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（由申请人提供）：众所周知，由外周或中枢神经系统损伤引起的慢性疼痛（神经性疼痛）对治疗具有抗性。维持任何类型的神经性疼痛数月或更长时间的机制知之甚少。脊髓损伤（SCI）大鼠模型的慢性疼痛最近已被证明依赖于伤害性感觉神经元（伤害感受器）的过度活跃，其中大部分疼痛起始活性产生于细胞体内。疼痛相关的伤害感受器超兴奋性和自发活动（SA）在体外的持续表达提供了一个特殊的机会，直接连接生化机制，维持慢性SCI疼痛的关键电生理活动。初步结果表明，由腺苷酸环化酶（AC），蛋白激酶A（PKA），A-激酶锚定蛋白（AKAP），并可能交换蛋白激活的cAMP（EPAC）复合物的持续信号传导起着至关重要的作用。虽然cAMP信号传导长期以来一直被认为对持续数小时至数天的急性疼痛很重要，但在维持持续数月的疼痛中的主要作用是出乎意料的。选择性抑制cAMP依赖性通路沿着不同步骤的药物阻断了SCI诱导的慢性SA，包括AKAP 5（AKAP 79/150）锚定复合物的抑制剂。背根神经节膜的生化研究揭示了SCI后AC调节的变化，表明在AC功能水平存在一种以前未知的机制，导致慢性疼痛。这些和相关的观察结果导致了这样的假设，即SCI后慢性伤害感受器SA和疼痛是通过1）AC调节的改变和2）AKAP 5支架大分子复合物来维持的，所述AKAP 5支架大分子复合物促进离子通道的cAMP依赖性PKA和EPAC调节。拟议的研究将利用两个PI实验室的互补优势，将体外生物化学、细胞生物学和电生理学与SCI后疼痛相关行为的体内测试相结合。实验将利用我们的发现，即在脊髓损伤水平以下的许多伤害感受器中，SCI诱导的SA明显与行为表达的超敏反应和疼痛有关。这将允许在解离的伤害感受器中使用电生理学和分子改变作为评估cAMP途径内信号分子的疼痛相关功能的研究的信息性终点。它还将允许汇集来自SCI动物的多个神经节，以促进生物化学和分子研究。将使用补充方法在整个动物中测试体外研究中公开的靶向大分子复合物的干预的预测行为和细胞效应，所述补充方法包括用于在伤害感受器中选择性表达破坏肽的新型病毒载体、靶向特定cAMP信号传导组分的敲减和抑制剂方法。从这些研究中获得的信息可能会导致重大的机制发现，这些发现可以指导未来的努力，通过靶向持续的细胞内信号传导来治疗慢性疼痛，这些信号传导维持了促进慢性疼痛的伤害感受器的过度活跃。