Opiates are important analgesic drugs, but their long-term use leads to the development of tolerance and addiction. Little is known about the biochemical mechanisms by which opiates exert their analgesic action or about the changes that accompany exposure to these drugs. The long-term objectives of our research are: to elucidate the signal transduction mechanisms involved in opiate receptor action, including the mechanism by which opiates affect neurotransmitter release; to determine the changes in these pathways following chronic opiate treatment and under withdrawal conditions; and to compare the above phenomena with those found for several other receptors. Our previous experiments along this line have shown that kappa-opiate receptors in spinal cord-dorsal root ganglion cocultures are negatively coupled to adenylate cyclase and to voltage-dependent Ca2+ channels. Chronic exposure to opiate agonists led both to a desensitization of the effector systems and to changes in several alpha-subunits of GTP-binding proteins. More recently, we found that opiate agonists reduced the phosphorylation of synapsin I (a synaptic vesicle-associated protein whose phosphorylation plays an important role in regulating neurotransmitter release) and that chronic opiate treatment increased the amount of synapsin I in the cells. These results shed new light on the mechanism(s) by which opiates could inhibit neurotransmitter release and on the mechanisms of opiate dependence and withdrawal. Utilizing cultured neurons and animal models, as well as biochemical and molecular biological tools, we intend to investigate the molecular mechanism(s) underlying the opiate-induced changes in synapsin and other synaptic vesicle proteins. This will include studies of (i) the regulation of synapsins (I and II) at the protein and mRNA levels, and (ii) the regulation of synapsin phosphorylation. We will correlate these functions with the effect of opiate treatments on neurotransmitter release and on various signal transduction mediators, e.g., cAMP, intracellular Ca2+ and the activities of kinases that participate in synapsin phosphorylation. Several other synaptic vesicle proteins will be similarly investigated (e.g., synaptophysin, synaptotagmin). These studies should further our understanding of opiate functions under normal, tolerant and withdrawal conditions. They should also increase our knowledge of the molecular events governing synaptic release of neurotransmitters. In addition, the results of these studies may provide tools that will contribute to new clinical strategies in opiate analgesia with diminished danger of addiction.
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