MOLECULAR MECHANISM OF ADENYLYL CYCLASE SUPERACTIVATION

腺苷酸环化酶超激活的分子机制

• 批准号: 6556723
• 负责人: HENRY I YAMAMURA
• 金额: $ 26.66万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6556723
• 关键词: CHO cells; adenylate cyclase; antisense nucleic acid; biological signal transduction; calcium ion; calmodulin dependent protein kinase; drug tolerance; enzyme activity; fluorimetry; gene expression; isozymes; opioid receptor; phenylamide; phosphorylation; piperazines; protein kinase A; protein kinase C; stimulant /agonist; transducin

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.

描述（由申请人提供）：通过 δ 阿片受体起作用的镇痛药具有较低的成瘾潜力，但与经典阿片类药物类似，在长期治疗后表现出耐受性。慢性阿片受体刺激会导致腺苷酸环化酶 (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，AC 同工酶 (AC VI) 在长期 5 阿片受体激动剂 (SNC 80) 处理下会被磷酸化。我们假设 AC VI 的磷酸化与腺苷酸环化酶的超激活有关，并且超激活与对慢性 δ 阿片类激动剂的耐受有关。在初步实验中，我们发现α-转导蛋白（一种假定的 G 蛋白 β-γ 亚基清除剂）可减弱 SNC 80 介导的 AC VI 慢性磷酸化和 AC 超激活。在本提案中，我们将使用其他独立的方法来研究 G 蛋白 β-γ-亚基在急性和慢性 SNC 80 治疗的细胞反应中的参与情况。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 介导的反应中的作用。随后我们将证明，长期 SNC 80 处理后释放的游离 β-γ 亚基可调节 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 hDOR/CHO 细胞中负责 AC VI 磷酸化的蛋白激酶的消耗也会减弱慢性 δ 阿片受体激动剂介导的 AC 超激活。更好地了解人类δ阿片受体药物耐受的分子机制应有助于开发具有更少副作用的长效镇痛药。