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Glioblastomas (GBMs) are the most common primary brain tumors characterized by strong invasiveness and angiogenesis. GBM cells and microenvironment secrete angiogenic factors and also express chemoattractant G protein-coupled receptors (GPCRs) to their advantage. We investigated the role of the vasoactive peptide urotensin II (UII) and its receptor UT on GBM angiogenesis and tested potential ligand/therapeutic options based on this system. On glioma patient samples, the expression of UII and UT increased with the grade with marked expression in the vascular and peri-necrotic mesenchymal hypoxic areas being correlated with vascular density. In vitro human UII stimulated human endothelial HUV-EC-C and hCMEC/D3 cell motility and tubulogenesis. In mouse-transplanted Matrigel sponges, mouse (mUII) and human UII markedly stimulated invasion by macrophages, endothelial, and smooth muscle cells. In U87 GBM xenografts expressing UII and UT in the glial and vascular compartments, UII accelerated tumor development, favored hypoxia and necrosis associated with increased proliferation (Ki67), and induced metalloproteinase (MMP)-2 and -9 expression in Nude mice. UII also promoted a “tortuous” vascular collagen-IV expressing network and integrin expression mainly in the vascular compartment. GBM angiogenesis and integrin αvβ3 were confirmed by in vivo99mTc-RGD tracer imaging and tumoral capture in the non-necrotic area of U87 xenografts in Nude mice. Peptide analogs of UII and UT antagonist were also tested as potential tumor repressor. Urotensin II-related peptide URP inhibited angiogenesis in vitro and failed to attract vascular and inflammatory components in Matrigel in vivo. Interestingly, the UT antagonist/biased ligand urantide and the non-peptide UT antagonist palosuran prevented UII-induced tubulogenesis in vitro and significantly delayed tumor growth in vivo. Urantide drastically prevented endogenous and UII-induced GBM angiogenesis, MMP, and integrin activations, associated with GBM tumoral growth. These findings show that UII induces GBM aggressiveness with necrosis and angiogenesis through integrin activation, a mesenchymal behavior that can be targeted by UT biased ligands/antagonists.

Overexpression of G protein-coupled receptors (GPCRs) in tumours is widely used to develop GPCR-targeting radioligands for solid tumour imaging in the context of diagnosis and even treatment. The human vasoactive neuropeptide urotensin II (hUII), which shares structural analogies with somatostatin, interacts with a single high affinity GPCR named UT. High expression of UT has been reported in several types of human solid tumours from lung, gut, prostate, or breast, suggesting that UT is a valuable novel target to design radiolabelled hUII analogues for cancer diagnosis. In this study, two original urotensinergic analogues were first conjugated to a DOTA chelator via an aminohexanoic acid (Ahx) hydrocarbon linker and then -hUII and DOTA-urantide, complexed to the radioactive metal indium isotope to successfully lead to radiolabelled DOTA-Ahx-hUII and DOTA-Ahx-urantide. The 111In-DOTA-hUII in human plasma revealed that only 30% of the radioligand was degraded after a 3-h period. DOTA-hUII and DOTA-urantide exhibited similar binding affinities as native peptides and relayed calcium mobilization in HEK293 cells expressing recombinant human UT. DOTA-hUII, not DOTA-urantide, was able to promote UT internalization in UT-expressing HEK293 cells, thus indicating that radiolabelled 111In-DOTA-hUII would allow sufficient retention of radioactivity within tumour cells or radiolabelled DOTA-urantide may lead to a persistent binding on UT at the plasma membrane. The potential of these radioligands as candidates to target UT was investigated in adenocarcinoma. We showed that hUII stimulated the migration and proliferation of both human lung A549 and colorectal DLD-1 adenocarcinoma cell lines endogenously expressing UT. In vivo intravenous injection of 111In-DOTA-hUII in C57BL/6 mice revealed modest organ signals, with important retention in kidney. 111In-DOTA-hUII or 111In-DOTA-urantide were also injected in nude mice bearing heterotopic xenografts of lung A549 cells or colorectal DLD-1 cells both expressing UT. The observed significant renal uptake and low tumour/muscle ratio (around 2.5) suggest fast tracer clearance from the organism. Together, DOTA-hUII and DOTA-urantide were successfully radiolabelled with 111Indium, the first one functioning as a UT agonist and the second one as a UT-biased ligand/antagonist. To allow tumour-specific targeting and prolong body distribution in preclinical models bearing some solid tumours, these radiolabelled urotensinergic analogues should be optimized for being used as potential molecular tools for diagnosis imaging or even treatment tools.

Vascular dysfunction is a common finding in type 2 diabetes, although the response to urotensin II (UII), a potent vasoconstrictor peptide, remains unclear. We investigated whether a UII-induced contraction was increased in the aortas from type 2 diabetic Goto-Kakizaki (GK) rats at the chronic stage. At 36 or 37 weeks of age (older group), a UII-induced contraction was seen in GK rats and was reduced by a Rho kinase inhibitor or urotensin receptor (UT) antagonist, whereas UII failed to induce a contraction in aortas from age-matched Wistar rats. In UII-stimulated aortas, the expression of Rho kinases, Rho A, and phosphorylated myosin phosphatase target subunit 1 did not change between the two groups; however, phosphorylation of extracellular-regulated kinase 1/2 and p38 mitogen-activated protein kinase (MAPK) was greater in GK than in Wistar rats. Compared to intact aortas, UII-induced contractions were slightly, but not significantly, increased by endothelial denudation of the aortas of Wistar rats at 24 weeks of age. At 6 weeks of age (young group), the UII-induced contractions were seen in GK and Wistar groups. The total expression and the membrane-to-cytosol ratio of the UT protein slightly decreased in Wistar aortas with aging but not in GK aortas. These results demonstrate that the UII-induced contraction gradually decreased with aging in Wistar rats and was preserved in type 2 diabetes. Although alterations of UII-induced contractions during aging and type 2 diabetes may be associated with kinase activities (MAPKs or Rho kinase) or receptor profiles, further investigations are necessary to clarify the mechanisms. [ABSTRACT FROM AUTHOR]

The vasoactive urotensin-II (UII), a cyclic undecapeptide widely distributed in cardiovascular, renal and endocrine systems, specifically binds the UII receptor (UT receptor), a G protein-coupled receptor (GPCR). The involvement of this receptor in numerous pathophysiological conditions including atherosclerosis, heart failure, hypertension, renal impairment and diabetes potentially makes it an interesting therapeutic target. To elucidate how UII binds the UT receptor through the identification of specific residues in transmembrane domains (TM) one (TM1) and two (TM2) that are involved in the formation of the receptor's binding pocket, we used the substituted-cysteine accessibility method (SCAM). Each residue of TM1 (V49 (1.30) to M76 (1.57) ) and TM2 (V88 (2.41) to H117 (2.70) ) was mutated, one by one, to a cysteine. The resulting mutants were then expressed in COS-7 cells and subsequently treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA treatment resulted in a significant binding inhibition of 125 I-UII to mutant I54C (1.35) in TM1 and mutants Y100C (2.53) , S103C (2.56) , F106C (2.59) , I107C (2.60) , T110C (2.63) and Y111C (2.64) in TM2. These results identify key structural residues in TM1 and TM2 that participate in the formation of the UT receptor binding pocket. Together with previous SCAM analysis of TM3, TM4, TM5, TM6 and TM7, these results have led us to identify residues within all 7 TMs that participate in UT's binding pocket and have enabled us to propose a model of this receptor's orthosteric binding site. [ABSTRACT FROM AUTHOR]

ABSTRACT Urotensin II (UII) and Urotensin II-related peptide (URP) are structurally related paralog peptides that exert peripheral and central effects. UII binding sites have been partly described in brain, and those of URP have never been reported. We exhaustively compared [125I]-UII and -URP binding site distributions in the adult rat brain, and found that they fully overlapped at the regional level. We observed UII/URP binding sites in structures lining ventricles, comprising the sphenoid nucleus and cell rafts scattered on a line joining the fourth ventricle and its lateral recess. After injection of UII and URP in the lateral ventricle, we observed c-Fos-positive cell nuclei in areas close to the fourth ventricle, indicating that these receptors are functional. Different c-Fos-containing cell populations were activated. They were all positive for vimentin and glial fibrillary acidic protein (GFAP), excluding the possibility of an ependymal nature. In conclusion, this study demonstrated that UII and URP binding sites are totally overlapping and that these sites were functional in regions bordering the fourth ventricle. These data support a role for UII/URP at the interface between brain parenchyma and cerebrospinal fluid. J. Comp. Neurol. 522:2634-2649, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: Urotensin-II (UII), a cyclic undecapeptide, selectively binds the urotensin-II receptor (UT receptor), a G protein-coupled receptor (GPCR) involved in cardiovascular effects and associated with numerous pathophysiological conditions including hypertension, atherosclerosis, heart failure, pulmonary hypertension and others. In order to identify specific residues in transmembrane domains (TM) three (TM3), four (TM4) and five (TM5) that are involved in the formation of the UT receptor binding pocket, we used the substituted-cysteine accessibility method (SCAM). Each residue in the F118(3.20) to S146(3.48) fragment of TM3, the L168(4.44) to G194(4.70) fragment of TM4 and the W203(5.30) to V232(5.59) fragment of TM5, was mutated, individually, to a cysteine. The resulting mutants were then expressed in COS-7 cells and subsequently treated with the positively charged sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA treatment resulted in a significant reduction in the binding of 125I-UII to TM3 mutants L126C(3.28), F127C(3.29), F131C(3.33) and M134C(3.36) and TM4 mutants M184C(4.60) and I188C(4.64). No loss of binding was detected following treatment by MTSEA for all TM5 mutants tested. In absence of a crystal structure of UT receptor, these results identify key determinants in TM3, TM4 and TM5 that participate in the formation of the UT receptor binding pocket and has led us to propose a homology model of the UT receptor. [Copyright &y& Elsevier]

Abstract: The present study, for the first time in ectothermic vertebrates, reports the immunoregulatory role of urotensins I and II (UI and UII). Urotensins decreased the phagocytosis and nitrite production by splenic phagocytes. On superoxide production, UI had stimulatory while UII showed inhibitory effect. UI exerted its effect on phagocytes through corticotrophin-releasing factor (CRF) receptor as its non-specific antagonist astressin completely blocked the effect of UI on phagocytosis, nitrite release and superoxide production. Among the antagonists for specific CRF receptor 1 and 2, only CRF receptor 1 antagonist NBI 27914 abolished the effect of urotensin I. On the other hand, UII mediated its effect through urotensin receptor (UT receptor) since its antagonist urantide antagonized the effect of UII on phagocytosis, superoxide and nitrite release. These findings provide the direct evidence on physiological role of UI and UII through CRF receptor 1 and UT receptor, respectively in control of fish immune responses. [ABSTRACT FROM AUTHOR]

Abstract: Accumulating evidence indicate that the neuropeptide urotensin II and urotensin II receptors are expressed in subsets of mammal spinal motoneurons. In fact, a role for the peptide in the regulation of motoneuron function at neuromuscular junction has been suggested, while roles for urotensin II at central synapses in spinal cord have never been addressed. We found that urotensin II receptors were closely associated with cholinergic terminals apposed to a subset of motoneuron and non-motoneuron cell bodies in the ventral horn of the adult mouse cervical spinal cord; urotensin II receptor was also expressed on non-cholinergic nerve terminals. In particular, urotensin II receptor appeared associated with both large cholinergic C-boutons and standard cholinergic terminals contacting some motoneuron perikarya. Cholinergic nerve terminals from mouse cervical spinal cord were equipped with functional presynaptic urotensin II receptors linked to excitation of acetylcholine release. In fact, functional experiments conducted on cervical spinal synaptosomes demonstrated a urotensin II evoked calcium-dependent increase in [3H]acetylcholine release pharmacologically verified as consistent with activation of urotensin II receptors. In spinal cord these actions would facilitate cholinergic transmission. These data indicate that, in addition to its role at the neuromuscular junction, urotensin II may control motor function through the modulation of motoneuron activity within the spinal cord. [ABSTRACT FROM AUTHOR]

Urotensin-II (U-II) is an 11-amino acid cyclic peptide which exerts its actions through a Gq protein-coupled receptor, UT. Much of the research focus of U-II is as a peptide of the periphery, particularly cardiovascular. Despite this, U-II was originally identified as a neuropeptide, and its expression is broad throughout the central nervous system. This brief review article catalogs the known sites of expression of UT within the CNS in the form of a diagrammatic rat brain atlas. Furthermore, the functional consequences of UT activation within specific brain regions are discussed along with the likely actions of synthetic UT ligands. Areas of high, medium, and low expression include the arcuate, paraventricular, and pedunculopontine tegmental nuclei, respectively. In the arcuate and paraventricular nuclei, where expression is high and moderate, U-II produces a pressor/tachycardic response in the former and a weaker response in the latter. Based on the known pharmacology of UT ligands (and assuming density is the primary determinant of efficacy in this case), we predict a weak response in the arcuate nucleus and possible antagonism of endogenous U-II response in the paraventricular nucleus by a low-efficacy partial agonist. These predicted responses lend themselves to relatively simple experimental verification. [ABSTRACT FROM AUTHOR]

Urotensin II (UII) is an II amino acid cyclic peptide originally isolated from the goby fish. The amino acid sequence of UlI is exceptionally conserved across most vertebrate taxa, sharing structural similarity to somatostatin. UII binds to a class of G protein-coupled receptor known as GPR14 or the urotensin receptor (UT). UII and its receptor, UT, are widely expressed throughout the cardiovascular, pulmonary, central nervous, renal, and metabolic systems. UII is generally agreed to be the most potent endogenous vasoconstrictor discovered to date. Its physiological mechanisms are similar in some ways to other potent mediators, such as endothelin-1. For example, both compounds elicit a strong vascular smooth muscle-dependent vasoconstriction via Ca2+ release. UII also exerts a wide range of actions in other systems, such as proliferation of vascular smooth muscle cells, fibroblasts, and cancer cells. It also 1) enhances foam cell formation, chemotaxis of inflammatory cells, and inotropic and hypertrophic effects on heart muscle; 2) inhibits insulin release, modulates glomerular filtration, and release of catecholamines; and 3) may help regulate food intake and the sleep cycle. Elevated plasma levels of UII and increased levels of UII and UT expression have been demonstrated in numerous diseased conditions, including hypertension, atherosclerosis, heart failure, pulmonary hypertension, diabetes, renal failure, and the metabolic syndrome. Indeed, some of these reports suggest that UII is a marker of disease activity. As such, the UT receptor is emerging as a promising target for therapeutic intervention. Here, a concise review is given on the vast physiologic and pathologic roles of UII. [ABSTRACT FROM AUTHOR]

Abstract: Urotensin-II (U-II) activates the U-II receptor (UT) to modulate a range of biological responses at both central and peripheral sites. Previous studies have demonstrated that the sequence Trp7-Lys8-Tyr9 of the cyclic portion of the peptide is crucial for biological activity. Here, we describe a focused structure–activity study of Tyr9 which has been replaced with a series of non-coded amino acids in the U-II(4–11) template. Thirteen analogs were synthesized and pharmacologically tested for intracellular calcium mobilization in HEK293 cells stably expressing the rat UT receptor. The results of this study demonstrated the following Tyr9 structure–activity features: (i) the position of the OH group of the side chain is not important for biological activity, (ii) the distance of the phenol moiety from the peptide backbone and its conformational freedom are crucial for UT receptor recognition, (iii) this position is important not only for receptor occupation but also for its activation since the 3,5-diiodoTyr9 chemical modification generated a potent partial agonist. This pharmacological activity of [3,5-diiodoTyr9]U-II(4–11) was confirmed in bioassay experiments performed using the rat thoracic aorta as a U-II sensitive preparation. [Copyright &y& Elsevier]

Urotensin II (UII) has been implicated widely in cardiovascular disease. The mechanism(s) through which it contributes to elevated blood pressure is unknown, but its emerging role as a regulator of mammalian renal function suggests that the kidney might be involved. The aim of this study was to determine the effect of UII on renal function in the spontaneously hypertensive rat (SHR). UII infusion (6 pmolµmin-1µ100 g body wt-1) in anesthetized SHR and control Wistar-Kyoto (WKY) rats produced marked reductions in glomerular filtration rate (ΔGFR WKY, n = 7, -0.3 ± 0.1 vs. SHR, n = 7, -0.6 ± 0.1 ml·min-1· 100 g body wt-1, P = 0.03), urine flow, and sodium excretion rates, which were greater in SHR by comparison with WKY rats. WKY rats also showed an increase in fractional excretion of sodium (ΔFENa +0.6 ± 0.1%, P = 0.02) in contrast to SHR in which no such change was observed (ΔFENa -0.6 ± 0.2%). Blockade of the UII receptor (UT), and thus endogenous UII activity, with urantide evoked an increase in GFR which was greater in SHR (+0.3 ± 0.1) compared with WKY rats (+0.1 ± 0.1 ml·min-1·100 g body wt-1, P = 0.04) and was accompanied by a diuresis and natriuresis. UII and UT mRNA expression were greater in the renal medulla than the cortex of both strains; however, expression levels were up to threefold higher in SHR tissue. SHR are more sensitive than WKY to UII, which acts primarily to lower GFR thus favoring salt retention in this model of hypertension. [ABSTRACT FROM AUTHOR]

Abstract: Urotensin II (UII) has been reported to modulate rapid eye movement (REM) sleep via activation of brainstem cholinergic neurons and REM sleep is regulated by locus coerleus (LC)–cerebrocortical noradrenergic neurons. We hypothesized that UII may activate LC–cerebrocortical noradrenergic neurons. To test this hypothesis, we have examined the effects of UII on norepinephrine release from rat cerebrocortical slices. In addition, the effect of the putative UT receptor antagonist [Pen5, DTrp7, Dab8]UII(4–11) (UFP-803) was assessed. We have compared this with other wakefulness-promoting neurotransmitters such as dopamine, glutamate, serotonin and histamine. We also studied the effects of UII and UFP-803 on intracellular Ca2+ ([Ca2+]i) in HEK293 cells stably expressing rat UT receptor (HEK293-rUT cells). UII produced a time- (peaking at ∼10min following stimulation with 10nM) and concentration-dependent increase in norepinephrine release with pEC50 and E max (% of basal) values of 8.78±0.17 (1.65nM) and 138±2%, respectively. UII also evoked dopamine, serotonin and histamine release with similar pEC50 values. UII increased glutamate release but only at high concentrations (<100nM) and this failed to saturate. UII markedly increased [Ca2+]i in HEK293-rUT cells in a concentration-dependent manner with pEC50 of 8.26±0.24. The UT antagonist UFP-803 reversed both UII-increased norepinephrine release from the cerebrocortical slices (pK B =8.98) and [Ca2+]i (pK B =8.87) in HEK293-rUT cells. Collectively these data suggest that UII evokes the release of norepinephrine via UT receptor activation and produces similar effects on other wakefulness-promoting neurotransmitters: these neurochemical actions of UII may be important for the control of the sleep–wake cycle. [Copyright &y& Elsevier]

Abstract: The importance of combining studies across vertebrates to provide insights into the functionality of hormone systems is considered, using recent advances in Urotensin II (UII) biology to illustrate this. The impact of genome analyses on understanding ligand and UII receptor (UT) structures is reviewed, noting their high conservation from fish to mammals. The early linkage of UII with fish osmoregulatory physiology drove our investigation of possible renal actions of UII in mammals. The kidney is a potential major source of UII in mammals and endogenous peptide appears to have tonal influence over renal excretion of water and electrolytes. Blockade of UII actions by administration of UT receptor antagonist, urantide, in anaesthetised rats, indicates that endogenous UII lowers renal filtration rates and excretion of water and ions. These effects are considered in relation to apparent association of UII with a number of human cardiovascular and renal disorders. Following up the sequencing of UT in mammals here we contrast the first fish UT sequences with those in other species. It is now evident that UT expression in fish osmoregulatory tissues, such as the gill and kidney, exhibits considerable plasticity in response to physiological challenge, providing an important component of the adaptive organismal responses. A number of areas of UII research, which will continue to benefit from moving questions between appropriate vertebrate groups, have been highlighted. These comparative approaches will yield improved understanding and further novel actions of this intriguing endocrine and paracrine system, so highly conserved across the vertebrate series. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Urotensin-II (U-II) is a vasoactive hormone that acts through a G-protein-coupled receptor named UT. Recently, we have shown, using the surface plasmon resonance technology that human U-II (hU-II) interacts with the hUT(281–300) fragment, a segment containing the extracellular loop III (EC-III) and short extensions of the transmembrane domains VI and VII (TM-VI and TM-VII). To further investigate the interaction of UT receptor with U-II, we have determined the solution structure of hUT(281–300) by high-resolution NMR and molecular modeling and we have examined, also using NMR, the binding with hU-II at residue level. In the presence of dodecylphosphocholine micelles, hUT(281–300) exhibited a type III β-turn (Q285–L288), followed by an α-helical structure (A289–L299), the latter including a stretch of transmembrane helix VII. Upon addition of hU-II, significant chemical shift perturbations were observed for residues located just on the N-terminal side of the β-turn (end of TM-VI/beginning of EC-III) and on one face of the α-helix (end of EC-III/beginning of TM-VII). These data, in conjunction with intermolecular NOEs, suggest that the initiation site of EC-III, as well as the upstream portion of helix VII, would be involved in agonist binding and allow to propose points of interaction in the ligand–receptor complex. [Copyright &y& Elsevier]

Abstract: In the present study we describe the synthesis and biological evaluation of 24 analogues of the urotensin II (U-II) fragment U-II(4-11) substituted in position 4 with coded and non-coded aromatic amino acids. All of the new analogues behaved as full U-II receptor (UT) agonists. Our results indicated that aromaticity is well tolerated, size, length and chirality of the side chain are not important, while substituents with a nitrogen atom are preferred. Thus acylation of U-II(5-11) with small groups bearing nitrogen atoms could be instrumental in future studies for the identification of novel potent UT receptor ligands. [Copyright &y& Elsevier]

Urotensin II (U-II) is a cyclic peptide that may be involved in cardiovascular dysfunction. In the present study, the acute effects of U-II on diastolic properties of the myocardium were investigated. Increasing concentrations of U-II (10−8 to 10−6 M) were added to rabbit papillary muscles in the absence ( n = 15) or presence of: (1) damaged endocardial endothelium (EE; n = 9); (2) U-II receptor antagonist, urantide (10−5 M; n = 7); (3) nitric oxide (NO) synthase inhibitor, NG-Nitro- l-Arginine (10−5 M; n = 9); (4) cyclooxygenase inhibitor, indomethacin (10−5 M; n = 8); (5) NO synthase and cyclooxygenase inhibitors, NG-Nitro- l-Arginine (10−5 M) and indomethacin (10−5 M), respectively, ( n = 8); or (6) protein kinase C (PKC) inhibitor, chelerythrine (10−5 M; n = 9). Passive length–tension relations were constructed before and after a single concentration of U-II (10−6 M; n = 3). U-II concentration dependently decreased inotropy and increased resting muscle length (RL). At 10−6 M, active tension decreased 13.8 ± 5.4%, and RL increased to 1.007 ± 0.001 L/ L max. Correcting RL to its initial value resulted in an 18.1 ± 3.0% decrease in resting tension, indicating decreased muscle stiffness, which was also suggested by the down and rightward shift of the passive length–tension relation. This effect remained unaffected by EE damage and PKC inhibition. In contrast, the presence of urantide and NO inhibition abolished the effects of U-II on myocardial stiffness, while cyclooxygenase inhibition significantly attenuated them. U-II decreases myocardial stiffness, an effect that is mediated by the urotensin-II receptor, NO, and prostaglandins. This represents a novel mechanism of acute neurohumoral modulation of diastolic function, suggesting that U-II is an important regulator of cardiac filling. [ABSTRACT FROM AUTHOR]

Several peptidic urotensin-II (UT) receptor antagonists exert ‘paradoxical’ agonist activity in recombinant cell- and tissue-based bioassay systems, likely the result of differential urotensin-II receptor (UT receptor) signal transduction/coupling efficiency between assays. The present study has examined this phenomenon in mammalian arteries and recombinant UT-HEK (human embryonic kidney) cells.BacMam-mediated recombinant UT receptor upregulation in HEK cells augmented agonist activity for all four peptidic UT ligands studied. The nominal rank order of relative intrinsic efficacy was U-II>urantide ([Pen5-DTrp7-Orn8]hU-II4–11)>SB-710411 (Cpa-c[DCys-Pal-DTrp-Lys-Val-Cys]-Cpa-amide)≫GSK248451 (Cin-c[DCys-Pal-DTrp-Orn-Val-Cys]-His-amide) (the relative coupling efficiency of recombinant HEK cells was cat>human≫rat UT receptor).The present study further demonstrated that the use of high signal transduction/coupling efficiency isolated blood vessel assays (primate>cat arteries) is required in order to characterize UT receptor antagonism thoroughly. This cannot be attained simply by using the rat isolated aorta, an artery with low signal transduction/coupling efficiency in which low-efficacy agonists appear to function as antagonists.In contrast to the ‘low-efficacy agonists’ urantide and SB-710411, GSK248451 functioned as a potent UT receptor antagonist in all native isolated tissues studied (UT receptor selectivity was confirmed in the rat aorta). Further, GSK248451 exhibited an extremely low level of relative intrinsic activity in recombinant HEK cells (4–5-fold less than seen with urantide). Since GSK248451 (1 mg kg−1, i.v.) blocked the systemic pressor actions of exogenous U-II in the anaesthetized cat, it represents a suitable peptidic tool antagonist for delineating the role of U-II in the aetiology of mammalian cardiometabolic diseases.British Journal of Pharmacology (2006) 148, 173–190. doi:10.1038/sj.bjp.0706716; published online 20 March 2006 [ABSTRACT FROM AUTHOR]

Urotensin II (UII) is a potent vasoactive hormone in mammals. However, despite its well-known effects on epithelial sodium transport in fish, little is known about its actions on the mammalian kidney. The aim of this study was to determine the effects of UII on renal function in the rat. Using standard clearance methods, the effects of rUII and the rat UII receptor (UT) antagonist, urantide, were studied. UII was measured in plasma and urine by radioimmunoassay. UII and UT were localized in the kidney by immunohistochemistry and mRNA expression quantified. Rat urinary [UII] was 1650-fold higher than that in plasma. Immunoreactive-UII was localized to the proximal tubules, outer and inner medullary collecting ducts (IMCD); UT receptor was identified in glomerular arterioles, thin ascending limbs, and IMCD. UII and UT mRNA expression was greater in the medulla; expression was higher still in spontaneously hypertensive rats (SHRs) associated with raised plasma (UII). Injection of rUII induced reductions in glomerular filtration rate (GFR), urine flow, and sodium excretion. Urantide infusion resulted in increases in these variables. Endogenous UII appears to contribute to the regulation of GFR and renal sodium and water handling in the rat. While hemodynamic changes predominate, we cannot rule out the possibility of a direct tubular action of UII. Increased expression of UII and UT in the SHR suggests that UII plays a role in the pathophysiology of cardiovascular disease.Kidney International (2006) 69, 1360–1368. doi:10.1038/sj.ki.5000290; published online 8 March 2006 [ABSTRACT FROM AUTHOR]

The novel urotensin-II (U-II) receptor (UT) ligand, [Pen5,DTrp7,Dab8]U-II(4–11) (UFP-803), was pharmacologically evaluated and compared with urantide in in vitro and in vivo assays.In the rat isolated aorta, UFP-803 was inactive alone but, concentration dependently, displaced the contractile response to U-II to the right, revealing a competitive type of antagonism and a pA2 value of 7.46.In the FLIPR [Ca2+]i assay, performed at room temperature in HEK293hUT and HEK293rUT cells, U-II increased [Ca2+]i with pEC50 values of 8.11 and 8.48. Urantide and UFP-803 were inactive as agonists, but antagonized the actions of U-II by reducing, in a concentration-dependent manner, the agonist maximal effects with apparent pKB values in the range of 8.45–9.05. In a separate series of experiments performed at 37°C using a cuvette-based [Ca2+]i assay and CHOhUT cells, urantide mimicked the [Ca2+]i stimulatory effect of U-II with an intrinsic activity (α) of 0.80, while UFP-803 displayed a small (α=0.21) but consistent residual agonist activity. When the same experiments were repeated at 22°C (a temperature similar to that in FLIPR experiments), urantide displayed a very small intrinsic activity (α=0.11) and UFP-803 was completely inactive as an agonist.In vivo in mice, UFP-803 (10 nmol kg−1) antagonized U-II (1 nmol kg−1)-induced increase in plasma extravasation in various vascular beds, while being inactive alone.In conclusion, UFP-803 is a potent UT receptor ligand which displays competitive/noncompetitive antagonist behavior depending on the assay. While UFP-803 is less potent than urantide, it displayed reduced residual agonist activity and as such may be a useful pharmacological tool.British Journal of Pharmacology (2006) 147, 92–100. doi:10.1038/sj.bjp.0706438; published online 7 November 2005 [ABSTRACT FROM AUTHOR]

SB-706375 potently inhibited [125I]hU-II binding to both mammalian recombinant and ‘native’ UT receptors (Ki 4.7±1.5 to 20.7±3.6 nM at rodent, feline and primate recombinant UT receptors and Ki 5.4±0.4 nM at the endogenous UT receptor in SJRH30 cells).Prior exposure to SB-706375 (1 μM, 30 min) did not alter [125I]hU-II binding affinity or density in recombinant cells (KD 3.1±0.4 vs 5.8±0.9 nM and Bmax 3.1±1.0 vs 2.8±0.8 pmol mg−1) consistent with a reversible mode of action.The novel, nonpeptidic radioligand [3H]SB-657510, a close analogue of SB-706375, bound to the monkey UT receptor (KD 2.6±0.4 nM, Bmax 0.86±0.12 pmol mg−1) in a manner that was inhibited by both U-II isopeptides and SB-706375 (Ki 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.SB-706375 was a potent, competitive hU-II antagonist across species with pKb 7.29–8.00 in HEK293-UT receptor cells (inhibition of [Ca2+]i-mobilization) and pKb 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 (Kapp∼20 nM).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 (Ki/IC50>1 μM). Accordingly, the contractile responses induced in isolated aortae by KCl, phenylephrine, angiotensin II and endothelin-1 were unaltered by SB-706375 (1 μM).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.British Journal of Pharmacology (2005) 145, 620–635. doi:10.1038/sj.bjp.0706229 Published online 25 April 2005 [ABSTRACT FROM AUTHOR]

Abstract: Urotensin-II is purported to influence pulmonary function by modulating smooth muscle tone/growth. In the present study, Northern blot and reverse transcription polymerase chain reaction (RT-PCR) analysis indicated the presence of UT receptor mRNA in cat trachea, bronchi and lung parenchyma. Urotensin-II contracted cat isolated trachea and bronchi with similar potencies (pEC50s 8.61±0.07–8.81±0.10). Contractile efficacies ranged from 19±9% to 63±11% KCl in the primary and secondary bronchi. The peptidic UT receptor antagonists BIM-23127, SB-710411 and GSK248451 (7.18±0.12, 7.52±0.08 and 9.05±0.16 cat recombinant UT pKis) inhibited urotensin-II-induced contraction of cat isolated trachea with pKbs 6.36±0.11, 6.74±0.07 and 9.27±0.12, respectively. As such, feline lung contains significant amounts of UT mRNA and this receptor appears to be functionally coupled to bronchoconstriction (the peptidic tool compound GSK248451 representing a sub-nanomolar inhibitor of such effects). These findings suggest that the cat represents a suitable species for future studies designed to assess the effects of the urotensin-II receptor on pulmonary (patho)physiology. [Copyright &y& Elsevier]

Abstract The peptide urotensin II (U-Il) is the cognate ligand of the 6-protein coupled receptor UT (formerly GPRI4). 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-Il to induce plasma extravasation in mice and attempted to characterize the receptor involved using the novel UT receptor ligand, [Orn8] U-Il. The Evans blue technique was used to quantify plasma extravasation. U-Il (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-Il were bell shaped with the maximal effect induced by I nmol/kg. [Orn8]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 [Orn8]U-II, the effects of I nmol/kg U-Il were always reduced and, in some tissues, abolished. The present findings demonstrate that U-Il 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. [ABSTRACT FROM AUTHOR]

Urotensin II (UII) is a piscine neuropeptide originally isolated from the teleost urophysis. The existence of UII in mammals has been demonstrated by cloning of the mammalian orthologs of UII precursor protein genes. While rat and mouse orthologs have been reported, only the tentative structures of UII peptides of these animals have been demonstrated, since prepro-UII proteins lack the typical processing sites in the amino-terminal region of the mature peptides. A novel peptide, UII-related peptide (URP), was discovered by monitoring UII-immunoreactivity in the rat brain, and its amino acid sequence was determined to be ACFWKYCV. cDNAs encoding rat, mouse, and human precursor proteins for URP were cloned and showed that the sequences of mouse and human URP peptides are identical to that for rat URP. URP was found to bind and activate the human or rat urotensin II receptors [GPR14, UT receptor (UTR)] and showed a hypotensive effect when administrated to anesthetized rats. The prepro-URP gene is expressed in several rat tissues, although with lower levels than the prepro-UII gene and, in the human, is expressed comparably to prepro-UII in several tissues except the spinal cord. These results suggest that URP is the endogenous and functional ligand for urotensin II receptor in the rat and mouse, and possibly in the human. [Copyright &y& Elsevier]

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 ([Pen5,DTrp7,Orn8]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 (CHOhUT). Indeed, urantide behaves as a competitive, potent (pA2 8.24), and pure antagonist in the rat aorta bioassay, while as an agonist (pEC50 8.11) in a calcium mobilization assay performed in CHOhUT cells. Urantide should be considered a low efficacy partial agonist. [Copyright &y& Elsevier]

Plasma and urinary levels of “urotensin(U)-II-like” substances determined in healthy human volunteers were 12.4 ± 0.6 ng/ml and 2.2 ± 0.3 ng/ml by RIA, an order of magnitude lower than that seen by RRA, 167.5 ± 9.5 ng/ml and 65.2 ± 4.3 ng/ml. HPLC demonstrated the existence of at least three prominent activity peaks in plasma and urine, the more hydrophobic of which did not co-elute with U-II, degradation products or URP. RRA and RIA recognized these peaks with contrasting efficacy. As such, published levels of “U-II-like” activity should be interpreted with caution until a better understanding is obtained regarding what species specific RIA and RRA assay reagents interact with. [Copyright &y& Elsevier]

The vasoactive peptide urotensin-II and its receptor, GPR14 (now known as UT receptor), are localised in the mammalian central nervous system. Accordingly, various centrally mediated effects of urotensin-II on behaviour, neuroendocrine hormones and neurochemistry have been described. To investigate neuroanatomical substrates for the central actions of urotensin-II, expression of the immediate early gene c-fos was examined following intracerebroventricular administration to rats. Urotensin-II increased Fos expression in the cingulate cortex and periaqueductal grey, suggesting important central roles for urotensin-II and its receptor. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

In this study we describe the ability of two human urotensin-ll (hU-II) derivatives [Pen[sub5].Orn[sub8]hU-11(4—11) and [Pen[sub5]DTrp[sub7].Onr[sub8]hU-11(4—11) (urantide) to block hU-II-induced contractions in the rat isolated thoracic aorta. Both compounds competitively antagonized hU-II- induced effects with PK[subB] = 7.4 ± 0.06 (n = I2) and pK[subB]= 8.3 ± 0.09 (n= 12). respectively. In contrast, neither [Pen[sup5].Orn[sup8]-hU-ll(4—11) nor urantide (1 μM each) was able to modify noradrenaline- or endothelin 1-induced contractile effects. At micromolar concentrations. [Pen[sup5].Orn[sup8]h.U-11(4—11) produced weak (≤ 25% of hU-ll maximum) agonist responses in the rat aorta. whereas urantide was totally uneffective as agonist up to 1 μM. In addition. [Pen[sup5].Orn[sub8]]hU-11(4-11 ) and urantide displaced [[sup125]I]urotensin II from specific binding at hU-ll recombinant receptors (UT receptors) transfected into CHO/K1 cells pK[sub1] = 7.7 ± 0.05, n=4 and pK[sub1] =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 lor exploring the (patho)physiological role of hU-ll in the mammalian cardiovascular system. [ABSTRACT FROM AUTHOR]

1 Urotensin-II (U-II) is among the most potent mammalian vasoconstrictors identified and may play a role in the aetiology of essential hypertension. Currently, only one mouse U-II receptor (UT) gene has been cloned. It is postulated that this protein is solely responsible for mediating U-II-induced vasoconstriction. 2 This hypothesis has been investigated in the present study, which assessed basal haemodynamics and vascular reactivity to hU-II in wild-type (UT +/.../ 1) and UT receptor knockout (UT/.../ 1) mice. 3 Basal left ventricular end-diastolic and end-systolic volumes/pressures, stroke volumes, mean arterial blood pressures, heart rales, cardiac outputs and ejection fractions in UT... mice and in UT... mice were similar. 4 Relative to UT... mouse isolated thoracic aorta, where hU-II was a potent spasmogen (pEC50 = 8.26±0.08) that evoked relatively little vasoconstriction (I7±2% 60mM KC1). vessels isolated from UT... mice did not respond to hU-II. However, in contrast, the superior mesenteric artery isolated from both the genotypes did not contract in the presence of hU-II. Reactivity to unrelated vasoconstrictors (phenylephrine, endothelin-1, KCl) and endothelium-dependent/independent vasodilator agents (carbachol, sodium nitroprusside) was similar in the aorta and superior mesenteric arteries isolated from both the genotypes. 5 The present study is the first to directly link hU-II-induced vasoconstriction with the UT receptor. Deletion of the UT receptor gene results in loss of hU-II contractile action with no 'nonspecific' alterations in vascular reactivity. However, as might be predicted based on the limited contractile efficacy recorded in vitro. the contribution that hU-II and its receptor make to basal systemic haemodynamics appears to be negligible in this species. [ABSTRACT FROM AUTHOR]

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 pEC50, 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 (pA2 = 7.54±0.14: n = 3) and rUT (pA2 = 7.70±0.05; n = 3) receptors. Furthermore. BIM-23127 reversed hU-II-induced contractile tone in the rat-isolated aorta with a pIC50 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. [ABSTRACT FROM AUTHOR]

Overexpression of G protein-coupled receptors (GPCRs) in tumours is widely used to develop GPCR-targeting radioligands for solid tumour imaging in the context of diagnosis and even treatment. The human vasoactive neuropeptide urotensin II (hUII), which shares structural analogies with somatostatin, interacts with a single high affinity GPCR named UT. High expression of UT has been reported in several types of human solid tumours from lung, gut, prostate, or breast, suggesting that UT is a valuable novel target to design radiolabelled hUII analogues for cancer diagnosis. In this study, two original urotensinergic analogues were first conjugated to a DOTA chelator via an aminohexanoic acid (Ahx) hydrocarbon linker and then -hUII and DOTA-urantide, complexed to the radioactive metal indium isotope to successfully lead to radiolabelled DOTA-Ahx-hUII and DOTA-Ahx-urantide. The 111In-DOTA-hUII in human plasma revealed that only 30% of the radioligand was degraded after a 3-h period. DOTA-hUII and DOTA-urantide exhibited similar binding affinities as native peptides and relayed calcium mobilization in HEK293 cells expressing recombinant human UT. DOTA-hUII, not DOTA-urantide, was able to promote UT internalization in UT-expressing HEK293 cells, thus indicating that radiolabelled 111In-DOTA-hUII would allow sufficient retention of radioactivity within tumour cells or radiolabelled DOTA-urantide may lead to a persistent binding on UT at the plasma membrane. The potential of these radioligands as candidates to target UT was investigated in adenocarcinoma. We showed that hUII stimulated the migration and proliferation of both human lung A549 and colorectal DLD-1 adenocarcinoma cell lines endogenously expressing UT. In vivo intravenous injection of 111In-DOTA-hUII in C57BL/6 mice revealed modest organ signals, with important retention in kidney. 111In-DOTA-hUII or 111In-DOTA-urantide were also injected in nude mice bearing heterotopic xenografts of lung A549 cells or colorectal DLD-1 cells both expressing UT. The observed significant renal uptake and low tumour/muscle ratio (around 2.5) suggest fast tracer clearance from the organism. Together, DOTA-hUII and DOTA-urantide were successfully radiolabelled with 111Indium, the first one functioning as a UT agonist and the second one as a UT-biased ligand/antagonist. To allow tumour-specific targeting and prolong body distribution in preclinical models bearing some solid tumours, these radiolabelled urotensinergic analogues should be optimized for being used as potential molecular tools for diagnosis imaging or even treatment tools. [ABSTRACT FROM AUTHOR]