ADENYLYL CYCLASE G BG STIMULATION AND OPIOID TOLERANCE

腺酰环化酶(AC)“超激活”长期以来在阿片类药物耐受模型中占据中心地位。封闭式申请将探索一个新的补充性假设，即阿片耐受性也是由胃肠道阿片受体激活后果的变化引起的。具体地说，假设慢性阿片类药物治疗诱导从阿片受体-Galphai抑制转变为Gbetagamma(Gi来源)对AC活动的刺激。这可能是由于Gbetagamma刺激的ACS的诱导和慢性吗啡后AC异构体特异性磷酸化的增加所致。在慢性吗啡治疗的组织和细胞系中，在没有外源性阿片类药物的情况下评估的某些AC亚型的刺激反应性显著降低。从抑制性阿片受体信号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继续存在，但这些异构体的“正常”活性将保持不变，即阿片类药物耐受性将随之而来。尽管肌间神经丛在阐明上述假说方面具有巨大的价值，但要证明这一假说，需要使用更简单的细胞培养系统，如CHO和HEK 293细胞系，这些细胞系稳定地转染了Mu阿片受体。其具体目的是：(1)确定慢性阿片类药物治疗对Gbetagamma刺激的AC异构体和AC蛋白编码水平的影响。(2)确定慢性阿片类药物治疗对抑制和刺激阿片受体-AC信号的影响。(3)确定慢性吗啡诱导的从抑制性阿片受体信号向刺激性阿片受体信号的转变是否通过增强AC的Gbetagamma兴奋来实现。(4)确定慢性吗啡后特异性AC异构体(S)(I、II、IV、VII)及其磷酸化增强的位点。G蛋白和阿片受体偶联的含量变化一直是试图阐明耐受的神经化学基础的主要焦点。慢性吗啡导致特定AC亚型的相对丰度和磷酸化状态发生变化，进而改变胃肠道阿片受体激活的后果，这一提法是新的。它代表了一种探索麻醉耐受性的新方法，可能会导致更有效的药物疗法来控制疼痛。

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.