MOLECULAR MECHANISM OF ADENYLYL CYCLASE SUPERACTIVATION

超出所提供的空间。通过5阿片受体起作用的镇痛剂具有低成瘾潜力，但与经典阿片类药物相似，在长期治疗后显示耐受性。慢性阿片受体刺激导致腺苷酸环化酶（AC）活性代偿性增加，称为AC超激活。我们已经证明，AC同工酶（AC VI）是磷酸化后，慢性5阿片受体激动剂（SNC 80）处理的CHO细胞转染人8阿片受体（hDOPJCHO）。我们推测腺苷酸环化酶的超活化与AC VI的磷酸化有关，而这种超活化与对慢性阿片受体激动剂的耐受有关。在初步的实验中，我们发现，o_-transducin，一个假定的G蛋白133，-亚基的清除剂，减弱慢性SNC 80介导的AC VI的磷酸化和AC超激活。在这个建议中，我们将使用其他独立的方法来研究G蛋白133亚基参与细胞对急性和慢性SNC 80治疗的反应。通过这些方法减弱AC超活化和AC VI磷酸化，将证实G蛋白133 β亚基在慢性SNC 80介导的再极化中的作用。随后，我们将证明，在慢性SNC 80处理后释放的游离133，-亚基调节hDOR/CHO细胞中第二信使调节蛋白激酶和Raf-1蛋白激酶的活性。最后，我们将证明，在hDOR/CHO细胞中负责AC VI磷酸化的蛋白激酶的消耗也减弱了慢性阿片类激动剂介导的AC超活化。更好地了解人类5阿片受体药物耐受性的分子机制应有助于开发副作用更少、作用更长的镇痛药。性能现场=

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===========================================