New developments in understanding the mechanisms of actions of biological messengers and their receptors require research carried out on many different levels of the organism, and making use of a wide range of scientific disciplines and methodologies. This program project proposes to carry out such a wide ranging study, with its primary, though not exclusive, focus on the opioid system. The various individual proposals in this project study opioid and other receptors from levels ranging from their molecular structure to their biochemical regulation and consequences to their biological functions. Approaches used range from the pharmacological (in vivo and in vitro manipulation), biochemical (receptor characterization and purification, receptor-channel coupling), chemical (structure-activity studies), immunological (mono- and polyclonal antibodies), molecular biological (gene expression and regulation of receptors or ligands) and neuroanatomical (characterization of storage site and release of neuropeptides). Studies of receptor structure will be carried out by Conti-Tronconi, Loh and Portoghese. Conti-Tronconi will prepare monoclonal antibodies to specific amino acid sequences of the nicotinic receptor, and use these antibodies to map agonist and antagonist binding sites, intra- and extra- membrane portions of the receptor, and tissue distribution of these receptors. Loh will apply a somewhat similar approach to a recently- cloned putative delta opioid receptor. Portoghese will further characterize the binding of opioid azines to brain membranes, which his previous research has determined involves conversion of the azine to a hydrazone, which then reacts with a neighboring phosphatide in the membrane. Law and Takemori will study opioid receptor regulation, and Eide will study regulation of neuropeptides. Law will study the regulation of the putative delta opioid receptor gene, which he and his collaborators recently cloned from NG108-15 neuroblastoma-glioma hybrid cells. Takemori will test the ability of selective irreversible mu and delta antagonists to block the development of tolerance. Elde will study processes involved in the storage and release of neuropeptides. Receptor function will be studied by Raftery, Holtzman and Lee. Raftery will study the cholinergic receptor as a sodium pump. Holtzman will further characterize the role of thiol:potein disulfide oxidoreductase in hormone action, which he recently found was activated by glucagon. Lee will use mono and polyclonal antibodies to explore the role of dynorphin in inducing analgesia and/or modulating morphine-induced analgesia, in both the brain and the spinal cord.
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