect of low concentrations of PDGF. ET-1 and ET-2 (10 nM) caused a 2-fold increase in the number of stained nuclei induced by 5 nM and 10 nM PDGF, whereas ET-3 and sarafotoxin S6b were less potent. These findings demonstrate that ET is a co-mitogen for rat vascular smooth muscle cells. The release of ET at sites of endothelial injury may therefore enhance the mitogenic action of locally acting PDGF on vascular smooth muscle cells and potentiate the proliferative response.

Anatomical localization and pharmacological activity of mature endothelins and their precursors in human vascular tissue.

OBJECTIVES: We developed four selective rabbit antisera in order to compare the distribution of immunoreactive mature endothelins and their precursors, proendothelin-1, proendothelin-2 and proendothelin-3, in the endothelium from human vascular tissue. Our second aim was to use in vitro pharmacological assays to test the vasoconstrictor actions of the mature endothelin and proendothelin peptides. METHODS: The antisera were shown to be selective by enzyme-linked immunosorbent assays. With these antisera, we detected immunoreactivity in serial cryostat sections from saphenous and mesenteric veins, and mesenteric and internal mammary arteries, using a peroxidase-antiperoxidase technique. In pharmacological experiments, segments of human coronary and mesenteric arteries were exposed to cumulative (0.06-60 nmol/l) concentrations of the endothelins and their precursors. RESULTS: Antisera directed against mature endothelin stained the cytoplasm of endothelial cells in all vessels tested. Immunoreactive proendothelin-1 and proendothelin-2 were also detected, but not proendothelin-3. Endothelin-1 and endothelin-2 were strongly vasoactive, with similar molar potencies, and caused a dose-related increase in contractile force in human coronary arteries (0.06-60 nmol/l). However, proendothelin-1 and proendothelin-2 were 100-fold and 1000-fold less vasoactive than their respective mature peptides. No contractile effect was seen with proendothelin-3 or endothelin-3 at the concentrations tested in human coronary arteries, and similar results were obtained with human mesenteric arteries. CONCLUSIONS: These results suggest that proendothelin-1 and proendothelin-2 must be converted to their corresponding mature peptides to produce vasoconstrictor activity in human vessels. Immunoreactive mature endothelin is widely distributed in human vascular endothelial cells and, if released, may produce endothelin-mediated vasoconstriction.

Myricerone caffeoyl ester (50-235) is a non-peptide antagonist selective for human ETA receptors.

OBJECTIVE: To assess the pharmacological profile of a novel non-peptide endothelin antagonist (50-235) at endothelin receptors in human vascular smooth muscle preparations using radiolabelled binding techniques and in vitro pharmacological assays. METHODS: The antagonist was investigated for its ability to inhibit specific [125I]-endothelin-1 binding to ETA and ETB receptors using cryostat sections of media of human coronary artery. Antagonism by 50-235 (1-30 mumol/l) of endothelin-1-induced vasoconstriction in isolated preparations of human coronary artery, saphenous vein and left internal mammary artery was also determined. RESULTS: In coronary artery 50-235 (10(-11) to 10(-4) mol/l) inhibited specifically bound [125I]-endothelin-1 (0.1 nmol/l) in a biphasic manner. The ratio of ETA:ETB receptor was 79:21. Increasing concentrations of 50-235 produced progressive rightwards displacements of the endothelin-1 dose-response curve in each of the three types of blood vessel. The dose-response curves were parallel and no attenuation of the maximum endothelin-1 response was observed suggesting that 50-235 was antagonizing endothelin-1 vasoconstriction in a competitive manner. The pA2 values determined by analysis of the Schild regression lines were 6.05 in coronary artery, 6.12 in saphenous vein and 6.18 in left internal mammary artery, and the slopes were not significantly different from unity. CONCLUSIONS: The antagonist 50-235 exhibits nanomolar affinity for human ETA receptors and 500-fold selectivity for ETA compared with ETB receptors. Its novel non-peptide structure demonstrates that the carbon-nitrogen bond is not crucial for endothelin antagonist activity, and might provide important information for the development of therapeutic agents for conditions in which endothelins may be pathophysiologically relevant.

Experimental hemiparkinsonism in the rat following chronic unilateral infusion of MPP+ into the nigrostriatal dopamine pathway--II. Differential localization of dopamine and cholecystokinin receptors.

The autoradiographical localization of dopamine D1, D2 and cholecystokinin receptors has been investigated in rat brain 6 months following unilateral infusion of 1-methyl-4-phenyl pyridinium ion (MPP+) (10 micrograms/day for 7 days) into the nigrostriatal dopamine pathway. Treatment with 1-methyl-4-phenyl pyridinium ion produced a marked depletion of dopamine cell bodies in the substantia nigra together with greater than 95% loss of tyrosine hydroxylase immunoreactivity in the striatum. Measurement of specific [3H]spiperone binding to D2 receptors indicated a 38% increase (P less than 0.01) in the maximal binding capacity of [3H]spiperone to striatal membrane homogenates and a 13% increase (P less than 0.05) in specific [3H]spiperone binding to striatal tissue sections, verifying striatal D2 receptor denervation supersensitivity. In contrast, MPP+ lesion of the nigrostriatal tract had no effect on the autoradiographical localization of striatal D1 or cholecystokinin receptors. In addition, there was a 38% loss (P less than 0.05) of D2 receptor binding sites in the substantia nigra pars compacta, whilst D1 receptors remained unchanged. Similar changes in dopamine and cholecystokinin receptor number were found following 6-hydroxydopamine lesion of the nigrostriatal dopamine pathway. These results provide further evidence that 1-methyl-4-phenyl pyridinium ion treatment in rats produces extensive destruction of the dopaminergic nigrostriatal tract and supports the differential anatomical localization of striatal and nigral D1, D2 and cholecystokinin receptors.

Characterization of the binding of endothelin ETB selective ligands in human and rat heart.

1. We determined competition binding characteristics of endothelin ETB receptor selective ligands in human left ventricle and compared these values to those obtained with rat left ventricle. Sarafotoxin S6c, ET-3, BQ788 and IRL2500 competed against [125I]-PD151242 (ETA selective radioligand) with low affinity in human left ventricle, confirming the ETB selectivity of these compounds. 2. ET-3 competed with moderate selectivity for ETB over ETA receptors in human left ventricle and with slightly higher selectivity in rat left ventricle (460 and 1,400 fold, respectively). There was a small difference in the affinity of ETA receptors for ET-3 (KD ETA in human left ventricle = 0.07 +/- 0.02 microM; KD ETA in rat left ventricle = 0.27 +/- 0.08 microM; P = 0.05) but no difference in the affinity of ETB receptors for this ligand (KD ETB in human left ventricle = 0.15 +/- 0.06 nM; KD ETB in rat left ventricle = 0.19 +/- 0.03 nM). 3. The selectivity of sarafotoxin S6c for ETB over ETA receptors in human left ventricle was 5,900 fold compared with 59,400 fold in rat left ventricle. The affinity of ETA receptors for sarafotoxin S6c was higher in human than in rat left ventricle (KD ETA = 2.00 +/- 0.20 microM and 3.50 +/- 0.26 microM, respectively; P = 0.03), while the affinity of ETB receptors for this ligand was higher in rat left ventricle (KD ETB = 0.06 +/- 0.02 nM) than in human left ventricle (KD ETB = 0.34 +/- 0.13 nM) (P = 0.02). The affinity of ETB receptors for sarafotoxin S6c in rat left ventricle determined in the absence or presence of GTP was the same indicating that differing affinity states of ETB receptors in human and rat left ventricle do not account for the variation observed between species. 4. There was no difference in the affinity of ETA receptors for BQ788 (KD ETA = 1.01 +/- 0.20 microM and KD ETA = 1.39 +/- 0.35 microM) or for the novel ETB selective antagonist. IRL2500 (KD ETA = 30.0 +/- 20.8 microM and KD ETA = 55.6 +/- 9.93 microM) in human and rat left ventricle, respectively. ETB receptors had a significantly higher affinity for BQ788 (KD ETB = 9.8 +/- 1.3 nM and KD ETB = 31.0 +/- 5.4 nM; P = 0.02) and IRL2500 (KD ETB = 78.2 +/- 9.7 nM and KD ETB = 300.0 +/- 75.1 nM; P = 0.03) in human and rat left ventricle, respectively. The synthetically synthesized ETB selective antagonist RES-701-1 (0.1 -3 microM) failed to inhibit [125I]-ET-1 binding in either tissue. 5. In conclusion, we have compared equilibrium dissociation constants for a number of ETB selective compounds in human and rat heart. The affinity of ETB receptors for sarafotoxin S6c, BQ788 and IRL2500 differed in human and rat left ventricle. No difference in affinity was detected for ET-3 binding at ETB receptors. Sarafotoxin S6c binding was unaffected by GTP indicating that the different receptor affinities in human and rat heart cannot be explained by differing ETB receptor affinity states. This study highlights the need to consider differences in binding characteristics that may arise from the use of tissues obtained from different species.

Endothelin receptor subtypes and their functional relevance in human small coronary arteries.

1. The potent constrictor peptide endothelin (ET) has been implicated in various cardiovascular disorders including myocardial infarction and atherosclerosis. We have investigated the nature of ET receptor subtypes present on human small coronary arteries. 2. Small coronary arteries were mounted in a wire-myograph for in vitro pharmacology. To investigate the ET receptor subtypes present in different segments of the coronary vascular tree, arteries were grouped according to internal diameter. Responses in arteries with small internal diameters (mean 316.7+/-7.9 microm; Group B) were compared to those in larger arteries (mean 586.2+/-23.1 microm; Group A). 3. ET-1 consistently and potently contracted arteries from Group A and B, with EC50 values of 1.7 (0.9-3.2) nM (n=15) and 2.3 (1.4-4.2) nM (n=14), respectively. No correlation was observed between ET-1 potency and internal diameter. The response to ET-1 was potently antagonized by the selective ET(A) receptor antagonist PD156707 in both Group A and Group B, yielding pA2 values of 8.60+/-0.12 (n=4-6) and 8.38+/-0.17 (n=4-6), respectively. Slopes from Schild regression were not significantly different from unity. 4. In contrast to ET-1, individual responses to ET-3 were variable. While all arteries from Group A responded to ET-3 (EC50 approximately 69 (23-210) nM) (n=12), no response was obtained in 5 of the 14 tested in Group B. Of those responding, many failed to reach a maximum at concentrations up to 1 microM. ET-1 was more potent than ET-3 in all arteries tested. A biphasic ET-3 response was observed in 8 arteries suggesting that a small ET(B) population was also present in some patients. The selective ET(B) receptor agonist sarafotoxin S6c had little or no effect up to 10 nM (n=4-6). 5. Responses to ET-1 and ET-3 were unaffected by removal of the endothelium in arteries from both groups suggesting a lack of functional, relaxant ET(B) receptors on endothelial cells (n=5). 6. Using autoradiography, specific high density binding of the non-selective, ET(A)/ET(B) ligand [125I]-ET-1 and selective ET(A) ligand [125I]-PD151242 was detected on the vascular smooth muscle layer of small intramyocardial coronary arteries (n=5). In contrast, little or no binding of the selective ET(B) receptor ligand [125I]-BQ3020 was observed (n=5). Similarly, [125I]-ET-1 binding to vascular smooth muscle was absent in the presence of the selective ET(A) receptor antagonist PD156707. 7. We conclude that human small epi- and intramyocardial coronary arteries express predominantly ET(A) receptors and it is these receptors which mediate ET-induced contractions. A constrictor ET(B) receptor population may exist in some patients. However, these receptors may have a limited role as contractions to ET-1 can be blocked fully by the selective ET(A) receptor antagonist PD156707.

Increased response to big endothelin-1 in atherosclerotic human coronary artery: functional evidence for up-regulation of endothelin-converting enzyme activity in disease.

Overproduction of the potent vasoconstrictor peptide endothelin-1 (ET-1) is implicated in the pathogenesis of coronary artery disease. In endothelium-denuded human coronary arteries the response to big ET-1 was significantly enhanced in atherosclerotic arteries (coronary artery disease, CAD; n=7) with an EC50 value of 96 nM (57- 161 nM, 95% C.I.) compared to 274 nM (205-365 nM) in non-diseased arteries (dilated cardiomyopathy, DCM; n=10) (Mann-Whitney U-test, P<0.05). Higher levels of immunoreactive endothelin (ET) could be detected by radioimmunoassay in bathing medium taken from CAD arteries than from DCM arteries (2.8+/-0.5 nM, n=5 vs 1.1+/-0.2 nM, n=7) (Student's two-tailed t-test, P<0.05). There were no differences in responses of arteries from either group to ET-1 (EC50 10 nM, CAD vs 14 nM, DCM). The enhanced response of atherosclerotic human coronary arteries to big ET-1 appears to be due to up-regulation of endothelin-converting enzyme (ECE) activity rather than to an augmented response of the arteries to ET-1. This non-endothelial ECE may therefore be an important therapeutic target in coronary artery disease.

ETA receptor-mediated constrictor responses to endothelin peptides in human blood vessels in vitro.

1. We have characterized the constrictor endothelin receptors present in human isolated blood vessels using ETA and ETB selective agonists and antagonists. 2. Monophasic dose-response curves were obtained for ET-1 with EC50 values of 6.8 nM in coronary artery, 3.9 nM in internal mammary artery, 17.4 nM in pulmonary artery, 14.5 nM in aorta and 3.2 nM in saphenous vein. In coronary artery, ET-2 was equipotent with ET-1 with an EC50 value of 5.7 nM. The non-selective peptide, sarafotoxin 6b, was 2-3 times less potent than ET-1 but the maximum responses to these two were comparable. 3. In each vessel ET-3 was much less active than ET-1. No response was obtained to ET-3 in aorta and pulmonary artery or in up to 50% of coronary artery, mammary artery and saphenous vein preparations. In those preparations that did respond, dose-response curves were incomplete at 300 nM. Variable contractions were also obtained with the ETB-selective agonist, sarafotoxin 6c (S6c). Where responses were detected, although S6c was more potent than ET-1 (EC50 values of 0.6-1.2 nM), the maximum response produced was always less than 20% of that to ET-1. 4. The synthetic ETB agonists, BQ3020 and [1,3,11,15Ala]-ET-1, were without effect in any of the five blood vessels at concentrations up to 3 microM. 5. ET-1-induced vasoconstriction was blocked by the ETA-selective antagonists, BQ123 and FR139317. Schild-derived pA2 values were 7.0, 7.4 and 6.9 for BQ123 and 7.6, 7.9 and 7.3 for FR139317 in coronary artery, mammary artery and saphenous vein, respectively, consistent with antagonism of ETA receptors. Slopes of the Schild regressions were not significantly different from one. Comparable values of pA2 were estimated for 3ftM BQ123 in aorta (7.4+/-0.5) and pulmonary artery (6.9) from the Gaddum-Schild equation.6. In conclusion we have shown that in human isolated blood vessels, ET-1 is more potent than ET-3 suggesting the presence of vasoconstrictor ETA receptors. This is supported by the lack of effect of the ETB agonists, BQ3020 and [1,3,1,1,15Ala]-ET-l and the ability of the ETA antagonists, BQ123 andFR139317 to block ET-1 responses. Some preparations did contract in response to low concentrations of the ETB-selective sarafotoxin 6c but responses were variable and the maximum was always much less than that to ET-1 in the same preparations. Therefore although constrictor ETB receptors were present on the smooth muscle of human blood vessels, vasoconstriction elicited by the endothelin peptides in vitro is via ETA receptor activation.

Characterization of the endothelin receptor selective agonist, BQ3020 and antagonists BQ123, FR139317, BQ788, 50235, Ro462005 and bosentan in the heart.

1. In this study we used ligand binding techniques to determine the affinity and selectivity of endothelin receptor agonists and antagonists in human left ventricle which expresses both ETA and ETB receptors, and compared these results with cardiovascular tissues from rat and porcine hearts. 2. The linear tripeptide antagonist, FR139317 competed for [125I]-ET-1 binding to human left ventricle with over 200,000 fold selectivity for the ETA receptor (KD ETA = 1.20 +/- 0.28 nM, KDETB = 287 +/- 93 microM). The ETA-selective non-peptide antagonist, 50235, competed with lower affinity and selectivity (KDETA = 162 +/- 61 nM, KDETB = 171 +/- 42 microM) in this tissue. BQ123 and FR139317 also showed high selectivity (greater than 20,000 fold) and affinity in rat (BQ123: KDETA = 1.18 +/- 0.16 nM, KDETB = 1370 +/- 1150 microM; FR139317: KDETA = 2.28 +/- 0.30 nM, KDETB = 292 +/- 114 microM) and pig heart (BQ123: KDETA = 0.52 +/- 0.05 nM, KDETB = 70.4 +/- 4.0 microM; FR139317: KDETA = 2.17 +/- 0.51 nM, KDETB = 47.1 +/- 5.7 microM) (n > or = 3 individuals +/- s.e.mean). 3. Although BQ3020 competed with over 1000 fold selectivity for the ETB subtype in human heart (KDETB = 1.38 +/- 0.72 nM, KDETA = 2.04 +/- 0.21 microM) the peptide inhibited only the binding of [125I]-ET-1 at concentrations greater than 100 nM in rat and porcine heart. This is in contrast to the data from the ETA-selective antagonists which indicated the presence of ETB sites in these tissues from animal hearts. 4. The peptide antagonist, BQ788, had a low, micromolar affinity (KD = 1.98 +/- 0.13 microM) using human left ventricle and no significant selectivity for the human ETB-subtype in this tissue. 5. The non-peptide ET antagonists, Ro462005 (KD = 50.3 +/- 9.5 microM) and bosentan (Ro470203; KD = 77.9 +/- 7.9 nM) competed monophasically for [125I]-ET-1 binding sites in human left ventricle. 6. The results show that the ETA antagonists, BQ123 and FR139317, are highly selective for ETA receptors in all cardiac tissues tested, whereas BQ788 has a low affinity and no selectivity in this human tissue. Further we showed that there are species differences in the binding of BQ3020 to the ETB receptors in the hearts derived from human, rat and pig.

Endothelin receptors in human coronary artery and aorta.

1. ETA and ETB-selective and non-selective ligands were used to define the endothelin receptors in the media (vascular smooth muscle layer) of human aorta and coronary artery. Saturation experiments with iodinated endothelin-1 (ET-1), endothelin-2 and sarafotoxin 6b (S6b) identified high affinity binding sites in aorta (KD [125I]-ET-1 0.33 +/- 0.02 nM (n = 9), KD [125I]-ET-2 1.04 +/- 0.23 nM (n = 5), KD [125I]-S6b 0.15 +/- 0.01 nM (n = 9 +/- s.e.mean)) and coronary artery (KD [125I]-ET-1 0.43 +/- 0.10 nM, KD [125I]-ET-2 0.71 +/- 0.17 nM, KD [125I]-S6b 0.27 +/- 0.03 nM (n = 3 +/- s.e.mean)). Hill coefficients (nH) approached unity in each case. 2. No specific binding was detectable with [125I]-ET-3 (4 pM-4 nM) in aorta. Unlabelled ET-3 competed monophasically with [125I]-ET-1 in aorta (KD, 8.21 +/- 1.62 nM, compared to unlabelled ET-1 KD, 0.60 +/- 0.20 nM) (n = 3 +/- s.e.mean). In coronary artery, the KD and Bmax values calculated from [125I]-ET-3 saturation experiments were 2.13 +/- 1.39 nM and 20.6 +/- 12.9 fmol mg-1 protein, respectively (n = 3 +/- s.e.mean). 3. ETA antagonists competed monophasically for [125I]-ET-1 (100 pM) binding sites with nanomolar or subnanomolar affinity in the aorta (KD BQ123, 0.47 +/- 0.13 nM; KD FR139317, 0.40 +/- 0.10 nM; KD PD151242, 2.09 +/- 0.48 nM) and coronary artery (KD FR139317, 0.41 +/- 0.13 nM; KD PD151242, 3.60 +/- 0.74 nM) (n = 3 +/- s.e.mean). However, two site fits were preferred on analysis of competition experiments with ETB-selective agonists versus [125I]-ET-1 in coronary artery (BQ3020: KDETA 0.96 +/- 0.14 microM, KD ETB 1.34 +/- 1.08 nM and sarafotoxin 6c: KD ETA 1.15 +/- 0.14 microM, KD ETB 1.77 +/- 0.72 nM) (n = 3 +/- s.e.mean). The selectivity of the agonists for ETB receptors (700 fold) was lower than reported in other species. 4. Sarafotoxin 6b (2 pM-2 microM) completely inhibited [125I]-ET-1 (100 pM) binding in aorta (KD 1.36 +/- 0.22 nM) (n = 3 +/- s.e.mean). The non-peptide compounds Ro462005 and bosentan, competed with [125I]-ET-1 binding in coronary artery with KD values of 0.19 +/- 0.04 microM and 2.94 +/- 0.95 nM, respectively (n = 3 +/- s.e.mean). 5. Inhibition of [125I]-ET-2 and [125I]-S6b binding by FR139317 was similar to the inhibition of [125I]-ET-1 binding in both arteries, being monophasic with KD values in the same range. 6. ETA receptors in coronary artery media were detected by [125I]-PD151242 (KD 0.23 +/- 0.04 nM, Bmax 10.1 +/- 1.2 fmol mg-1 protein) (n = 3 +/- s.e.mean). [125I]-BQ3020, an ETB-selective radioligand, indicated the presence of a smaller population of ETB receptors in this tissue (KD 0.60 +/- 0.31 nM, Bmax 4.5 +/- 2.1 fmol mg-1 protein) (n = 3 +/- s.e.mean). 7. Autoradiography with [125I]-PD151242 and [125I]-BQ3020 confirmed the predominance of ETA receptors in the media of both arteries. 8. The results of this study indicate that ETA receptors predominate in the vascular smooth muscle of human cardiac arteries, with a small and variable population of ETB receptors detectable in the coronary artery.