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Background: Circulating levels of chemerin are significantly higher in hypertensive patients and positively correlate with blood pressure. Chemerin activates chemokine-like receptor 1 (CMKLR1 or ChemR23) and is proposed to activate the "orphan" G-protein-coupled receptor 1 (GPR1), which has been linked with hypertension. Our aim was to localize chemerin, CMKLR1, and GPR1 in the human vasculature and determine whether 1 or both of these receptors mediate vasoconstriction., Methods and Results: Using immunohistochemistry and molecular biology in conduit arteries and veins and resistance vessels, we localized chemerin to endothelium, smooth muscle, and adventitia and found that CMKLR1 and GPR1 were widely expressed in smooth muscle. C9 (chemerin149-157) contracted human saphenous vein (pD 2 =7.30±0.31) and resistance arteries (pD 2 =7.05±0.54) and increased blood pressure in rats by 9.1±1.0 mm Hg at 200 nmol. Crucially, these in vitro and in vivo vascular actions were blocked by CCX832, which we confirmed to be highly selective for CMKLR1 over GPR1. C9 inhibited cAMP accumulation in human aortic smooth muscle cells and preconstricted rat aorta, consistent with the observed vasoconstrictor action. Downstream signaling was explored further and, compared to chemerin, C9 showed a bias factor=≈5000 for the G i protein pathway, suggesting that CMKLR1 exhibits biased agonism., Conclusions: Our data suggest that chemerin acts at CMKLR1, but not GPR1, to increase blood pressure. Chemerin has an established detrimental role in metabolic syndrome, and these direct vascular actions may contribute to hypertension, an additional risk factor for cardiovascular disease. This study provides proof of principle for the therapeutic potential of selective CMKLR1 antagonists., (© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Apelin peptide analogues displaying bias towards G protein signalling pathways have beneficial cardiovascular actions compared with the native peptide in humans in vivo. Our aim was to determine whether small molecule agonists could retain G protein bias. We have identified a biased small molecule, CMF-019, and characterised it in vitro and in vivo. In competition radioligand binding experiments in heart homogenates, CMF-019 bound to the human, rat and mouse apelin receptor with high affinity (pKi=8.58±0.04, 8.49±0.04 and 8.71±0.06 respectively). In cell-based functional assays, whereas, CMF-019 showed similar potency for the Gαi pathway to the endogenous agonist [Pyr(1)]apelin-13 (pD2=10.00±0.13 vs 9.34±0.15), in β-arrestin and internalisation assays it was less potent (pD2=6.65±0.15 vs 8.65±0.10 and pD2=6.16±0.21 vs 9.28±0.10 respectively). Analysis of these data demonstrated a bias of ∼400 for the Gαi over the β-arrestin pathway and ∼6000 over receptor internalisation. CMF-019 was tested for in vivo activity using intravenous injections into anaesthetised male Sprague-Dawley rats fitted with a pressure-volume catheter in the left ventricle. CMF-019 caused a significant increase in cardiac contractility of 606±112mmHg/s (p<0.001) at 500nmol. CMF-019 is the first biased small molecule identified at the apelin receptor and increases cardiac contractility in vivo. We have demonstrated that Gαi over β-arrestin/internalisation bias can be retained in a non-peptide analogue and predict that such bias will have the therapeutic benefit following chronic use. CMF-019 is suitable as a tool compound and provides the basis for design of biased agonists with improved pharmacokinetics for treatment of cardiovascular conditions such as pulmonary arterial hypertension., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Radioligand binding is widely used to characterize receptors and determine their anatomical distribution, particularly the superfamily of seven transmembrane-spanning G protein-coupled receptors for both established transmitters such as endothelin-1 and an increasing number of orphan receptors recently paired with their cognate ligands. Three types of assay are described. In saturation experiments, tissue sections, cultured cells, or homogenates are incubated with an increasing concentration of a radiolabeled ligand, which can be a labeled analog of a naturally occurring transmitter, hormone, or synthetic drug. Analysis using iterative nonlinear curve-fitting programs, such as KELL, measures the affinity of the labeled ligand for a receptor (equilibrium dissociation constant, K ( D )), receptor density (B (max)), and Hill slope (nH). The affinity and selectivity of an unlabeled ligand to compete for the binding of a fixed concentration of a radiolabeled ligand to a receptor are determined using a competition binding assay. Kinetic assays measure the rate of association to or dissociation from a receptor from which a kinetic K ( D ) may be derived. Quantitative autoradiography and image analysis is a sensitive technique to detect low levels of radiolabeled ligands and determine the anatomical distribution of receptors in sections that retain the morphology of the tissue. The measurement of bound radioligand within discrete regions of autoradiographical images using -computer-assisted image analysis is described.

Background and Purpose: The aim of this study was to determine whether the apelin/APJ system is altered in human cardiovascular disease by investigating whether the expression of apelin or its receptor is altered at the protein level., Experimental Approach: Radioligand binding studies were used to determine apelin receptor density in human cardiac tissues. Apelin peptide levels in cardiovascular tissues were determined by radioimmunoassay. In vitro pharmacology was used to assess vasoactive properties of apelin in human coronary artery. Localization of apelin and its receptor in coronary artery was determined using immunohistochemistry., Key Results: Apelin receptor density was significantly decreased in left ventricle from patients with dilated cardiomyopathy or ischaemic heart disease compared with controls, but apelin peptide levels remained unchanged. Apelin was up-regulated in human atherosclerotic coronary artery and this additional peptide localized to the plaque, colocalizing with markers for macrophages and smooth muscle cells. Apelin potently constricted human coronary artery., Conclusions and Implications: We have detected changes in the apelin/APJ system in human diseased cardiac and vascular tissue. The decrease in receptor density in heart failure may limit the positive inotropic actions of apelin, contributing to contractile dysfunction. The contribution of the increased apelin levels in atherosclerotic coronary artery to disease progression remains to be determined. These data suggest a potential role for the apelin/APJ system in human cardiovascular disease.

We have examined whether differential expression of UT receptors in cardiovascular tissues from rats and humans may account for the diverse vascular actions reported for urotensin-II. We found UT immunoreactivity ubiquitously expressed in arterial and venous smooth muscle and cardiomyocytes in both species, however, compared to human, levels of UT immunoreactivity in rat vascular endothelial cells was below the level for detection. In rat skeletal muscle cells UT receptor localized to the sarcolemma, a pattern comparable to that for isoforms of nitric oxide synthase suggesting that urotensin-II mediated hindquarter vasodilatation may involve release of nitric oxide from skeletal muscle fibers.

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.

The G protein-coupled receptor GPR54 (also designated KISS1) is activated by cleavage products of the KiSS1 protein, the kisspeptins (KP), to act as a molecular switch for puberty. Additionally, KP are potent inhibitors of tumor metastasis and play a role in placentation, both processes involving angiogenesis. Our aim was to investigate whether GPR54 and KP are expressed within normal and diseased human vasculature and what their functional role may be. RT-PCR screening of human blood vessels revealed a discrete localization of GPR54 mRNA in smooth muscle of vessels with the same developmental origins, aorta, coronary artery, and umbilical vein, a pattern confirmed by immunocytochemistry and radioligand binding. Novel ligand [(125)I]KP-13 exhibited saturable and high-affinity binding in aorta smooth muscle sections (dissociation constant K(D) = 0.2 +/- 0.03 nM), and using confocal microscopy, we found colocalization of receptor and peptide to vascular endothelial cells and to the atherosclerotic plaque of coronary artery. RIA detected 13.04 +/- 2.94 and 20.50 +/- 5.00 fmol/g KP in human coronary artery and aorta, respectively. KP-10, KP-13, and KP-54 acted as vasoconstrictors with comparable potency and efficacy in isolated rings of coronary artery (negative logarithm of the EC(50) and maximal response, respectively, as follows: KP-10, 7.89 +/- 0.24 and 33.7 +/- 17.0; KP-13, 8.66 +/- 0.88 and 35.1 +/- 7.9; KP-54, 8.86 +/- 1.11 and 25.7 +/- 5.5) and umbilical vein (negative logarithm of the EC(50) and maximal response, respectively, as follows: KP-10, 8.44 +/- 022 and 24.3 +/- 3.7; KP-13, 8.43 +/- 0.88 and 28.4 +/- 8.6; KP-54, 8.93 +/- 0.39 and 36.9 +/- 5.2). In conclusion, we have detected expression of both peptide and receptor in aorta, coronary artery, and umbilical vein and have shown for the first time that the KP are vasoconstrictors in humans, suggesting a previously undescribed role for GPR54 and KP in the cardiovascular system.

The aim of the study was to measure regional haemodynamic responses to 6 h infusions of human urotensin II (hUII), to identify possible mediators of the effects observed, and to relate the findings to the distribution of urotensin II receptors (UT receptors). Male, Sprague-Dawley rats had pulsed Doppler flow probes and intravascular catheters implanted for measurement of regional haemodynamics in the conscious, freely moving state. Infusions of saline (0.4 ml h(-1)) or hUII (30, 300 and 3,000 pmol kg(-1) h(-1)) were given i.v. for 6 h, and the effects of pretreatment with indomethacin (5 mg kg(-1) h(-1)), N(G)-nitro-L-arginine methyl ester (L-NAME, 3 mg kg(-1) h(-1)) or propranolol (1 mg kg(-1); 0.5 mg kg(-1) h(-1)) on responses to hUII (300 pmol kg(-1) h(-1) for 6 h) were assessed. Cellular localisation of UT receptor-like immunoreactivity was determined in relevant tissues. hUII caused dose-dependent tachycardia and hindquarters vasodilatation, accompanied by a slowly developing rise in blood pressure. Haemodynamic effects of hUII were attenuated by propranolol or L-NAME and abolished by indomethacin. UT receptor-like immunoreactivity was detected in skeletal and vascular smooth muscle. The findings indicate that in conscious rats, infusions of hUII cause vasodilatation, which, of the vascular beds monitored, is selective for the hindquarters and dependent on cyclooxygenase products and nitric oxide. The pressor effect of hUII under these conditions is likely to be due to an increase in cardiac output, possibly due to a positive inotropic effect. UT receptor-like immunoreactivity present in skeletal muscle is consistent with the haemodynamic pattern.

Endothelin (ET)-1 and thromboxane (Tx) levels are increased in human atherosclerosis. One of the aims of this study was to understand how receptors for a peptide mediator (ET-1) with a long physiological half life, would differ from a lipid mediator (TxA(2)), with a short physiological half life, in human coronary artery disease (CAD). Secondly, to determine if receptor protein is present in human coronary artery vascular smooth muscle for the recently adopted peptide orphan receptors for urotensin-II, apelin and ghrelin. The ET(A) receptor subtype predominated in the medial smooth muscle layer of both non-diseased coronary artery (NCA) and CAD. However, this subtype was present at relatively low density in the proliferated intimal layer of CAD. The ET(B) receptor protein was not altered with CAD, compared with NCA. Tx receptor density was significantly (P<0.05) increased in both the media and intima of CAD, compared with NCA. There was no alteration in receptor density, on the medial smooth muscle for urotensin-II and apelin with CAD. Interestingly, receptor density for the novel vasodilator peptide ghrelin was significantly (P<0.05) increased (approx. 4 fold) with CAD, compared with NCA. The alteration of receptor density with disease for Tx and ghrelin provides novel therapeutic targets for the treatment of atherosclerosis. In conclusion, while some GPCR are altered, others remain unchanged with human atherosclerosis. The increase in vasoconstrictor Tx receptor density with disease suggests the importance of Tx receptor antagonism. Intriguingly, the increase in receptor density for the novel vasodilator ghrelin, identified from post-genomic research, may potentially be beneficial with human atherosclerosis.

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.

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.