| Opioid drugs are widely used for general analgesia, but have severe adverse effects limiting their clinical application. A major goal of pharmacology is to design new drugs without these side effects but retaining the analgesic actions. This requires understanding the molecular basis of opioid actions. Opioids initiate their actions by binding to specific receptors. However, the factors regulating the genes that encode these receptors have still not been determined.; I have characterized a cell line, P19 embryonal carcinoma cells, expresses all three opioid receptor types in different differentiation stages. The mu-opioid receptor is expressed only in P19 cells induced to differentiate by treatment with retinoic acid, not in undifferentiated cells, while delta- and kappa-opioid receptors are expressed in both differentiated and undifferentiated cells. The cells expressing opioid receptors are neurons and tend to form aggregates. The mu-opioid receptor expressing cells also express delta-opioid receptor and glutamic acid decarboxylase, indicating they possess the GABAergic phenotype.; The ability to activate expression of the mu-opioid receptor experimentally makes this cell line a suitable model for studying the initial appearance of mu-opioid receptor in the developing neural system. Furthermore, since the mu-opioid receptor is expressed only in aggregated cells, I have also evaluated the role of aggregation in activating expression of this receptor. Experiments in which aggregates were either enhanced or disrupted demonstrate that the expression of mu-opioid receptor is closely correlated to the state of aggregation. Furthermore, treatment of cultures with an antibody to neural cell adhesion molecule (NCAM) reduced the expression level of mu-opioid receptor but did not affect aggregation, indicating the involvement of NCAM in the pathway mediating expression of the mu-opioid receptor gene. Other components of this pathway identified include phospholipase C, arachidonic acid, N and L type calcium channels and CaM kinase II. NCAM's effect was specific to the mu-opioid receptor, not affecting either GAD or the delta-opioid receptor gene. My studies thus suggests NCAM is actively regulating mu-opioid receptor gene expression in development through cell to cell contact and activating the intracellular messenger pathways. |
