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Endocrinology, doi:10.1210/en.2006-0818
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Endocrinology Vol. 148, No. 1 140-147
Copyright © 2007 by The Endocrine Society

Kisspeptins Are Novel Potent Vasoconstrictors in Humans, with a Discrete Localization of Their Receptor, G Protein-Coupled Receptor 54, to Atherosclerosis-Prone Vessels

Emma J. Mead, Janet J. Maguire, Rhoda E. Kuc and Anthony P. Davenport

Clinical Pharmacology Unit, University of Cambridge, Centre for Clinical Investigation, Addenbrooke’s Hospital, Cambridge CB2 2QQ, United Kingdom

Address all correspondence and requests for reprints to: Dr. Anthony P. Davenport, Clinical Pharmacology Unit, University of Cambridge, Level 6, Centre for Clinical Investigation, Box 110, Addenbrooke’s Hospital, Cambridge, CB2 2QQ, United Kingdom. E-mail apd10{at}medschl.cam.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 [125I]KP-13 exhibited saturable and high-affinity binding in aorta smooth muscle sections (dissociation constant KD = 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 EC50 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 EC50 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE G PROTEIN-COUPLED receptor GPR54 (1) (previously designated AXOR12 or hOT7T175) was recently paired with its cognate ligands kisspeptin (KP)-54 (metastin), KP-13, and KP-10, all endogenous cleavage products of the KiSS1 protein (2, 3, 4, 5) (Fig. 1Go), which have been isolated in human tissue (5). GPR54 has been shown to be a molecular switch of puberty in humans and mice, possibly acting directly to release GnRH from hypothalamic neurons (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16). In Western societies where blood pressure increases throughout life, during pubertal growth, the rate of increase of blood pressure accelerates compared with pre- and post-pubertal growth (17). Current reports focus on determining the molecular mechanisms by which GPR54 acts as a switch for puberty; however, before pairing with GPR54, KP had also been identified as potent inhibitors of metastasis in a variety of cancer types (18) and as invasion inhibitors of primary human trophoblasts (19). Additionally, KP have been shown to down-regulate the activity of matrix metalloproteinase (MMP)-9 and MMP-2 (20), both of which act as extracellular proteases enabling angiogenesis in cancer metastasis and placentation (21) and have a role in the formation of atherosclerotic plaques (22, 23). KP are also present in the plasma, circulating at low picomolar levels (10, 24), with an associated decrease in obese women suffering polycystic ovary syndrome compared with normal-weight women with the same syndrome.


Figure 1
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  		FIG. 1. The structure of the KP pre-pro-protein KP-145 and its cleavage products KP-54, KP-13, and KP-10. Amino acids in black are the common sequence that the KP antibody was raised against. SP, Signal peptide.

 
The role of the KP in the cardiovascular system remains unknown, but their identification as circulating peptides in plasma, linked to obesity, and the association of KP and GPR54 with physiological processes involving angiogenesis, led us to hypothesize that both receptor and peptide may be expressed in normal and diseased human vasculature. We tested this hypothesis using a systematic PCR screening approach of GPR54 to identify expression in a panel of arteries and veins. This resulted in detection of an unusual distribution in vessels that originate from the aorta-gonad-mesonephros and are prone to atherosclerotic plaque formation, with subsequent immunocytochemistry (ICC) studies localizing both peptide and receptor to the atherosclerotic plaque of the coronary artery. We further addressed expression of GPR54 in the vessels at the protein level, using a novel radioligand to define the pharmacological criteria of saturable, specific, and high-affinity binding. RIA and ICC revealed a potential source of KP in vascular endothelial cells. Because reported potencies for activation of GPR54 by KP cleavage products varied (http://www.iuphar-db.org/GPCR/ReceptorDisplayForward?receptorID=2989), we compared responses to KP-10, KP-13, and KP-54 in isolated human coronary artery and umbilical vein and showed for the first time that KP function as potent vasoconstrictors in these tissues, with a response comparable to angiotensin (Ang)-II in coronary artery.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The peptides KP-54 and KP-13 were custom synthesized by Severn Biotech (Kidderminster, UK). KP-10 and the structurally unrelated peptides Ang-II, urotensin (U)-II, somatostatin (SST)-14, and nociceptin were from the Peptide Institute (Osaka, Japan). Apelin-13 was from Bachem (St. Helens, UK) and ghrelin from Phoenix Pharmaceuticals (Belmont, CA). [125I]KP-13 (2000 Ci/mmol) (Amersham Biosciences, GE Healthcare, Little Chalfont, UK) was prepared from unlabeled KP-13 by direct iodination with sodium [125I]iodide using the chloramine-T method. The rabbit anti-GPR54 (375–398, human) serum and rabbit anti-KP-10 [KP(45–54)-NH2, human] were from Phoenix Pharmaceuticals. AlexaFluor 488-conjugated goat antirabbit serum and AlexaFluor 568-conjugated goat antimouse serum were from Molecular Probes (Leiden, The Netherlands). All molecular biology reagents, including primers, were from Invitrogen (Paisley, UK) All other reagents were from Sigma-Aldrich (Poole, UK).

Human tissue collection
Human tissues were collected with local ethical approval and informed consent. Aorta and coronary artery were from the explanted hearts of patients transplanted for dilated cardiomyopathy or ischemic heart disease (Table 1Go). Left internal mammary artery, saphenous vein, and radial artery were from patients receiving coronary artery bypass grafts. For immunocytochemical and radioligand binding studies, tissues were snap-frozen immediately after surgery and stored at –70 C until used. Tissues for in vitro pharmacology studies were transferred into pots containing Krebs’ solution, stored at 4 C, and used within 24 h of surgery. Human umbilical veins were collected post parturition in PBS at 4 C and used within 24–48 h. Myometrium was collected immediately post parturition and frozen until used. Human umbilical vein endothelial cells (HUVEC) were cultured as previously described (25) to subconfluency.


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  		TABLE 1. Summary of patient details

 
RNA analysis
Tissue was cleaned of connective tissue in an RNase-free environment and the endothelium removed by forceful stokes of a razor blade (26). Tissue (10 mg) was homogenized, using the FastPrep instrument (Q-BIOgene, Cambridge, UK). mRNA was extracted using the Invitrogen Micro-Fast Track 2.0 kit according to the manufacturer’s protocol. Synthesis of cDNA was carried out using the Invitrogen SuperScript III First-Strand Synthesis System for RT-PCR and oligo-DT primer. PCR of cDNA was performed using primers specific for ß-actin and GPR54 (5' primer, 5'-CTCGCTGGTCATCTACGTCA-3'; 3' primer, 5'-CCAGTTGTAGTTCGGCAGGT-3'). Conditions were 98 C for 1 min, followed by 30 cycles of 97 C for 1 min, 59 C for 30 sec, 72 C for 1 min, and a final extension at 72 C for 10 min. PCR products were separated by electrophoresis on a 1% agarose gel with ethidium bromide and sizes estimated by reference to a 100-bp DNA molecular weight standard. The integrity of cDNA samples was determined by detection of ß-actin expression, using primers spanning an intronic region in the ß-actin gene, to detect genomic DNA (gDNA) contamination. Expression of GPR54 has previously been reported in human uterine tissue; we therefore used myometrium as a positive control tissue to determine that GPR54 primers were able to detect GPR54 mRNA in previously published regions of expression (3). A positive ß-actin control and a negative control, in which cDNA was omitted, were included for each PCR.

Fluorescence dual-labeling immunocytochemistry
Cryostat cut sections (30 µm) of human arteries and veins were thaw mounted onto poly-L-lysine-coated slides and stored at –70 C until required. Before use, slides were transferred into slide racks and air dried overnight at room temperature; subsequently, tissue was fixed in acetone (4 C) for 10 min. Nonspecific protein interaction was blocked by 2 h incubation in 5% nonimmunized goat serum (GS) in PBS. To localize GPR54 and KP, tissues were then incubated for 48 h at 4 C in 1:100 rabbit anti-GPR54 (375–398, human) serum or rabbit anti-KP-10 [KP(45–54)-NH2, human] serum in PBS/Tween 20 (T) and 3% nonimmunized GS with one of the cell-specific marker antibodies. Negative controls were carried out in adjacent sections by omission of the primary antisera. Subsequent to washing in PBS/T (three times for 5 min each at 4 C), sections were incubated with a secondary antibody solution containing AlexaFluor 488-conjugated goat antirabbit serum (1:200) and AlexaFluor 568-conjugated goat antimouse serum (1:200) in PBS/T with 3% GS for 1 h at 22 C. After a final wash in PBS/T (three times for 5 min each at 4 C), sections were mounted in Vectasheild mounting medium, and visualization was carried out using laser-scanning confocal microscopy (Leica Microsystems, Heidelberg, Germany) as previously described (27).

[125I]KP-13 binding studies
Unless otherwise stated, all binding studies were carried out using 10-µm sections of human aorta smooth muscle thaw mounted onto poly-L-lysine-coated slides and stored at –70 C until required. Additionally, three sections were collected into microcentrifuge tubes to allow measurement of protein. Sections were thawed and preincubated with phosphate binding buffer (in mM: 50 NaH2PO4, 100 NaCl; 5 EDTA, 5 MgCl2, 1 protease inhibitor cocktail, pH 7.4 at 22 C) for 15 min to remove endogenous KP and degradative enzymes and subsequently incubated for 2 h with 0.2 nM [125I]KP-13 (custom synthesis; Amersham Biosciences) in binding buffer. Unlabeled KP-13 (1 µM) (Severn Biotech) defined nonspecific binding in adjacent sections. Equilibrium was broken by washing three times for 5 min each in an excess of 50 mM Tris (4 C, pH 7.4) buffer. Sections were wiped from slides using filter paper and transferred to a counting tube, and binding of [125I]KP-13 was detected using a {gamma}-counter to measure disintegrations per minute (DPM). Protein concentrations were determined using the Bio-Rad DC 96-well microtiter plate system.

Receptor autoradiography
Sections were incubated with 0.5 nM [125I]KP-13, and nonspecific binding was determined by inclusion of 10 µM KP-13. Washed sections were apposed to radiation-sensitive film (Kodak BioMax MR-1), with iodine standards, for 4 d. Binding was quantified by computer-assisted densitometry using the Quantimet 970 system as previously described (28).

Specificity of [125I]KP-13 binding
Sections were incubated in the presence and absence of 1 µM structurally related peptides KP-54 (Severn Biotech), KP-13, and KP-10 as well as the structurally unrelated vasoactive peptides Ang-II, U-II, SST-14, apelin-13, ghrelin, and nociceptin.

Association and dissociation studies
Tissue sections were incubated with [125I]KP-13 for increasing times (0–240 min) for association assays, or for 2 h followed by increasing wash periods (0–1320 min) for dissociation assays.

Saturation and competition binding assays
Saturation studies were carried out using increasing concentrations (80 pM to 5 nM) of [125I]KP-13 in binding buffer. Competition assays were carried out in the presence of the GPR54 agonist KP-10 or KP-13 (20 pM to 500 nM). The nonlinear curve-fitting programs in the KELL package (Biosoft, http://www.biosoft.com), EBDA (Equilibrium Binding Data Analysis), and LIGAND were used to analyze the data, and results were normalized to protein content in femtomoles per milligram protein. EBDA was used to convert DPM to molar concentrations of ligand and perform initial data analysis to provide estimates of equilibrium dissociation constant (KD) and maximum binding (Bmax) using Scatchard transformation and Hill slopes calculated separately. Files created by EBDA were used in conjunction with estimates of binding parameters by LIGAND to fit data to a one- or multiple-binding-site model using a weighted, nonlinear curve-fitting routine, which was iteratively refined to more accurately estimate KD and Bmax than linear (Scatchard) transformations alone can determine. The program indicates whether a one-site fit is a statistically better fit than a two-site model using the F test, which takes into account the improvement in the goodness of fit that accompanies an increase in the number of parameters to be fitted in the two-site model compared with a one-site fit.

Extraction of KP from human plasma, human aorta, and human coronary artery
Human blood (n = 4, male) was collected into tubes containing EDTA and centrifuged at 2000 x g for 10 min at 4 C. Plasma was removed, pooled, and acidified in 75% ethanol containing 25% 0.1 M HCl and spun at 2000 x g for 10 min at 4 C. Human aorta (n = 8) and human coronary artery (n = 7) samples were homogenized using the FastPrep instrument in 1 ml/g tissue in 0.5 M acetic acid and boiled for 15 min, and samples were spun at 30,000 x g for 30 min at 4 C. Plasma and tissue supernatants were extracted using Oasis silica-based columns (Waters, Elstree, UK), activated by methanol and washed in deionized H2O before addition of sample. Subsequent to washing twice in 0.1% trifluoroacetic acid, samples were eluted in 1 ml 80% MeOH/0.1% trifluoroacetic acid. Samples were dried overnight in an evacuated centrifuge and reconstituted in RIA buffer (40 mM Na2HPO4, 10 mM NaH2PO4, 8 mM sodium azide, 0.025% gelatin, and 0.05% Tween 20, pH 7.4 at 22 C). Recovery of KP (mean ± SEM, 93 ± 7%) was detected using plasma samples spiked with 10 pM synthetic KP-13 and was performed for each assay.

RIA
Rabbit anti-KP-10 [KP(45–54)-NH2] serum was used in the KP RIA at a final dilution of 1/30,000 in RIA buffer. The assay consisted of 100 µl standard (0–10 nM synthetic KP-13) or extracted sample and 100 µl KP-10 antiserum, diluted in RIA buffer. Tubes were incubated overnight at 4 C and 100 µl [125I]KP-13 (10,000–15,000 cpm), diluted in RIA buffer, added. Tubes were incubated for another 24 h at 4 C, free and bound KP was separated by addition of 250 µl activated charcoal in 1% dextran solution (Sigma), followed by centrifugation at 3000 x g for 10 min at 4 C, and supernatant was decanted. The remaining [125I]KP-13 in the pellet was detected using a {gamma}-counter to measure DPM. For specificity studies, the assay contained 10 nM structurally related peptides KP-54 and KP-10 as well as the structurally unrelated vasoactive peptides Ang-II, U-II, SST-14, apelin-13, ghrelin, and nociceptin. The detection limit of the assay was 2.8 fmol/tube, with the linear portion of the curve ranging from 8–200 fmol/tube with a slope of –1.23 and IC50 of 34 fmol/tube (published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). The intra- and interassay variations were calculated using values from pooled human plasma and were 2.7 ± 0.1 and 6.1 ± 0.4%, respectively.

In vitro pharmacology: constrictor responses to KP-10, KP-13, and KP-54 in human artery and vein
Human coronary, radial, and internal mammary arteries and umbilical and saphenous veins were cleaned of connective tissue and cut into 4-mm rings. The lumen was gently rubbed using blunt forceps to remove the endothelial cell layer (26). Rings were mounted in 5-ml organ baths containing oxygenated (95% O2/5% CO2) Krebs’ solution (in mM/liter: 90 NaCl, 45 NaHCO3, 5 KCl, 0.5 MgSO4·7H2O, 1 Na2HPO4·2H2O, 2.25 CaCl2, 5 fumaric acid, 5 glutamic acid, 10 glucose, and 5 sodium pyruvate, pH 7.4 at 37 C). Isometric tension was recorded using F30 transducers (Hugo Sachs Elecktronik, Freiburg, Germany), and output was to a chart recorder and to the MP100 data acquisition system (BIOpac Systems Inc., Santa Barbara, CA) for subsequent analysis. After a 1-h equilibration period, optimal basal tension and tissue viability were determined by recording the response to 0.1 M KCl at incrementally increasing levels of basal tension until no further increase in developed isometric force was obtained. The maximal KCl response was taken as the KCl control. After another 1-h equilibration period, absence of the endothelial layer was verified by lack of significant (<15%) relaxation to 1 µM acetylcholine after preconstriction with 10 µM phenylephrine. In umbilical vein, absence of endothelium was verified histologically. Cumulative concentration response curves were then constructed to KP-10, KP-13, and KP-54 (1 pM to 100 nM), and when no additional increase in response was obtained, experiments were terminated by the addition of 0.1 M KCl (post-KCl) to determine the maximal contractility of each vessel ring and viability at the end of the experiment. Agonist responses were normalized to the maximal post-KCl response. Data were analyzed using iterative curve-fitting software (FigP; Biosoft, Cambridge, UK), to determine the maximal response (Emax) to each agonist and EC50.

Data and statistical analysis
Data are expressed as mean ± SEM. Emax, the negative logarithm of the EC50 (pD2), and tissue viability [basal resting tension, maximal constriction to 0.1 M KCl at optimal resting tension (expressed as millinewtons per millimeter) and ratio of post-KCl to maximal pre-KCl response] between tissues treated with the three different KP were compared using one-way ANOVA. A P value < 0.05 was considered statistically significant, and n values are the number of patients from which tissues were obtained. Binding study data were analyzed using the iterative, nonlinear curve-fitting programs EBDA and LIGAND in the KELL package (Biosoft, Cambridge, UK). Pooled KD, Bmax, and Hill slope were expressed as mean ± SEM and normalized to protein concentrations. Mann Whitney U Test was used to compare KD, derived from competition assays. RIA variance is expressed as mean ± SD, except where noted.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of GPR54 mRNA in human vasculature
We investigated the expression of GPR54 mRNA across a panel of vascular tissues as an initial indicator of GPR54 localization. We confirmed that all samples were free of gDNA contamination and had maintained their integrity by screening using ß-actin primers spanning an intronic region in the ß-actin gene (Fig. 2Go). We detected discrete expression of GPR54 mRNA in smooth muscle of the developmentally related vessels, aorta, coronary artery, and umbilical vein, but not in other vessels screened, including radial artery (Fig. 2Go and supplemental data).


Figure 2
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  		FIG. 2. Expression of GPR54 mRNA in smooth muscle of human umbilical vein (UV) (n = 3), coronary artery (CA) (n = 3), and aorta (A) (n = 4). Myometrium (M) (n = 3) was a control tissue. ß-Actin detection of samples determined integrity of cDNA and absence of gDNA. The PCR was carried out in the absence of cDNA (–), and ß-actin detection in a myometrium sample was used as a positive control (+). PCR product size was determined using a 100-bp standard DNA ladder (L). GPR54 was not detected in other human large conduit vessels screened, including radial artery (RA), saphenous vein, and mammary artery.

 
Immunocytochemical localization of GPR54 and KP protein
Extensive BLAST (29) searching of the human genome revealed no sequences likely to cross-react with either the peptide or receptor antibody. We were able to detect a single band, of the appropriate size, by performing a Western blot using the GPR54 antibody and membrane preparations from human aorta, in which preabsorption of the rabbit anti-GPR54 with the immunizing peptide (GPR54 375–398, 10 µM) attenuated the specific band (supplemental data). The KP-10 antibody cross-reacted with KP-10, KP-13, and KP-54 (Fig. 1Go, and see RIA specificity results); however we were able to prevent staining by preabsorption of the antibody to synthetic KP-10 (supplemental data). In agreement with our mRNA distribution, we detected GPR54-like immunoreactivity in the smooth muscle of histologically normal human aorta, coronary artery, and umbilical vein and in small coronary arteries (supplemental data) using light microscopy but not in other vessels tested. We used fluorescent dual-labeling ICC and confocal laser scanning microscopy to further investigate the cellular and subcellular localization of GPR54, which confirmed GPR54-like immunoreactivity in smooth muscle cells of aorta, coronary artery, and umbilical vein (Fig. 3Go, top). Additionally, both KP-like immunoreactivity and GPR54-like immunoreactivity colocalized to endothelial cells in coronary artery and umbilical vein (Fig. 3Go, bottom). Furthermore, we identified peptide and receptor-like immunoreactivity in cultured HUVEC (Fig. 4Go), but at the cellular level, KP-like immunoreactivity was not spatially colocalized to Weibel-Palade bodies that store von Willebrand factor (vWF) in endothelial cells. In addition to localization to histologically normal vessels, and consistent with discrete localization in atherosclerosis-prone vessels, expression of GPR54 and the KP was further localized to atherosclerotic plaques of diseased coronary artery (Fig. 5Go).


Figure 3
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  		FIG. 3. Top, Representative photomicrographs showing confocal images of fluorescent dual-labeling ICC of smooth muscle {alpha}-actin (SM{alpha}A) and GPR54-like immunoreactivity in smooth muscle of human coronary artery (CA) (n = 4) (A–C), umbilical vein (UV) (n = 4) (D–F), and aorta (n = 4) (G–I). GPR54, shown in green, colocalized with SM{alpha}A, shown in red, in all vessels, resulting in yellow staining in the overlay. Scale bar, 150 µm. Bottom, Representative photomicrographs showing confocal images of fluorescent dual-labeling ICC of endothelial cell marker vWF and GPR54/KP-like immunoreactivity in endothelial cells of human coronary artery (CA) (n = 4) (GPR54 in A–C; KP in D–F) and umbilical vein (UV) (n = 4) (GPR54 in G–I; KP in J–L). GPR54/KP, shown in green, colocalized with vWF, shown in red, in all vessels, resulting in yellow staining in the overlay. Scale bar, 150 µm.

 

Figure 4
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  		FIG. 4. Representative photomicrographs showing confocal images of fluorescent dual-labeling ICC of endothelial cell marker vWF and GPR54/KP-like immunoreactivity in HUVEC (GPR54 in A–D; KP in E–H). GPR54/KP are shown in green, vWF is shown in red, and nuclear marker 4',6-diamidino-2-phenylindole (DAPI) is in blue. Both peptide and receptor are expressed in cultured endothelial cells; however, KP-like immunoreactivity did not spatially colocalize with the inducible secretory granules containing vWF. Scale bar, 50 µm.

 

Figure 5
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  		FIG. 5. Representative photomicrographs showing confocal images of fluorescent dual-labeling ICC of macrophage marker CD68 and GPR54/KP-like immunoreactivity in the atherosclerotic plaque of human coronary artery (CA) (n = 4) (GPR54 in A–C; KP in D–F). GPR54 and KP are shown in green, and CD68 is shown in red. Phase-contrast images of normal and diseased coronary artery are given in G and H to show structure of vessels. Ad, Adventitia; EC, endothelial cell; SM, smooth muscle. Scale bar, 150 µm.

 
Localization of specific [125I]KP-13 binding in human aorta, coronary artery, and umbilical vein
Using our radioligand, [125I]KP-13, we confirmed specific expression of GPR54 in the smooth muscle of human aorta, coronary artery, and umbilical vein. We measured the highest density of specific binding in aorta (213.9 ± 45.8 amol ligand bound/mm2 of tissue), with lower levels detected in coronary artery and umbilical vein (32.6 ± 11.5 and 32.3 ± 2.8 amol/mm2, respectively).

[125I]KP-13 binding characterization in human aortic smooth muscle
Because the highest density of binding was observed in aortic smooth muscle, all further characterization of our ligand was carried out in this tissue. At a fixed concentration of 1 µM, KP-13 and KP-10 competed significantly (P < 0.05) for 0.2 nM [125I]KP-13 binding, whereas structurally unrelated vasoactive peptides did not compete for binding, confirming specificity of ligand binding (supplemental data). Additionally, KP-54 did not compete significantly for binding, possibly because of differential folding of this larger peptide under these assay conditions. In saturation experiments, over the concentration range tested, [125I]KP-13 bound monophasically with saturable, subnanomolar affinity (KD, 0.20 ± 0.03 nM) and a density of binding (Bmax, 7.65 ± 0.95 fmol/mg protein) comparable to other orphan receptors recently paired with their cognate ligand, such as ghrelin (Bmax, 17.5 ± 2.5 fmol/mg protein) (30). Hill coefficients were close to unity (nH, 1.12 ± 0.09), confirming binding with a single affinity. Binding of [125I]KP-13 was time dependent with an association rate constant (kobs) of 0.164 ± 0.054 min–1 and a half-time for association (t1/2) of 4 min. Binding was reversible at 4 C, with a dissociation rate constant of 0.0020 ± 0.0006 min–1 from a single site and a half-rate for dissociation (t1/2) of 301 min.

[125I]KP-13 competition binding in human aorta
To determine the ability of KP-10 to compete with [125I]KP-13 binding at the GPR54 receptor, competition binding experiments were performed. KP-10 and KP-13 competed monophasically for [125I]KP-13 binding with comparable KD of 0.03 ± 0.007 nM and 0.26 ± 0.12 nM, respectively (P > 0.05, Mann Whitney U test).

Detection of KP in human coronary artery and aorta by RIA
To confirm expression of the KP in coronary artery and aorta, we developed a RIA. The KP antibody cross-reacted 100% with all of the KP fragments, and structurally unrelated vasoactive peptides were below the level of detection in this assay. We detected 0.83 ± 0.19 pM in pooled plasma. In agreement with ICC detection of KP in coronary artery and aorta, we detected 13.04 ± 2.94 and 20.50 ± 5.00 fmol/g tissue, respectively.

Constrictor responses to KP in endothelium-denuded coronary artery and umbilical vein
We observed no significant difference between basal resting tension and viability of the tissue at the start of the experiment (pre-KCl response) or at the end of the experiment (ratio of post-KCl to pre-KCl response) in vessel rings belonging to three different treatment groups (Table 2Go) in either of the vessels. The three KP constricted with comparable potency and efficacy (P > 0.05) (Fig. 6Go, A–F, and Table 2Go). No response was detected in radial artery, mammary artery, or saphenous vein to any of the peptides, consistent with the lack of detectable levels of GPR54 at the molecular and protein level


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  		TABLE 2. Endothelium-independent vasoconstriction of human coronary artery and umbilical vein

 

Figure 6
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  		FIG. 6. KP-10, KP-13, and KP-54 are vasoconstrictors of human coronary artery and umbilical vein. Graphs show one representative curve for each vasoconstrictor response in endothelium-denuded coronary artery (A–C) and umbilical vein (D–F) by KP-10 (bullet), KP-13 ({blacksquare}), and KP-54 ({blacktriangleup}).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we report a previously undescribed function of KP as potent endothelium-independent vasoconstrictors of human coronary artery and umbilical vein and show for the first time an unusual, discrete expression of GPR54 in the human cardiovascular system. We observed expression of GPR54 mRNA in aorta, coronary artery, and umbilical vein but not in other vessels screened by RT-PCR. Immunocytochemical and autoradiographic localization of GPR54 was in agreement with our reported mRNA expression in smooth muscle. This discrete localization is intriguing and has not previously been reported for a GPR. One possible explanation is that these particular blood vessels are developmentally related. GPR54 has been detected in the gonads and aorta, which have a common developmental origin in the aorta-gonad-mesonephros. The aorta is the first discernible vessel formed in the embryo, and the umbilical vein forms the connection between the dorsal aorta and placenta (31), with the coronary artery later branching into the aorta (32). Therefore, expression of GPR54 in placenta, which arises from the gonadal region, and in aorta, umbilical vein, and coronary artery is consistent with the developmental origins of these tissues. Interestingly, whereas the umbilical vein, as a neonatal tissue, is functional for too short a time period to develop atherosclerosis, the aorta and coronary artery are particularly prone to the formation of atherosclerotic plaques, with coronary fatty streaks beginning to appear in adolescence (33). Confocal microscopy revealed that both the KP and GPR54 are expressed in the atherosclerotic plaque of coronary artery.

Radioligand binding studies have previously been carried out on cells artificially expressing this receptor; however, no radioligand has been described as a tool to characterize GPR54, in native tissue, according to the pharmacological criteria of saturable, specific, and reversible binding. We were able to use our ligand [125I]KP-13 to detect specific binding in aorta, coronary artery, and umbilical vein with none of the structurally unrelated vasoactive peptides tested able to compete for binding confirming the selectivity of the radioligand for a novel receptor. Furthermore, characterization of this ligand revealed that in human tissue, [125I]KP-13 binding is time dependent, dissociable, and with subnanomolar affinity, comparable to that previously determined in cell lines (3, 5). Receptor autoradiography showed the highest density of receptor in aorta, and saturation analysis confirmed that GPR54 is expressed at a comparable level to other orphan GPR recently paired with their cognate ligands in human aorta (30).

We have confirmed the presence of KP in plasma at comparable levels to that previously reported by ELISA and RIA (10, 19); however, the source of circulating peptide remains unclear. Our detection of KP in coronary artery and aorta by RIA and of KP-like immunoreactivity in endothelial cells of coronary artery and umbilical vein by confocal microscopy suggests an endothelial cell source of KP, potentially acting as a paracrine regulator of vascular tone within these vessels. Overspill from endothelial cells may contribute to circulating levels of KP in plasma, which are similar to that reported for other endothelial-derived peptides such as endothelin (34). We did not detect colocalization of KP-like immunoreactivity to the Weibel-Palade bodies, which are thought to release vasoactive peptides such as endothelin from the regulated pathway (35), therefore suggesting release of KP by the constitutive pathway in this cell type.

KP-10, KP-13, and KP-54 are potent vasoconstrictors; in coronary artery, their responses were comparable to what we observe for the potent vasoactive peptide Ang-II (pD2, 8.87 ± 0.19; Emax, 31.7 ± 4.5; n = 37; Maguire, J. J., unpublished data). The observed potency was within the range previously determined in cell lines artificially expressing the receptor (3, 4), and in agreement with these studies, the shortest KP peptide tested, KP-10, had comparable potency and efficacy to the full-length KP-54 sequence; therefore, the biological activity of these peptides is highly likely to reside within the extreme C terminus.

Before this study, the primary functional roles described for the KP and GPR54 were as a molecular switch for puberty, as metastasis inhibitors, and as components of placental invasion into the uterus (6). The mechanism through which the KP act as inhibitors of metastasis has been proposed as an attenuation of cell motility by stimulation of stress fiber formation (5) and through inhibition of the collagenases MMP-2 and MMP-9 (36, 37, 38). The discovery that the KP are potent vasoconstrictors and are expressed in vasculature makes them possible targets in cancer therapy (39), not only as antiangiogenic factors but also with the potential to modulate blood flow to tumors. Significantly, the identification of KP as vasoconstrictors of umbilical vein implies that the role of KP in pregnancy is more complex than inhibition of trophoblast invasiveness alone.

We have detected discrete localization of GPR54 to aorta, coronary artery, and umbilical vein, with both peptide and receptor expressed in the atherosclerotic plaque. Therefore, in coronary artery, the vasoconstrictor action of KP that we have described may serve to locally exacerbate impaired blood flow in vessels that are themselves prone to atherosclerosis and already have a reduced lumen (40). In conclusion, in addition to established endocrine actions, we have discovered a novel function of GPR54 and KP mediating vasoconstriction, with a discrete localization to atherosclerosis-prone vessels.


   Acknowledgments
 
We thank Jean Chadderton and the theater and consultant staff of Papworth hospital, the staff of the Rosie Maternity Unit at Addenbrooke’s hospital for tissue collection, and Michaela Watts for collection of human blood. We also thank Michael Ashby for expertise in RIA, Susan Monteith for RT-PCR advice, and William Colledge for commenting on this manuscript.


   Footnotes
 
This work was supported by the British Heart Foundation; E.M. is the recipient of the A. J. Clark studentship funded by the British Pharmacological Society.

Author disclosures: The authors have nothing to declare.

First Published Online October 5, 2006

Abbreviations: Ang, Angiotensin; DPM, disintegrations per minute; gDNA, genomic DNA; GPR, G protein-coupled receptor; GS, goat serum; HUVEC, human umbilical vein endothelial cell(s); ICC, immunocytochemistry; KP, kisspeptin(s); MMP, matrix metalloproteinase; SST, somatostatin; T, Tween 20; U, urotensin; vWF, von Willebrand factor.

Received June 19, 2006.

Accepted for publication September 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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