MOLECULAR STUDIES OF THE OPIATE RECEPTOR

The goal of the proposed research is the characterization of the Mu, Delta, and K opiate receptors of the brain and the elucidation of the molecular mechanism of opiate action. Opiate receptors are membrane-associated proteinaceous structures present in mammalian nervous tissue which provide the sites of action for opiates and endogenous peptides, possible neurotransmitters or hormones involved in pain perception. Purification of these receptors will involve affinity chromatography, ion-exchange chromatography, HPLC and immunoaffinity chromatography. Characterization of the solubilized receptors will emphasize the structural features of the opiate binding sites, receptor subtype size, subunit composition, conformation, and mechanism of action. Standard hybridization methods will be used in an attempt to isolate monoclonal antibodies directed against the Mu, Delta and K receptors of the brain. The monoclonal antibodies will be used 1) for further purification of the brain opiate receptors, 2) for the elucidation of the molecular basis of the receptor subtype heterogeneity, and 3) ultimately to search for the genes encoding the opiate receptors. The uniqueness of our approach involves our Mu receptor preparation, which has been purified 500-fold. The goal of our second project is the elucidation of the neuroanatomical patterns of K and Sigma receptors using light microscopy autoradiography. Visualization of these receptors will be carried out a) in the presence of Mu and Delta blocking ligands, or b) after selective protection of these sites during Beta-CNA inactivation. The goal of a third project is to determine the functional role of Mu and K opiate receptors. The appearance of Mu and K receptor sites in the developing animal will be determined and correlated with the ontogenetic patterns for Mu and K-opiate induced analgesia. The goal of our fourth project is the purification and identification of an endogenous ligand for the phencyclidine/Sigma receptors. Brain extracts have been prepared and will be purified by successive steps involving preparative and analytic-scale HPLC. Such studies are hoped to contribute significantly to the understanding of how the brain works with regard to pain perception and endogenous psychosis and how, on the molecular level, opiates and opioid peptides exert their physiological effects including tolerance. Such an understanding of narcotic action should expedite the design of therapeutic agents which are strong analgesics but do not produce dependence, and facilitate the production and use of agents of value in the therapy of patients dependent upon narcotic drugs.