Physiological Implications of Opioid Receptor Regulation

描述（由申请人提供）：吗啡作为缓解疼痛的金标准，在临床上受到一些不良特性的限制，包括耐受性、依赖性和呼吸抑制的发作。阿片类镇痛药，如吗啡，主要通过激活mu阿片受体（muOR）来调节其生物学效应。muOR是一种G蛋白偶联受体（GPCR），受GPCR激酶（GRK）磷酸化和随后在受体脱敏过程中与β -抑制素结合的调节。基因消融β - arrestin2 （β - aarr2）在小鼠中具有看似矛盾的效果；它会增强和延长吗啡镇痛，并显著降低吗啡耐受性。此外，这些小鼠的身体依赖性没有变化；相比之下，几乎消除了呼吸抑制。吗啡在这个小鼠模型中的作用接近一个假设的，最佳的阿片类镇痛药用于临床使用。在β aarr2基因敲除（β aarr2- ko）小鼠中，与吗啡镇痛相关的脑区中muOR与G蛋白的偶联升高，这可能有助于增强镇痛反应。然而，β aarr2在吗啡诱导的呼吸抑制中的作用尚不清楚。此外，虽然吗啡镇痛在β - r2- ko小鼠中增强，但对其他阿片类药物（如芬太尼和美沙酮）的反应没有改变。我们假设GRKs和β -拘捕素调节muORs，这种调节的特异性由船上的阿片激动剂决定。因此，muOR水平上的这些调节差异可能是吗啡、芬太尼、美沙酮和丁丙诺啡等多种临床相关阿片类药物产生不同药理作用的基础。通过研究缺乏单个GRKs （GRK2、GRK3、GRK4、GRK5和GRK6）或betarr2的小鼠品系中阿片介导的行为和生理反应，我们有独特的机会来评估GRKs和betarr2对体内阿片反应的贡献。神经化学变化将在这些动物身上进行评估，同时进行行为研究，重点是受体运输、受体脱敏和下游神经适应性变化。HEK-293细胞将被用作模型系统，以进一步阐明观察到的体内现象的分子机制。本研究的总体目标是在确定药物对生理和病理条件的特异性作用时，对muOR调节有更深入的了解。为此，这些研究重点研究了GRKs和parrestins在阿片耐受性（AIM I）、依赖性（AIM II）和阿片副作用（AIM III）的发展中对muOR调控的贡献。阐明muOR调控的复杂性可能指向微调受体反应，以提高镇痛疗效，限制滥用责任和消除阿片类药物治疗的不良副作用。

DESCRIPTION (provided by applicant): Morphine, the gold standard for pain relief, is clinically limited by several adverse properties including tolerance, dependence and the onset of respiratory suppression. Opiate analgesics, such as morphine, mediate their biological effects mainly via activation of the mu opioid receptor (muOR). The muOR, a G protein coupled receptor (GPCR), is regulated by GPCR kinase (GRK) phosphorylation and subsequent binding of betaarrestins in a process known as receptor desensitization. The genetic ablation of betaarrestin2 (betaarr2)in mice has seemingly paradoxical effects; it leads to enhanced and prolonged morphine analgesia and dramatically reduces morphine tolerance. Moreover, these mice display no change in physical dependence; in contrast, respiratory suppression is practically eliminated. The effects of morphine in this mouse model approach a hypothetical, optimal opiate analgesic for clinical use. The coupling of the muOR to G proteins is elevated in brain regions associated with morphine analgesia in the betaarr2knockout (betaarr2-KO) mice which may contribute to the enhanced analgesic responses. However, the role of betaarr2 in morphine-induced respiratory suppression is unclear. Furthermore, while morphine analgesia is enhanced in the betaarr2-KO mice, lresponses to other opiates such as fentany and methadone, are unaltered. We have hypothesized that GRKs and betaarrestins regulate muORs and the specificity of this regulation is determined by the opiate agonist onboard. These regulatory differences at the level of the muOR may thereby underlie the diverse pharmacological effects produced by a wide-range of clinically relevant opiates such as morphine, fentanyl, methadone and buprenorphine. We have the unique opportunity to evaluate the contributions of GRKs and betarr2 to opiate responses in vivo by studying opiatemediated behaviors and physiological responses in strains of mice lacking individual GRKs (GRK2, GRK3, GRK4, GRK5, and GRK6) or betarr2. Neurochemical alterations will be assessed in these same animals in parallel to the behavioral studies with a focus on receptor trafficking, receptor desensitization, and downstream neuroadaptive changes. HEK-293 cells wil be used as a model system to further elucidate the molecular mechanisms underlying the observed in vivo phenomena. The overall objective of this study is to gain a greater understanding of muOR regulation in determining the specificity of drug effects on physiological and pathological conditions. Toward this goal, these studies focus on examining the contribution of GRKs and parrestins to muOR regulation in the development of opiate tolerance (AIM I), dependence (AIM II), and the side effects induced by opiates (AIM III). Illuminating the intricacies of muOR regulation may point to fine-tuning receptor responsiveness to increase analgesic efficacy, limit abuse liabilityand eliminate adverse side effects in developing opiate pharmaceutical therapies.