Prolonged agonist exposure leads to desensitization of receptor functions in many receptor systems. Although this phenomenon is widespread and well documented, very little information is available regarding the molecular mechanisms involved. A good example with important clinical implications is the desensitization to opiates. Opiates are important analgesic drugs. However, their long-term use leads to the development of tolerance and addition. Little is known about the biochemical mechanisms by which opiates exert their analgesic action and the changes accompanying exposure to these drugs. The main objectives of this research proposal are: to elucidate the signal transduction mechanisms involved in opiate receptor action (with special emphasis on the kappa receptor); to determine the changes in the regulatory pathways following chronic opiate treatment; and to compare the above mechanisms and changes with those found with several other receptors. Our initial 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 kappa opiate agonists has lead to desensitization of opiate actions on both effector systems, and in addition to a large reduction of 2 to 3 fold in the amount of one of specific subunits of the GTP-binding proteins. Utilizing cultured neurons, control and opiate addicted animals, as well as biochemical, electrophysiological and molecular biological tools, we intend to investigate the molecular mechanisms underlying this desensitization process and reveal the relationship between the desensitization and the regulation of GTP binding proteins. This will include: studies of the regulation of GTP-binding protein subunit at the protein and mRNA levels; regulation of GTP-binding protein phosphorylation; modulation of protein kinase C activity; regulation of intracellular Ca2+ concentration and of Ca2+ channel density, etc. These studies should further our understanding of opiate functions under normal and tolerant (desensitized) conditions, could provide tools that should contribute to new clinical strategies in opiate analgesia with diminished danger of addiction and, in addition, should increase our knowledge of the molecular events governing the phenomenon of desensitization of receptor functions.
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