Biochemical Evidence for Orphanin FQ/Nociceptin Receptor Heterogeneity in Mouse Brain

doi:10.1006/bbrc.1996.5867

Biochemical and Biophysical Research Communications

Volume 230, Issue 2, 13 January 1997, Pages 462-465

https://doi.org/10.1006/bbrc.1996.5867 Get rights and content

Abstract

A recently identified novel peptide, orphanin FQ/nociceptin (OFQ/N), is an endogenous ligand for a unique member of the cloned opioid receptor family. Saturation studies in mouse brain membranes reveal curvilinear Scatchard plots with both a higher (K_D3.8 pM, B_max31.6 fmol/mg protein) and a lower (K_D896 pM, B_max233 fmol/mg protein) affinity site in brain tissue, compared to only a single site in transfected CHO cells (K_D36 pM). Competition studies confirm the high affinity of OFQ/N for this site, but shallow Hill slopes suggest heterogeneity. Traditional opioids have poor affinity for this receptor and OFQ/N and its fragments do not label traditional opioid receptors. In brain homogenates, both OFQ/N and OFQ/N(1-11) inhibit forskolin-stimulated camp accumulation with IC₅₀values of 1 nM or less, an action which is readily reversed by opioid antagonists. OFQ/N(1-7) shows little activity. Together, these studies suggest the presence of heterogeneous, functionally active OFQ/N receptors in mouse brain.

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Cited by (116)

Quantitative study of [Tyr <sup>10</sup>]nociceptin/orphanin FQ (1-11) at NOP receptors in rat periaqueductal gray and expressed NOP receptors in HEK293 cells
2012, Life Sciences
The nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor was reported to be functionally heterogeneous. We investigated if [Tyr¹⁰]N/OFQ(1-11), a peptide ligand reported to selectively bind to the high affinity site of ¹²⁵I-[Tyr¹⁴]N/OFQ in rodent brains, can be a tool for revealing the NOP receptor heterogeneity. We have previously founded an NOP receptor subset insensitive to Ro 64-6198 and (+)-5a Compound, two non-peptide NOP agonists, in rat ventrolateral periaqueductal gray (vlPAG) neurons. Here, we examined if [Tyr¹⁰]N/OFQ(1-11) differentiated (+)-5a Compound-sensitive and -insensitive vlPAG neurons. Certain mu-opioid (MOP) receptor ligands highly competing with [Tyr¹⁰]N/OFQ(1-11) in binding studies also showed high affinity at expressed heteromeric NOP–MOP receptors. We also examined if [Tyr¹⁰]N/OFQ(1-11) distinguished heteromeric NOP–MOP receptors from homomeric NOP receptors.
The NOP receptor activity was evaluated by G-protein coupled inwardly rectifying potassium (GIRK) currents in rat vlPAG slices, and by inhibition of cAMP accumulation in HEK293 cells expressing NOP receptors or co-expressing NOP and MOP receptors.
In vlPAG neurons, [Tyr¹⁰]N/OFQ(1-11), like N/OFQ, induced GIRK currents through NOP receptors. It was less potent (EC₅₀: 8.98 μM) but equi-efficacious as N/OFQ. [Tyr¹⁰]N/OFQ(1-11) displayed different pharmacological profiles as (+)-5a Compound, and was effective in both (+)-5a Compound-sensitive and -insensitive neurons. In NOP-expressing HEK293 cells and NOP- and MOP-co-expressing cells, [Tyr¹⁰]N/OFQ(1-11) displayed similar concentration–response curves in decreasing cAMP accumulation.
[Tyr¹⁰]N/OFQ(1-11) is an NOP full agonist and less potent than N/OFQ. However, it can neither reveal the functional heterogeneity of NOP receptors in vlPAG neurons nor differentiate heteromeric NOP–MOP and homomeric NOP receptors.
Antinociceptive effects of spinally administered nociceptin/orphanin FQ and its N-terminal fragments on capsaicin-induced nociception
2011, Peptides
Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand for the N/OFQ peptide (NOP) receptors, has been shown to be metabolized into some fragments. We examined to determine whether intrathecal (i.t.) N/OFQ (1–13), (1–11) and (1–7) have antinociceptive activity in the pain-related behavior after intraplantar injection of capsaicin. The i.t. administration of N/OFQ (0.3–1.2 nmol) produced an appreciable and dose-dependent inhibition of capsaicin-induced paw-licking/biting response. The N-terminal fragments of N/OFQ, (1–13) and (1–11), were antinociceptive with a potency lower than N/OFQ. Calculated ID₅₀ values (nmol, i.t.) were 0.83 for N/OFQ, 2.5 for N/OFQ (1–13) and 4.75 for N/OFQ (1–11), respectively. The time-course effect revealed that the antinociceptive effects of these N-terminal fragments lasted longer than those of N/OFQ. Removal of amino acids down to N/OFQ (1–7) led to be less potent than N/OFQ and its fragments, (1–13) and (1–11). Antinociception induced by N/OFQ or N/OFQ (1–13) was reversed significantly by i.t. co-injection of [Nphe¹]N/OFQ (1–13)NH₂, a peptidergic antagonist for NOP receptors, whereas i.t. injection of the antagonist did not interfere with the action of N/OFQ (1–11) and (1–7). Pretreatment with the opioid receptor antagonist naloxone hydrochloride did not affect the antinociception induced by N/OFQ and its N-terminal fragments. These results suggest that N-terminal fragments of N/OFQ are active metabolites and may modulate the antinociceptive effect of N/OFQ in the spinal cord. The results also indicate that N/OFQ (1–13) still possess antinociceptive activity through NOP receptors.
Quantitative study of the antagonistic effect of (-)-cis-1-Methyl-7-[[4-(2, 6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten- 5-ol (SB-612111) on nociceptin/orphanin FQ-mediated potassium channel activation in rat periaqueductal gray slices
2011, European Journal of Pharmacology
Nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor, a non-opioid branch of the opioid receptor family, shows structural similarities to traditional opioid receptors but binds opioid with very poor affinity. This receptor has been implicated in many physiological functions including pain regulation. This study quantitatively investigated the effect of (−)-cis-1-Methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1 -yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111), a novel non-peptide ligand of NOP receptor, on the native NOP receptors in the midbrain ventrolateral periaqueductal gray (vlPAG), a crucial region for pain regulation. SB-612111 concentration-dependently antagonized N/OFQ-induced G-protein coupled inwardly rectifying K⁺ (GIRK) current in vlPAG neurons. The IC₅₀ value of SB-612111 estimated from dose–response curves is 87.7 ± 1.2 nM. SB-612111 had no intrinsic agonistic activity and did not affect the GIRK current induced by [D-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin, a mu-opioid receptor agonist, when tested at concentrations of up to 1 μM. It is concluded that SB-612111 is a pure, potent and selective antagonist of NOP receptors that mediate GIRK channel activation in the vlPAG neurons.
Discovery of {1-[4-(2-{hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl}-1H- benzimidazol-1-yl)piperidin-1-yl]cyclooctyl}methanol, systemically potent novel non-peptide agonist of nociceptin/orphanin FQ receptor as analgesic for the treatment of neuropathic pain: Design, synthesis, and structure-activity relationships
2010, Bioorganic and Medicinal Chemistry
Neuropathic pain is a serious chronic disorder caused by lesion or dysfunction in the nervous systems. Endogenous nociceptin/orphanin FQ (N/OFQ) peptide and N/OFQ peptide (NOP) receptor [or opioid-receptor-like-1 (ORL1) receptor] are located in the central and peripheral nervous systems, the immune systems, and peripheral organs, and have a crucial role in the pain sensory system. Indeed, peripheral or intrathecal N/OFQ has displayed antinociceptive activities in neuropathic pain models, and inhibitory effects on pain-related neurotransmitter releases and on synaptic transmissions of C- and Aδ-fibers. In this study, design, synthesis, and structure–activity relationships of peripheral/spinal cord-targeting non-peptide NOP receptor agonist were investigated for the treatment of neuropathic pain, which resulted in the discovery of highly selective and potent novel NOP receptor full agonist {1-[4-(2-{hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl}-1H-benzimidazol-1-yl)piperidin-1-yl]cyclooctyl}methanol 1 (HPCOM) as systemically (subcutaneously) potent new-class analgesic. Thus, 1 demonstrates dose-dependent inhibitory effect against mechanical allodynia in chronic constriction injury-induced neuropathic pain model rats, robust metabolic stability and little hERG potassium ion channel binding affinity, with its unique and potentially safe profiles and mechanisms, which were distinctive from those of N/OFQ in terms of site-differential effects.
Nociceptin and its metabolite attenuate U0126-induced memory impairment through a nociceptin opioid peptide (NOP) receptor-independent mechanism
2010, Neurobiology of Learning and Memory
Nociceptin binds to nociceptin opioid peptide (NOP) receptors. We reported that although high doses of nociceptin impaired memory function and that these effects were mediated via NOP receptors, low doses of nociceptin attenuated the memory impairment, and these attenuating effects were not mediated via NOP receptors. Even very low doses of nociceptin were biologically active and suggested a certain binding site for this peptide, but the mechanism underlying this attenuating effect has not yet been elucidated. In the present study, we investigated the effect of an intrahippocampal injection (i.h.) of nociceptin on memory impairment induced by U0126, a MEK inhibitor, and Rp-cAMPS, a PKA inhibitor in a step-down type passive avoidance test. U0126 (2.63 nmol/mouse, i.h.) impaired memory formation and training-dependent phosphorylation of ERK2 in the hippocampus. Co-administration of nociceptin (10 fmol/mouse) significantly attenuated memory impairment, while it did not attenuate the inhibition of training-dependent phosphorylation of ERK2 induced by U0126. On the other hand, nociceptin did not attenuate memory impairment induced by Rp-cAMPS (0.448 nmol/mouse, i.h.). Nociceptin (1 fmol/mouse) also attenuated U0126 (5.26 nmol/mouse)-induced memory impairment in NOP receptor knockout mice. Nociceptin was reported to metabolize into fragments (1–13) and (14–17) in vivo, which showed pharmacological activities without affecting NOP receptors. Our findings showed that nociceptin (14–17) (1 fmol/mouse) also attenuated U0126-induced memory impairment, while nociceptin (1–13) (0.1–10 fmol/mouse) did not attenuate memory impairment. These results suggest a novel action site or mechanism for the attenuating effects of nociceptin and its metabolite, and the sequence of nociceptin (14–17) is a critical structure.
Selective and high affinity labeling of neuronal and recombinant nociceptin receptors with the hexapeptide radioprobe [<sup>3</sup>H]Ac-RYYRIK-ol
2009, Neurochemistry International
The synthetic hexapeptide Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-ol (Ac-RYYRIK-ol) represents a highly potent and selective partial agonist ligand for the nociceptin/orphanin FQ (N/OFQ) peptide receptor (nociceptin receptor, NOPr). Ac-RYYRIK-ol has been labeled with tritium yielding [³H]Ac-RYYRIK-ol with exceptionally high specific radioactivity of 94 Ci/mmol. The radioprobe is chemically stable even at 24 °C in ethanol solution for at least 4 days. No significant decomposition of the [³H]ligand occurred under the condition of the binding experiments indicating a fine enzymatic stability of the peptide. Radioreceptor binding studies were conducted using native neuronal NOPr preparation of rat brain membrane fractions and recombinant human nociceptin receptor (_hNOPr) preparations from cultured Chinese Hamster Ovary (CHO) cells stably expressing _hNOPr. Specific binding of the compound was reversible, saturable and of high affinity. No cross-reaction with the opioid receptors was observed suggesting superior NOPr selectivity of the ligand. Monophasic isotherm curves obtained in radioligand binding saturation and homologous displacement experiments indicated the presence of single binding sites in both preparations. Average densities of the [³H]Ac-RYYRIK-ol recognition sites were 237 and 749 fmol/mg protein in rat brain and transfected cells, respectively. Equilibrium affinity values (K_ds) were determined by three independent way providing identical results. In rat brain membranes K_ds of 0.3–1.3 nM were found depending upon the assay type. In homologous competition studies performed on _hNOP-CHO cell membranes almost the same binding affinities were measured for Ac-RYYRIK-ol either with [³H]Ac-RYYRIK-ol (K_i 2.8 nM) or with [³H](Leu¹⁴)nociceptin (2.3 nM). A number of NOPr and opioid ligands were screened in heterologous displacement experiments and displayed a rank order of affinity profile being consistent with fairly good NOPr selectivity of the sites labeled by [³H]Ac-RYYRIK-ol. Taken together, the high molar activity, improved chemical and biological stability and the capability of the selective and high affinity labeling make this novel radioprobe available for further exploring the biochemical pharmacology and receptor-ligand interaction of the NOP receptor.

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Regular ArticleBiochemical Evidence for Orphanin FQ/Nociceptin Receptor Heterogeneity in Mouse Brain

Abstract

Regul. Pept.

FEBS Lett.

Gene

Mol. Brain Res.

FEBS Lett.

FEBS Lett.

Regul. Pept.

Trends Pharmacol. Sci.

Mol. Pharmacol.

Synapse

Science

Regular Article
Biochemical Evidence for Orphanin FQ/Nociceptin Receptor Heterogeneity in Mouse Brain