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

doi:10.1016/j.ejphar.2004.07.089

European Journal of Pharmacology

Volume 498, Issues 1–3, 13 September 2004, Pages 83-86

https://doi.org/10.1016/j.ejphar.2004.07.089 Get rights and content

Abstract

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 (CHO_hUT). 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 CHO_hUT cells. Urantide should be considered a low efficacy partial agonist.

Introduction

Urotensin-II is a cyclic undecapeptide originally isolated from goby fish urophysis (Bern and Lederis, 1969). Five years ago, urotensin-II was identified as the natural ligand of an orphan G-protein coupled receptor (Ames et al., 1999) now referred to as UT receptor (Douglas and Ohlstein, 2000). The urotensin-II/UT receptor system seems to play an important role in cardiovascular functions (Douglas et al., 2004) although central nervous effects of urotensin-II have also been described (Gartlon et al., 2001, Matsumoto et al., 2004). Specific and selective UT receptor antagonists should provide useful tools for investigating the biological role(s) of the urotensin-II/UT receptor system.

Structure–activity studies revealed that the cyclic portion (Cys⁵–Cys¹⁰) of the peptide is crucial for biological activities, and the sequence Trp⁷–Lys⁸–Tyr⁹ has been shown to be the most important for UT receptor occupation and activation (Brkovic et al., 2003, Flohr et al., 2002). The replacement of Lys⁸ with Orn⁸ lead to the identification of the low potency UT receptor partial agonist [Orn⁸]urotensin-II (Camarda et al., 2002). Grieco et al. (2002) replaced Cys⁵ with penicillamine in the octapeptide urotensin-II(4–11), thus generating [Pen⁵]urotensin-II(4–11) that behaved as a potent UT receptor agonist. Recently, these chemical modifications were combined with Trp⁷→DTrp⁷ substitution, leading to identification of [Pen⁵,DTrp⁷,Orn⁸]urotensin-II(4–11) (urantide). This peptide binds with high affinity to the human recombinant UT receptor and behaves as a pure, potent, and competitive UT receptor antagonist in the rat aorta bioassay (Patacchini et al., 2003).

In the present study, in order to characterize the pharmacological features of urantide, its actions were carefully reassessed in the rat aorta bioassay and compared in a calcium mobilization assay performed on CHO cells stably expressing the human UT receptor (CHO_hUT).

Section snippets

Rat aorta bioassay

All experimental protocols were approved by the ethics committee of the University of Ferrara. Male Sprague–Dawley rats (250–300 g; from Morini, Reggio Emilia, Italy) were decapitated under anaesthesia and the thoracic aortae were isolated and cut in helicoidal strips. The endothelium was rubbed and tissues were placed in organ baths containing Krebs solution at 37 °C and pH 7.4. A tension of 1 g was applied to the tissues and after 1 hour of stabilization, 1 μM noradrenaline (used as standard

Urantide antagonizes the contractile effects of urotensin-II in the rat thoracic aorta

As previously reported, urotensin-II was able to contract thoracic aortic strips in a concentration-dependent manner with high potency (pEC₅₀ 8.31±0.11). Urantide, up to 10 μM, did not evoke any contractile effect. However, at 10, 100 and 1000 nM, urantide produced a concentration dependent and parallel rightward shift of the concentration–response curve to urotensin-II, without modifying its maximal effects (Fig. 1, top panel). Schild analysis of the data is compatible with a competitive type

Discussion

Urantide has been recently reported to be a potent ligand of the UT receptor (Patacchini et al., 2003). This peptide binds with nanomolar affinity (pK_i 8.3) to the recombinant hUT and antagonizes (pK_B 8.3) the contractile effects of urotensin-II in the rat aorta without showing any residual agonist activity (Patacchini et al., 2003). Our findings confirm these data. Indeed, urantide (i) binds with similar high affinity (pK_i 7.9–9.0) to mouse, rat, and human UT receptors (Douglas, unpublished

Acknowledgements

This study was supported by funds from the University of Ferrara and from the UHL-NHS Trust.

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Urantide represents a potent UT receptor antagonist since it is about 50- to 100-fold more potent than any other compounds tested in the rat isolated aorta. Despite of the potent UT receptor antagonist activity in the rat aorta bioassay, urantide shows residual agonist activity at human recombinant assay in a calcium mobilization test [76]. In order to develop a selective antagonist, chemical modifications led to the generation of the peptide [Pen5, DTrp7,Dab8]U-II [4–11], also known as UFP-803, closely related to the urantide sequence [77].
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We hypothesize that urantide itself, may increase NO releasing directly or indirectly by elevating Ca2+ concentration in endothelium of the arteries. Because some studies have demonstrated that urantide also has a low efficacy partial agonist in a calcium mobilization in Chinese hamster ovary (CHO) cells [31] and elevated Ca2+ concentration could stimulate NO releasing from vascular endothelium [32]. Moreover, a link between NO pathway and UII has been recently described that inhaled nitroglycerin reduced pulmonary artery UII levels, indicating NO, on the other hand, also regulates UII metabolism [33], that the specific mechanism is still unknown.
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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 ∼10 min following stimulation with 10 nM) and concentration-dependent increase in norepinephrine release with pEC₅₀ and E_max (% of basal) values of 8.78 ± 0.17 (1.65 nM) 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 (<100 nM) 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.
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Short communicationUrantide mimics urotensin-II induced calcium release in cells expressing recombinant UT receptors

Abstract

Introduction

Section snippets

Rat aorta bioassay

Urantide antagonizes the contractile effects of urotensin-II in the rat thoracic aorta

Discussion

Acknowledgements

Trends Pharmacol. Sci.

J. Biol. Chem.

Neurosci. Lett.

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

Nature

A reference preparation for the study of active substances in the caudal neurosecretory system of teleosts

J. Endocrinol.

Functional and binding characterizations of urotensin II-related peptides in human and rat urotensin II-receptor assay

J. Pharmacol. Exp. Ther.

A new ligand for the urotensin II receptor

Br. J. Pharmacol.

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