MOLECULAR MECHANISM OF ADENYLYL CYCLASE SUPERACTIVATION

超出提供的空间。通过5阿片受体作用的止痛药成瘾潜力低，但与经典阿片类药物相似，在长期治疗后表现出耐受性。慢性阿片受体刺激导致腺酰环化酶(AC)活性的代偿性增加，称为AC超激活。我们已经证明，在转染人8阿片受体(HDOPJCHO)的CHO细胞中，AC同工酶(AC VI)在慢性5阿片激动剂(SNC80)作用下被磷酸化。我们推测AC VI的磷酸化参与了腺酰环化酶的过度激活，而过度激活则参与了对8种慢性阿片类激动剂的耐受。在初步实验中，我们发现G蛋白133-亚基的清除剂-转导素能减弱慢性SNC80介导的AC VI的磷酸化和AC的过度激活。在这项提案中，我们将使用其他独立的方法来研究G蛋白133亚单位参与细胞对急性和慢性SNC80治疗的反应。通过这些方法抑制AC的超激活和AC VI的磷酸化，将证实G蛋白133亚基在慢性SNC80介导的复极中的作用。随后，我们将证明，在慢性SNC80处理后，释放的133，-亚基调节hDOR/CHO细胞中第二信使调节的蛋白激酶和Raf-1蛋白激酶的活性。最后，我们将证明，耗尽hDOR/CHO细胞中负责AC VI磷酸化的蛋白激酶也可以减弱慢性8阿片激动剂介导的AC过度激活。更好地了解人类5阿片受体耐药的分子机制应该有助于开发副作用更少的长效止痛药。表演网站========================================Section End===========================================

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