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

描述(申请人提供)：细胞外ATP是痛觉神经元(伤害性感受器)传递伤害性刺激的信使。感觉神经元特异的ATP门控离子通道P2X3的克隆引发了对ATP直接激活伤害性感受器能力的深入研究。然而，我们有新的证据表明，感觉神经元也表达G蛋白偶联受体家族(P2Y受体)的成员，该受体对ATP或相关化合物做出反应，并参与伤害性信号传递。这些受体在神经元中的特性很差：在已知的8个家族成员中，只有P2Y1和P2Y2在感觉神经元中被评估，并且两者都与伤害性信号转导有关。根据P2Y受体对信号转导通路的偶联作用可分为两类，我们假设GQ偶联受体具有镇痛作用，而GI偶联受体具有镇痛作用。这项建议包括三个特定的目标，旨在确定哪些P2Y家族成员参与伤害性信号传递，并可能成为治疗疼痛干预的有用靶点。具体目标1将使用定量PCR、免疫组织化学和Wester blotting技术来确定感觉神经元中哪些P2Y受体表达，以及表达是否在炎性损伤反应中发生变化。具体目标2将使用钙成像和后免疫细胞化学来表征GQ偶联和GI偶联的P2Y受体在分离的感觉神经元中的兴奋和抑制作用。具体目标3将在体内检测P2Y ADP受体对行为疼痛反应阈值的贡献，并将确定在急性和持续性炎症性疼痛模型中，对P2Y受体的药物或基因操作是否具有止痛作用。这些研究将证明一个新的受体家族对感觉神经元信号转导的贡献，并将为核苷酸在持续性疼痛环境中作为信号分子的机制提供有价值的见解。本提案中的实验将检验这样的假设，即P2Y家族的核苷酸受体成员是伤害感受器敏感性的强大调节者，在持续性疼痛的维持中发挥重要作用。我们将直接测试操纵这些受体的可能性，包括使用已经开发的拮抗剂用于非疼痛相关综合征的临床治疗，在持续性疼痛的动物模型中是一种有效的止痛治疗。

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.