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

doi:10.1016/j.ejphar.2004.03.059

European Journal of Pharmacology

Volume 492, Issues 2–3, 25 May 2004, Pages 113-116

https://doi.org/10.1016/j.ejphar.2004.03.059 Get rights and content

Abstract

SB-710411 (Cpa-c[d-Cys-Pal-d-Trp-Lys-Val-Cys]-Cpa-amide) inhibited [¹²⁵I]urotensin-II rat and monkey UT receptor binding (pK_is 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 (pK_b 6.54±0.05), this ligand functioned as an agonist at the monkey UT receptor (pEC₅₀ 6.56±0.35, E_max 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, E_maxs 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.

Introduction

Urotensin-II and its G-protein-coupled receptor, UT, are believed to play a role in the (patho)physiological regulation of mammalian cardiovascular function (Douglas, 2003). The search for a definitive role for urotensin-II and its receptor in the control of cardiovascular homeostasis would be greatly assisted by the development of selective UT receptor antagonists. To this end, an increasing number of UT receptor antagonists have been described recently (see Douglas, 2003). One such putative antagonist is SB-710411 (Cpa-c[d-Cys-Pal-d-Trp-Lys-Val-Cys]-Cpa-amide), a cyclic somatostatin analogue (Coy et al., 2000). SB-710411 inhibits urotensin-II-induced contraction in the rat isolated aorta (K_b∼500 nM; Behm et al., 2002). However, as with most putative peptidic antagonists described to date, little is known about the pharmacology of this ligand in other species. Such a consideration is important, potentially, since rodent and primate UT receptor orthologues exhibit significant sequence differences at the amino acid level (Elshourbagy et al., 2002).

Whereas mouse and rat UT receptors are ∼93% identical, the homology between rodent and primate UT receptors is considerably lower at ∼76% identity (monkey and human UT receptors are 97% identical; Elshourbagy et al., 2002). These sequence differences raise the distinct possibility that the pharmacological effects of any given UT receptor modulator might differ between primate and non-primate species. Support for this contention comes from a recent observation that the UT receptor ligand BIM-23042 (d-2-Nal-c[Cys-Tyr-d-Trp-Lys-Val-Cys]-2-Nal-amide) exhibits differential agonist/antagonist activity across several UT receptor orthologues (Herold et al., 2002). In order to address this issue, the present study examined the pharmacological effects of SB-710411 at rat and monkey recombinant UT receptors. Further, the intrinsic activity of this ligand was also evaluated in monkey isolated tissues.

Section snippets

Radioligand binding studies in recombinant cells

[¹²⁵I]Urotensin-II competition binding studies were performed with membranes prepared from human embryonic kidney (HEK-293) cells stably transfected with rat or monkey UT receptors using a scintillation proximity assay as described previously Ames et al., 1999, Elshourbagy et al., 2002. Non-specific binding was defined using 1 μM unlabeled urotensin-II. Competition binding curves were analyzed by nonlinear regression: K_i=(IC₅₀/([S]/K_D))+1, where [S] is the concentration of [¹²⁵I]urotensin-II

Radioligand binding studies in recombinant cells

Urotensin-II and SB-710411 both competed for [¹²⁵I]urotensin-II binding at the recombinant rat UT receptor (pK_is 9.26±0.14 and 7.50±0.07, respectively; n=3, Fig. 1A). As with the rat UT receptor, urotensin-II and SB-710411 were both able to inhibit [¹²⁵I]urotensin-II binding at the recombinant monkey UT receptor with pK_is of 8.78±0.12 and 6.82±0.06, respectively (n=3, Fig. 1B).

Inositol phosphate formation in recombinant cells

SB-710411 failed to stimulate inositol phosphate formation in rat recombinant UT-HEK293 cells (n=4, Fig. 1C). Indeed,

Discussion

SB-710411 acts as a ligand for both the recombinant rat and monkey UT receptors (30–150 nM affinities, 56- and 87-fold less potent than urotensin-II, respectively). However, the present study reveals that the functional behaviour of SB-710411 at these two UT receptor orthologues differs radically.

As was predicted, based on the previous observation that SB-710411 is an antagonist of urotensin-II-induced contraction in the rat isolated aorta (Behm et al., 2002), SB-710411 inhibited urotensin-II

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2023, Biochemical Pharmacology
The urotensinergic system, involved in the development and/or progression of numerous pathological conditions, is composed of one G protein-coupled receptor (UT) and two endogenous ligands known as urotensin II (UII) and urotensin II-related peptide (URP). These two structurally related hormones, which exert common and divergent effects, are thought to play specific biological roles. In recent years, we have characterized an analog termed urocontrin A (UCA), i.e. [Pep⁴]URP, which is capable of discriminating the effects of UII from URP. Such an action could allow the delineation of the respective functions of these two endogenous ligands. In an effort to define the molecular determinants involved in this behavior and to improve the pharmacological profile of UCA, we introduced modifications from urantide, considered for some time as a lead compound for the development of UT antagonists, into UCA and assessed the binding, contractile activity and G protein signaling of these newly developed compounds. Our results show that UCA and its derivatives exert probe-dependent effects on UT antagonism, and we have further identified [Pen², Pep⁴]URP as a G_q biased ligand with an insurmountable antagonism in our aortic ring contraction assay.
Towards Targeting the Urotensinergic System: Overview and Challenges
2019, Trends in Pharmacological Sciences
The urotensinergic system, comprised of a G protein-coupled receptor (UT) and two endogenous ligands named urotensin II (UII) and urotensin II-related peptide (URP), has garnered significant attention due to its involvement in the initiation and/or the evolution of various diseases. Accordingly, multiple studies using animal models have demonstrated that UT antagonists may have utility as potential therapeutic agents for treating atherosclerosis, pulmonary arterial hypertension, heart failure, and cancer. Unfortunately, clinical investigations of UT antagonist candidates showed limited efficacy in humans. This system, which has yet to be effectively targeted, therefore remains to be therapeutically exploited. Here, we discuss various hypotheses that could explain the in vivo failure of UT antagonists.
Noradrenaline release in rodent tissues is inhibited by interleukin-1β but is not affected by urotensin II, MCH, NPW and NPFF
2011, Pharmacological Reports
Citation Excerpt :
It was tempting to assume that urotensin II, like angiotensin II (which acts via Gq protein-coupled AT1 receptors [1]), might possess a dual vasopressor effect, namely via a postsynaptic site of action and additionally via a facilitatory effect on the release of noradrenaline that in turn contributes to the overall effect on the vessel in vivo. However, unlike angiotensin II (which served as a positive control [37]), urotensin II did not facilitate noradrenaline release in a concentration range of 1–1000 nM, including its Ki of 1.4 nM in a radioligand binding study in the rat kidney [10] and an EC50 of 1.9 nM in contraction experiments in the rat aorta [3]. In the concentration range used in the present study, urotensin II led to an increase in the release of noradrenaline and other amines in rat cerebrocortical slices [23].
We studied whether noradrenaline release is affected by interleukin-1β and the neuropeptides urotensin II, melanin-concentrating hormone (MCH), neuropeptide W (NPW) and neuropeptide FF (NPFF). Rodent tissues preincubated with [³H]noradrenaline were superfused, and the effect of peptides on the electrically-evoked tritium overflow (“noradrenaline release”) was studied. In mouse brain cortex, interleukin-1β at 0.3 nM and the prostaglandin E₂ analogue sulprostone at 3 nM inhibited noradrenaline release by about 40%; the effect of interleukin-1β developed gradually, whereas the effect of sulprostone occurred promptly. Urotensin II at 0.001–1 µM did not affect noradrenaline release in rat kidney cortex, whereas 0.01 µM angiotensin II increased it (positive control). MCH at 0.01–1 µM did not alter noradrenaline release in the rat brain cortex, and NPW 1 µM did not affect noradrenaline release in the mouse hypothalamus or hippocampus. In each model, 0.1 µM sulprostone inhibited noradrenaline release (positive control). NPFF and the NPFF₂ receptor agonist dNPA (1 µM) did not affect noradrenaline release in the mouse atria; the inhibitory effect of the δ opioid receptor agonist 1 µM DPDPE on noradrenaline release in this tissue was not altered by NPFF or dNPA at 0.32 µM but was counteracted by the δ opioid antagonist naltrindole at 0.001 µM. In conclusion, interleukin-1β inhibits noradrenaline release in the mouse cortex; the effect develops gradually, suggesting that it affects protein biosynthesis. Noradrenergic neurons in various tissues from rodents are devoid of presynaptic receptors for urotensin II, MCH, NPW and NPFF. Finally, an interaction between a δ opioid agonist and NPFF could not be detected.
Urotensin II and urotensin II-related peptide (URP) in cardiac ischemia-reperfusion injury
2008, Peptides
Circulating urotensin II (UII) concentrations and the tissue expression of its cognate receptor (UT) are elevated in patients with cardiovascular disease (CVD). The functional significance of elevated plasma UII levels in CVD is unclear. Urotensin-related peptide (URP) is a paralog of UII in that it contains the six amino acid ring structures found in UII. Although both peptides are implicated as bioactive factors capable of modulating cardiovascular status, the role of both UII and URP in ischemic injury is unknown. Accordingly, we provide here the first report describing the direct cardiac effects of UII and URP in ischemia-reperfusion injury. Isolated perfused rat hearts were subjected to no-flow global ischemia for 45 min after 30 min preconditioning with either 1 nM rUII or 10 nM URP. Both rUII- and URP-induced significant vasodilation of coronary arteries before (both P < 0.05) and after ischemia (both P < 0.05). Rat UII alone lowered contractility prior to ischemia (P = 0.053). Specific assay of perfusate revealed rUII and URP both significantly inhibited reperfusion myocardial creatine kinase (CK) release (P = 0.012 and 0.036, respectively) and atrial natriuretic peptide (ANP) secretion (P = 0.025). Antagonism of the UT receptor with 1 μM palosuran caused a significant increase in perfusion pressure (PP) prior to and post-ischemia. Furthermore, palosuran significantly inhibited reductions in both PP and myocardial damage marker release induced by both rUII and URP. In conclusion, our data suggests rUII and URP reduce cardiac ischemia-reperfusion injury by increasing flow through the coronary circulation, reducing contractility and therefore myocardial energy demand, and inhibiting reperfusion myocardial damage. Thus, UII and URP present as novel peptides with potential cardioprotective actions.
[Orn<sup>5</sup>]URP acts as a pure antagonist of urotensinergic receptors in rat cortical astrocytes
2008, Peptides
Cultured rat astrocytes, which express functional urotensin II (UII)/UII-related peptide (URP) receptors (UT), represent a very suitable model to investigate the pharmacological profile of UII and URP analogs towards native UT. We have recently designed three URP analogs [d-Trp⁴]URP, [Orn⁵]URP and [d-Tyr⁶]URP, that act as UT antagonists in the rat aortic ring bioassay. However, it has been previously reported that UII/URP analogs capable of inhibiting the contractile activity of UII possess agonistic activity on UT-transfected cells. In the present study, we have compared the ability of URP analogs to compete for [¹²⁵I]URP binding and to modulate cytosolic calcium concentration ([Ca²⁺]_c) in cultured rat astrocytes. All three analogs displaced radioligand binding: [d-Trp⁴]URP and [d-Tyr⁶]URP interacted with high- and low-affinity sites whereas [Orn⁵]URP only bound high-affinity sites. [d-Trp⁴]URP and [d-Tyr⁶]URP both induced a robust increase in [Ca²⁺]_c in astrocytes while [Orn⁵]URP was totally devoid of activity. [Orn⁵]URP provoked a concentration-dependent inhibition of URP- and UII-evoked [Ca²⁺]_c increase and a rightward shift of the URP and UII dose–response curves. The present data indicate that [d-Trp⁴]URP and [d-Tyr⁶]URP, which act as UII antagonists in the rat aortic ring assay, behave as agonists in the [Ca²⁺]_c mobilization assay in cultured astrocytes, whereas [Orn⁵]URP is a pure selective antagonist in both rat aortic ring contraction and astrocyte [Ca²⁺]_c mobilization assays.
Evaluation of no-wash calcium assay kits: Enabling tools for calcium mobilization
2007, Journal of Biomolecular Screening
The no-wash calcium assay kits developed by Molecular Devices Corporation have greatly enhanced the throughput of cell-based calcium mobilization high-throughput screening (HTS) assays and enabled screening using nonadherent cells. The fluorescent imaging plate reader (FLIPR) Calcium 3 Assay Kit, optimal for targets that have proteins or peptides as agonists, has 2 potential drawbacks: 1) a significant downward spike in fluorescence signal upon liquid transfer that can be the same magnitude as the agonist response, making data analysis difficult; and 2) medium removal is required for some targets, which essentially reintroduces a wash step. Several no-wash products were introduced in 2005. The authors compare the Fluo-4 NW Calcium Assay Kit and the BD™ Calcium Assay Kit with the FLIPR Calcium 3 Assay Kit using human native rhabdomyosarcoma cells expressing the urotensin-II receptor (UT). The BD™ Calcium Assay Kit gives the best performance in the true no-wash mode, in which both agonist and antagonist activity are easily quantified. Although these new products provide additional options for measuring calcium mobilization, the different results observed with each kit, using the UT receptor as an example, suggest that one should characterize all dyes against each target in a systematic way prior to choosing one for HTS.

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¹: Current address: RheoGene, Inc., Norristown, PA 19403, USA.

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

Abstract

Introduction

Section snippets

Radioligand binding studies in recombinant cells

Radioligand binding studies in recombinant cells

Inositol phosphate formation in recombinant cells

Discussion

Reg. Pep.

Curr. Opin. Pharmacol.

Hepatology

Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14

Nature

Pharmacological characterization of SB-710411 (Cpa-c[d-Cys-Pal-d-Trp-Lys-Val-Cys]-Cpa-amide), a novel peptidic urotensin-II receptor antagonist

Br. J. Pharmacol.