Jean-Claude Meunier

ORL1, a novel member of the opioid receptor family : cloning, functional expression and localization

Selective PCR amplification of human and mouse genomic DNAs with oligonucleotides encoding highly conserved regions of the δ‐opioid and somatostatin receptors generated a human DNA probe (hOP01, 761 bp) and its murine counterpart (mOP86, 447 bp). hOP01 was used to screen a cDNA library from human brainstem. A clone (named hORL1) was isolated, sequenced and found to encode a protein of 370 amino acids whose primary structure displays the seven putative membrane‐spanning domains of a G protein‐coupled membrane receptor. The hORL1 receptor is most closely related to opioid receptors not only on structural (sequence) but also on functional grounds: hORLl is 49–50% identical to the murine μ‐, δ‐ and κ‐opioid receptors and, in CHO‐K1 cells stably transfected with a pRc/CMV:hORLl construct, ORL1 mediates inhibition of adenylyl cyclase by etorphine, a ‘universal’ (nonselective) opiate agonist. Yet, hORLl appears not to be a typical opioid receptor. Neither is it a somatostatin or σ (N‐allylnormetazocine) receptor. mRNAs hybridizing with synthetic oligonucleotides complementary to mOP86 are present in many regions of the mouse brain and spinal cord, particularly in limbic (amygdala, hippocampus, septum, habenula,⋯) and hypothalamic structures. We conclude that the hORL1 receptor is a new member of the opioid receptor family with a potential role in modulating a number of brain functions, including instinctive behaviours and emotions.

Nociceptin stimulates locomotion and exploratory behaviour in mice.

The recently characterized heptadecapeptide nociceptin, the endogenous agonist of the orphan opioid receptor-like 1 (ORL1 receptor), has been tested for its effects on locomotion and exploratory behaviour in mice. I.c.v. administration of as little as 10 ng of nociceptin/animal stimulated locomotor activity. This effect was dose-dependent, increasing in intensity up to 100 ng and in duration for doses in the range of 1000-10000 ng. The stimulation of horizontal locomotion elicited by 100 ng nociceptin was accompanied by a stimulation of the vertical component of locomotion. These effects were not reversed by high doses (1.5 and 4.5 mg/kg s.c.) of the opioid receptor antagonist naloxone. Increasing doses of the dopamine D2 receptor antagonist haloperidol (0.1-0.5 mg/kg i.p.) as well as of the dopamine D1 receptor antagonist SCH 23390 [R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine hydrochloride] (0.0075-0.03 mg/kg s.c.) reversed this effect, suggesting that nociceptin exerts its motor-stimulant actions by increasing central dopaminergic transmission. Nociceptin was also found to increase the number of head dips in the hole-board test, indicating that the peptide stimulates exploratory behaviour.

Autoradiographic localization of [3H]nociceptin binding sites in the rat brain

The binding sites of nociceptin (also named orphanin FQ), the endogenous ligand of ORL1 (opiate receptor like 1), were localized in rat brain, using an autoradiographic procedure. High levels of binding were observed in the cingulate, retrosplenial, perirhinal, insular and occipital cortex, anterior and posteromedial cortical amygdaloid nuclei, basolateral amygdaloid nucleus, amygdaloid complex, posterior hippocampus, dorsal endopiriform, central medial thalamic, paraventricular, rhomboid thalamic, suprachiasmatic, ventromedial hypothalamic nuclei, mammillary complex, superficial gray layer of the superior colliculus, locus coeruleus, dorsal raphe nucleus. More moderate labelling was observed in the prefrontal, fronto-parietal, temporal, piriform cortex, dentate gyrus, anterior olfactory nucleus, olfactory tubercle, shell of nucleus accumbens, claustrum, lateral septum, laterodorsal thalamic, medial habenular, subthalamic, reuniens thalamic nuclei, subiculum, periaqueductal grey matter and pons. A lower binding site density was observed in the anterior and medial hippocampus, olfactory bulb, caudate putamen, the core of the nucleus accumbens, medial septum, ventrolateral, ventroposterolateral and mediodorsal thalamic nuclei, lateral and medial geniculate nuclei, hypothalamic area, substantia nigra, ventral tegmentum area and interpedoncular nucleus. A moderate and similar labelling was found in the dorsal and ventral horn of the spinal cord. No labelling was apparent in the corpus callosum. Thus, it appears that the ORL1 receptor is particularly abundant in the cerebral cortex, limbic system of the rat brain and some areas involved in pain perception.

Distribution of the nociceptin and nocistatin precursor transcript in the mouse central nervous system

The distribution of prepronociceptin messenger RNA, the recently identified endogenous ligand of the ORL1 receptor (opioid receptor-like-1), has been studied in the adult mouse central nervous system using in situ hybridization. Prepronociceptin is a new peptide precursor that generates, upon maturation, at least three bioactive peptides: nociceptin, noc2 and the recently described nocistatin. Considering both the density of labeled neurons per region and their intensity of labeling, the distribution of prepronociceptin messenger RNA-containing neurons can be summarized as follows: the highest level of prepronociceptin messenger RNA expression was detected in the septohippocampal nucleus, bed nucleus of the stria terminalis, central amygdaloid nucleus, and in selective thalamic nuclei such as the parafascicular, reticular, ventral lateral geniculate and zona incerta. High to moderate levels of prepronociceptin messenger RNA expression were detected in the lateral, ventral and medial septum, and were evident in brainstem structures implicated in descending antinociceptive pathways (e.g., the gigantocellular nucleus, raphe magnus nucleus, periaqueductal gray matter), and also observed in association with auditory relay nuclei such as the inferior colliculi, lateral lemniscus nucleus, medioventral preolivary nucleus and lateral superior nucleus. A moderate level of prepronociceptin messenger RNA expression was observed in the medial preoptic nucleus, ventromedial preoptic nucleus, periventricular nucleus, pedonculopontine tegmental nucleus, solitary tract nucleus and spinal trigeminal nucleus. A weak level of prepronociceptin messenger RNA expression was present in some areas, such as the cerebral cortex, endopiriform cortex, hippocampal formation, medial amygdaloid nucleus, anterior hypothalamic area, medial mammillary hypothalamic nuclei, retrorubral field and substantia nigra pars compacta. No labeled cells could be found in the caudate-putamen, nucleus accumbens and ventral tegmental area. The present data confirm that nociceptin is expressed in a broad array of regions of the central nervous system. In good correlation with the presently known physiological actions of nociceptin, they include, amongst others, brain areas conveying/integrating pain and auditory sensory afferences.

Comparison of the structure‐activity relationships of nociceptin and dynorphin A using chimeric peptides

The aim of the present study was to delineate the functional domains of nociceptin (noc), a neuropeptide which is structurally related to dynorphin A (dyn). The binding and biological potencies towards the nociceptin (ORL1) and dynorphin A (κ‐opioid) receptors of twenty dyn/noc and noc/dyn hybrid peptides were compared with those of the parent heptadecapeptides. Replacement of as many as eleven residues in the C‐terminus of dynorphin by the corresponding nociceptin sequence has no significant effect on binding and biological activity towards the κ‐opioid receptor. In marked contrast, replacement of as few as six residues (RKLANQ) in the C‐terminus of nociceptin by the corresponding dynorphin sequence (LKWDNQ) dramatically impairs both affinity and activity towards the ORL1 receptor. This clearly indicates that the two neuropeptides have different functional architectures, despite the dual structural homology of both ligands and receptors. Moreover, the recombinant peptide approach led us to identify hybrids whose sequences differ only at positions 5 and 6 and displaying opposite or no receptor selectivity. One contains the dynorphin Leu5‐Arg6 sequence and prefers the κ‐opioid receptor, whereas the other comprises the nociceptin Thr5‐Gly6 sequence and prefers the ORL1 receptor. A third, containing the mixed dynorphin/nociceptin Leu5‐Gly6 sequence, does not discriminate between the two types of receptor.

Autoradiographic localization of [3H]nociceptin binding sites from telencephalic to mesencephalic regions of the mouse brain

The binding sites of [3H]nociceptin (also named Orphanin FQ), the endogenous ligand of the ORL1 (opiate receptor like 1) receptor, were localized in the central nervous system of the mouse using an autoradiographic procedure. A high density of binding sites was seen in the cerebral cortex, paraventricular nucleus of the thalamus, amygdaloid complex, suprachiasmatic nucleus, medial thalamus and medial geniculate nucleus. Moderate binding was observed in the nucleus accumbens, lateral septum, lateral thalamus, hippocampus, periaqueductal grey matter and pons. Finally, low levels of binding were seen in the striatum, olfactory tubercle, hypothalamus and substantia nigra. Thus, it appears that the ORL1 receptor is particularly abundant in the cerebral cortex and limbic system of the mouse brain.

The nociceptin (ORL1) receptor: molecular cloning and functional architecture

Nociceptin and the ORL1 receptor share high sequence similarity with opioid peptides, particularly dynorphin A, and their receptors. However, nociceptin and dynorphin A may use distinct molecular pathways to bind and activate their cognate receptors. Activation of the kappa-opioid receptor by dynorphin A is thought to require interactions of its N-terminal hydrophobic domain (Y(1)GGF) with the receptor opioid binding pocket, located within the transmembrane helix bundle, while activation of the ORL1 receptor appears to require interactions of the positively charged core (R(8)KSARK) of nociceptin with the negatively charged second extracellular receptor loop.

Replacement of Gln280 by His in TM6 of the human ORL1 receptor increases affinity but reduces intrinsic activity of opioids

The ORL1 ( pioid eceptor‐ ike) receptor is the G protein‐coupled receptor whose amino acid sequence is closest to those of opioid receptors. Residues that are conserved in ORL1 and the three types of opioid receptor, but also a residue, His in the sixth putative transmembrane (TM6) helix, which is present in all opioid receptor types but absent in ORL1, appear to play a key role in receptor recognition and/or activation. Here we have sought to create an opioid binding pocket in the non‐opioid ORL1 receptor by replacing residue Gln280 in its TM6 by the corresponding His residue of opioid receptors. The mutation affects neither the affinity of nociceptin ‐ the natural ORL1 agonist ‐ for the receptor, nor the potency of nociceptin to inhibit adenylyl cyclase via ORL1. In contrast, we find that a few opioid ligands, the agonists lofentanil, etorphine and dynorphin A, and especially the antagonists diprenorphine and nor‐BNI, bind the mutant Q280H receptor with substantially (5‐ to > 100‐fold) higher apparent affinity than they do the wild‐type receptor. Moreover, lofentanil and etorphine no longer act as pure agonists, as they do at the native ORL1 receptor, but are endowed with clear antagonist properties at the mutant receptor. The mutation Q280H, which increases affinity while decreasing intrinsic activity of opioids at ORL1, emphasizes the importance of the His residue for opioid recognition and activation.

Orphan neuropeptide NocII, a putative pronociceptin maturation product, stimulates locomotion in mice.

NOCII is a heptadecapeptide whose sequence lies immediately downstream of nociceptin, the newly discovered natural agonist of the ORL1 receptor, in pronociceptin, nociceptins precursor polypeptide. Since the sequence of NocII is framed by putative convertase excision sites and it totally conserved across murine and human species, we have sought to determine whether this orphan neuropeptide might by physiologically significant, i.e. endowed with central biological activity in vivo. Intracerebroventricular administration of 10 and 100 ng of NocII increased locomotion in mice. However, unlike nociceptin, which stimulates both the horizontal and vertical (rearing) components of locomotion, NocII affected only the horizontal component. The motor stimulant action of NocII appears to depend largely on dopamine transmission since it is totally reversed by the D1 or the D2 dopamine receptor antagonists SCH 23390 and haloperidol. NocII does not modify the number of explored holes in the hole board test, indicating that, unlike nociceptin, the orphan peptide does not affect exploratory behavior in mice.

Nociceptin-induced apparent hyperalgesia in mice as a result of the prevention of opioid autoanalgesic mechanisms triggered by the stress of an intracerebroventricular injection

Summary— The effects on nociperception of nociceptin/Orphanin FQ (noc/OFQ), the endogenous ligand of the ORL1 (opioid receptor like 1) receptor, have been evaluated in mice upon intracerebroventricular injection of 10 to 10 000 ng doses of the peptide. In the hot plate test (55 °C) the licking, rearing and jump latencies were significantly reduced by noc/OFQ (100–250 ng). Noc/OFQ (100–1000 ng) also reduced the latency to tail withdrawal in the tail flick test. In the formalin test (injection in a hind paw of a formalin solution), noc/OFQ (100 ng) increased significantly the duration of paw licking and/or biting at the earliest period of observation. In the writhing test, the number of writhes evoked by intraperitoneal administration of dilute acetic acid was not modified by noc/OFQ at doses in the range of 10–1000 ng, but was decreased by 10 000 ng. The reduction in jump latency in the hot plate test was observed even when mice were pretreated with morphine (2 mg/kg, sc). The analgesic effect of acetorphan (5 mg/kg, iv) was also reduced by nociceptin (100 ng); on the other hand the hyperalgesic effect of naloxone (4.5 mg/kg, sc) was not additive with that of nociceptin (100 ng). Comparing in various tests the nociceptive thresholds of uninjected mice to that of saline icv injected mice, it appeared that the latter injection induced an increase in these thresholds which was prevented by nociceptin. It is suggested that nociceptin displays hyperalgesic effects by preventing autoanalgesic (opioidergic) mechanisms triggered by the stress elicited by intracerebroventricular injection.