The objective of this research is to investigate the life cycle and mechanism of action of opioid receptors in cultured neuroblastoma hybrid cells. The questions that will be addressed include: 1) What are the sites of receptor synthesis and processing within the cell? 2) How are they transported to the cell surface (which is the locus for most of the binding sites and their major site of action in signal transduction)? 3) How are they transported back into the cell? 4) Are they internalized for functional reasons as well as for degradation? The rodent neuroblastoma cells to be studied have been demonstrated to contain large amounts of a single class of opioid receptor. They are far simpler than brain in the types of receptor present, and they are more manipulatable. Opioid receptors from the cell surface will be separated from those found in various organelles within the cells by subcellular fractionation techniques. Changes in receptor concentration, ligand binding properties, and molecular weight will be assessed in the different cellular compartments as a function of time after treatment of the cells with agents that either inhibit the processing of the receptor or enhance their synthesis. Once suitably large populations of intracellular receptors are identified as being either newly synthesized enroute to the cell surface or sites being internalized, their properties will be compared. Kinetic and steady state binding studies will be performed on the receptor populations to provide information on their mechanism of signal transduction. Knowledge of receptor synthesis, action and turnover will contribute to the ultimate elucidation of the molecular mechanisms of drug dependence and tolerance. Since morphine, heroin and other related drugs of abuse act at opioid receptors of the central and peripheral nervous system, such knowledge will have an impact on rational approaches to the treatment of drug dependence.
