ADENYLYL CYCLASE G BG STIMULATION AND OPIOID TOLERANCE

腺苷酸环化酶 G BG 刺激和阿片类药物耐受性

• 批准号: 6515664
• 负责人: ALAN R GINTZLER
• 金额: $ 24.58万
• 依托单位: SUNY DOWNSTATE MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2004-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6515664
• 关键词: G protein; adenylate cyclase; drug tolerance; isozymes; morphine; opioid receptor; phosphorylation; receptor coupling; tissue /cell culture

Adenylyl cyclase (AC) 'superactivation' has long held a central position in models of opioid tolerance. The enclosed application will explore a new, complementary hypothesis that opioid tolerance also results from changes in the consequences of opioid receptor activation of Gi. Specifically, it is postulated that chronic opioid treatment induces a shift from opioid receptor- Galphai inhibition to Gbetagamma (Gi-derived) stimulation of AC activity. This would result from the induction of Gbetagamma-stimulated ACs and increases in AC isoform-specific phosphorylation after chronic morphine. In chronic morphine-treated tissue and cell lines, stimulatory responsiveness of some AC isoforms, assessed in the absence of exogenous opioid, is significantly reduced. The switch from inhibitory to stimulatory opioid receptor signaling would compensate for this attenuated activity. Consequently, despite the continued presence of inhibitory concentrations of opioid, 'normal' activity of these isoforms would be maintained, i.e., opioid tolerance would ensue. Although the myenteric plexus has been of enormous value in formulating the above hypothesis, its proof will require the use of simpler, cell culture systems such as CHO and HEK 293 cell lines, stably transfected with the mu opioid receptor. The specific aims are: (1) Determine the effect of chronic opioid treatment on levels of mRNA encoding Gbetagamma-stimulated AC isoforms and AC protein. (2) Determine the effect of chronic opioid treatment on inhibitory vs stimulatory opioid receptor-AC signaling. (3) Determine if the chronic morphine-induced shift from inhibitory to stimulatory opioid receptor signaling is mediated via augmented Gbetagamma stimulation of AC. (4) Determine the specific AC isoform(s) (I, II, IV VII) and sites therein that manifest augmented phosphorylation following chronic morphine. Altered content of G proteins and opioid receptor coupling thereto has been a predominant focus of attempts to elucidate neurochemical underpinnings of tolerance. The formulation that chronic morphine induces changes in the relative abundance and phosphorylation state of specific AC isoforms which in turn alters the consequences of opioid receptor activation of Gi is novel. It represents a new approach to probing narcotic tolerance which could result in more effective pharmacotherapies for managing pain.

腺苷酸环化酶（AC）“超激活”长期以来在阿片类药物耐受性模型中占据中心地位。所附的申请将探索一个新的，互补的假设，阿片耐受性也由阿片受体激活Gi的后果的变化引起。具体来说，假设慢性阿片类药物治疗诱导阿片受体- Galphai抑制向gbetagama （gi衍生）刺激AC活性的转变。这可能是由于慢性吗啡后，β - γ刺激的AC和AC异构体特异性磷酸化的增加。在慢性吗啡处理的组织和细胞系中，在缺乏外源性阿片样物质的情况下，一些AC亚型的刺激反应性显着降低。从抑制性到刺激性阿片受体信号的转换将补偿这种减弱的活性。因此，尽管阿片类药物的抑制浓度持续存在，但这些同种异构体的“正常”活性将得到维持，即阿片类药物耐受性将随之而来。尽管肌丛在形成上述假设方面具有巨大的价值，但其证明将需要使用更简单的细胞培养系统，如CHO和HEK 293细胞系，稳定地转染mu阿片受体。具体目的是：(1)确定慢性阿片类药物治疗对gbetagama刺激的AC亚型和AC蛋白编码mRNA水平的影响。(2)确定慢性阿片治疗对抑制性和刺激性阿片受体- ac信号传导的影响。(3)确定慢性吗啡诱导的阿片受体信号从抑制性到刺激性的转变是否通过增强的谷丙酰胺刺激AC介导。(4)确定慢性吗啡后特异性AC异构体（I， II， IV， VII）及其中表现出增强磷酸化的位点。G蛋白含量的改变和与之耦合的阿片受体一直是试图阐明耐受性的神经化学基础的主要焦点。慢性吗啡诱导特异性AC亚型的相对丰度和磷酸化状态的变化，从而改变Gi的阿片受体激活的后果，这一说法是新的。它代表了一种探索麻醉耐受性的新方法，可以导致更有效的药物治疗来管理疼痛。