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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 K i of 1.75nM compared with 0.20nM 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 52nM compared with 24nM 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 2143nM 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. [Copyright &y& Elsevier]

Neuropeptide Y, one of the most abundant brain peptides, has been found to modulate several important biological functions via a family of G-protein coupled receptors. To investigate the localization of functional NPY receptor subtypes in the rat brain, we performed agonist-induced [35S]GTPγS autoradiography. The Y1/Y4/Y5 agonist Leu31, Pro34-NPY increased [35S]GTPγS binding in several brain areas with a regional distribution consistent with that produced when labeling adjacent sections with [125I]-Leu31, Pro34-PYY. The Y1 selective antagonist BIBP3226 antagonized the Leu31, Pro34-NPY stimulated increase in [35S]GTPγS binding in all areas examined. The Y2 agonist C2-NPY stimulated [35S]GTPγ binding in numerous brain areas with a regional distribution similar to the binding observed with [125I]-PYY 3-36. No increase in [35S]GTPγS binding above basal was observed in any brain area evaluated using Y4 and Y5 selective agonists. This study demonstrates abundant Y1 and Y2 receptor activation in the rat brain, while evidence for functional Y4 and Y5 receptors was not observed. [Copyright &y& Elsevier]