Mechanisms of cAMP signaling that drive spontaneous activity in nociceptors

驱动伤害感受器自发活动的 cAMP 信号传导机制

• 批准号: 9318602
• 负责人: Carmen W. Dessauer
• 金额: $ 32.93万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-15 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9318602
• 关键词: A kinase anchoring protein; Ache; Acute; Acute Pain; Address; Adenylate Cyclase; Adult; Affect; Afferent Neurons; American; Animals; Behavior; Behavioral; Binding; Biochemical; Biochemistry; Calmodulin; Cells; Cellular biology; Chronic; Complex; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Effectiveness; Electrophysiology (science); Esthesia; Future; Gene Expression; Goals; Hour; Hyperactive behavior; Hyperreflexia; Hypersensitivity; In Vitro; Inflammation; Injury; Ion Channel; Joints; Knock-out; Laboratories; Lead; Life; Link; Macromolecular Complexes; Maintenance; Measures; Membrane; Methods; Modeling; Molecular; Nature; Neuraxis; Nociception; Nociceptors; Pain; Pathway interactions; Peptides; Peripheral; Persistent pain; Play; Productivity; Protein Isoforms; Protein Kinase Inhibitors; Proteins; Rattus; Regulation; Resistance; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Spinal Anesthesia; Spinal Ganglia; Spinal Injuries; Spinal cord injury; Spinal cord injury patients; System; TRPV1 gene; Techniques; Testing; Viral; Viral Vector; Walking; Wheelchairs; Work; chronic pain; clinically relevant; cost; discount; experimental study; expression vector; follow-up; in vitro activity; in vivo; inhibitor/antagonist; insight; intervention effect; knock-down; neuronal cell body; novel; painful neuropathy; protein complex; protein kinase inhibitor; public health relevance; scaffold; spinal cord injury pain; spontaneous pain; targeted agent; transcriptome sequencing

 DESCRIPTION (provided by applicant): Chronic pain caused by injury to the peripheral or central nervous system (neuropathic pain) is notoriously resistant to treatment. The mechanisms that maintain any type of neuropathic pain for months or longer are poorly understood. Chronic pain in a rat model of spinal cord injury (SCI) has recently been shown to depend upon hyperactivity in nociceptive sensory neurons (nociceptors), with much of the pain-initiating activity generated within the cell bodies. The continued expression of pain-linked nociceptor hyper excitability and spontaneous activity (SA) in vitro provides a special opportunity to link biochemical mechanisms directly to electrophysiological activity critical for maintaining chronic SCI pain. Preliminary results indicate that continuing signaling by complexes containing adenylyl cyclase (AC), protein kinase A (PKA), and A-kinase anchoring proteins (AKAPs), and possibly exchange protein activated by cAMP (EPAC) plays a vital role. While cAMP signaling has long been known to be important for acute pain lasting hours to days, a major role in maintaining pain lasting months is unexpected. Agents selectively inhibiting different steps along cAMP-dependent pathways blocked chronic SCI-induced SA, including inhibitors of AKAP5 (AKAP79/150)-anchored complexes. Biochemical studies of membranes from dorsal root ganglia revealed a change in AC regulation after SCI, suggesting the existence of a previously unknown mechanism at the level of AC function that contributes to chronic pain. These and related observations led to the hypothesis that chronic nociceptor SA and pain after SCI are maintained by 1) alterations in AC regulation and 2) AKAP5-scaffolded macromolecular complexes that facilitate cAMP-dependent PKA and EPAC regulation of ion channels. The proposed studies will exploit complementary strengths of the two PIs' laboratories by combining in vitro biochemistry, cell biology, and electrophysiology coordinated with in vivo tests of pain-related behavior after SCI. Experiments will take advantage of our findings that robust SCI-induced SA in numerous nociceptors below the spinal injury level is clearly linked to behaviorally expressed hypersensitivity and pain. This will allow the use of electrophysiological and molecular alterations in dissociated nociceptors as informative endpoints for studies evaluating pain-related functions of signaling molecules within the cAMP pathway. It will also allow pooling of multiple ganglia from SCI animals to facilitate biochemical and molecular studies. Predicted behavioral and cellular effects of interventions targeting macromolecular complexes disclosed in the in vitro studies will be tested in the whole animal using complementary approaches, including a novel viral vector for expression of disrupting peptides selectively in nociceptors, an knockdown and inhibitor methods targeting specific cAMP signaling components. Information gained from these studies may lead to major mechanistic discoveries that could guide future efforts to treat chronic pain by targeting the persistent intracellular signaling that maintains hyperactivity in nociceptors that promotes chronic pain.

 描述（由适用提供）：众所周知，由周围或中枢神经系统受伤引起的慢性疼痛（神经性疼痛）对治疗具有抵抗力。几个月或更长时间维持任何类型的神经性疼痛的机制知之甚少。最近已显示出大鼠脊髓损伤模型（SCI）模型中的慢性疼痛取决于伤害性感觉神经元（伤害感受器）中的多动症，其中大部分疼痛激发活性在细胞体内产生。在体外，疼痛连接的伤害感受器超令人兴奋和赞助者的自发活动（SA）的持续表达为直接将生物化学机制与维持慢性SCI疼痛至关重要的电生理活性联系起来提供了特殊的机会。初步结果表明，含有含有腺苷环酶（AC），蛋白激酶A（PKA）和A-激酶锚定蛋白（AKAP）的复合物继续信号传导，并可能由CAMP激活（EPAC）激活的蛋白质起着至关重要的作用。虽然cAMP信号长期以来一直在持续数小时至几天的急性疼痛中很重要，但在持续疼痛持续几个月中的主要作用是出乎意料的。沿cAMP依赖性途径的不同步骤有选择性地阻断了慢性SCI诱导的SA，包括AKAP5的抑制剂（AKAP79/150）锚定的复合物。对背根神经节机制的生化研究表明，SCI后AC调节发生了变化，这表明在AC功能水平上存在一种先前未知的机制，这会导致慢性疼痛。这些和相关的观察结果导致了以下假设：慢性伤害感受器SA和SCI后的疼痛通过1）AC调节的改变以及2）AKAP5损坏的大分子复合物的改变，从而促进了依赖CAMP依赖的PKA和EPAC调节离子通道。提出的研究将通过结合体外生物化学，细胞生物学和电生理学与SCI后疼痛相关行为的体内测试来利用这两个PIS实验室的互补优势。实验将利用我们的发现，即在脊柱损伤水平以下的许多伤害感受器中强大的SCI诱导的SA显然与行为表达的超敏反应和疼痛有关。这将允许在解散的伤害感受器中使用电生理和分子改变，作为评估cAMP途径中信号分子疼痛功能的研究的信息终点。它还将允许从SCI动物中汇集多个神经节，以促进生化和分子研究。在体外研究中披露的靶向大分子复合物的干预措施的预测行为和细胞作用将使用完整的方法在整个动物中进行测试，包括一种新型病毒载体，用于在Nocteptors中有选择地破坏Petides，敲低和抑制剂方法靶向特定的cAMP信号组件。从这些研究中获得的信息可能会导致重大的机械发现，这些发现可以指导未来的努力来治疗慢性疼痛，以靶向持续的细胞内信号传导，从而在伤害感受器中保持过度活跃，从而促进慢性疼痛。