Elsevier

Neuropeptides

Volume 40, Issue 2, April 2006, Pages 95-105
Neuropeptides

Distribution of nociceptin and Ro64-6198 activated [35S]-GTPγS binding in the rat brain

https://doi.org/10.1016/j.npep.2005.11.002Get rights and content

Abstract

The peptide, nociceptin, was discovered as the endogenous ligand for the opioid-like receptor, ORL1. Since its discovery, this peptide has been shown to modulate the perception of pain, modulate feeding and produce behavioral effects in rodent models of mood disorders. Recently, the non-peptide agonist {(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4,5]decan-4-one} (Ro64-6198) of the ORL1 receptor has been reported in the literature. In the present study, we compared the distribution and potency of Ro64-6198 with nociceptin for their ability to stimulate [35S]-GTPγS binding to sections of rat brain. In initial studies, Ro64-6198 inhibited 125I-nociceptin binding to the hORL1 receptors with a Ki of 1.75 nM compared with 0.20 nM for nociceptin. To assess agonist potency in a whole cell assay, a cell line expressing the hORL1 receptor and Gα15 was created and used for calcium mobilization studies. In this assay system, Ro64-6198 increased intracellular calcium with an EC50 of 52 nM compared with 24 nM for nociceptin. Having verified the agonist properties of Ro64-6198, we then assessed the potency and distribution of ORL1 receptor activation in rat brain sections. In dose–response studies, Ro64-6198 increased [35S]-GTPγS binding to a variety of brain regions with EC50 values ranging from 84.9 to 2143 nM depending on the brain regions evaluated. These potencies were similar to that seen for nociceptin, but substantially lower than values established using [125I] nociceptin binding to the cloned human ORL1 receptor. In general, the brain distribution of agonist stimulated [35S]-GTPγS binding was similar when either Ro64-6198 or nociceptin were used. Using these techniques, we have demonstrated, for the first time that Ro64-6198 activates [35S]-GTPγS binding to rat brain sections and this compound stimulates a similar population of receptors as nociceptin.

Introduction

The ORL1 receptor was identified in 1994 as the fourth opioid receptor using homology cloning techniques (Bunzow et al., 1994, Chen et al., 1994, Fukuda et al., 1994, Mollereau et al., 1994, Wang et al., 1994). This G-protein-coupled receptor had significant sequence homology with the classical opioid receptors (μ, κ and δ). Nociceptin (also termed orphanin FQ), a 17 amino acid peptide, was subsequently identified in 1995 as the endogenous ligand for this receptor (Meunier et al., 1995, Reinscheid et al., 1995). While nociceptin has a significant sequence homology with dynorphin A, it lacks the N-terminal tyrosine required for activity at the classical opioid receptors. Through the ORL1 receptor, nociceptin produces an inhibition of adenylate cyclase activity via inhibitory G protein (Gi) coupling. In addition, agonist stimulation of the ORL1 receptor modulates calcium channels in the hippocampus (Knoflach et al., 1996) and rostral ventral medulla (Vaughan et al., 2001) and potassium channels in the rat dorsal raphe, locus ceruleus, periacquaductal gray and hypothalamus (Connor et al., 1996, Vaughan and Christie, 1996, Vaughan et al., 1997, Wagner et al., 1998). Through these mechanisms, nociceptin can produce both inhibitory and disinhibitory actions in the central nervous system depending on the regional and synaptic localization.

Nociceptin and ORL1 receptors have a broad distribution in the central nervous system and, therefore, may be involved in a variety of brain functions. Intracerebroventricular or site directed injections of nociceptin into the brain has been reported to suppress spatial learning (Sandin et al., 1997), affect locomotion (Florin et al., 1996), increase food intake (Pomonis et al., 1996), reduce anxiety-like behaviors and responses to stressful stimuli (Jenck et al., 1997, Griebel et al., 1999). In addition, intracerebroventricularly administered nociceptin is reported to be pro-nociceptive and enhance the formalin-induced pain response (Zhu et al., 1997, Suaudeau et al., 1998, Wang et al., 1999). Recently, a non-peptide agonist of the ORL1 receptor has been described. This compound, {(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4,5]decan-4-one} (Ro64-6198, Fig. 1A) appears to be a selective and brain penetrant agonist of the ORL1 receptor in rodents. This compound produces anxiolytic-like effects in rodent models (Dautzenberg et al., 2001, Ciccocioppo et al., 2002), however, somewhat higher doses have been reported to produce mechanism-related neurological deficits (Higgins et al., 2001).

ORL1 receptors have been localized in the brain using a variety of techniques. Using autoradiographic methods, high levels of radiolabeled nociceptin binding have been observed in areas such as the cerebral cortex, amygdala, hypothalamus and hippocampus. Like other Gi/Go protein-coupled receptors, activation of the ORL1 receptor increases the binding of the non-hydrolyzable GTP analog, [35S]-guanylyl-5′-O-(γ-thio)-triphosphate ([35S]-GTPγS) (Adapa and Toll, 1997, Sim and Childers, 1997, Narita et al., 1999). The distribution of nociceptin enhanced [35S]-GTPγS binding to the rat mouse and guinea pig brain has been reported by several groups (Shimohira et al., 1997, Sim and Childers, 1997, Narita et al., 1999, Sim-Selley et al., 2003). In general, the enhanced [35S]-GTPγS binding closely mimics the distribution of ORL1 receptors as described utilizing direct ligand binding techniques. To date, the functional effects of Ro64-6198 in this paradigm have not been explored. Therefore, in the present study, we compared the distribution of nociceptin and Ro64-6198 enhanced [35S]-GTPγS in the rat brain to evaluate the specificity and potency of the non-peptide agonist.

Section snippets

Receptor binding assay

Receptor binding assays were conducted in a final volume of 200 μl containing 100 pM [125I]-nociceptin (New England Nuclear, Wilmington, DE) and 50 μl of cell membrane homogenate made from a stable HEK293 cell line expressing the human nociceptin receptor (Receptor Biology, Inc., Beltsville, MD). The incubation buffer contained 50 mM Tris–HCl, 1 mM MgCl2, 1 mM EDTA and 0.1% BSA (Sigma) (pH 7.4) and 5.0 μg cell protein/well with or without various concentrations of nociceptin (Peninsula, San Carlos,

Biochemical evaluation

Initial biochemical experiments were conducted to assess the potency and intrinsic activity of the non-peptide compound. Binding studies were conducted using 125I-nociceptin to the human ORL1 receptor expressed in HEK293 cells (Fig. 1B). In these studies, nociceptin was found to have a Ki of 0.20 nM while Ro64-6198 had a Ki of 1.75 nM. To assess the functional potency of the agonists, a Ca2+ mobilization assay was developed by co-transfecting cells with the ORL1 receptor and the promiscuous G

Discussion

Nociceptin has been proposed to play an important role in many centrally mediated actions. In the present study, we characterized nociceptin and a non-peptide agonist Ro64-6198 using in vitro binding and functional assays. The non-peptide agonist Ro64-6198 exhibited somewhat lower potency in both binding and functional assays when compared to nociceptin. Interestingly, both Ro64-6198 and nociceptin had substantially lower potencies in the functional assay when compared to binding. Similar data

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