[Orn⁵]URP acts as a pure antagonist of urotensinergic receptors in rat cortical astrocytes

Abstract

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.

Introduction

Urotensin II (UII) is a cyclic peptide initially isolated from the caudal neurosecretory system of teleost fish [30]. UII has been subsequently identified in the brain of amphibians [11] and mammals [13], [14]. Recently, a paralog of UII termed UII-related peptide (URP, Table 1) has been characterized in mouse, rat and human [35]. Comparative genomics studies have shown that UII and URP are two members of a family of regulatory peptides that also comprises somatostatin and cortistatin [36]. Indeed, all four peptides possess a disulfide bridge and share, within their cyclic region, the Phe-Trp-Lys motif that is essential for their biological activities [12], [36].

UII and URP are the endogenous ligands of the previously orphan G-protein-coupled receptor GPR 14 [2], [23], [27], [29], [35] now renamed UT receptor. Exposure of UT-transfected cells to UII or URP causes activation of phospholipase C and an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_c) [16], [35]. The UT receptor is most similar to members of the somatostatin receptor family, notably sst4 [25], and it has been shown that somatostatin and cortistatin can, to a certain extent, increase [Ca²⁺]_c in UT-transfected cells [23]. Based on the structural homology between somatostatin receptors and UT, it is likely that the Lys residue within the cyclic region of UII and URP interacts with the conserved Asp residue in transmembrane domain 3 of UT, as previously shown for somatostatin and its receptors [28], [34].

Structure–activity relationship studies have shown the importance of the conserved hexapeptide ring of UII and URP in the biological activity of both peptides [22]. In particular, point substitution of each individual amino acid of the Trp-Lys-Tyr tripeptide by their d-enanthiomers suppresses the binding affinity and/or the contractile activity of UII [21] and URP [9]. Concurrently, molecular modeling under NMR constraints has revealed the occurrence of an inverse γ-turn centered on the Trp⁴-Lys⁵-Tyr⁶ triad of URP that is likely to play a crucial role in the biological activity of the peptide [9].

So far, the signaling mechanisms associated with UT activation have mainly been investigated on cell lines transfected with recombinant receptors [1], [16], [33], [37]. Recently, we have found that cultured rat astrocytes constitutively express the UT gene and possess high-affinity UII binding sites [8]. Exposure of cultured astrocytes to UII activates phospholipase C and increases [Ca²⁺]_c in a concentration-dependent manner [8]. Cultured rat astrocytes thus represent a suitable model in which to investigate the pharmacological characteristics of UII and URP analogs towards native UT receptors.

Owing to the potential implication of the urotensinergic system in pathophysiological conditions, notably in renal and cardiovascular diseases, several groups are currently trying to develop selective UT antagonists [3], [5], [10]. In this context, we have recently designed three URP analogs (Table 1) that were found to decrease ([d-Tyr⁶]URP) or suppress ([d-Trp⁴]URP and [Orn⁵]URP) UII-evoked contraction of rat aortic rings [9]. Paradoxically, some antagonists devoid of intrinsic contractile activity may behave as agonists at recombinant receptors, as determined by measuring their ability to induce calcium mobilization [4], [6], [7]. It is thus necessary to characterize the pharmacological profile of potential antagonists not only on UT-transfected cells but also on cells expressing native UT receptors. The aim of the present study was to examine the ability of [d-Trp⁴]URP, [Orn⁵]URP and [d-Tyr⁶]URP to interact with naturally occurring UII binding sites in rat cortical astrocytes [8] and to investigate their agonistic and antagonistic activities by using a calcium mobilization assay.