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

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.