Pro- and Anti-Nociceptive Actions of P2y Nucleotide Receptors in Sensory Neurons

描述（由申请人提供）：细胞外ATP是疼痛感受神经元（伤害感受器）传递伤害性刺激的信使。感觉神经元特异性ATP门控离子通道P2 X3的克隆引起了对ATP直接激活伤害感受器的能力的深入研究。然而，我们有新的证据表明，感觉神经元也表达一个G蛋白偶联受体（P2 Y受体）家族的成员，该家族对ATP或相关化合物作出反应，并有助于伤害性信号传导。这些受体在神经元中的特征很差：在8个已知的家族成员中，只有P2 Y1和P2 Y2在感觉神经元中进行了评价，并且两者都涉及伤害性信号转导。P2 Y受体可基于其与信号转导途径的偶联分为2组;我们假设Gq偶联受体是促痛觉的，而Gi偶联受体是镇痛的。该提案包括3个特定目的，旨在确定哪些P2 Y家族成员有助于伤害性信号传导，并可能成为疼痛治疗干预的有用靶点。具体目标1将使用定量PCR，免疫组织化学和Wester印迹技术来确定哪些P2 Y受体在感觉神经元中表达，以及表达是否响应于炎症损伤而变化。具体目标2将使用钙成像和事后免疫细胞化学表征分离的感觉神经元中Gq偶联和Gi偶联P2 Y受体的兴奋和抑制作用。具体目标3将检查P2 Y ADP受体对体内行为疼痛反应阈值的贡献，并将确定P2 Y受体的药理学或遗传操作是否在急性和持续性炎性疼痛模型中具有镇痛作用。这些研究将证明一个新的受体家族对感觉神经元信号传导的贡献，并将为核苷酸在持续性疼痛的设置中作为信号分子的机制提供有价值的见解。本提案中的实验将验证以下假设，即核苷酸受体的P2 Y家族成员是伤害感受器敏感性的强大调节剂，在维持持续性疼痛中起重要作用。痛苦我们将直接测试操纵这些受体的可能性，包括使用已经在开发中的非疼痛相关综合征的临床治疗的拮抗剂，是持续性疼痛的动物模型中的有效镇痛治疗。

DESCRIPTION (provided by applicant): Extracellular ATP is a messenger in the transduction of noxious stimuli by pain-sensing neurons (nociceptors). The cloning of the sensory neuron-specific ATP-gated ion channel P2X3 generated intense investigation into the ability of ATP to directly activate nociceptors. However, we have new evidence that sensory neurons also express members of a family of G protein-coupled receptors (P2Y receptors) that respond to ATP or related compounds and contribute to nociceptive signaling. These receptors are poorly characterized in neurons: of the 8 known family members only P2Y1 and P2Y2 have been evaluated in sensory neurons and both have been implicated in nociceptive signal transduction. P2Y receptors can be divided into 2 groups based on their coupling to signal transduction pathways; we hypothesize that Gq-coupled receptors are proalgesic while Gi- coupled receptors are analgesic. This proposal consists of 3 Specific Aims designed to identify which P2Y family members contribute to nociceptive signaling and may be useful targets for therapeutic intervention in pain. Specific Aim 1 will use quantitative PCR, immunohistochemistry and Wester blotting techniques to determine which P2Y receptors are expressed in sensory neurons and whether expression changes in response to inflammatory injury. Specific Aim 2 will characterize excitatory and inhibitory actions of Gq-coupled and Gi-coupled P2Y receptors in dissociated sensory neurons using calcium imaging and post-hoc immunocytochemistry. Specific Aim 3 will examine the contribution of P2Y ADP receptors to behavioral pain response thresholds in vivo and will determine whether pharmacological or genetic manipulation of P2Y receptors is analgesic in models of acute and persistent inflammatory pain. These studies will demonstrate the contributions of a new family of receptors to sensory neuron signaling and will provide valuable insight into the mechanisms through which nucleotides act as signaling molecules in the setting of persistent pain.Experiments in this proposal will test the hypothesis that members of the P2Y family of nucleotide receptors are powerful regulators of nociceptor sensitivity that play an important role in the maintenance of persistent pain. We will directly test the possibility that manipulation of these receptors, including the use of antagonists already in development for clinical treatment of non-pain-related syndromes, is an effective analgesic treatment in animal models of persistent pain.