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@25 - new agonists, antagonists, inhibitors and emerging research frontiers: IUPHAR Review 12.

Since the discovery of endothelin (ET)-1 in 1988, the main components of the signalling pathway have become established, comprising three structurally similar endogenous 21-amino acid peptides, ET-1, ET-2 and ET-3, that activate two GPCRs, ETA and ETB . Our aim in this review is to highlight the recent progress in ET research. The ET-like domain peptide, corresponding to prepro-ET-193-166 , has been proposed to be co-synthesized and released with ET-1, to modulate the actions of the peptide. ET-1 remains the most potent vasoconstrictor in the human cardiovascular system with a particularly long-lasting action. To date, the major therapeutic strategy to block the unwanted actions of ET in disease, principally in pulmonary arterial hypertension, has been to use antagonists that are selective for the ETA receptor (ambrisentan) or that block both receptor subtypes (bosentan). Macitentan represents the next generation of antagonists, being more potent than bosentan, with longer receptor occupancy and it is converted to an active metabolite; properties contributing to greater pharmacodynamic and pharmacokinetic efficacy. A second strategy is now being more widely tested in clinical trials and uses combined inhibitors of ET-converting enzyme and neutral endopeptidase such as SLV306 (daglutril). A third strategy based on activating the ETB receptor, has led to the renaissance of the modified peptide agonist IRL1620 as a clinical candidate in delivering anti-tumour drugs and as a pharmacological tool to investigate experimental pathophysiological conditions. Finally, we discuss biased signalling, epigenetic regulation and targeting with monoclonal antibodies as prospective new areas for ET research.

The Concise Guide to PHARMACOLOGY 2013/14: overview.

The Concise Guide to PHARMACOLOGY 2013/14 provides concise overviews of the key properties of over 2000 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties from the IUPHAR database. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.12444/full. This compilation of the major pharmacological targets is divided into seven areas of focus: G protein-coupled receptors, ligand-gated ion channels, ion channels, catalytic receptors, nuclear hormone receptors, transporters and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. A new landscape format has easy to use tables comparing related targets. It is a condensed version of material contemporary to late 2013, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in previous Guides to Receptors & Channels. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and GRAC and provides a permanent, citable, point-in-time record that will survive database updates.

Chemokine receptor CCR5: from AIDS to atherosclerosis.

There is increasing recognition of an important contribution of chemokines and their receptors in the pathology of atherosclerosis and related cardiovascular disease. The chemokine receptor CCR5 was initially known for its role as a co-receptor for HIV infection of macrophages and is the target of the recently approved CCR5 antagonist maraviroc. However, evidence is now emerging supporting a role for CCR5 and its ligands CCL3 (MIP-1α), CCL4 (MIP-1ß) and CCL5 (RANTES) in the initiation and progression of atherosclerosis. Specifically, the CCR5 deletion polymorphism CCR5delta32, which confers resistance to HIV infection, has been associated with a reduced risk of cardiovascular disease and both CCR5 antagonism and gene deletion reduce atherosclerosis in mouse models of the disease. Antagonism of CCL5 has also been shown to reduce atherosclerotic burden in these animal models. Crucially, CCR5 and its ligands CCL3, CCL4 and CCL5 have been identified in human and mouse vasculature and have been detected in human atherosclerotic plaque. Not unexpectedly, CC chemokines have also been linked to saphenous vein graft disease, which shares similarity to native vessel atherosclerosis. Distinct roles for chemokine-receptor systems in atherogenesis have been proposed, with CCR5 likely to be critical in recruitment of monocytes to developing plaques. With an increased burden of cardiovascular disease observed in HIV-infected individuals, the potential cardiovascular-protective effects of drugs that target the CCR5 receptor warrant greater attention. The availability of clinically validated antagonists such as maraviroc currently provides an advantage for targeting of CCR5 over other chemokine receptors.

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.

Evaluation of translocator protein quantification as a tool for characterising macrophage burden in human carotid atherosclerosis.

Macrophage presence within atherosclerotic plaque is a feature of instability and a risk factor for plaque rupture and clinical events. Activated macrophages express high levels of the translocator protein/peripheral benzodiazepine receptor (TSPO/PBR). In this study, we investigated the potential for quantifying plaque inflammation by targeting this receptor. TSPO expression and distribution in the plaque were quantified using radioligand binding assays and autoradiography. We show that cultured human macrophages expressed 20 times more TSPO than cultured human vascular smooth muscle cells (VSMCs), the other abundant cell type in plaque. The TSPO ligands [3H](R)-1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoquinoline carboxamide ([3H](R)-PK11195) and [3H]N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)acetamide ([3H]-DAA1106) bound to the same sites in human carotid atherosclerotic plaques in vitro, and demonstrated significant correlation with macrophage-rich regions. In conclusion, our data indicate that radioisotope-labelled DAA1106 has the potential to quantify the macrophage content of atherosclerotic plaque.

Emerging pharmacology and physiology of neuromedin U and the structurally related peptide neuromedin S.

Neuromedin U (NMU) has been paired with the G-protein-coupled receptors (GPRs) NMU(1) (formerly designated as the orphan GPR66 or FM-3) and NMU(2) (FM-4 or hTGR-1). Recently, a structurally related peptide, neuromedin S (NMS), which shares an amidated C-terminal heptapeptide motif, has been identified in both rat and human, and has been proposed as a second ligand for these receptors. Messenger RNA encoding NMU receptor subtypes shows differential expression: NMU(1) is predominantly expressed in peripheral tissues, particularly the gastrointestinal tract, whereas NMU(2) is abundant within the brain and spinal cord. NMU peptide parallels receptor distribution with highest expression in the gastrointestinal tract and specific structures within the brain, reflecting its major role in the regulation of energy balance. The NMU knockout mouse has an obese phenotype and, in agreement, the Arg165Trp amino acid variant of NMU-25 in humans, which is functionally inactive, co-segregated with childhood-onset obesity. Emerging physiological roles for NMU include vasoconstriction mediated predominantly via NMU(1) with nociception and bone remodelling via NMU(2). The NMU system has also been implicated in the pathogenesis of septic shock and cancers including bladder carcinoma and acute myeloid leukaemia. Intriguingly, NMS is more potent at NMU(2) receptors in vivo where it has similar central actions in suppression of feeding and regulation of circadian rhythms to NMU. Taken together with its vascular actions, NMU may be a functional link between energy balance and the cardiovascular system and may provide a future target for therapies directed against the disorders that comprise metabolic syndrome.

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).

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.

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.

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.

Thromboxane receptor density is increased in human cardiovascular disease with evidence for inhibition at therapeutic concentrations by the AT(1) receptor antagonist losartan.

1. The aim of this study was to establish how thromboxane receptors (TP) respond to the increase in levels of plasma thromboxane observed in both cardiac (cardiomyopathy, ischaemic heart disease and pulmonary hypertension) and vascular disease (atherosclerosis of coronary artery disease and accelerated atherosclerosis of saphenous vein grafts). 2. The agonist radioligand [(125)I]-BOP, bound rapidly to TP receptors in normal human cardiovascular tissue, displaying high affinity in left ventricle (K(D) 0.23 +/- 0.06 nM, B(max) 28.4 +/- 5.7 fmol mg(-1) protein) and reversibility with a t(1/2) of 10 min (n = five individuals +/- s.e.mean). 3. In the heart, TP receptor density in the right ventricle of primary pulmonary hypertensive patients were significantly increased (66.6 +/- 6 fmol mg(-1) protein) compared to non-diseased right ventricle (37.9 +/- 4.1 fmol mg(-1) protein, n = six individuals +/- s.e.mean, P<0.05). 4. In diseased vessels, TP receptor densities were significantly increased (3 fold in the intimal layer) in atherosclerotic coronary arteries, saphenous vein grafts with severe intimal thickening (n = 8-12 individuals, P<0.05) and aortic tissue (n=5 - 6 individuals, P<0.05), compared with normal vessels. 5. Losartan, tested at therapeutic doses, competed for [(125)I]-BOP binding to human vascular tissue, suggesting that some of the anti-hypertensive effects of this AT(1) receptor antagonist could also be mediated by blocking human TP receptors. 6. The differential distribution of TP receptors in the human cardiovascular system and the alteration of receptor density, accompanying the increase in endogenous thromboxane levels in cardiovascular disease, suggest that TP receptors represent a significant target for therapeutic interventions and highlights the importance for the development of novel selective antagonist for use in humans.

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.

[125I-His(9)]-ghrelin, a novel radioligand for localizing GHS orphan receptors in human and rat tissue: up-regulation of receptors with athersclerosis.

1. Ghrelin is the recently identified endogenous ligand for the cloned growth hormone secretagogue receptor (GHS-R). We have characterized for the first time the binding of human [125I-His(9)]-ghrelin to normal human and rat tissue and demonstrated expression of this 'orphan' receptor that has previously been predicted to exist from mRNA. Furthermore, we have discovered that [125I-His(9)]-ghrelin density is significantly increased in atherosclerosis. 2. [125I-His(9)]-Ghrelin bound to non-diseased human heart (left ventricle) with an association rate constant (k(obs)) of 0.16+/-0.004 min(-1), a dissociation rate constant of 0.068+/-0.0005 min(-1) (kinetically derived K(D) of 0.1 nM; n=5 individuals+/-s.e.mean), a K(D) of 0.43+/-0.08 nM and B(max) of 7.8+/-0.9 fmol mg(-1) protein (n=6 individual+/-s.e.mean). 3. Specific [125I-His(9)]-ghrelin binding was to the human vasculature including aorta, coronary, pulmonary, arcuate arteries in the kidney and saphenous veins. In rat tissues, binding sites were also localized to the vasculature in peripheral tissues as well as the granular layer of the cerebellum in the CNS. 4. [125I-His(9)]-Ghrelin binding was significantly up-regulated (3 - 4 fold) in both atherosclerotic coronary arteries and saphenous vein grafts with advanced intimal thickening, compared with normal vessels (P<0.05). 5. Our results suggest that the native receptor for [125I-His(9)]-ghrelin may be widely distributed in the human cardiovascular system. Furthermore, changes in the density of this proposed ghrelin receptor implicates this new transmitter system in the development of atherosclerosis and may therefore represent a novel therapeutic target in the treatment of cardiovascular disease.

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.

Physiological antagonism of endothelin-1 in human conductance and resistance coronary artery.

The ability of four endogenous vasodilators, nitric oxide (NO; 0.01 - 30 microM), atrial (ANP), brain (BNP) and C-type (CNP) natriuretic peptide (0.1 - 300 nM), to reverse endothelin-1 (ET-1; 10 nM) constrictions in human resistance and conductance coronary arteries (CA) in vitro was investigated. ET-1 (0.1 - 300 nM) constricted resistance CA more potently than conductance CA (P<0.05; EC(50) values 2.98 nM (95% CI: 1.49 - 5.95 nM and 8.58 (4.72 - 15.6 nM) respectively)). The NO-donor diethylamine NONOate fully reversed the ET-1 constriction in conductance CA (E(MAX) 127+/-9.16%), however only partial reversal was observed in resistance CA (E(MAX) 78.8+/-8.13; P<0.05). The soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (100 microM) reduced the maximum response to diethylamine NONOate to 76.9+/-14.4% in conductance CA (P<0.05), but had no effect on resistance CA (E(MAX) 77.2+/-18.4%). There was no difference between responses to ANP in conductance and resistance CA (EC(50) values 4.25 nM (0.84 - 21.4 nM) and 18.4 nM (2.92 - 116 nM), E(MAX) 53.1+/-14.7% and 48.6+/-11.8% respectively). BNP was a more potent vasodilator of conductance than resistance CA. In conductance CA the mean EC(50) value was 2.4 nM (0.74 - 7.75 nM), E(MAX) 54.5+/-14.9%. Concentration-response curves to BNP were incomplete in resistance CA. Concentration-response curves to CNP were incomplete in both conductance and resistance CA. The greater potency of ET-1 in resistance vessels may exacerbate the effects of increased circulating levels of the peptide in disease. Only NO could fully reverse ET-1 mediated constrictions in conductance CA, and none of the dilators tested could completely counteract constrictions in resistance CA.

Depressor and regionally-selective vasodilator effects of human and rat urotensin II in conscious rats.

The regional haemodynamic effects of rat or human urotensin II (U-II) 3, 30, 300 and 3000 pmol kg(-1), i.v.) were assessed in separate groups of conscious, unrestrained, male, Sprague-Dawley rats (n=8 in each). Rat and human U-II had similar effects. At a dose of 3 pmol kg(-1), neither peptide had any significant action, while at a dose of 30 pmol kg(-1), there was a transient mesenteric vasodilatation (significant only for rat U-II). At doses of 300 and 3000 pmol kg(-1), there were dose-dependent tachycardias, and mesenteric and hindquarters hyperaemic vasodilatations. Thus, in conscious rats, the predominant cardiovascular action of rat and human U-II is vasodilatation. This is in contrast to recent findings with human U-II in non-human primates, but is consistent with effects on human isolated resistance vessels.