The long range goal of this project is to study the cannabinoid receptor- ligand interactions at the cellular and molecular levels. To accomplish this goal, we use receptor radioligand binding assays based on [3H]CP- 55940, a potent bicyclic cannabinoid agonist, and analyses of the G protein-regulated inhibition of adenylate cyclase as the signal transduction pathway utilized by the cannabinoid receptor in brain. Our primary source of tissue is rat brain, because of the abundance of cannabinoid receptors that are predominantly if not entirely the CB1 subtype located in that tissue, and their importance in the regulation of cannabimimetic biological effects including antinociception. We will also utilize the N18TG2 neuroblastoma cell as a model for CB1 receptors because it is a rapidly proliferating cloned cell line that possesses significant neuronal phenotypic properties and well-characterized signal transduction pathways. Differentiated HL6O macrophages will be used to examine signal transduction by CB2 receptors. CB1 and CB2 receptors transfected into CHO cells will also be utilized. The aims of the continuation of this project are to: A. Continue characterization of novel ligands, affinity ligands and positron emission tomography (PET) scanning ligands for CB1 and CB2 receptors, B. Purify, characterize, and identify the cannabinoid receptor binding activity in brain, C. Characterize the structure-activity relationship profiles for cannabinoid, aminoalkylindole and eicosanoid ligands for the CB2 receptor, and D. Develop a CB1 receptor radioligand binding assay using an antagonist ligand. It is expected that these studies will elucidate the receptor-ligand points of interaction that may differ between the CB1 and CB2 subtypes such that selective agonists for these receptors can be found or developed. The development of an antagonist ligand binding assay for the CB1 subtype will facilitate study of this receptor by allowing many types of investigations not previously feasible using an agonist assay.