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.

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).

EPR spectra of photosystem I and other iron protein components in intact cells of cyanobacteria.

Electron paramagnetic resonance (EPR) spectra were recorded of whole filaments of the cyanobacteria Nostoc muscorum and Anabaena cylindrica. Signals due to manganese were removed by freezing and thawing the cells in EDTA. EPR spectra were assigned on the basis of their g values, linewidths, temperature dependence and response to dithionite and light treatments. The principal components identified were: (i) rhombic Fe3+ (signal at g = 4.3), probably a soluble storage form of iron; (ii) iron-sulfur centers A and B of Photosystem I; (iii) the photochemical electron acceptor 'X' of Photosystem I; this component was also observed for the first time in isolated heterocysts; (iv) soluble ferredoxin which was present at a concentration of 1 molecule per 140 +/- 20 chlorophyll molecules; (v) a membrane-bound iron-sulfur protein (g = 1.92). A signal g = 6 in the oxidized state was probably due to an unidentified heme compound. During deprivation of iron the rhombic Fe3+, centers A, B and X of Photosystem I, and soluble ferredoxin were all observed to decrease.

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.

Interactions between ubiquinones and vitamins in membranes and cells.

The interaction between ubiquinones and vitamin E was studied in the inner membranes of rat liver mitochondria, liposomes and human erythrocyte plasma membranes. Free radicals were produced by addition of exogenous oxidants, and their reaction with chromanols and ubiquinone was followed by ESR and HPLC. Membranes were made deficient in ubiquinone but sufficient in alpha-tocopherol and were reconstituted with added ubiquinone. With these membrane preparations it was shown that (i) in the inner mitochondrial membranes there is a requirements for ubiquinone in the enzymatic recycling of vitamin E; (ii) succinate-ubiquinone reductase incorporated in liposomes cannot protect vitamin E in the absence of ubiquinone and (iii) in human erythrocyte plasma membranes protection against the loss of vitamin E can be provided by NADH-cytochrome-b5-dependent enzymatic recycling. We conclude that ubiquinonols (ubisemiquinones) reduce vitamin E through electron transport.

EPR characterization of soluble fragments of succinate dehydrogenase from mutant strains of Bacillus subtilis.

Succinate dehydrogenase is a membrane-bound metallo-flavo-enzyme containing a bi- (S-1), a tri- (S-3) and a tetranuclear (S-2) iron-sulfur cluster. The catalytic portion of the enzyme contains two distinct subunits designated Fp and Ip. Using concentrated extracts from mutant strains of Bacillus subtilis it was demonstrated, by using low temperature EPR, that cluster S-2 can be assembled in a soluble succinate dehydrogenase. In a mutant with a truncated Ip subunit which lacks 7 of the 11 conserved cysteine residues, cluster S-1 lacked the spin relaxation properties attributable to an adjacent cluster S-2. These data are consistent with a model where one or more cysteine residues from the middle set of 4 conserved cysteines in the Ip subunit are ligands to the tetranuclear cluster.

Neutrophil antioxidant capacity during the respiratory burst: loss of glutathione induced by chloramines.

Low-molecular weight antioxidants in rat peritoneal neutrophils undergo rapid redox recycling, so measurements were made of their initial content and subsequent changes during the respiratory burst, when superoxide formation is maximized. Endogenous vitamin E, ascorbate and total glutathione (reduced + oxidized) were not significantly changed during 30 min of respiratory burst, which was stimulated by phorbol 12-myristate 13-acetate (PMA). When de novo synthesis of glutathione was inhibited by buthionine-[S,R] sulfoximine (BSO), the glutathione content rapidly decreased in activated neutrophils but not in resting cells. The lost total glutathione was recovered neither from the incubation medium nor as a protein-bound form, which suggests that irreversible oxidation of glutathione occurs. Furthermore, the glutathione loss continues even 30 min after PMA stimulation, when the respiratory burst has almost ceased. The decrease of glutathione was prevented by added catalase, or by addition of NaN3 or KCN which inhibits myeloperoxidase (MPO). Superoxide dismutase had no protective effects. These findings suggest the involvement of an MPO-H2O2-halide system in the accelerated consumption of glutathione during the respiratory burst. Additional studies showed that neutrophil-derived chloramines found in the extracellular medium could lead to intracellular glutathione loss. Incubation of resting cells with chemically produced membrane permeable monochloramine in the presence of BSO resulted in a decrease of glutathione, whereas membrane-impermeable taurine-chloramine was less effective. We conclude that chloramines are responsible for accelerated glutathione turnover in neutrophils during the respiratory burst. Permeable extracellular chloramines derived from the respiratory burst activity, such as monochloramine, can reenter cells and react with thiols.

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.

Cyclic peptides as selective tachykinin antagonists.

Twenty homodetic cyclic peptides based on the C-terminal sequence of substance P were prepared (Table I) by a combination of solid-phase techniques and cyclizations using azide coupling procedures. Incorporation of dipeptide mimics based on substituted gamma-lactams were used in some cases to restrict their conformational mobility. Five of these cyclic peptides were shown to have high tachykinin antagonist activity (pA2 > 6) at NK-2 receptors (rat vas deferens). The two most potent of this series, XVII, cyclo(Gln-Trp-Phe-Gly-Leu-Met) (pA2 = 8.1), and I cyclo(Gln-Trp-Phe(R)Gly[ANC-2]Leu-Met) (pA2 = 6.7), were selective for NK-2 receptors compared with the other tachykinin receptors (Table II).

Regional effects of pertussis toxin in vivo and in vitro on GABAB receptor binding in rat brain.

Agonist binding to GABAB receptors modulates the activity of the guanine nucleotide binding proteins, Go and Gi. These G proteins are ADP-ribosylated by pertussis toxin and this prevents them from coupling to the GABAB receptor resulting in a reduction in high-affinity GABAB binding. GTP, which binds to a different site on the G protein alpha subunit, also reduces the affinity of the receptor for the G protein, and this can be used as a "marker" for G protein-GABAB receptor linkage. We have examined GABAB binding site distribution in rat brain after unilateral intrahippocampal pertussis toxin injection in vivo, and after incubating brain slices in pertussis toxin in vitro, using the technique of receptor autoradiography. The effect of pertussis toxin was compared with that of GTP gamma S on GABAB binding. Intrahippocampal pertussis toxin administration reduced GABAB but not GABAA receptor binding and the effects appeared to be limited by pertussis toxin diffusion. More widespread reductions in GABAB binding were seen after incubation of brain slices in vitro but the extent varied in different brain regions. No reduction was detected in the corpus striatum. GABAB binding was also reduced in membranes prepared from cerebral cortex, hippocampus and cerebellum but there was no significant reduction in the corpus striatum after pertussis toxin treatment. GTP gamma S reduced GABAB binding to a similar extent in all areas studied irrespective of their sensitivity to pertussis toxin suggesting that while GABAB binding sites are linked to G proteins throughout the rat brain, those in the corpus striatum may be predominantly pertussis toxin insensitive.

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.

Endothelin receptor expression and pharmacology in human saphenous vein graft.

1. We have investigated the expression and pharmacology of endothelin (ET) receptors in human aortocoronary saphenous vein grafts. 2. Subtype-selective ligands were used to autoradiographically identify ET(A) ([125I]-PD151242) and ET(B)([125I]-BQ3020) receptors. In graft saphenous vein ETA receptors predominated in the media, with few ET(B) receptors identified. Neither subtype was detected in the thickened neointima. 3. The ratio of medial ET(A):ET(B) receptors was 75%: 25% in both graft and control saphenous vein. 4. ET-1 contracted control (EC50 2.9 nM) and graft (EC50 4.5 nM) saphenous vein more potently than diseased coronary artery (EC50 25.5 nM). 5. In all three blood vessels ET-1 was 100 times more potent than ET-3 and three times more potent than sarafotoxin 6b (S6b). Little or no response was obtained in any vessel with the ET(B) agonist sarafotoxin 6c (S6c). 6. The ET(A) antagonist PD156707 (100 nM) blocked ET-1 responses in all three vessels with pKb values of approximately 8.0. 7. For individual graft veins the EC50 value for ET-1 and 'age' of graft in years showed a significant negative correlation. 8. In conclusion there is no alteration in ET receptor expression in the media of saphenous veins grafted into the coronary circulation compared to control veins. ETA receptors predominantly mediate the vasoconstrictor response to ET-1 in graft vein, with no apparent up-regulation of ET(B) receptors. The sensitivity of the graft vein to ET-1 increased with graft 'age', suggesting that these vessels may be particularly vulnerable to the increased plasma ET levels that are detected in patients with cardiovascular disease.

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.

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.

Endothelin converting enzyme (ECE) activity in human vascular smooth muscle.

1. We have characterized the human smooth muscle endothelin converting enzyme (ECE) present in the media of the endothelium-denuded human umbilical vein preparation. 2. Endothelin-1 (ET-1) and ET-2 were potent constrictors of umbilical vein with EC50 values of 9.2 nM and 29.6 nM, respectively. ET-1 was at least 30 times more potent than ET-3 suggesting the presence of constrictor ETA receptors. Little or no response was obtained to the ETB-selective agonist sarafotoxin 6c. These data suggest that endothelin-mediated vasoconstriction is via ETA receptors in this preparation. 3. Autoradiographical visualization of endothelin receptors with subtype selective ligands confirmed the predominance of the ETA receptor in the media of umbilical vein. High density of binding was obtained with the ETA selective [125I]-PD151242, with much lower levels detected with the ETB selective [125I]-BQ3020. 4. Big ET-1 (EC50 = 42.7 nM) and big ET-2(1-38) (EC50 = 99.0 nM) were less potent than ET-1 and ET-2, respectively. Big ET-2(1-38) was more potent than its isoform big ET-2(1-37) with concentration-response curves to big ET-2(1-37) incomplete at 300 nM. No response was obtained to big ET-3 at concentrations up to 700 nM. The C-terminal fragments, big ET-1(22-38) and big ET-2(22-38) were inactive. 5. Responses to ET-1 were unaffected by either the neutral endopeptidase (NEP) inhibitor thiorphan (10(-5) M) or by the dual NEP/ECE inhibitor phosphoramidon (10(-5) M). Big ET-1 was also unaffected by thiorphan but antagonized in a concentration-dependent manner by phosphoramidon (10(-5) M and 10(-4) M). 6. Addition of all four big endothelin peptides to human umbilical vein preparations resulted in detectable amounts of ET-IR in the bathing medium. Therefore, although big ET-3 was functionally inactive this reflects the low potency of ET-3 at the ETA receptor rather than the lack of ability of this smooth muscle ECE to convert big ET-3 to ET-3. 7. To conclude we have demonstrated the presence of a phosphoramidon-sensitive ECE on the smooth muscle layer of the human umbilical vein which can convert big ET-1, big ET-2(1-37), big ET-2(1-38) and big ET-3 to their mature biologically active forms. The precise subcellular localization of this enzyme and its physiological relevance remains to be determined.

[(125)I]-(Pyr(1))Apelin-13 is a novel radioligand for localizing the APJ orphan receptor in human and rat tissues with evidence for a vasoconstrictor role in man.

1. We have determined the binding characteristics of [(125)I]-(Pyr(1))Apelin-13, a putative ligand for the APJ orphan receptor in human cardiovascular and rat tissue and investigated the functional properties of (Pyr(1))Apelin-13 in human saphenous vein. 2. The binding of [(125)I]-(Pyr(1))Apelin-13 to sections of human heart tissue was time dependent and rapid at 23 degrees C. Data were fitted to a single site model with an association rate constant (k(obs)) of 0.115 min(-1). [(125)I]-(Pyr(1))Apelin-13 also dissociated from a single site with a dissociation rate constant of 0.0105 min(-1). 3. In saturation binding experiments [(125)I]-(Pyr(1))Apelin-13 bound to human left ventricle with a K(D) value of 0.35+/-0.08 nM, B(max) of 4.3+/-0.9 fmol mg(-1) protein with a Hill slope of 0.97+/-0.04 and to the right atria with a K(D) of 0.33+/-0.09 nM, B(max) of 3.1+/-0.6 fmol mg(-1) protein and a Hill slope of 0.93+/-0.05. 4. [(125)I]-(Pyr(1))Apelin-13 binding sites were localized using autoradiography to human cardiovascular tissue, including coronary artery, aorta and saphenous vein grafts. In rat tissue a high density of receptors were localized to the molecular layer of the rat cerebellum, rat lung, rat heart and low levels in the rat kidney cortex. 2. (Pyr(1))Apelin-13 potently contracted human saphenous vein with a pD(2) value of 8.4+/-0.2 (n=8). The maximum response elicited by the peptide was 22.6+/-6% of 100 mM KCl. 6. We provide the first evidence of APJ receptor expression, relative densities and functional properties of (Pyr(1))Apelin-13 in human cardiovascular tissue.