The nociceptin/orphanin FQ receptor ligand acetyl-RYYRIK-amide exhibits antagonistic and agonistic properties

Abstract

The hexapeptide acetyl-RYYRIK-amide (Ac-RYYRIK-NH₂) has recently been reported to act as partial agonist of the nociceptin/orphanin FQ (noc/OFQ) receptor expressed in CHO cells. In addition, this peptide acts as a competitive antagonist of noc/OFQ-stimulated GTPγ³⁵S binding in rat brain membranes as well as of the noc/OFQ-evoked chronotropic effect in rat cardiomyocytes. In contrast to this antagonism, in the present study, Ac-RYYRIK-NH₂ was found to behave as an agonist at noc/OFQ receptors, affecting spontaneous locomotor activity. When administered intracerebroventricularly (i.c.v.), noc/OFQ and Ac-RYYRIK-NH₂ inhibited spontaneous locomotor activity in mice with ID₅₀ of 1.1 and 0.07 nmol, respectively. Co-administration of both peptides lead to additive effects. The higher potency of Ac-RYYRIK-NH₂ could not be clearly explained by differential metabolism, because in vivo microdialysis in rat striatum and in vitro metabolic inactivation by rat and mouse brain membranes revealed extensive inactivation of both peptides. Similar to Ac-RYYRIK-NH₂, [Phe¹psi(CH₂-NH)Gly²]noc/OFQ(1–13)-NH₂ ([F/G]NC(1–13)NH₂) inhibited the noc/OFQ-stimulated GTPγ³⁵S binding in rat brain membranes (Schild constant 3.83 nM) and mouse brain sections, although several reports have shown that this peptide exhibits agonist activity of noc/OFQ in the CNS. Changes in the optimum conditions of the in vitro assay for GTP binding increased low partial agonism of Ac-RYYRIK-NH₂ in GTP binding response. To explain the discrepancy between the in vitro antagonism of G protein coupling of the noc/OFQ receptor and in vivo agonism of Ac-RYYRIK-NH₂ and of [F/G]NC(1–13)NH₂, it is suggested that low partial agonism of receptor/G protein coupling in native systems may be sufficient to evoke full biologic responses. The extent of partial agonism for GTP binding and of coupling reserve may vary in different systems, thus explaining why [F/G]NC(1–13)NH₂ and Ac-RYYRIK-NH₂ were reported to exhibit antagonist, partial agonist, or even full agonist properties, depending on the system studied.

Introduction

The heptadecapeptide FGGFTGARKSARKLANQ, named nociceptin [15] or orphanin FQ [22], is the natural ligand of the opioid receptor like (ORL1) receptor, a G protein-coupled receptor (GPCR) representing an additional member of the opioid receptor family. Although nociceptin/orphanin FQ (noc/OFQ) shows some structural analogy to opioid peptides, particularly dynorphin A, and acts at the molecular and cellular levels in almost the same way as the μ-, δ-, and κ-opioids do, it does not activate classic opioid receptors, and their associated pharmacological effects differ (reviewed in refs. [8], [16], [28]).

In order to accurately define the physiological roles of noc/OFQ, specific and high-affinity antagonists are required. A truncated analog of noc/OFQ, [Phe¹psi(CH₂-NH)Gly²]noc/OFQ(1–13)-NH₂ ([F/G]NC(1–13)NH₂), was found to act as a selective competitive antagonist of noc/OFQ in two peripheral noc/OFQ-sensitive preparations, the electrically stimulated guinea-pig ileum and mouse vas deferens [12]. Furthermore, this peptide blocked the noc/OFQ-mediated inhibition of the rat rostral ventrolateral medulla neurons [7] and increase in potassium conductance in amygdaloid neurons [14]. However, in other assay systems, especially for effects at central sites, the peptide was found to act as partial [6], [25] or even full agonist [3], [5], [11], [13], [25], [30], [31] of noc/OFQ (for a detailed analysis of the pharmacological profile of this compound see ref. 4).

From a combinatorial library of acetylated hexapeptide amides, some compounds were identified as high-affinity ligands for the ORL1 receptor transfected into CHO cells [10]. These peptides exhibited varying partial agonist activities in CHO cells [10]. It has been shown that at high levels of expression, recombinant receptors amplify any positive intrinsic activity of a receptor ligand. Hence, in vitro or in vivo antagonists may become partial agonists [18], [26]. Based on the reverse conclusion, the hexapeptide amide with the lowest activity on the ORL1 receptor in CHO cells, acetyl-RYYRIK-amide (Ac-RYYRIK-NH₂) [10], was chosen to test for antagonistic activity at the noc/OFQ receptor in an in vitro system [2]. Indeed, in rat brain preparations Ac-RYYRIK-NH₂ competitively antagonized the noc/OFQ-evoked coupling of the receptor to G proteins, as measured in GTPγS binding assays [2]. The model of receptor G protein coupling implicates that antagonizing this coupling should lead to the antagonism of all other biologic activities of noc/OFQ dependent on G protein activation. In agreement with this suggestion, Ac-RYYRIK-NH₂ competitively antagonized the chronotropic effect of noc/OFQ in rat cardiomyocytes [2]. Because of the discrepancies in the literature between the antagonism and mainly central agonism of [F/G]NC(1–13)NH₂ noted above, in the present study, we have investigated the behavior of Ac-RYYRIK-NH₂ in an in vivo assay for central activity. In apparent contrast to the G protein coupling model, this peptide was found to act as a potent agonist of the noc/OFQ receptor for inhibiting spontaneous locomotor activity in mice, and, at the same time, the central agonist [F/G]NC(1–13)NH₂ (like Ac-RYYRIK-NH₂) was shown to competitively antagonize the noc/OFQ-stimulated GTPγS binding in brain membranes.