Adenylyl cyclase signaling in persistent pain

慢性疼痛每年影响超过5000万美国人，导致非常严重的疾病。 个人和社会成本。更让人进退两难的是， 止痛药在过去十年里几乎翻了两番。疼痛的临床挑战 有证据表明慢性疼痛与急性疼痛在机制上不同， 疼痛，因此，深入了解分子和细胞机制的基础上， 向慢性疼痛的转变是改善和扩大治疗选择的根本。 痛觉过敏启动是一个令人信服的模型过渡到慢性疼痛，其中最初的 伤害解决，但使动物处于第二次伤害诱导的启动状态， 极大地延长了疼痛反应。虽然以前的研究已经探讨了发展， 持续的机械超敏反应，我们已经调整了这个模型，以检查持续的机械超敏反应。 由表达热门控离子通道TRPV 1的伤害感受器介导的超敏反应， 可以在一系列炎症和神经性疾病中引起疼痛。拟议的实验 这里将测试腺苷酸环化酶同种型AC 2对热痛觉过敏引发的贡献， 它以前在感觉神经元中没有被表征，并且对 通过Gi/o偶联受体如阿片受体的抑制。初步分析表明， AC 2在TRPV 1表达神经元中高度表达，并且是以下表现所必需的： 热痛觉过敏启动。具体目标1将检查AC 2基因缺失的影响， 对基线时行为伤害性阈值的药理学抑制，急性 痛觉过敏和在痛觉过敏启动的情况下。具体目标2将决定是否 AC 2和下游效应子Epac通过AKAP家族成员在功能上偶联。 支架蛋白的免疫共沉淀-质谱，并将确定 AKAP基因缺失对Epac信号传导和伤害感受器功能的影响 使用基于CRISPR/Cas9的方法。具体目标3将决定 AC 2和AKAP基因缺失通过Epac依赖性PKC磷酸化介导的Epac功能 热痛觉过敏的底物分析和行为评估。该提案将使用 创新办法，探索有助于发展 持续性痛觉过敏

Chronic pain affects more than 50 million Americans per year, resulting in extraordinary personal and societal costs. Adding to the dilemma, deaths involving prescription opiate analgesics have almost quadrupled in the last ten years. The clinical challenge of pain management is underscored by evidence that chronic pain is mechanistically distinct from acute pain, therefore a thorough understanding of the molecular and cellular mechanisms underlying the transition to chronic pain is fundamental to improving and expanding treatment options. Hyperalgesic priming is a compelling model of the transition to chronic pain in which an initial injury resolves, but leaves the animal in a primed state in which a second insult induces a greatly prolonged pain response. While previous studies have explored the development of sustained mechanical hypersensitivity, we have adapted this model to examine sustained hypersensitivity mediated by nociceptors expressing the heat-gated ion channel TRPV1, which can drive pain in a range of inflammatory and neuropathic conditions. Experiments proposed here will test the contribution to heat hyperalgesic priming of adenylyl cyclase isoform AC2, which has not been previously characterized in sensory neurons, and which is insensitive to inhibition by Gi/o-coupled receptors such as opioid receptors. Preliminary analysis indicates that AC2 is highly expressed in TRPV1-expressing neurons and required for the manifestation of heat hyperalgesic priming. Specific Aim 1 will examine the impact of AC2 gene deletion and pharmacological inhibition on behavioral nociceptive thresholds at baseline, in acute hyperalgesia and in the setting of hyperalgesic priming. Specific Aim 2 will determine whether AC2 and the downstream effector Epac are functionally coupled through AKAP family member scaffolding proteins by co-immunoprecipitation-mass spectrometry, and will determine the impact of deleting identified AKAP genes on Epac signaling and nociceptor function in vitro using a CRISPR/Cas9-based approach. Specific Aim 3 will determine the consequences for Epac function of AC2 and AKAP gene deletion through Epac-dependent PKC phospho- substrate profiling and behavioral assessment of heat hyperalgesia. This proposal will use innovative approaches to explore novel mechanisms contributing to the development of persistent hyperalgesia.