ADENYLYL CYCLASE G BG STIMULATION AND OPIOID TOLERANCE

腺苷酸环化酶（AC）“超激活”长期以来在阿片类药物耐受模型中占据着核心地位。 随附的申请将探索一个新的、补充性的假设，即阿片类药物耐受性也是由于 Gi 的阿片类药物受体激活后果的变化造成的。 具体来说，假设长期阿片类药物治疗会诱导从阿片类受体 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 Gbetagamma 刺激的 AC 的诱导以及 AC 异构体​​特异性磷酸化的增加所致。 在慢性吗啡处理的组织和细胞系中，在不存在外源性阿片类药物的情况下评估的一些 AC 亚型的刺激反应性显着降低。 从抑制性阿片受体信号转为刺激性阿片受体信号传导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，但这些亚型的“正常”活性将得以维持，即阿片类药物耐受性将随之发生。 尽管肌间丛在提出上述假设方面具有巨大价值，但其证明需要使用更简单的细胞培养系统，例如稳定转染μ阿片受体的CHO和HEK 293细胞系。 具体目标是： (1) 确定长期阿片类药物治疗对编码 Gbetagamma 刺激的 AC 亚型和 AC 蛋白的 mRNA 水平的影响。 (2) 确定长期阿片类药物治疗对抑制性和刺激性阿片受体-AC 信号传导的影响。 (3) 确定吗啡诱导的慢性阿片受体信号从抑制性向刺激性转变是否是通过 AC 的 Gbetagamma 刺激增强来介导的。 (4) 确定慢性吗啡后磷酸化增强的特定 AC 亚型（I、II、IV VII）及其位点。 G蛋白及其偶联的阿片受体含量的改变一直是试图阐明耐受性的神经化学基础的主要焦点。 慢性吗啡会诱导特定 AC 异构体​​的相对丰度和磷酸化状态发生变化，进而改变阿片受体激活 Gi 的结果，这一说法是新颖的。 它代表了一种探索麻醉耐受性的新方法，可能会产生更有效的治疗疼痛的药物疗法。

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.