EXCEED THE SPACE PROVIDED. Analgesics that act through the 5 opioid receptor have low addictive potential but similarly to classical opiates, display tolerance after long-term treatment. Chronic opioid receptor stimulation leads to a compensatory increase in adenylyl cyclase (AC) activity, called AC superactivation. We have demonstrated that an AC isoenzyme (AC VI) is phosphorylated upon chronic 5opioid agonist (SNC 80) treatment in CHO cells transfected with the human 8 opioid receptor (hDOPJCHO). We hypothesize that phosphorylation of AC VI is involved in adenylyl cyclase superactivation, and that superactivation is involved in tolerance to chronic 8 opioid agonists. In preliminary experiments we found that o_-transducin, a putative scavenger of G protein 133,- subunits, attenuated both chronic SNC 80 mediated phosphorylation of AC VI and AC superactivation. In this proposal we will use other, independent methods, to study the involvement of G protein 133,-subunitsin cellular responses to acute- and chronic SNC 80 treatment. Attenuation of AC superactivation and AC VI phosphorylation by these methods will confirm the role of G protein 133,-subunitsin chronic SNC 80-mediated respolises. Subsequently we will show that free 133,-subunitsr,eleased upon chronic SNC 80 treatment, regulate the activity of second messenger regulated protein kinases and Raf-1 protein kinase in hDOR/CHO cells. Finally, we will demonstrate that depletion of the protein kinase responsible for phosphorylation of AC VI in hDOR/CHO cells also attenuates chronic 8 opioid agonist mediated AC superactivation. Better understanding of the molecular mechanisms of drug tolerance at the human 5 opioid receptor should aid in the development of longer acting analgesics with fewer side effects. PERFORMANCE SITE ========================================Section End===========================================
