Quantitative study of [(pF)Phe4,Arg14,Lys15]nociceptin/orphanin FQ-NH2 (UFP-102) at NOP receptors in rat periaqueductal gray slices

doi:10.1016/j.ejphar.2007.10.006

European Journal of Pharmacology

Volume 579, Issues 1–3, 28 January 2008, Pages 110-115

https://doi.org/10.1016/j.ejphar.2007.10.006 Get rights and content

Abstract

The nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor is a novel member of the opioid receptor family with little affinity for traditional opioids. This receptor and its endogenous ligand, N/OFQ, are widely distributed in the brain and are implicated in many physiological functions including pain regulation. [(pF)Phe⁴,Arg¹⁴,Lys¹⁵]N/OFQ-NH₂ (UFP-102) is a newly developed peptide agonist of NOP receptors. In this study, we quantitatively investigated the effect of UFP-102 at native NOP receptors of the ventrolateral periaqueductal gray (PAG), a crucial midbrain area involved in pain regulation and enriched with NOP receptors, using blind patch-clamp whole-cell recording technique in rat brain slices. UFP-102, like N/OFQ, induced an outward current in ventrolateral PAG neurons and increased the membrane current elicited by a hyperpolarization ramp from − 60 to − 140 mV. The current induced by UFP-102 was characterized with inward rectification and had a reversal potential near the equilibrium potential of K⁺ ions, indicating that UFP-102 activates G-protein coupled inwardly rectifying K⁺ channels. The effect of UFP-102 was concentration-dependent with the maximal effect similar to that of N/OFQ. The EC₅₀ value was 11 ± 2 nM, which is 5 fold lower than that of N/OFQ. The effect of UFP-102 was not affected by naloxone while competitively antagonized by UFP-101 ([Nphe¹,Arg¹⁴,Lys¹⁵]N/OFQ-NH₂), a potent NOP receptor antagonist, with a pA₂ value of 6.7. These results suggest that UFP-102 is a full agonist at the postsynaptic NOP receptors of the midbrain of rats and is 5 fold more potent than N/OFQ.

Introduction

A new member of the opioid receptor family was identified in 1994 when opioid receptors were cloned. It was named initially as ORL1, opioid-like orphan receptor, because it is highly homologous to classical opioid receptors but has little affinity for traditional opioids (Mollereau et al., 1994). Its endogenous agonist was simultaneously identified by two groups and was termed nociceptin (Meunier et al., 1995) or orphanin FQ (Reinscheid et al., 1995). This receptor was, thereafter, renamed after its endogenous ligand as nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor and was classified as a non-opioid branch of the opioid receptor family (NC-IUPHAR, 2004). N/OFQ has been implicated in many physiological or pathological functions, including pain regulation, stress response, feeding, locomotor activity, learning and memory, pituitary functions, and immune and cardiovascular controls (Darland et al., 1998, Peluso et al., 1998, Calo' et al., 2000, Mogil and Pasternak, 2001, Chiou et al., 2007). Heterogeneity of NOP receptors has been suggested from the findings that there are splicing variants of NOP receptor transcripts and more than one specific binding site of N/OFQ in the rodent brain (Mathis et al., 1997). Furthermore, functional heterogeneity of NOP receptors has been revealed by a receptor ligand, Ro 64-6198 (Chiou et al., 2004, Kuzmin et al., 2004). Hence, the development and characterization of NOP receptor ligands would be of help in revealing the physio/pathological roles of N/OFQ and clarifying the possible diversity of NOP receptors.

[(pF)Phe⁴,Arg¹⁴,Lys¹⁵]N/OFQ-NH₂ (UFP-102) is a novel peptide agonist of NOP receptors developed by substituting the 4th, 14th and 15th amino acid of N/OFQ with (pF)Phe, Arg and Lys, respectively (Guerrini et al., 2005). These modifications make UFP-102 more potent than N/OFQ in several assays performed in vitro on expressed recombinant NOP receptors (CHO_hNOP) and native receptors of several peripheral preparations and cortex synaptosomes (Carra et al., 2005). It also displayed longer duration of action than N/OFQ in vivo (Carra et al., 2005, Economidou et al., 2006). In this study, we have quantitatively investigated the pharmacological characteristics of UFP-102 at the postsynaptic NOP receptors of rat brain slices containing the midbrain periaqueductal gray (PAG), which is enriched with dense NOP receptors (Anton et al., 1996).

Experiments were conducted in the ventrolateral region of the PAG, which is a crucial site for morphine-induced supraspinal analgesia (Yaksh et al., 1976) and also the action site that N/OFQ reverses the antinociceptive effect of morphine (Morgan et al., 1997). Previous studies have shown that N/OFQ activates inwardly rectifying K⁺ channels, which are coupled by G-protein (Ikeda et al., 1997), in most of the tested neurons in the ventrolateral PAG (Vaughan and Christie, 1997, Chiou, 1999, Chiou, 2001, Chiou and Fan, 2002, Chiou et al., 2002, Chiou et al., 2004, Chiou et al., 2005). The effect of UFP-102 at the NOP receptors of ventrolateral PAG neurons was, therefore, quantified by the increment of this G-protein coupled inwardly rectifying K⁺ (GIRK) current. The potency and efficacy of UFP-102 were compared with those of N/OFQ.

Section snippets

Materials and methods

All animal experiments were approved by the Institutional Animal Care and Use Committee of College of Medicine, National Taiwan University. All efforts were made to minimize the number of animals used. The dissection of PAG slices, electrophysiological recordings, and data analysis were similar to that described in the previous report (Chiou et al., 2005).

Coronal midbrain slices (400 μm) containing the PAG were dissected from 9–17 day-old rats (Wistar strain) and equilibrated in the artificial

UFP-102 activated inwardly rectifying K⁺ channels.

UFP-102, at 3–3000 nM, shifted the holding current (I_hold in Fig. 1A) outwardly and increased the membrane current elicited by hyperpolarization ramps from − 60 to − 140 mV voltage-dependently in ventrolateral PAG neurons (Fig. 1B). Among 78 recorded neurons, 73 (94%) neurons were responsive to UFP-102. The current increase at more negative potentials was greater than that at less negative potentials. Thus, the current–voltage (I–V) relationship of UFP-102-induced current, which was obtained by

Discussion

In the present study, we demonstrated that UFP-102 activates GIRK channels through NOP receptor activation in rat ventrolateral PAG neurons. UFP-102 acts as a selective full agonist and is 5 times more potent than N/OFQ. To the best of our knowledge, this is the first quantitative study of UFP-102 on postsynaptic NOP receptors in brain slices containing a pain-relevant region.

The current induced by UFP-102 has a reversal potential resembling the equilibrium potential of K⁺ ions and was

Acknowledgements

This study was supported by the grants from National Health Research Institutes, Taiwan (NHRI-EX95-9506NI and NHRI-EX96-9506NI), National Science Council, Taiwan (NSC 95-2320-B002-095-MY3) and National Bureau of Controlled Drugs, Department of Health, Taiwan (DOH96-NNB-1027).

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Nociceptin (orphanin FQ) is a 17-residue neuropeptide hormone with roles in both nociception and analgesia. It is an opioid-like peptide that binds to and activates the G-protein-coupled receptor opioid receptor-like-1 (ORL-1, NOP, orphanin FQ receptor, kappa-type 3 opioid receptor) on central and peripheral nervous tissue, without activating classic delta-, kappa-, or mu-opioid receptors or being inhibited by the classic opioid antagonist naloxone. The three-dimensional structure of ORL-1 was recently published, and the activation mechanism is believed to involve capture by ORL-1 of the high-affinity binding, prohelical C-terminus. This likely anchors the receptor-activating N-terminus of nociception nearby for insertion in the membrane-spanning helices of ORL-1. In search of higher agonist potency, two lysine and two aspartate residues were strategically incorporated into the receptor-binding C-terminus of the nociceptin sequence and two Lys(i) → Asp(i + 4) side chain–side chain condensations were used to generate lactam cross-links that constrained nociceptin into a highly stable α-helix in water. A cell-based assay was developed using natively expressed ORL-1 receptors on mouse neuroblastoma cells to measure phosphorylated ERK as a reporter of agonist-induced receptor activation and intracellular signaling. Agonist activity was increased up to 20-fold over native nociceptin using a combination of this helix-inducing strategy and other amino acid modifications. An NMR-derived three-dimensional solution structure is described for a potent ORL-1 agonist derived from nociceptin, along with structure–activity relationships leading to the most potent known α-helical ORL-1 agonist (EC₅₀ 40 pM, pERK, Neuro-2a cells) and antagonist (IC₅₀ 7 nM, pERK, Neuro-2a cells). These α-helix-constrained mimetics of nociceptin(1–17) had enhanced serum stability relative to unconstrained peptide analogues and nociceptin itself, were not cytotoxic, and displayed potent thermal analgesic and antianalgesic properties in rats (ED₅₀ 70 pmol, IC₅₀ 10 nmol, s.c.), suggesting promising uses in vivo for the treatment of pain and other ORL-1-mediated responses.
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This paper is the 31st consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2008 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (Section 2), and the roles of these opioid peptides and receptors in pain and analgesia (Section 3); stress and social status (Section 4); tolerance and dependence (Section 5); learning and memory (Section 6); eating and drinking (Section 7); alcohol and drugs of abuse (Section 8); sexual activity and hormones, pregnancy, development and endocrinology (Section 9); mental illness and mood (Section 10); seizures and neurologic disorders (Section 11); electrical-related activity and neurophysiology (Section 12); general activity and locomotion (Section 13); gastrointestinal, renal and hepatic functions (Section 14); cardiovascular responses (Section 15); respiration and thermoregulation (Section 16); and immunological responses (Section 17).
1-Benzyl-N-[3-[spiroisobenzofuran-1(3H),4′-piperidin-1-yl]propyl]pyrrolidine-2-carboxamide (Compound 24) antagonizes NOP receptor-mediated potassium channel activation in rat periaqueductal gray slices
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Nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor, the fourth member of opioid receptor family, shares 60–70% homology with traditional opioid receptors but displays little affinity for opioids. This receptor was implicated in many neurological functions and its functional heterogeneity has been proposed. Therefore, it is imperative to develop and characterize new ligands for NOP receptors. 1-Benzyl-N-[3-[spiroisobenzofuran-1(3H),4′-piperidin-1-yl]propyl]pyrrolidine-2-carboxamide (Compound 24) is a new non-peptide ligand of NOP receptor having antagonistic actions in cloned and peripheral NOP receptors. In this study, we quantitatively characterized its effect on the native NOP receptors in the midbrain slices containing ventrolateral periaqueductal gray (vlPAG), a region with dense NOP receptors and involved in pain regulation. In vlPAG neurons, N/OFQ induced G-protein-coupled inwardly rectifying potassium (GIRK) current through NOP receptors. Compound 24, at 0.3–10 μM, attenuated N/OFQ-induced GIRK current concentration-dependently. The antagonistic potency of Compound 24 in vlPAG neurons (IC₅₀: 2.6 ± 0.6 μM) was, however, lower than that obtained in mouse vas deferens preparations or expressed human NOP receptors. The action kinetic of Compound 24 was slower than [Nphe¹, Arg¹⁴, Lys¹⁵]N/OFQ-NH₂ (UFP-101), a peptide antagonist, in the same preparation. Compound 24 had no intrinsic agonistic activity at NOP receptors at the concentration up to 10 μM. However, at concentrations higher than 3 μM, it also attenuated the GIRK current induced by [D-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin, a mu-opioid receptor agonist. It is concluded that Compound 24 acts as a pure antagonist at the native NOP receptors in the vlPAG with moderate potency and selectivity.
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Quantitative study of [(pF)Phe4,Arg14,Lys15]nociceptin/orphanin FQ-NH2 (UFP-102) at NOP receptors in rat periaqueductal gray slices

Abstract

Introduction

Section snippets

Materials and methods

UFP-102 activated inwardly rectifying K+ channels.

Discussion

Acknowledgements

J. Bioorg. Med. Chem.

Neuroscience

Neuropharmacology

Neuropharmacology

Eur. J. Pharmacol.

Trends Neurosci.

Peptides

Brain Res. Mol. Brain Res.

Biochem. Biophys. Res. Commun.

FEBS Lett.

J. Neuroimmunol.

Peptides

Brain Res.

Immunohistochemical localization of ORL-1 in the central nervous system of the rat

J. Comp. Neurol.

Pharmacology of nociceptin and its receptor: a novel therapeutic target

Br. J. Pharmacol.

UFP-101. A peptide antagonist selective for the Nociceptin/orphanin FQ receptor

CNS Drug Rev.

[(pF)Phe4,Arg14,Lys15]N/OFQ-NH2 (UFP-102), a highly potent and selective agonist of the nociceptin/orphanin FQ receptor

J. Pharmacol. Exp. Ther.

Hyperpolarization by opioids acting on mu-receptors of a sub-population of rat periaqueductal gray neurones in vitro

Br. J. Pharmacol.

Quantitative study of [(pF)Phe⁴,Arg¹⁴,Lys¹⁵]nociceptin/orphanin FQ-NH₂ (UFP-102) at NOP receptors in rat periaqueductal gray slices

UFP-102 activated inwardly rectifying K⁺ channels.

[(pF)Phe⁴,Arg¹⁴,Lys¹⁵]N/OFQ-NH2 (UFP-102), a highly potent and selective agonist of the nociceptin/orphanin FQ receptor