Opioids, including drugs like morphine and heroin as well as endogenous opioid peptides, bind to receptors that belong to the superfamily of G-protein-coupled receptors. The best studied actions of opioids are mediated through the Gi/o family of transducers, where the effectors include inhibition of adenylyl cyclase, activation of potassium conductance and inhibition of calcium channels. Since its inception, the overall goal of this project has been to characterize the G-protein-coupled signal transduction pathways for opioid receptors in brain. During the past funding period, this goal was significantly aided by the development of agonist-stimulated [35S]GTPgS binding as a measure of receptor activation of transducers. These studies showed that the neuroanatomical resolution of this method could be dramatically extended with in vitro [35S]GTPgS autoradiography in brain sections. This development allowed us to address questions which cannot be addressed by any other technique, and explore the regional specificity of agonist efficacy. In the future planned studies for this project, this technique will be combined with other approaches to provide a molecular understanding of mechanisms of opioid signal transduction in neurons. First, the concept that opioid agonist efficacy varies in different brain regions will be tested with a combination of full and partial agonists assayed for maximal efficacy in stimulating [35S]GTPgS binding and in receptor/transducer amplification. Second, novel GTP analogs and procedures will be developed to extend the method of [35S]GTPgS binding to allow for increased anatomical resolution and to determine G-protein subtypes coupled to opioid receptor types in different regions. Third, [35S]GTPgS autoradiography will be performed in animal models such as opioid receptor transgenic knockout mice to determine whether these genetic manipulations have altered specific receptor-G-protein coupling in different brain areas. Fourth, the concepts learned by assay of [35S]GTPgS binding will be extended to a downstream target of the opioid receptor second messenger system: forskolin-stimulated expression of pro-enkephalin mRNA in mu receptor-transfected C-6 glioma cells. Finally, mechanisms of chronic opioid action on receptor-G-protein interactions will be examined in both brain sections and in mu receptor-transfected NG108-15 cells.
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