Mechanisms of cAMP signaling that drive spontaneous activity in nociceptors

项目总结： 外周或中枢神经系统损伤引起的慢性疼痛(神经病理性疼痛) 臭名昭著的抗拒治疗，而驱动和/或维持慢性疼痛的机制仍然存在 不清楚。我们已经证明，严重损伤后慢性伤害性感受器的过度兴奋性是由 CAMP信号通过多个cAMP效应器，包括PKA、EPAC和HCN通道。这些 AKAP介导的AC与AKAP形成复合体增强了通路，并显示出显著的串扰 使用RAS/MAPK信号。CAMP和RAS介导的通路在血浆中开始激活 膜(PM)，并且对PM内的脂类聚集唯一敏感我们还展示了 脊髓损伤减少了GαI对AC的抑制，导致阿片类药物在背根节的效力降低 神经元。这种敏感度的降低可以通过一夜之间在幼稚动物的DRG神经元中模拟出来 暴露于神经营养因子或通过5分钟的温和去极化接近放电 严重损伤后背根神经节神经元的阈值。重要的是，伤害性感受器的过度兴奋和减少 由损伤、神经营养因子或急性去极化诱导的阿片类药物效力 通过抑制RAS依赖的信号或质膜中脂类的重组而逆转。 我们假设，在许多损伤模型中发生的持续去极化会导致改变。 在PM的脂质组织中，导致ERK信号的增加和阿片类药物反应的减少。发布 神经营养因子加强了这些通路，并与cAMP信号一起，驱动 伤害性感受器过度兴奋和慢性疼痛状态。为了解决这些假设，我们提出了三个假设 目标。1)确定C-Raf降低MOR-GαI抑制AC的机制，2)确定 去极化和脊髓损伤引起的RAS激活和伤害性感受器超兴奋性的机制 去极化与cAMP、C-Raf和细胞信号之间相互作用的功能后果 厄克。重要的是，我们的模型确定了多个FDA批准的药物，这些药物可以同时增强 严重创伤后内源性阿片类反应和阻断伤害性感受器的过度兴奋性。

Project Summary: Chronic pain caused by injury to the peripheral or central nervous system (neuropathic pain) is notoriously resistant to treatment, while the mechanisms that drive and/or maintain chronic pain remain unclear. We have shown that chronic nociceptor hyperexcitability after severe injury is maintained by cAMP signaling through multiple cAMP effectors, including PKA, EPAC and HCN channels. These pathways are enhanced by AKAP-mediated complex formation with AC and show significant cross-talk with Ras/MAPK signaling. Activation of cAMP- and Ras-mediated pathways initiate at the plasma membrane (PM) and are uniquely sensitive to clustering of lipids within the PM. We have also shown that spinal cord injury reduces AC inhibition by Gαi, resulting in reduced potency of opioids in DRG neurons. This reduced sensitivity can be mimicked in DRG neurons from naïve animals by overnight exposure to neurotrophic factors or by a 5 min, modest depolarization that approaches the firing threshold of DRG neurons after severe injury. Importantly, nociceptor hyperexcitability and reductions in opioid potency, induced by either injury, neurotrophic factors or acute depolarization, can be reversed by inhibition of Ras-dependent signaling or reorganization of lipids in the plasma membrane. We hypothesize that the sustained depolarization that occurs in many injury models drives alterations in PM lipid organization, leading to increased ERK signaling and decreased opioid responses. Release of neurotrophic factors reinforce these pathways and, in conjunction with cAMP signaling, drives nociceptor hyperexcitability and a chronic pain state. To address these hypotheses, we propose three Aims. 1) Determine the mechanism for reduced MOR-Gαi inhibition of AC by C-Raf, 2) Define the mechanism of Ras activation and nociceptor hyperexcitability by depolarization and SCI, and 3) Define functional consequences of interactions among depolarization and cell signaling by cAMP, C-Raf, and ERK. Importantly, our model identifies multiple FDA-approved drugs that could simultaneously enhance endogenous opioid responses and block nociceptor hyperexcitability after severe injury.