Immunocytochemical localization of the urotensin-II receptor, UT, to rat and human tissues: Relevance to function

peptides 29 (2008) 735–742

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Immunocytochemical localization of the urotensin-II receptor, UT, to rat and human tissues: Relevance to function Janet J. Maguire *, Rhoda E. Kuc, Matthias J. Kleinz, Anthony P. Davenport Clinical Pharmacology Unit, University of Cambridge, Level 6 Centre for Clinical Investigation, Box 110 Addenbrooke’s Hospital, Cambridge CB2 2QQ, UK

article info

abstract

Article history:

We have examined whether differential expression of UT receptors in cardiovascular

Received 4 April 2007

tissues from rats and humans may account for the diverse vascular actions reported for

Received in revised form

urotensin-II. We found UT immunoreactivity ubiquitously expressed in arterial and venous

14 August 2007

smooth muscle and cardiomyocytes in both species, however, compared to human, levels of

Accepted 14 August 2007

UT immunoreactivity in rat vascular endothelial cells was below the level for detection. In

Published on line 26 August 2007

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

Keywords:

vasodilatation may involve release of nitric oxide from skeletal muscle fibers. # 2007 Elsevier Inc. All rights reserved.

Urotensin-II UT receptor Human cardiovascular system Immunocytochemistry Endothelial cells Rat skeletal muscle

1.

Introduction

Urotensin-II (U-II) is a 12 amino acid peptide that was originally discovered in the urophysis of the teleost fish, Gillichthys mirabilis [35]. A gene encoding a human form of U-II was subsequently cloned [9] and the G protein-coupled receptor UT, a human homologue of the rat orphan receptor GPR14/SENR [31,41], identified as the endogenous receptor for the human undecapeptide U-II (hU-II) [2]. In fish, U-II has a variety of endocrine functions and has been demonstrated to constrict fish smooth muscle [4,35]. The fish peptide, containing the characteristic cyclic hexapeptide that is essential for the biological activity of U-II from all species [28], also shows vasoconstrictor properties in the blood vessels from mammals [16,20], suggesting an evolutionary conserved role for this

receptor system in the regulation of cardiovascular function. This is supported by the discovery of genes for UT and U-II in a number of mammalian and non-mammalian species including the flounder [26], frog [9], mouse [8], pig [33] and cat [3]. Messenger RNA (mRNA) encoding both UT [2,11,25] and U-II [2,8,12] is abundantly expressed in the cardiovascular tissue of rats and humans, with the highest levels of UT mRNA expression observed, unexpectedly, in skeletal muscle [11]. Accordingly, UT receptors have been identified, using radioligand binding, in human myocardium, blood vessels and skeletal muscle [29] as well as in rat blood vessels [20,29]. U-II has been reported to be the most potent vasoconstrictor of mammalian blood vessels discovered [2] and also elicits endothelium-dependent vasodilatation. Localization of the peptide to vascular endothelial cells of healthy [2,30] and

* Corresponding author. Tel.: +44 1223 762579; fax: +44 1223 762576. E-mail address: [email protected] (J.J. Maguire). 0196-9781/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2007.08.021

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diseased [12,30] human blood vessels using immunocytochemistry suggest that U-II acts as an autocrine/paracrine mediator at least in the vasculature. Whether U-II induces vasoconstriction or vasodilatation appears to depend on the species (for example, U-II contracts human arteries and veins in vitro [29] but is arterioselective in monkey [2]) and the vascular bed investigated and on the presence or absence of a functional endothelium, for review see refs. [10,28]. In the conscious rat, in vivo, the effect of infused U-II is an increase in heart rate, blood pressure and vascular conductance in the hindquarters, effects attenuated by the nitric oxide synthase (NOS) inhibitor L-NAME and the non-selective cyclo-oxygenase inhibitor indomethacin [15]. To examine whether differential expression of UT receptors in cardiovascular tissues from rats and humans may account for the diversity of vascular actions observed for U-II we have investigated the distribution of UT in a representative panel of rat tissues using standard immunocytochemistry. In skeletal muscle, additional experiments were performed to localize NOS isoforms and the endothelial cell marker von Willebrand factor for comparison. We employed fluorescent double labeling confocal microscopy to identify the specific cell types expressing UT in rat compared to human tissue and in human umbilical vein endothelial cells (HUVECs) to establish morphological evidence for the involvement of either the synthetic or secretory pathways in the production and release of endothelial U-II.

2.

Materials and methods

2.1.

Materials

Rabbit anti-UT receptor (human) antibody was purchased from Lifespan Biosciences (Seattle, WA, USA). UT receptor blocking peptide was from MBL International Corporation (Woburn, MA, USA). Human urotensin-II and rabbit anti-human urotensin-II antibody were from Peptide Institute Inc. (Osaka, Japan). Mouse anti-NOS I (brain/neuronal NOS), anti-NOS III (endothelial NOS) and anti-NOS II (inducible NOS) monoclonal antibodies were from Affiniti Research Products Ltd. (Exeter, UK). Mouse antihuman von Willebrand factor (vWF) and mouse anti-human smooth muscle a-actin monoclonal antibodies, secondary antibodies, rabbit-PAP complex and horseradish-peroxidaseconjugated swine anti-rabbit antibody were from DAKO (Glostrup, Denmark). AlexaFluor 488 conjugated goat antirabbit serum and AlexaFluor 568 conjugated goat anti-mouse serum were obtained from Molecular Probes (Leiden, The Netherlands). Vectashield mounting medium containing 40 ,6diamio-2-phenylindole hydrochloride (DAPI) was from Vector Laboratories (Burlingame, CA, USA) and DePeX-Gurr mounting medium from BDH Laboratory Supplies (Poole, UK). Iodinated human urotensin-II (2000 Ci mmol1) was from Amersham Pharmacia Biotech (Amersham, UK). All other reagents were from Sigma–Aldrich Company Ltd. (Poole, UK).

2.2.

Tissue collection

Human tissues were obtained with local ethical approval. Left ventricular (n = 3) and atrial (n = 3) myocardium were

from donor hearts for which there was no suitable recipient. Coronary artery was from patients transplanted for ischemic heart disease (n = 1) or donor hearts not required for further transplantation (n = 2). Saphenous vein (n = 3) and mammary artery (n = 3) were from patients receiving coronary artery bypass grafts. Histologically normal pieces of kidney (n = 4) and lung (n = 4) were from patients undergoing nephrectomy and lobectomy, respectively, for non-obstructive carcinoma. Histologically normal adrenal (n = 3) was from patients undergoing adrenalectomy for phaeochromocytoma. Human umbilical vein endothelial cells (HUVECs) were a gift from Dr. Jun Wang (Department of Medicine, University of Cambridge) and were cultured and grown to sub-confluency as previously described [37]. Rat brain, spinal cord, heart, kidney, mesentery, lung and hindquarter (n = 3–8) were from male Sprague–Dawley rats (300–350 g, Charles River, Wilmington, MA, USA) euthanized using CO2 inhalation. All tissues were snap frozen in liquid nitrogen; tissues and cells were stored at 70 8C until required.

2.3.

Immunocytochemistry

The site-directed rabbit anti-human UT antibody was raised against amino acids 233–258 (rlarayrr sqrasfkrar rpgaralr) in the third cytoplasmic domain of the human UT receptor sequence and exhibits cross reactivity with rat UT (rat 233–258 rlaraywl sqqasfkqtr rlpnprvl). This was confirmed in pilot experiments, where the staining pattern obtained in rat tissues was consistent with reported mRNA distribution [31,41]. A BLAST-p search [1] of publicly available human and rat peptide libraries retrieved no other human or rat peptide with significant sequence similarity. By Western blotting, this antibody (1:300 dilution) detected proteins of the expected size (43 kDa) for the UT receptor in homogenates of rat heart and skeletal muscle that was abolished by pre-absorption with the blocking peptide (Fig. 1). We have previously characterized the rabbit anti-human U-II antibody in human tissues [30]. Cryostat-cut sections of rat tissues (10 mm) were dried overnight at room temperature and fixed in ice-cold acetone for 10 min. To visualize UT-like immunoreactivity (UT-LI), sections were first incubated with 5% non-immunized swine serum (SS) in phosphate-buffered saline (PBS), for 1 h at 22 8C, to block non-specific protein interactions then with the rabbit anti-UT antibody (1:200 dilution in PBS containing

Fig. 1 – Western blot showing bands of the expected size (43 kDa) for the UT receptor in homogenates of rat skeletal muscle (lane 2) and heart (lane 3) that were abolished by pre-absorption with the blocking peptide (lanes 4 and 5, respectively). Lanes (1 and 6) are molecular weight markers (kDa).

peptides 29 (2008) 735–742

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Fig. 2 – Photomicrographs showing UT-LI in rat tissues. UT-LI (solid arrowheads) was present in cardiomyocytes from rat left ventricle (A) and skeletal muscle (skm) cells (B). Vascular smooth muscle cells in arteries (a) and veins (v) of rat hindquarter (B), kidney (C) and mesentery (D and H) stained positively for UT-LI. UT-LI was absent or below the level of detection in vascular endothelial cells (H) (open arrow head). As a negative control, the primary antiserum was omitted in adjacent tissue sections (E–G) (scale bar A–H = 50 mm). UT-LI (solid arrowheads) localised to regions of the sarcolemma in rat hindquarter skeletal muscle (I) with similar expression pattern for NOS I (J). These differ from the distribution of the endothelial cell marker vWF (K). Primary antiserum for UT was pre-absorbed for 18 h with the blocking peptide (L) and was omitted in an adjacent tissue section as a negative control (M) (scale bar M = 200 mm).

Fig. 3 – Photomicrographs showing UT-LI in rat tissues. UT-LI is shown as green fluorescence (A and D), the vascular smooth muscle marker a-actin as red fluorescence (B and E), with overlays of the two color channels and the nuclear marker DAPI (blue fluorescence) (C and F). UT-LI (solid arrowheads) was present in rat cardiac myocytes and co-localized with a-actin in vascular smooth muscle cells of intramyocardial vessels (A and C). Little or no UT-LI was present in vascular endothelial cells (open arrowhead) which can be identified by their cell nuclei, in blue (C). UT-LI was also present in rat skeletal muscle, where it exhibited a striated pattern on the cell surface (D and F) (scale bar = 50 mm). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Fig. 4 – Photomicrographs showing UT-LI (A–L) in human vascular tissues and UT-LI (M–O) and U-II-LI (P–R) in HUVECs. In human tissues (A–L), UT-LI is shown as green fluorescence, a-actin or vWF as red fluorescence and the right hand columns show overlays of the two color channels including the nuclear marker DAPI (blue fluorescence). UT-LI (white arrowheads) was present in vascular smooth muscle cells of epicardial coronary (A–C, J–L) and mammary (G–I) artery and co-localized with a-actin (C). In contrast to marked staining in surrounding myocytes, a low level of UT-LI was identified in vascular smooth muscle cells of small intramyocardial coronary artery (D and F). UT-LI was also observed in vWF-positive vascular endothelial cells of mammary (G–I) and coronary (J–L) artery. In HUVECs (M–R) UT/U-II-LI is shown as green fluorescence, vWF as red fluorescence and the right column shows overlays of the two color channels including the nuclear marker DAPI (blue fluorescence). UT-LI (M–O) and U-II-LI (P–R) (white arrowheads) is present in small secretory vesicles of the endothelial

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0.1% Tween-20 (PBS/T) and 1% SS) for 72 h at 4 8C. Adjacent sections were incubated without the primary antibody as a negative control or additional sections were incubated with primary antibody that had been pre-absorbed for 18 h with the blocking peptide. Tissue sections were washed (3  5 min, 4 8C) in PBS/Tween-20 and incubated with swine anti-rabbit antibody (1:200 dilution in 1% SS PBS/T), for 1 h at room temperature. After repeated washing, sections were incubated with rabbit peroxidase/anti-peroxidase (PAP) complex (1:400 dilution in 1% SS PBS/T) for 1 h at room temperature, washed again and finally incubated for 3 min with 3,30 -diaminobenzidine (2.5%) in 0.05 M Tris– HCl buffer containing 0.3% hydrogen peroxide. The chromogenic reaction was stopped by immersion of sections in distilled water. Sections were dehydrated using a graded alcohol series, submerged in xylene for 1 h to clear, mounted using DePeX-Gurr mounting medium and examined using a standard bright field microscope. Images were captured using a U-TV1-X digital camera (Olympus UK Ltd., London, UK) and AnalySis software (Soft Imaging System GmbH, Mu¨nster, Germany). In sections of hindquarter, experiments were repeated using mouse antiNOS I, anti-NOS III, anti-NOS II or anti-vWF monoclonal primary antibodies, rabbit anti-mouse secondary antibody, mouse anti-PAP and non-immunized rabbit serum, as appropriate.

2.4.

Fluorescent double staining/confocal microscopy

To investigate the precise cellular distribution, fluorescent double staining for UT-LI and the vascular smooth muscle marker a-actin or the endothelial cell marker vWF was carried out in those rat tissues and human tissues (previously reported [30]) showing expression of UT-LI in the standard immunocytochemistry experiments. Cryostat cut sections of rat and human tissues (30 mm) were dried and fixed in ice-cold acetone. Sections were blocked with 5% non-immunized goat serum (GS) in PBS (1 h, room temperature) then incubated in 1% GS PBS/T containing rabbit anti-UT antiserum (1:100 dilution), mouse anti-vWF monoclonal antibody (1:50 dilution) or mouse anti-smooth muscle a-actin (1:100 dilution), for 48 h at 4 8C. Sections were washed in cold PBS/T then incubated with the secondary antibody solution containing both AlexaFluor 488 conjugated goat anti-rabbit serum and AlexaFluor 568 conjugated goat anti-mouse serum, each at 1:100 dilution, in 1% GS PBS/T. To establish the intracellular localization of receptor and ligand in endothelial cells, experiments were repeated using HUVECs with rabbit antiUT and rabbit anti-U-II (1:100 in 1% GS PBS/T) antibodies in combination with the mouse anti-vWF antibody. After repeated washing, tissue sections or cells were mounted using Vectashield mounting medium containing DAPI. Confocal imaging was performed using a Leica TCS-NT-UV confocal laser-scanning microscope (Leica Microsystems, Heidelberg, Germany).

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2.5. Autoradiographical visualisation of [125I]-urotensin-II binding To additionally verify the UT distribution obtained using the receptor antibody described above we carried out autoradiographical localization of [125I]-U-II binding using cryostat-cut sections (10 mm) of rat thoracic aorta, spinal cord, brain, heart, kidney, lung and skeletal muscle. Sections were pre-incubated for 1 h in 20 mM Tris–HCl buffer, pH 7.4, containing 5 mM MgCl2 and 0.2% BSA and then incubated with 0.25 nM [125I]-U-II for 1 h. Non-specific binding was determined using human UII (1 mM). Sections were washed for 10 min in 50 mM Tris–HCl, pH 7.4, at 4 8C, dried and apposed, with standards, to radiation sensitive film for 3 days. The resulting autoradiograms were analyzed using computer-assisted densitometry [29].

3.

Results

3.1.

Immunocytochemistry

In rat heart, UT-LI was localized to cardiomyocytes of the left and right atrium and ventricle (Fig. 2A). UT-LI was also detected in hindquarter skeletal muscle and in smooth muscle cells forming the medial layer of blood vessels from the rat heart, hindquarters (Fig. 2B), kidney (Fig. 2C) and mesentery (Fig. 2D and H). UT-LI was absent or below the level of detection in endothelial cells lining these blood vessels (Fig. 2H). In rat skeletal muscle UT-LI appeared to localize to regions of the sarcolemma (Fig. 2I) a distribution similar to that obtained with the NOS isoforms, particularly NOS I (Fig. 2J), but distinct from the endothelial cell marker vWF (Fig. 2K). UT-LI in rat tissues was abolished by pre-absorption of the UT antibody with the blocking peptide (Fig. 2L) and staining was absent when the antibody was omitted (Fig. 2E–G, M).

3.2.

Fluorescent double staining/confocal microscopy

Confocal laser scanning microscopy confirmed the presence of UT-LI in rat cardiomyocytes (Fig. 3A–C) and skeletal muscle (Fig. 3D–F). UT-LI also co-localized with a-actin in vascular smooth muscle cells of blood vessels in rat heart and skeletal muscle (Fig. 3). UT-LI in endothelial cells lining rat vessels was absent or below the level for detection (Fig. 3C). In human tissues UT-LI was identified in vascular smooth muscle cells of epicardial coronary artery (Fig. 4A–C, J–L), LIMA (Fig. 4G–I) and saphenous vein (not shown) and also in cardiac myocytes (Fig. 4D and F). Lower levels of UT-LI were observed in small intramyocardial coronary arteries (Fig. 4D and F) and in renal, adrenal and pulmonary vessels (not shown). UT-LI was also present in vascular endothelial cells lining human LIMA (Fig. 4G–I), saphenous vein (not shown) and coronary artery (Fig. 4J–L). In HUVECs, confocal microscopy revealed both UT-LI (Fig. 4M–O) and U-II-LI (Fig. 4P–R) abundantly expressed in

cell cytoplasm. Neither UT-LI (O) nor U-II-LI (R) shows spatial co-localization with vWF in rod like structures (scale bar A– L = 25 mm; scale bar M–R = 20 mm). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Fig. 5 – Autoradiographical analysis of [125I]-U-II binding in rat tissues. Specific density of binding was expressed as amol mmS2 and values are mean W S.E.M. from 3 to 5 animals. [125I]-U-II binding was identified in rat tissues including heart, kidney, skeletal muscle, brain and spinal cord. Highest density of receptors was in the abducens and lateral septal nuclei of the brain.

small secretory vesicles of the cytoplasm and accumulated in structures closely associated with the nuclear surface. Colocalization with vWF to Weibel-Palade bodies was not observed for either UT-LI or U-II-LI.

3.3.

Receptor autoradiography

In peripheral and central rat tissues quantitative autoradiography indicated that highest binding density of [125I]-U-II binding was observed in the abducens (215.4  61.7 amol mm2, n = 6) and lateral septal (168.1  2.7 amol mm2, n = 3) nuclei of the brain. Moderate levels were expressed, for example, in the dorsal root ganglion of spinal cord (85.6  9.1 amol mm2, n = 3) and kidney medulla (72.1  24.4 amol mm2, n = 3) with lower levels in heart, vasculature such as the aorta (25.0  4.5 amol mm2, n = 6) and skeletal muscle (Fig. 5).

4.

Discussion

U-II has been shown, in vitro and in vivo, to contract [5,23,27– 30,34] and relax [39] human arteries and veins and to elicit effects on human heart [36] actions presumed to be due to stimulation of UT receptors. We have now substantiated this hypothesis by demonstrating the presence of UT-LI in human cardiovascular tissues, confirming the ubiquitous localization of UT receptor protein to vascular smooth muscle and endothelial cells of human large and small diameter arteries, veins and cultured HUVECs and to human cardiomyocytes. Using immunocytochemistry and receptor autoradiography we have also demonstrated a similar pattern of UT receptor expression in rat cardiovascular tissues and skeletal muscle, with the exception that we did not observe endothelial cell localization of UT-LI in rat blood vessels. Our observations are

in agreement with the widespread expression of UT mRNA in the cardiovascular system and skeletal muscle of rats and humans [2,11,25] and the presence of UT-LI and binding of [125I]-U-II in rat skeletal muscle [15,28] and rat heart [17]. For our UT receptor autoradiography we used rat brain as a control tissue as we have previously demonstrated that radiolabeled urotensin-II detects highest levels of receptor density in the abducens nucleus and spinal cord [29]. In this study we also detected [125I]-U-II binding in lateral septal nucleus, cortex, cerebellum and thalamic nuclei consistent with the recent detailed report of UT receptor expression in rat brain by Je´gou et al. [21]. The expression pattern for UT-LI that we find may explain the variability of vasoactive actions observed for U-II in human isolated blood vessel preparations. The localization of receptor protein to endothelial cells and smooth muscle cells is consistent with reports of both vasoconstriction [27,29,30,34] and vasodilatation [39] to U-II. The observed response is presumably dependent on the balance of these actions, with the presence of a functional endothelium being critical. In healthy blood vessels activation of endothelial UT receptors may result in release of dilators such as nitric oxide and prostacyclin, whereas in endothelium denuded blood vessels U-II is a potent vasoconstrictor by direct activation of UT receptors on vascular smooth muscle. Vasoconstriction is likely to be the predominant action of the peptide in conditions of endothelium dysfunction as suggested by the observation that in normal subjects iontophoresed U-II elicits a vasodilator response in the skin microcirculation; in contrast in patients with chronic heart failure a dose-dependent vasoconstriction was obtained [24]. These opposing actions to U-II are comparable to those reported for ET-1, although it is not yet clear whether U-II plays a role in the maintenance of normal vascular tone. Our data in coronary artery showing expression of receptor protein in both epicardial coronary arteries and small intramyocardial vessels suggests a role in the regulation of blood flow to the heart and is consistent with the observed vasoconstrictor response in vitro of U-II in large and small diameter human endothelium-denuded coronary arteries [29,30]. Surprisingly, despite reports of endothelium-dependent vasodilator actions in rat blood vessels in vitro [6] levels of UTLI observed in rat vascular endothelial cells appeared to be less than in human vascular tissues, or were below the level for detection. This was unexpected as the predominant response to infusion of U-II in rat in vivo was hypotension [13,14,18] that was sensitive to inhibitors of nitric oxide and assumed to be due to endothelial release of endogenous dilators. However, the response to U-II is critically dependent on the mode of administration, as bolus infusion of U-II in non-anaesthetized, freely moving rats has been shown to evoke tachycardia, increased vascular conductance in the mesenteric and hind quarter vasculature and a drop in mean arterial blood pressure (MABP) [13,14] while continuous administration of U-II, over several hours, resulted in tachycardia and increased MABP [15], effects most likely to be mediated by central activation of the sympathetic nervous system by U-II [15,19]. Interestingly, these actions were accompanied by increased vascular conductance only in the rat hindquarters that was partially attenuated by co-administration of L-NAME, indomethacin

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and propranolol. In these animals U-II infusion was without effect on blood flow in the renal and mesenteric beds. We were unable to detect UT-LI to endothelial cells from the rat blood vessels that we examined suggesting that if present they are expressed at relatively low levels compared to receptor expression levels in adjacent smooth muscle cells or cardiomyocytes. Endothelium-dependent vasodilator responses to UII have been reported in rat arteries in vitro and to rat cultured neurovascular endothelial cells [38] suggesting a greater degree of heterogeneity of UT receptor expression in rat peripheral and central vasculature compared to human vasculature. One other report has demonstrated using an antibody raised against rat GPR14 that immunoreactivity to the protein, while present in cardiomyocytes, does not co-localize to coronary endothelial cells in rat heart [17]. In contrast in our study UT-LI was clearly identified on the sarcolemmal surface of rat skeletal muscle fibers, a pattern reminiscent of that expressed by NOS isoforms, particularly the constitutive enzyme, NOS I. We hypothesize, therefore, that stimulation of UT on skeletal muscle by infused U-II, resulting in a rise in intracellular Ca2+ [42] within the muscle cells, may lead to calcium-dependent activation of NOS I and cyclo-oxygenase enzymes [7]. These are abundantly expressed in rat skeletal muscle [22,40] and activation leads to the formation and diffusion of NO and vasodilator prostanoids that contribute to autoregulation of skeletal muscle blood flow and hence L-NAME and indomethacin sensitive increases in vascular conductance. Finally, we have obtained evidence that U-II-LI was present in secretory vesicles in the cytoplasm of human endothelial cells in situ and cultured HUVECs. U-II-LI in vascular endothelial cells did not spatially co-localize with von Willebrand factor, suggesting that U-II is produced and secreted via a constitutive pathway [32] and that it is not released from the Weibel-Palade bodies of the regulated pathway. The detection of U-II-LI in HUVEC further supports previous reports showing the presence of U-II-LI in human vascular endothelial cells in situ [12,30]. Whether, physiologically, U-II is released abluminally onto the underlying smooth muscle to contribute to vascular tone remains to be determined. In summary, we have demonstrated UT-LI in vascular smooth muscle cells and endothelial cells from human large conduit arteries and veins as well as in human cardiac myocytes. Compared to human, levels of UT-LI present in rat vascular endothelial cells were low. We confirmed that in rats, UT-LI is widely expressed in vascular smooth muscle cells from different vascular beds, in skeletal muscle and in cardiac myocytes. In human endothelial cells U-II-LI localized small cytoplasmic vesicles suggesting that there may be continuous synthesis and secretion of U-II in healthy arteries and veins. Taken together, these findings highlight some species differences between localization of endothelial UT receptor in rat and human vasculature and complement the hypothesis that the U-II system may be important in the regulation of cardiovascular function in health and disease.

Acknowledgments Supported by grants from the British Heart Foundation, Cambridge European and Isaac Newton Trusts. We thank

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the Consultant and theatre staff of Papworth Hospital for collection of human tissues. HUVECs were a kind gift from Dr. Jun Wang (Department of Medicine, University of Cambridge).

references

[1] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215:403–10. [2] Ames RS, Sarau HM, Chambers JK, Willette RN, Aiyar NV, Romanic AM, et al. Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14. Nature 1999;401:282–6. [3] Behm DJ, Doe CP, Johns DG, Maniscalco K, Stankus GP, Wibberley A, et al. Urotensin-II: a novel systemic hypertensive factor in the cat. Naunyn Schmiedebergs Arch Pharmacol 2004;369:274–80. [4] Bern HA, Pearson D, Larson BA, Nishioka RS. Neurohormones from fish tails: the caudal neurosecretory system. I. ‘‘Urophysiology’’ and the caudal neurosecretory system of fishes. Recent Prog Horm Res 1985;41:533–52. [5] Bo¨hm F, Pernow J. Urotensin II evokes potent vasoconstriction in human in vivo. Br J Pharmacol 2002;135:25–7. [6] Bottrill FE, Douglas SA, Hiley R, White R. Human urotensinII is an endothelium-dependent vasodilator in rat small arteries. Br J Pharmaol 2000;130:1865–70. [7] Clifford PS, Hellsten Y. Vasodilatory mechanisms in contracting skeletal muscle. J Appl Physiol 2004;97:393–403. [8] Coulouarn Y, Jegou S, Tostivint H, Vaudry H, Lihrmann I. Cloning, sequence analysis and tissue distribution of the mouse and rat urotensin II precursors. FEBS Lett 1999;457:28–32. [9] Coulouarn Y, Lihrmann I, Jegou S, Anouar Y, Tostivint H, Beauvillain JC, et al. Cloning of the cDNA encoding the urotensin II precursor in frog and human reveals intense expression of the urotensin II gene in motoneurons of the spinal cord. Proc Natl Acad Sci USA 1998;95:15803–8. [10] Douglas SA, Dhanak D, Johns DG. From ‘gills to pills’: urotensin-II as a regulator of mammalian cardiorenal function. Trends Pharmacol Sci 2004;25:76–85. [11] Douglas SA, Naselsky D, Ao Z, Disa J, Herold CL, Lynch F, et al. Identification and pharmacological characterization of native, functional human urotensin-II receptors in rhabdomyosarcoma cell lines. Br J Pharmacol 2004;142:921– 32. [12] Douglas SA, Tayara L, Ohlstein EH, Halawa N, Giaid A. Congestive heart failure and expression of myocardial urotensin II. Lancet 2002;359:1990–7. [13] Gardiner SM, March JE, Kemp PA, Bennett T. Bolus injection of human UII in conscious rats evokes a biphasic haemodynamic response. Br J Pharmacol 2004;143:422–30. [14] Gardiner SM, March JE, Kemp PA, Davenport AP, Bennett T. Depressor and regionally selective vasodilator effects of human and rat urotensin II in conscious rats. Br J Pharmacol 2001;132:1625–9. [15] Gardiner SM, March JE, Kemp PA, Maguire JJ, Kuc RE, Davenport AP, et al. Regional heterogeneity in the haemodynamic responses to urotensin II infusion in relation to UT receptor localization. Br J Pharmacol 2006;147:612–21. [16] Gibson A. Complex effects of Gillichthys urotensin II on rat aortic strips. Br J Pharmacol 1987;91:205–12. [17] Gong H, Wang YX, Zhu YZ, Wang WW, Wang MJ, Yao T, et al. Cellular distribution of GPR14 and the positive inotropic role of urotensin II in the myocardium in adult rat. J Appl Physiol 2004;97:2228–35.

742

peptides 29 (2008) 735–742

[18] Hagesawa K, Kobayashi Y, Kobayashi H. Vasodepressor effect of urotensin-II in rats. Neuroendocrinol Lett 1992;14:357–63. [19] Hood SG, Watson AM, May CN. Cardiac actions of central but not peripheral urotensin II are prevented by betaadrenoceptor blockade. Peptides 2005;26:1248–56. [20] Itoh H, McMaster D, Lederis K. Functional receptors for fish neuropeptide urotensin II in major rat arteries. Eur J Pharmacol 1988;149:61–6. [21] Je´gou S, Cartier D, Dubessy C, Gonzalez BJ, Chatenet D, Tostivint H, et al. Localization of the urotensin II receptor in the rat central nervous system. J Comp Neurol 2006;495:21–36. [22] Kobzik L, Reid MB, Bredt DS, Stamler JS. Nitric oxide in skeletal muscle. Nature 1994;372:546–8. [23] Leslie SJ, Denvire M, Webb DJ. Human urotensin II causes vasoconstriction in the human skin microcirculation. Circulation 2000;102(Suppl. 11):542. [24] Lim M, Honisett S, Sparkes CD, Komesaroff P, Kompa A, Krum H. Differential effects of urotensin II on vascular tone in normal subjects and patients with chronic heart failure. Circulation 2004;109:1212–4. [25] Liu Q, Pong SS, Zeng Z, Zhang Q, Howard AD, Williams Jr DL, et al. Identification of urotensin II as the endogenous ligand for the orphan G-protein-coupled receptor GPR14. Biochem Biophys Res Commun 1999;266:174–8. [26] Lu W, Dow L, Gumusgoz S, Brierley MJ, Warne JM, McCrohan CR, et al. Coexpression of corticotropinreleasing hormone and urotensin I precursor genes in the caudal neurosecretory system of the euryhaline flounder (Platichthys flesus): a possible shared role in peripheral regulation. Endocrinology 2004;145:5786–97. [27] MacLean MR, Alexander D, Stirrat A, Gallagher M, Douglas SA, Ohlstein EH, et al. Contractile responses to human urotensin-II in rat and human pulmonary arteries: effect of endothelial factors and chronic hypoxia in the rat. Br J Pharmacol 2000;130:201–4. [28] Maguire JJ, Davenport AP. Is urotensin-II the new endothelin? Br J Pharmacol 2002;137:579–88. [29] Maguire JJ, Kuc RE, Davenport AP. Orphan-receptor ligand human urotensin II: receptor localization in human tissues and comparison of vasoconstrictor responses with endothelin-1. Br J Pharmacol 2000;131:441–6. [30] Maguire JJ, Kuc RE, Wiley KE, Kleinz MJ, Davenport AP. Cellular distribution of immunoreactive urotensin-II in human tissues with evidence of increased expression in atherosclerosis and a greater constrictor response of small compared to large coronary arteries. Peptides 2004;25:1767–74.

[31] Marchese A, Heiber M, Nguyen T, Heng HH, Saldivia VR, Cheng R, et al. Cloning and chromosomal mapping of three novel genes, GPR9, GPR10, and GPR14, encoding receptors related to interleukin 8, neuropeptide Y, and somatostatin receptors. Genomics 1995;29:335–44. [32] Mayadas T, Wagner DD, Simpson PJ. von Willebrand factor biosynthesis and partitioning between constitutive and regulated pathways of secretion after thrombin stimulation. Blood 1989;73:706–11. [33] Mori M, Sugo T, Abe M, Shimomura Y, Kurihara M, Kitada C, et al. Urotensin II is the endogenous ligand of a Gprotein-coupled orphan receptor, SENR (GPR14). Biochem Biophys Res Commun 1999;265:123–9. [34] Paysant J, Rupin A, Simonet S, Fabiani JN, Verbeuren TJ. Comparison of the contractile responses of human coronary bypass grafts and monkey arteries to human urotensin-II. Fundam Clin Pharmacol 2001;15: 227–31. [35] Pearson D, Shively JE, Clark BR, Geschwind II, Barkley M, Nishioka RS, et al. Urotensin II: a somatostatin-like peptide in the caudal neurosecretory system of fishes. Proc Natl Acad Sci USA 1980;77:5021–4. [36] Russell FD, Molenaar P, O’Brien DM. Cardiostimulant effects of urotensin-II in human heart in vitro. Br J Pharmacol 2001;132:5–9. [37] Russell FD, Skepper JN, Davenport AP. Endothelin peptide and converting enzymes in human endothelium. J Cardiovasc Pharmacol 1998;31(Suppl. 1):S19–21. [38] Spinazzi R, Albertin G, Nico B, Guidolin D, Di Liddo R, Rossi GP, et al. Urotensin-II and its receptor (UT-R) are expressed in rat brain endothelial cells, and urotensin-II via UT-R stimulates angiogenesis in vivo and in vitro. Int J Mol Med 2006;18:1107–12. [39] Stirrat A, Gallagher M, Douglas SA, Ohlstein EH, Berry C, Kirk A, et al. Potent vasodilator responses to human urotensin-II in human pulmonary and abdominal resistance arteries. Am J Physiol Heart Circ Physiol 2001;280:H925–8. [40] Sudbo J, Reith A, Florenes VA, Nesland JM, Ristimaki A, Bryne M. COX-2 expression in striated muscle under physiological conditions. Oral Dis 2003;9:313–6. [41] Tal M, Ammar DA, Karpuj M, Krizhanovsky V, Naim M, Thompson DA. A novel putative neuropeptide receptor expressed in neural tissue, including sensory epithelia. Biochem Biophys Res Commun 1995;209:752–9. [42] Tzanidis A, Hannan RD, Thomas WG, Onan D, Autelitano DJ, See F, et al. Direct actions of urotensin II on the heart: implications for cardiac fibrosis and hypertrophy. Circ Res 2003;93:246–53.