Structure-activity relationship study of position 4 in the urotensin-II receptor ligand U-II(4-11)

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.     

Development and pharmacological characterization of "caged" urotensin II analogs

Urotensin-II (U-II) is a cyclic 11-amino acid peptide known as a potent mammalian vasoconstrictor. To study some purported intracellular actions of U-II, masked analogs of this peptide, becoming biologically active only upon UV exposure, were developed. Those analogs described as "caged" were derivatized with a photolabile 4,5-dimethoxynitrobenzyl group on the side chain of Lys-8 or Tyr-9. Both caged analogs of U-II showed a major decrease in their affinity towards the UT receptor. Nevertheless, upon UV irradiation, the native and biologically active U-II peptide was recovered. Thus, this work describes the development of new "caged" U-II derivatives and demonstrates that vasoactivity of U-II can be controlled by masking and unmasking two key residues.     

Urotensin II is a new chemotactic factor for UT receptor-expressing monocytes

Urotensin II (U-II), a vasoactive cyclic neuropeptide which activates the G protein-coupled receptor UT receptor, exerts various cardiovascular effects and may play a role in the pathophysiology of atherosclerosis. In this study, we report that the UT receptor is expressed and functional on human PBMC and rat splenocytes. PBMC surface expression of the UT receptor was mainly found in monocytes and NK cells, also in a minority of B cells, but not in T cells. Stimulation of monocytes with LPS increased UT receptor mRNA and protein expression. Cloning and functional characterization of the human UT receptor gene promoter revealed the presence of NF-kappaB-binding sites involved in the stimulation of UT receptor gene expression by LPS. Activation of the UT receptor by U-II induced chemotaxis with maximal activity at 10 and 100 nM. This U-II effect was restricted to monocytes. Analysis of the signaling pathway involved indicated that U-II-mediated chemotaxis was related to RhoA and Rho kinase activation and actin cytoskeleton reorganization. The present results thus identify U-II as a chemoattractant for UT receptor-expressing monocytes and indicate a pivotal role of the RhoA-Rho kinase signaling cascade in the chemotaxis induced by U-II.     

Characterization of urotensin-II receptor structural domains involved in the recognition of U-II, URP, and urantide

Urotensin-II (U-II) and urotensin-II-related peptide (URP) are potent vasoconstrictors, and this action is mediated through a G protein-coupled receptor identified as UT. This receptor is expressed abundantly in the mammalian cardiovasculature, and the effects of U-II and URP can be blocked with urantide, a selective antagonist. Thus, we carried out a study with the aim to characterize the conformational arrangement of the three extracellular loops of UT as well as the transmembrane domains III and IV. Secondary structures of the synthetic receptor fragments were determined using circular dichroism (CD) spectroscopy in a variety of solvent and micelle conditions. Spectra showed that all receptor segments but not the extracellular loop I exhibited a propensity for adopting the alpha-helix folding. Furthermore, using surface plasmon resonance (SPR) technology, we measured the binding affinities of the ligands, U-II, URP, and urantide toward the UT extracellular segments. SPR data showed that both U-II and URP bind extracellular loops II and III with similar affinities, whereas none of these two ligands were able to interact with the extracellular loop I. Moreover, the binding of urantide was observed only with the second extracellular loop. These results imply that U-II and URP but not urantide would bind to UT according to a common pattern. Also, the correlation of the CD spectral information with the affinity data suggested that the adoption of a helical geometry in UT, by extracellular loops II and III, might be essential for favoring the binding of ligands.     

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 Kd'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 quasi-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.     

Structure-activity relationships of urotensin II and URP

Urotensin II (U-II) and urotensin II-related peptide (URP) are the endogenous ligands for the orphan G-protein-coupled receptor GPR14 now renamed UT. At the periphery, U-II and/or URP exert a wide range of biological effects on cardiovascular tissues, airway smooth muscles, kidney and endocrine glands, while central administration of U-II elicits various behavioral and cardiovascular responses. There is also evidence that U-II and/or URP may be involved in a number of pathological conditions including heart failure, atherosclerosis, renal dysfunction and diabetes. Because of the potential involvement of the urotensinergic system in various physiopathological processes, there is need for the rational design of potent and selective ligands for the UT receptor. Structure-activity relationship studies have shown that the minimal sequence required to retain full biological activity is the conserved U-II(4-11) domain, in particular the Cys5 and Cys10 residues involved in the disulfide bridge, and the Phe6, Lys8 and Tyr9 residues. Free alpha-amino group and C-terminal COOH group are not necessary for the biological activity, and modifications of these radicals may even increase the stability of the analogs. Punctual substitution of native amino acids, notably Phe6 and Trp7, by particular residues generates analogs with antagonistic properties. These studies, which provide crucial information regarding the structural and conformational requirements for ligand-receptor interactions, will be of considerable importance for the design of novel UT ligands with increased selectivity, potency and stability, that may eventually lead to the development of innovative drugs.     

Solution structure of urotensin-II receptor extracellular loop III and characterization of its interaction with urotensin-II

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 beta-turn (Q285-L288), followed by an -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 beta-turn (end of TM-VI/beginning of EC-III) and on one face of the -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.     

Endogenous urotensin II selectively modulates erectile function through eNOS

Background

Urotensin II (U-II) is a cyclic peptide originally isolated from the neurosecretory system of the teleost fish and subsequently found in other species, including man. U-II was identified as the natural ligand of a G-protein coupled receptor, namely UT receptor. U-II and UT receptor are expressed in a variety of peripheral organs and especially in cardiovascular tissue. Recent evidence indicates the involvement of U-II/UT pathway in penile function in human, but the molecular mechanism is still unclear. On these bases the aim of this study is to investigate the mechanism(s) of U-II-induced relaxation in human corpus cavernosum and its relationship with L-arginine/Nitric oxide (NO) pathway.

Methodology/Principal Findings

Human corpus cavernosum tissue was obtained following in male-to-female transsexuals undergoing surgical procedure for sex reassignment. Quantitative RT-PCR clearly demonstrated the U-II expression in human corpus cavernosum. U-II (0.1 nM–10 µM) challenge in human corpus cavernosum induced a significant increase in NO production as revealed by fluorometric analysis. NO generation was coupled to a marked increase in the ratio eNOS phosphorilated/eNOS as determined by western blot analysis. A functional study in human corpus cavernosum strips was performed to asses eNOS involvement in U-II-induced relaxation by using a pharmacological modulation. Pre-treatment with both wortmannin or geldanamycinin (inhibitors of eNOS phosphorylation and heath shock protein 90 recruitment, respectively) significantly reduced U-II-induced relaxation (0.1 nM–10 µM) in human corpus cavernosum strips. Finally, a co-immunoprecipitation study demonstrated that UT receptor and eNOS co-immunoprecipitate following U-II challenge of human corpus cavernosum tissue.

Conclusion/Significance

U-II is endogenously synthesized and locally released in human corpus cavernosum. U-II elicited penile erection through eNOS activation. Thus, U-II/UT pathway may represent a novel therapeutical target in erectile dysfunction.     

Behavioral actions of urotensin-II

Urotensin-II (U-II) and urotensin-II-related peptide (URP) have been identified as the endogenous ligands of the orphan G-protein-coupled receptor GPR14 now renamed UT. The occurrence of U-II and URP in the central nervous system, and the widespread distribution of UT in the brain suggest that U-II and URP may play various behavioral activities. Studies conducted in rodents have shown that central administration of U-II stimulates locomotion, provokes anxiety- and depressive-like states, enhances feeding activity and increases the duration of paradoxical sleep episodes. These observations indicate that, besides the endocrine/paracrine activities of U-II and URP on cardiovascular and kidney functions, these peptides may act as neurotransmitters and/or neuromodulators to regulate various neurobiological activities.     

Pro-angiogenic activity of Urotensin-II on different human vascular endothelial cell populations

Urotensin-II (U-II), along its receptor UT, is widely expressed in the cardiovascular system, where it exerts regulatory actions under both physiological and pathological conditions. In the present study, human vascular endothelial cells (EC) from one arterious and three venous vascular beds were used to investigate in vitro their heterogeneity in terms of expression of U-II and UT and of angiogenic response to the peptide. Real-time PCR and immunocytochemistry demonstrated the expression of UT, as mRNA and protein, in all the EC populations investigated. U-II, on the contrary, was detectable only in EC from aorta and umbilical vein. U-II did not affect the proliferation rate of adult human EC, but induced a moderate proliferative effect on EC from human umbilical vein. When tested in the Matrigel assay, however, all EC exhibited a strong angiogenic response to the peptide, comparable to that of fibroblast growth factor-2 (FGF-2) and it was not associated to an increased expression of vascular endothelial growth factor (VEGF) and/or its receptors. The angiogenic effect of U-II was abolished by the UT antagonist palosuran. Overall, these data suggest that U-II, in addition to the well known role in the regulation of cardiovascular function, also exert a specific angiogenic activity.     

Cardiorenovascular effects of urotensin II and the relevance of the UT receptor

Urotensin II (U-II) is a vasoactive peptide with many potent effects in the cardiorenovascular system. U-II activates a G-protein-coupled receptor termed UT. UT and U-II are highly expressed in the cardiovascular and renal system. Patients with various cardiovascular diseases show high U-II plasma levels. It was demonstrated that elevated U-II plasma levels and increased UT expression seem to play a role in heart failure, end-stage renal disease and atherosclerosis. U-II induces potent changes in vascular tone regulation. In addition, U-II stimulates vascular smooth muscle cell proliferation and cardiomyocyte hypertrophy. Currently several pharmaceutical companies are developing compounds to control the U-II/UT system. There are preclinical and some clinical studies showing potential benefits of inhibiting U-II function in renal disease, heart failure, and diabetes. This article will review both pre- and clinical data concerning cardiorenovascular effects of U-II.     

Urotensin II and atherosclerosis

Urotensin II, through its interaction with its UT receptor, is a potent vasoactive peptide in humans and in several animal models. Recent studies have demonstrated elevated plasma U-II levels in patients with atherosclerosis and coronary artery disease. U-II is expressed in endothelial cells, smooth muscle cells and infiltrating macrophages of atherosclerotic human coronary arteries. UT receptor expression is up-regulated by inflammatory stimuli. Activation of UT receptor by U-II stimulates endothelial and smooth muscle cell proliferation and monocytes chemotaxis. Therefore, in addition to its primary vasoactive effect, these observations suggest a role of U-II and UT receptor in the initiation and/or progression of atherosclerosis.     

Biological properties and functional determinants of the urotensin II receptor

The urotensin II receptor (UT) is a member of the G protein-coupled receptor (GPCR) family and binds the cyclic undecapeptide urotensin II (U-II) as well as the octapeptide urotensin II-related peptide (URP). The active UT mediates pleiotropic effects through various signal transduction pathways, including coupling to G proteins and activating the mitogen-activated protein kinase pathway. Several highly conserved residues and motifs of class A GPCRs that are important for activity are found in UT. This review highlights some of the putative roles of these motifs in the binding, activation and desensitization of UT.     

Identification and characterization of binding sites for human urotensin-II in Sprague-Dawley rat renal medulla using quantitative receptor autoradiography

Urotensin-II (U-II), a ligand for the G-protein-coupled receptor UT, has been characterized as the most potent mammalian vasoconstrictor identified to date. Although circulating levels of U-II are altered in lower species (e.g., fish) upon exposure to hypo-osmotic stress, little is known about the actions of this cyclic undecapeptide within the kidney, an organ that plays a pivotal role in the control of cardiovascular homeostasis, influencing both cardiac preload (plasma volume) and after load (peripheral resistance). The present study reports the identification of specific, high affinity [125I]hU-II binding sites in Sprague-Dawley rat kidney outer medulla by autoradiography and also through membrane radioligand binding (Kd 1.9 +/- 0.9 nM and Bmax 408 +/- 47 amol mm(-2) and Kd 1.4 +/- 0.3 nM and Bmax 51.3 +/- 7.8 fmol mg(-1) protein, respectively). Differences were observed in the binding characteristics within rat strains. Compared to the Sprague-Dawley, Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rat kidney outer medulla displayed low density < 20 fmol mg(-1) protein and low affinity (> 1 microM) [125I]hU-II binding sites. Thus, the relative contribution of specific U-II binding sites to the physiological actions of U-II in the control of cardiorenal homeostasis is worthy of further investigation.     

Urotensin-II and cardiovascular remodeling

Urotensin-II (U-II), a cyclic undecapeptide, and its receptor, UT, have been linked to vascular and cardiac remodeling. In patients with coronary artery disease (CAD), it has been shown that U-II plasma levels are significantly greater than in normal patients and the severity of the disease is increased proportionally to the U-II plasma levels. We showed that U-II protein and mRNA levels were significantly elevated in the arteries of patients with coronary atherosclerosis in comparison to healthy arteries. We observed U-II expression in endothelial cells, foam cells, and myointimal and medial vSMCs of atherosclerotic human coronary arteries. Recent studies have demonstrated that U-II acts in synergy with mildly oxidized LDL inducing vascular smooth muscle cell (vSMC) proliferation. Additionally, U-II has been shown to induce cardiac fibrosis and cardiomyocyte hypertrophy leading to cardiac remodeling. When using a selective U-II antagonist, SB-611812, we demonstrated a decrease in cardiac dysfunction including a reduction in cardiomyocyte hypertrophy and cardiac fibrosis. These findings suggest that U-II is undoubtedly a potential therapeutic target in treating cardiovascular remodeling.     

Ligands and signaling of the G-protein-coupled receptor GPR14, expressed in human kidney cells.

Activation of the urotensin II (U-II) receptor, GPR14, leads to an increase in Ca(2+), activation of phospholipase A(2) (PLA(2)) and an increase in arachidonic acid. The signaling pathway for guanylin peptides in the kidney involves an unknown G-protein coupled receptor which activates PLA(2) and increases arachidonic acid as well. To test if guanylin peptides could be, as U-II, agonists for the GPR14 receptor in the kidney, we used HEK293 and CHO cells transfected with hGPR14 (HEK293+hGPR14, CHO+hGPR14, respectively). Effects of guanylin peptides and U-II were studied by slow-whole-cell patch-clamp analysis and microfluorimetric measurements of intracellular Ca(2+). Guanylin peptides and U-II depolarized HEK293+hGPR14 significantly more than wild type cells. These effects were inhibited in the presence of Ba(2+) or PLA(2) inhibition (AACOCF(3)), suggesting that guanylin peptides and U-II increase arachidonic acid and inhibit ROMK channels in these cells. However, only U-II was capable to increase the cellular Ca(2+), suggesting different mechanism of GPR14 activation by guanylin peptides and U-II. This signaling pathway of U-II involves PKC, because U-II effects in HEK293+hGPR14 cells were inhibited by calphostin C. Guanylin peptides activate PLA(2) and inhibit ROMK channels in HEK293 cells transfected with the human GPR14 receptor. Since GPR14 is present in mouse and human CCD it is a candidate for the guanylate cyclase independent receptor for guanylin peptides.     

Human urotensin-II enhances plasma extravasation in specific vascular districts in Wistar rats

Plasma extravasation (PE) was measured in adult Wistar rats by injecting Evans blue dye (EB) (20 mg kg-1) intravenously in the absence or presence of human urotensin II (U-II) (0.1-10 nmol kg-1). A consistent increase of PE was observed in specific organs (e.g., aorta, from 28.1 +/- 2.4 to 74.6 +/- 3.6 micro g EB g-1 dry tissue; P < 0.001) after an administration of 4.0 nmol kg-1 (a preselected optimal dose) of U-II. The effects of U-II (4.0 nmol kg-1) were compared with those of endothelin-1 (ET-1) (1.0 nmol kg-1). In the thoracic aorta and pancreas, U-II was active, while ET-1 was not. The two agents were equivalent in the heart and kidney, whereas, in the duodenum, ET-1 was more active than U-II. Increases of plasma extravasation induced by U-II, but not by ET-1, were reduced after treatment with [Orn8]U-II (0.3 micromol kg-1). This latter antagonist did not show any significant residual agonistic activity in vivo in the rat. Other specific receptor antagonists for ET-1, such as BQ-123 (endothelin type A (ETA) receptor) and BQ-788 (endothelin type B (ETB) receptor), and for the platelet activating factor (PAF), such as BN50730, failed to modify the action of U-II. The present study is the first report describing the modulator roles of U-II on vascular permeability in specific organs. Moreover, the action of U-II appears specific, since it is independent of the ET-1 and PAF signalling pathways.     

Urotensin-II-stimulated expression of pro-angiogenic factors in human vascular endothelial cells

Urotensin-II (U-II) is an endogenous peptide recently characterized as a "nonclassic" pro-angiogenic cytokine. In fact, human vascular endothelial cells express the U-II receptor and exhibit a strong in vitro angiogenic response to the peptide, which was specifically triggered by the binding of U-II to its receptor and involved the activation of ERK1/2 and PI3K/Akt signaling pathways. Moreover, available studies, designed to investigate the pro-angiogenic effect quite shortly following U-II stimulation, suggested that the angiogenic action of the peptide was direct and not associated with an increased expression of vascular endothelial growth factor (VEGF) and/or its receptors. In the present study, the expression of three pro-angiogenic factors, namely VEGF, endothelin-1, and adrenomedullin, was studied in human umbilical vein endothelial cells (HUVEC) for longer times of U-II stimulation. RT-PCR and Western blot indicated that in HUVEC, exposed for at least 24h to U-II, the expression of the three angiogenic molecules was significantly increased at both mRNA and protein level, opening the possibility that U-II, not only could exert a direct stimulation of an angiogenic phenotype in endothelial cells quite shortly following exposure to the peptide, but could also further enhance the process indirectly by inducing in the cells a delayed production of other pro-angiogenic factors. Interestingly, a preliminary analysis of the time course of the in vitro capillary-like pattern formation was consistent with this view, suggesting a two phase temporal dynamics of the process.