Adenylyl cyclase signaling in persistent pain

慢性疼痛每年影响5000多万美国人，导致非同寻常的 个人和社会成本。雪上加霜的是，与处方阿片类药物有关的死亡 在过去的十年里，止痛药几乎翻了两番。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是不同的，这一点强调了管理。 因此，对疼痛的分子和细胞机制有深入的了解 向慢性疼痛的过渡是改善和扩大治疗选择的基础。 痛觉过敏启动是一种令人信服的向慢性疼痛过渡的模型，在这种模型中，最初的 受伤会痊愈，但会让动物处于一种预备状态，在这种状态下，第二次侮辱会导致 大大延长了疼痛反应时间。虽然之前的研究已经探索了 持续性机械过敏症，我们已经调整了这个模型来检查持续性 表达热门离子通道TRPV1的伤害性感受器介导的超敏反应 在一系列炎症和神经性疾病中会引起疼痛。建议进行的实验 这里将测试腺酰环化酶异构体AC2在热痛过敏启动中的作用， 它以前没有在感觉神经元中被表征，并且对它不敏感 阿片受体等Gi/o偶联受体的抑制作用。初步分析表明， AC2在TRPV1表达的神经元中高表达，是肿瘤发生所必需的 热痛敏引爆剂。具体目标1将检查AC2基因缺失和 急性发作时药物抑制对行为伤害性阈值的影响 痛觉过敏症和在痛觉过敏启动的背景下。具体目标2将决定是否 AC2和下游效应器EPAC通过AKAP家族成员在功能上偶联 通过免疫共沉淀-质谱学分析支架蛋白，将确定 AKAP基因缺失对EPAC信号转导和伤害性感受器功能的影响 使用基于CRISPR/CAS9的方法。具体目标3将决定以下方面的后果 EPAC依赖的PKC磷酸化途径在AC2和AKAP基因缺失中的作用 热痛觉过敏的底物分析和行为评估。这项提案将使用 探索有助于发展的新机制的创新办法 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.