MOLECULAR MECHANISM OF ADENYLYL CYCLASE SUPERACTIVATION

描述（由申请方提供）：通过δ阿片受体起作用的镇痛剂具有低成瘾性，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，在用人δ阿片受体（hDOR/CHO）转染的CHO细胞中，慢性阿片类激动剂（SNC 80）处理后，AC同工酶（AC VI）被磷酸化。我们推测，腺苷酸环化酶的超活化与AC VI的磷酸化有关，而这种超活化与慢性δ阿片受体激动剂的耐受有关。在初步实验中，我们发现，α-transducin，一个假定的清道夫G蛋白β-γ-亚基，衰减慢性SNC 80介导的磷酸化AC VI和AC超活化。在这个提议中，我们将使用其他独立的方法来研究G蛋白β-γ-亚基参与急性和慢性SNC 80治疗的细胞反应。通过这些方法减弱AC超活化和AC VI磷酸化将证实G蛋白β-γ亚基在慢性SNC 80介导的再极化中的作用。随后，我们将表明，在慢性SNC 80处理后释放的游离β-γ-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耗竭也减弱慢性δ阿片受体激动剂介导的AC超活化。更好地了解人类δ阿片受体的药物耐受性的分子机制应有助于开发副作用更少的长效镇痛药。

DESCRIPTION (provided by applicant):Analgesics that act through the delta 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 5 opioid agonist (SNC 80) treatment in CHO cells transfected with the human delta opioid receptor (hDOR/CHO). We hypothesize that phosphorylation of AC VI isinvolved in adenylyl cyclase superactivation, and that superactivation is involved in tolerance to chronic delta opioid agonists. In preliminary experiments we found that alpha-transducin, a putative scavenger of G protein beta-gamma-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 beta-gamma-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 beta-gamma-subunits in chronic SNC 80-mediated respolises. Subsequently we will show that free beta-gamma-subunits, released 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 delta opioid agonist mediated AC superactivation. Better understanding of the molecular mechanisms of drug tolerance at the human delta opioid receptor should aid in the development of longer acting analgesics with fewer side effects.