ADENYLYL CYCLASE G BG STIMULATION AND OPIOID TOLERANCE

腺苷酸环化酶（AC）的“超激活”长期以来一直在阿片类药物耐受模型中占据中心地位。 所附的申请将探索一种新的补充假设，即阿片类药物耐受性也是由Gi的阿片类药物受体激活的后果的变化引起的。 具体而言，假设慢性阿片类药物治疗诱导从阿片类受体- Galphai抑制转变为AC活性的Gbetagamma（Gi衍生）刺激。这可能是由于慢性吗啡后，γ-氨基丁酸刺激的AC的诱导和AC亚型特异性磷酸化的增加。 在慢性吗啡处理的组织和细胞系中，在不存在外源性阿片样物质的情况下评估的一些AC亚型的刺激反应性显著降低。 从抑制性阿片受体信号传导到刺激性阿片受体信号传导的转换将补偿这种减弱的活性。因此，尽管持续存在抑制浓度的阿片样物质，这些亚型的“正常”活性将得以维持，即，阿片类药物耐受性会随之产生。 虽然肌间神经丛在形成上述假设中具有巨大的价值，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，用μ阿片受体稳定转染。 具体目标是：（1）确定慢性阿片样物质治疗对编码γ-射线刺激的AC亚型和AC蛋白的mRNA水平的影响。 (2)确定慢性阿片类药物治疗对抑制性与刺激性阿片受体-AC信号传导的影响。(3)确定慢性吗啡诱导的阿片受体信号传导从抑制性转变为刺激性是否通过AC的增强γ射线刺激介导。 (4)确定特定的AC亚型（I，II，IV VII）和其中的位点，其在慢性吗啡后表现出磷酸化增强。 改变G蛋白和阿片受体偶联到其上的内容已经是试图阐明耐受性的神经化学基础的主要焦点。 慢性吗啡诱导特定AC同种型的相对丰度和磷酸化状态的变化，这进而改变Gi的阿片样物质受体活化的结果的制剂是新颖的。 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治疗来管理疼痛。

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.