UT Receptors Research Articles

Presence of urotensin-II receptors at the cell nucleus: Specific tissue distribution and hypoxia-induced modulation

Urotensin II (UII) and its receptor UT, are widely expressed in the cardiovascular and central nervous system, where they exert regulatory actions under both physiological and pathological conditions. Our study, aimed at investigating the presence of functional nuclear UT in various rat and monkey tissues as well as in human cell lines, demonstrated for the first time by Western blot analysis and confocal immunofluorescence a tissue-specific nuclear expression of this receptor (heart and central nervous system). This nuclear UT was further characterized pharmacologically through radioligand binding studies using specific ligands of the urotensinergic system, as well as somatostatin. In 2D-gel experiments, we observed the presence of different post-translational modifications between membrane and nuclear UT receptors in brain extracts. Transcription initiation assays showed de novo RNA synthesis caused by UII and Urotensin-related peptide (URP) which were inhibited by an UT antagonist urantide. In hypoxic/ischemic conditions, UT receptors were differentially modulated in regard to subcellular localization. Thus, the unique regiospecificity of the nuclear UT receptor along with its particular modulation under hypoxic conditions could indicate a specific and complementary physiological role that could be correlated with pro-angiogenic and/or neuromodulatory actions of UII, both in the cardiovascular and central nervous system.

Palosuran inhibits binding to primate UT receptors in cell membranes but demonstrates differential activity in intact cells and vascular tissues

The recent development of the UT ligand palosuran (1-[2-(4-benzyl-4-hydroxy-piperidin-1-yl)-ethyl]-3-(2-methyl-quinolin-4-yl)-urea sulphate salt) has led to the proposition that urotensin-II (U-II) plays a significant pathological role in acute and chronic renal injury in the rat. In the present study, the pharmacological properties of palosuran were investigated further using a series of radioligand binding and functional bioassays. Palosuran functioned as a 'primate-selective' UT ligand in recombinant cell membranes (monkey and human UT K(i) values of 4 +/- 1 and 5 +/- 1 nM), lacking appreciable affinity at other mammalian UT isoforms (rodent and feline K(i) values >1 microM). Paradoxically, however, palosuran lost significant (10- to 54-fold) affinity for native and recombinant human UT when radioligand binding was performed in intact cells (K(i) values of 50 +/- 3 and 276 +/- 67 nM). In accordance, palosuran also exhibited diminished activity in hUT (human urotensin-II receptor)-CHO (Chinese hamster ovary) cells (IC50 323 +/- 67 nM) and isolated arteries (K(b)>10 microM in rat aorta; K(b)>8.5 microM in cat arteries; K(b)>1.6 microM in monkey arteries; K(b) 2.2 +/- 0.6 microM in hUT transgenic mouse aorta). Relative to recombinant binding K(i) values, palosuran was subjected to a 392- to 690-fold reduction in functional activity in monkey isolated arteries. Such phenomena were peculiar to palosuran and were not apparent with an alternative chemotype, SB-657510 (2-bromo-N-[4-chloro-3-((R)-1-methyl-pyrrolidin-3-yloxy)-phenyl]-4,5-dimethoxybenzenesulphonamide HCl). Collectively, such findings suggest that caution should be taken when interpreting data generated using palosuran. The loss of UT affinity/activity observed in intact cells and tissues cf. membranes offers a potential explanation for the disappointing clinical efficacy reported with palosuran in diabetic nephropathy patients. As such, the (patho)physiological significance of U-II in diabetic renal dysfunction remains uncertain.

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

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.

New insights into urotensin endocrinology: From fish to man

Theurotensins(UIandUII)werefirstidentifiedinthecau-dal neurosecretory system (CNSS) of fish, a unique neuroen-docrine structure located in the terminal segments of thespinalcord.Morerecently,homologuestobothpeptideshavebeenidentifiedinmammalsand,whileresearchcontinuesintotheroleoftheCNSSinfish,interestintheactionsoftheuro-tensins in other vertebrates, including man, has grown.Steve Douglas reviewed the history of UII research inmammals, following the identification of its ‘‘orphan’’ G-protein coupled UT receptor, GPR14. Both UII and UTreceptor are localised in regions of the mammalian brain,as well as in non-neural tissues such as cardiovascular andrenal tissues. The potent vasoconstrictor actions of UII ledto the search for antagonists that might be effective in dis-ease states such as hypertension, congestive heart failureand diabetes mellitus. Current animal studies give causefor optimism that this ‘‘from gills to pills’’ approach willlead to novel and improved clinical tools, such as in patientswhose hypertension cannot currently be controlled. Forexample, UT receptor antagonists have been shown to behighly protective against heart failure in animal models.Continuing on this theme, Nick Ashton and Alaa Abdel-Razikreported renal effects of UII in the rat. UT receptors arepresent in renal medulla and UII appears in relatively highconcentrations in urine, suggesting that it is activelysecreted. Both haemodynamic and tubular actions of UIIare indicated, leading to reduced urinary output, and theconverse effects were seen with a UT receptor antagonist,urantide.Isabelle Lihrmann gave a comparative overview of UII,UII-related peptide (URP) and UT receptor distributionthroughout the vertebrates. mRNA expression profilesfor UII appear to be well conserved throughout the verte-brate series from dipneusts to mammals, with the highestlevel of expression in spinal cord, localised to motoneuronsin the ventral horn. A similar distribution is seen for URPexpression, apart from some differential expression in cra-nial nuclei. The UT receptor is widely expressed in theCNS and peripheral tissues, including skeletal muscle. Inaddition to central actions of the peptides with respect tocardiovascular control, these findings perhaps support alocomotory role for UII. In addition a role for URP inarousal, via the reticular activating system, was proposed.Jean-Claude Le Mevel reported investigations into thecentral actions of UI and UII. The two peptides had differ-ential effects when injected intracerebroventricularly introut. UI, but not UII, caused increases in blood pressureand ventilatory output. Both peptides enhanced locomotoractivity (swimming), though effects of UII were more longlasting. These findings suggest that the urotensins haveimportant regulatory functions within the brain, whichmay also be of relevance to mammalian studies.A comparative genomics study by Herve Tostivint,examined the relationship between UII/URP and somato-statin (SS)-related peptides, previously considered to beunrelated gene products. These peptides display a similarcyclic structure and precursor organisation throughoutthe vertebrates. Analysis of predicted gene duplicationsand chromosomal locations has led to an evolutionarymodel supporting a single ancestral gene superfamily forUII- and SS-related genes, raising questions regarding theevolution of UII and its function in lower vertebrates.TheCNSSofteleost fish waspresented asa model systeminwhichtoexamineneurosecretorymechanismsand uroten-sin physiology. In zebrafish (Caroline Parmentier), neurose-cretory Dahlgren cells display immunoreactivity for both UIand UII. Most cells do not colocalise the two peptides.However, in some, UI and UII are colocalised even at thelevel of secretory terminals and vesicles. Detailed morpho-logical observations indicate further secretory roles for theCNSS that have yet to be defined. With its characterisedgenome, the zebrafish provides the genetic tools with whichto dissect CNSS function and other functional aspects ofurotensin-related physiology. Sarah Alderman described atissue mapping study of the expression patterns of UI, cor-ticotropin-releasing factor (CRF), and CRF-binding proteininadultanddevelopingzebrafish. All threeshowwidespreadbut highly localised distribution within the CNS, including

Seasonal changes in peptide, receptor and ion channel mRNA expression in the caudal neurosecretory system of the European flounder ( Platichthys flesus)

The caudal neurosecretory system (CNSS) of the euryhaline flounder Platichthys flesus has suggested roles in osmoregulatory, reproductive and nutritional adaptation, as fish migrate between seawater (winter) and brackish/freshwater (summer) environments. This study examined seasonal changes in mRNA expression profile of functionally important genes in the CNSS. cDNAs encoding neuropeptides, receptors and ion channels were cloned by reverse transcriptase polymerase chain reaction (RT-PCR) and screening of a flounder CNSS cDNA library. The expression profile of cloned genes was determined by real-time RT-PCR at 2-month intervals throughout the year in CNSS from seawater-adapted fish. Plasma cortisol (measured by radioimmunoassay) showed a peak in April, the time of spawning. Expression levels of mRNA for peptides urotensins I and II (UI, UII) and corticotropin releasing factor (CRF) all showed a seasonal cycle, with lowest expression in April and highest in August–October. The expression of CRF2(UI), UT(UII) and CRF1 receptors was not correlated with corresponding peptide expression. Receptors for potential neuromodulators of CNSS activity also displayed a seasonal mRNA expression profile. Glucocorticoid, 5-hydroxytryptamine, κ-opioid and glutamate receptor expression peaked around April, suggesting that modulation of electrical activity of the neurosecretory Dahlgren cells is of particular importance at this time. Expression of mRNA for L-type Ca 2+ and Ca-activated K + channels was lower during the summer months. These channels underlie electrical bursting activity in Dahlgren cells. Ion channel mRNA expression was also lower in CNSS from flounder fully adapted to freshwater as opposed to seawater, consistent with previously reported observations of reduced bursting activity in Dahlgren cells from freshwater-adapted CNSS. These findings support the hypothesis that the CNSS is functionally reprogrammed to cope with changes in physiological challenge as fish migrate between sea and estuaries in winter and spring.

Relationship Between Hypertension and Atherosclerosis: From a Viewpoint of the Most Potent Vasoconstrictor Human Urotensin II

Human urotensin II (U-II), the most potent vasoconstrictor peptide identified to date, and its receptor (UT) are involved in etiology of hypertension. In hypertensive patients, U-II induces vasoconstriction in forearm brachial artery infusion studies. Recent studies demonstrated elevated plasma U-II concentrations in patients with hypertension, diabetes mellitus, atherosclerosis, and coronary artery disease. U-II is expressed in endothelial cells, macrophages, macrophagederived foam cells, and myointimal and medial vascular smooth muscle cells (VSMCs) of atherosclerotic human coronary arteries. UT receptors are present in VSMCs of human coronary arteries, thoracic aorta and cardiac myocytes. Lymphocytes are the most active producers of U-II, whereas monocytes and macrophages are the major cell types expressing UT receptors, with relatively little receptor expression in foam cells, lymphocytes, and platelets. U-II accelerates foam cell formation by up-regulation of acyl-coenzyme A:cholesterol acyltransferase-1 in human monocyte-derived macrophages, and stimulates cell growth and up-regulates type 1 collagen expression in human endothelial cells. U-II also activates NADPH oxidase and plasminogen activator inhibitor-1 in human VSMCs, and stimulates VSMC proliferation with synergistic effects observed when combined with oxidized LDL, reactive oxygen species and serotonin. These findings suggest that U-II plays key roles in accelerating the development of atherosclerosis, and hence coronary artery disease.

Regional heterogeneity in the haemodynamic responses to urotensin II infusion in relation to UT receptor localisation

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.

Human Urotensin II as a Link between Hypertension and Coronary Artery Disease

Hypertension is a well-known risk factor for atherosclerosis, but the molecular mechanisms that link elevated blood pressure to the progression of atherosclerosis remain unclear. Human urotensin II (U-II), the most potent endogenous vasoconstrictor peptide identified to date, and its receptor (UT receptor) are involved in the etiology of essential hypertension. In patients with essential hypertension, U-II infused into the forearm brachial artery has been shown to induce vasoconstriction. Recent studies have demonstrated elevated plasma U-II concentrations in patients with essential hypertension, diabetes mellitus, atherosclerosis, and coronary artery disease. U-II is expressed in endothelial cells, macrophages, macrophage-derived foam cells, and myointimal and medial vascular smooth muscle cells (VSMCs) of atherosclerotic human coronary arteries. UT receptors are present in VSMCs of human coronary arteries, the thoracic aorta and cardiac myocytes. Lymphocytes are the most active producers of U-II, whereas monocytes and macrophages are the major cell types expressing UT receptors, with relatively little receptor expression in foam cells, lymphocytes, and platelets. U-II accelerates foam cell formation by up-regulation of acyl-coenzyme A:cholesterol acyltransferase-1 in human monocyte-derived macrophages. In human endothelial cells, U-II promotes cell proliferation and up-regulates type 1 collagen expression. U-II also activates nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and plasminogen activator inhibitor-1 in human VSMCs, and stimulates VSMC proliferation with synergistic effects observed when combined with oxidized low-density lipoprotein, lysophosphatidylcholine, reactive oxygen species or serotonin. These findings suggest that U-II plays key roles in accelerating the development of atherosclerosis, thereby leading to coronary artery disease.

The Urotensin-II Receptor Antagonist Palosuran Improves Pancreatic and Renal Function in Diabetic Rats

Urotensin-II (U-II) is a cyclic peptide that acts through a specific G-protein-coupled receptor, UT receptor. Urotensin-II and UT receptors have been described in pancreas and kidney, but their function is not well understood. We studied the effects of chronic treatment of diabetic rats with the orally active selective U-II receptor antagonist palosuran. Streptozotocin treatment causes pancreatic beta-cell destruction and leads to the development of hyperglycemia, dyslipidemia, and renal dysfunction. Long-term treatment of streptozotocin-induced diabetic rats with palosuran improved survival, increased insulin, and slowed the increase in glycemia, glycosylated hemoglobin, and serum lipids. Furthermore, palosuran increased renal blood flow and delayed the development of proteinuria and renal damage. The U-II system is unique in that it plays a role both in insulin secretion and in the renal complications of diabetes. Urotensin receptor antagonism might be a new therapeutic approach for the treatment of diabetes.

Nonpeptidic urotensin‐II receptor antagonists I: in vitro pharmacological characterization of SB‐706375

1. SB-706375 potently inhibited [(125)I]hU-II binding to both mammalian recombinant and 'native' UT receptors (K(i) 4.7+/-1.5 to 20.7+/-3.6 nM at rodent, feline and primate recombinant UT receptors and K(i) 5.4+/-0.4 nM at the endogenous UT receptor in SJRH30 cells). 2. Prior exposure to SB-706375 (1 microM, 30 min) did not alter [(125)I]hU-II binding affinity or density in recombinant cells (K(D) 3.1+/-0.4 vs 5.8+/-0.9 nM and B(max) 3.1+/-1.0 vs 2.8+/-0.8 pmol mg(-1)) consistent with a reversible mode of action. 3. The novel, nonpeptidic radioligand [(3)H]SB-657510, a close analogue of SB-706375, bound to the monkey UT receptor (K(D) 2.6+/-0.4 nM, B(max) 0.86+/-0.12 pmol mg(-1)) in a manner that was inhibited by both U-II isopeptides and SB-706375 (K(i) 4.6+/-1.4 to 17.6+/-5.4 nM) consistent with the sulphonamides and native U-II ligands sharing a common UT receptor binding domain. 4. SB-706375 was a potent, competitive hU-II antagonist across species with pK(b) 7.29-8.00 in HEK293-UT receptor cells (inhibition of [Ca(2+)](i)-mobilization) and pK(b) 7.47 in rat isolated aorta (inhibition of contraction). SB-706375 also reversed tone established in the rat aorta by prior exposure to hU-II (K(app) approximately 20 nM). 5. SB-706375 was a selective U-II antagonist with >/=100-fold selectivity for the human UT receptor compared to 86 distinct receptors, ion channels, enzymes, transporters and nuclear hormones (K(i)/IC(50)>1 microM). Accordingly, the contractile responses induced in isolated aortae by KCl, phenylephrine, angiotensin II and endothelin-1 were unaltered by SB-706375 (1 microM). 6. In summary, SB-706375 is a high-affinity, surmountable, reversible and selective nonpeptide UT receptor antagonist with cross-species activity that will assist in delineating the pathophysiological actions of U-II in mammals.

Urotensin II-induced hypotensive responses in Wistar–Kyoto (Wky) and spontaneously hypertensive (Shr) rats

Human urotensin II (hU-II) is a potent vasoactive peptide which modulates some of the functions of the cardiovascular and other systems. The in vivo mechanism of action by which hU-II may influence blood pressure in developmental and pathological conditions, is poorly understood. Herein, the blood pressure effects of hU-II (0.1–10 nmol/kg) injected intravenously (i.v.) were studied on ketamine/xylazine anesthetized male WKY and SHR rats aged 4 and 8 weeks. hU-II elicited dose-dependent decreases in mean arterial pressure in both strains of animals. The hypotensive responses to hU-II were, however, significantly higher in SHR rats, independently of age. Four-week-old SHR rats (which are normotensive) were, however, less responsive than their hypertensive 8-week-old counterparts. A series of pharmacological inhibitors were used to identify putative endogenous (endothelial) factors that might account for the hU-II-mediated hypotension in 8-week-old SHR. These include the non-selective nitric oxide synthase inhibitor l-NAME (5 μmol/kg), the non-selective cyclooxygenase inhibitor meclofenamate (16 μmol/kg), the voltage-sensitive and ATP-sensitive K +-channel inhibitors, 4-aminopyridine (5 μmol/kg) and glybenclamide (10 μmol/kg), the cytochrome P450 CYP2C9 inhibitor sulfaphenazole (15 μmol/kg), the cytoskeletal fixation agent phalloidin (15 μmol/kg), the endothelin ET B receptor antagonist BQ-788 (35 μmol/kg), the bradykinin B 2 receptor antagonist HOE 140 (0.5 μmol/kg), the angiotensin AT 2 antagonist PD 123319 (10 μmol/kg) and the UT receptor antagonist urantide (10 μmol/kg). These agents were administered i.v. either at 2.5, 10 or 40 min prior hU-II injection (10 nmol/kg). Among these inhibitors, sulfaphenazole and phalloidin were able to reduce hU-II-induced hypotension. This suggests that the vasodepressor effect of hU-II is mediated by UT receptors and relies in part on the release of epoxide related products; increased microvascular permeability may also contribute to the blood pressure lowering effect of hU-II. Since urantide blocks the constrictor effects of hU-II on isolated aorta, but is inactive against the hypotensive action of hU-II in vivo, the results presented in this paper provide, for the first time, evidence for the existence of two different functional sites for hU-II.

Characterization of functional urotensin II receptors in human skeletal muscle myoblasts: comparison with angiotensin II receptors

The properties of urotensin II (U-II) receptor (UT receptor) and angiotensin II (ANG II) receptor (AT receptor) in primary human skeletal myoblasts (HSMM) and differentiated skeletal myotubes (HSMMT) were characterized. Radiolabeled U-II and ANG II bound specifically to HSMM with K d's of 0.31 nM (2311 receptors/cell) and 0.61 nM (18,257 receptors/cell), respectively. The cyclic segment of U-II peptide, CFWKYC, was the minimal sequence required for binding, with the WKY residues essential. Inhibitor studies suggested AT1 is the predominant ANG II receptor. After radioligand binding, under conditions designed to minimize receptor internalization, half the bound U-II was resistant to acid washing suggesting that U-II binds tightly to its receptor in a qusai-irreversible fashion. The AT1 receptor-bound radioligand was completely removed under the same conditions. RT-PCR detected the expression of mRNAs for UT and AT1 receptors. Western blotting showed that U-II and ANG II signaled via ERK1/2 kinase. UT receptor was not lost upon differentiation into myotubes since both mRNA for UT receptor and U-II binding were still present. ANG II receptors were also present as shown by ANG II-induced calcium mobilization.

Urotensin II stimulates plasma extravasation in mice via UT receptor activation

The peptide urotensin II (U-II) is the cognate ligand of the G-protein coupled receptor UT (formerly GPR14). A role in the regulation of cardiovascular functions has been proposed for this novel peptide/receptor system. In the present study, we evaluated the ability of U-II to induce plasma extravasation in mice and attempted to characterize the receptor involved using the novel UT receptor ligand, [Orn(8)]U-II. The Evans blue technique was used to quantify plasma extravasation. U-II (0.01, 0.1, 1 and 10 nmol/kg) dose-dependently stimulated plasma extravasation in airways, gastrointestinal and urogenital tract tissues from mice, but not in the skin. In most tissues, the dose/response curves to U-II were bell shaped with the maximal effect induced by 1 nmol/kg. [Orn(8)]U-II at 30 nmol/kg was per se either inactive or produced a non-significant increase in plasma extravasation; in the presence of 30 nmol/kg [Orn(8)]U-II, the effects of 1 nmol/kg U-II were always reduced and, in some tissues, abolished. The present findings demonstrate that U-II promotes plasma extravasation in various mouse vascular regions via activation of UT receptors. The mouse plasma extravasation assay will be a useful tool in future studies aimed at characterizing the pharmacological features of novel UT receptor ligands in vivo.

Urantide mimics urotensin-II induced calcium release in cells expressing recombinant UT receptors

Urotensin-II is the natural ligand of the UT receptor. This novel system is involved in the regulation of cardiovascular functions. Recently, a urotensin-II analog ([Pen 5,DTrp 7,Orn 8]urotensin-II(4–11)) named urantide, has been proposed as a selective and potent UT receptor antagonist. In order to pharmacologically characterize this new compound, urantide was tested on the native UT receptors of the rat aorta and on the human recombinant receptors expressed in CHO cells (CHO hUT). Indeed, urantide behaves as a competitive, potent (p A 2 8.24), and pure antagonist in the rat aorta bioassay, while as an agonist (pEC 50 8.11) in a calcium mobilization assay performed in CHO hUT cells. Urantide should be considered a low efficacy partial agonist.

Differential agonistic and antagonistic effects of the urotensin-II ligand SB-710411 at rodent and primate UT receptors

SB-710411 (Cpa-c[d-Cys-Pal-d-Trp-Lys-Val-Cys]-Cpa-amide) inhibited [125I]urotensin-II rat and monkey UT receptor binding (pKis 7.50±0.07 and 6.82±0.06). However, whereas SB-710411 antagonized urotensin-II-induced inositol phosphate formation at the rat UT receptor (pKb 6.54±0.05), this ligand functioned as an agonist at the monkey UT receptor (pEC50 6.56±0.35, Emax 5.27±0.65-fold over basal). Indeed, in contrast to the rat UT receptor (and rat isolated arteries), SB-710411 exhibited intrinsic activity in monkey arteries acting as an efficacious vasoconstrictor (pEC50s 5.03±0.18 to 5.71±0.21, Emaxs 101±4 to 218±58% KCl). These data demonstrate that caution must be taken when extrapolating the pharmacology of a specific ligand(s) between the rodent and primate UT receptors.

Urantide: an ultrapotent urotensin II antagonist peptide in the rat aorta.

In this study we describe the ability of two human urotensin-II (hU-II) derivatives [Pen5,Orn8]hU-II(4-11) and [Pen5,DTrp7,Orn8]hU-II(4-11) (urantide) to block hU-II-induced contractions in the rat isolated thoracic aorta. Both compounds competitively antagonized hU-II- induced effects with pKB=7.4+/-0.06 (n=12) and pKB=8.3+/-0.09 (n=12), respectively. In contrast, neither [Pen5,Orn8]hU-II(4-11) nor urantide (1 microm each) was able to modify noradrenaline- or endothelin 1-induced contractile effects. At micromolar concentrations, [Pen5,Orn8]hU-II(4-11) produced weak (< or =25% of hU-II maximum) agonist responses in the rat aorta, whereas urantide was totally uneffective as agonist up to 1 microm. In addition, [Pen5,Orn8]hU-II(4-11) and urantide displaced [125I]urotensin II from specific binding at hU-II recombinant receptors (UT receptors) transfected into CHO/K1 cells (pKi=7.7+/-0.05, n=4 and pKi=8.3+/-0.04, n=4, respectively). To our knowledge, urantide is the most potent UT receptor antagonist so far described, and might represent a useful tool for exploring the (patho)physiological role of hU-II in the mammalian cardiovascular system.

The neuromedin B receptor antagonist, BIM-23127, is a potent antagonist at human and rat urotensin-II receptors.

The functional activity of the peptidic neuromedin B receptor antagonist BIM-23127 was investigated at recombinant and native urotensin-II receptors (UT receptors). Human urotensin-II (hU-II) promoted intracellular calcium mobilization in HEK293 cells expressing the human UT (hUT) or rat UT (rUT) receptors with pEC(50) values of 9.80+/-0.34 (n=6) and 9.06+/-0.32 (n=4), respectively. While BIM-23127 alone had no effect on calcium responses in either cell line, it was a potent and competitive antagonist at both hUT (pA(2)=7.54+/-0.14; n=3) and rUT (pA(2)=7.70+/-0.05; n=3) receptors. Furthermore, BIM-23127 reversed hU-II-induced contractile tone in the rat-isolated aorta with a pIC(50) of 6.66+/-0.04 (n=4). In conclusion, BIM- 23127 is the first hUT receptor antagonist identified to date and should not be considered as a selective neuromedin B receptor antagonist.

A new ligand for the urotensin II receptor.

The cyclic peptide human urotensin II (U-II) has been recently recognized as the endogenous ligand of an orphan GPCR, subsequently named the UT receptor. No synthetic ligands are available for investigating this novel peptide-receptor system. A novel UT receptor ligand, [Orn(8)]U-II, was synthesized and evaluated in calcium functional assays performed on HEK293 cells expressing the recombinant rat and human UT receptor and in the rat aorta bioassay. [Orn(8)]U-II behaves as a full agonist (pEC(50) approximately equal 8) at both human and rat UT receptors in the FlipR calcium assay eliciting similar maximal effects as the natural ligand U-II. On the contrary, in the rat aorta bioassay, [Orn(8)]U-II behaves as a competitive and selective antagonist (pA(2)=6.56) showing however a small but consistent residual agonist activity. It is therefore proposed that [Orn(8)]U-II is a partial agonist at UT receptors.

Molecular and pharmacological characterization of genes encoding urotensin-II peptides and their cognate G-protein-coupled receptors from the mouse and monkey.

Urotensin-II (U-II) and its receptor (UT) represent novel therapeutic targets for management of a variety of cardiovascular diseases. To test such hypothesis, it will be necessary to develop experimental animal models for the manipulation of U-II/UT receptor system. The goal of this study was to clone mouse and primate preproU-II and UT for pharmacological profiling. Monkey and mouse preproU-II genes were identified to encode 123 and 125 amino acids. Monkey and mouse UT receptors were 389, and 386 amino acids, respectively. Genomic organization of mouse genes showed that the preproU-II has four exons, while the UT receptor has one exon. Although initially viewed by many exclusively as cardiovascular targets, the present study demonstrates expression of mouse and monkey U-II/UT receptor mRNA in extra-vascular tissue including lung, pancreas, skeletal muscle, kidney and liver. Ligand binding studies showed that [125I]h U-II bound to a single sites to the cloned receptors in a saturable/high affinity manner (Kd 654+/-154 and 214+/-65 pM and Bmax of 1011+/-125 and 497+/-68 fmol mg-1 for mouse and monkey UT receptors, respectively). Competition binding analysis demonstrated equipotent, high affinity binding of numerous mammalian, amphibian and piscine U-II isopeptides to these receptors (Ki=0.8 - 3 nM). Fluorescein isothiocyanate (FITC) labelled U-II, bound specifically to HEK-293 cells expressing mouse or monkey UT receptor, confirming cell surface expression of recombinant UT receptor. Exposure of these cells to human U-II resulted in an increase in intracellular [Ca2+] concentrations (EC50 3.2+/-0.8 and 1.1+/-0.3 nM for mouse and monkey UT receptors, respectively) and inositol phosphate (Ip) formation (EC50 7.2+/-1.8 and 0.9+/-0.2 nM for mouse and monkey UT receptors, respectively) consistent with the primary signalling pathway for UT receptor involving phospholipase C activation.