MOLECULAR MECHANISM OF ADENYLYL CYCLASE SUPERACTIVATION

说明(申请人提供)：通过增量阿片受体作用的止痛药成瘾潜力低，但类似于经典阿片类药物，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，在慢性5阿片激动剂(SNC80)作用下，在转染人Delta阿片受体(hDOR/CHO)的CHO细胞中，AC同工酶(AC VI)被磷酸化。我们假设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慢性增量阿片类激动剂的耐受。在初步实验中，我们发现，作为G蛋白β-伽马亚基的清除剂，α-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β-伽马亚基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β-γ亚基在慢性SNC80介导的复极中的作用。随后，我们将展示在慢性SNC80处理后释放的游离β-伽马亚基，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增量阿片激动剂介导的AC过度激活。更好地了解人类Delta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。

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.