Trafficking-Dependent Signaling of Pain by Protease-Activated Receptors

项目总结 慢性疼痛是一个尚未得到解决的重大医学问题。从急性转化为急性转化的基础机制 对(生理性)到慢性(病理性)疼痛的了解很少，目前的治疗方法也不够充分。这个 Proposal研究了与肠易激综合征和炎症性肠病相关的结肠疼痛。 在损伤和炎症过程中被激活的蛋白水解酶可以通过分解蛋白水解酶来向伤害性感受器发出信号。 激活的受体-2(PAR2)，激活瞬时受体电位通道并诱导长时间的 过度兴奋和伤害性。尽管蛋白水解酶和PAR2与结肠疼痛有关，但 持续性蛋白酶诱导疼痛的信号机制还远不清楚，PAR2是否是一种 慢性疼痛的治疗目标是不确定的。前提是PAR2唯一适合传输持久 由于蛋白分解(催化)激活的不可逆机制，受体对 来自内体的信号，以及动员细胞内感受器储存的有效机制。因此， 内体PAR2拮抗剂和运动抑制药可有效缓解疼痛。这些概念将 在完整的小鼠、小鼠和人结肠的分离片段以及培养的伤害性神经元中进行检测。 方法将包括：伤害性行为评估，伤害性感受器的电生理分析 激活，以及生物物理和成像方法，以评估伤害感受器中PAR2的运输和信号传递。 表达荧光PAR2的小鼠将被用于研究PAR2的贩运。靶向缺失PAR2的小鼠 伤害性感受器和内体PAR2独特的脂结合拮抗剂将被用来确定 PAR2在伤害性感受器的内吞体内是治疗持续性结肠疼痛的靶点。这些研究将提供 对慢性疼痛的机制和治疗的见解。它们对治疗靶向有影响 G蛋白偶联受体；这个由1000个成员组成的受体家族是40%药物的靶标。因为有很多人 激活的受体内化并继续发出信号，有效的治疗需要内脏靶向药物。

PROJECT SUMMARY Chronic pain is a major unmet medical problem. The mechanisms that underlie the transition from acute (physiological) to chronic (pathological) pain are poorly understood and current therapies are inadequate. The proposal investigates colonic pain, with relevance to irritable bowel syndrome and inflammatory bowel disease. Proteases that are activated during injury and inflammation can signal to nociceptors by cleaving protease- activated receptor-2 (PAR2), which activates transient receptor potential channels and induces long-lasting hyperexcitability and nociception. Although proteases and PAR2 have been implicated in colonic pain, the signaling mechanisms that underlie persistent protease-induced pain are far from clear, and whether PAR2 is a therapeutic target for chronic pain is uncertain. The premise is that PAR2 is uniquely suited to transmit persistent nociception due to the irreversible mechanism of proteolytic (catalytic) activation, the capacity of the receptor to signal from endosomes, and the efficient mechanisms that mobilize intracellular receptor stores. Accordingly, antagonists of endosomal PAR2 and inhibitors of mobilization provide effective pain relief. These concepts will be examined in intact mice, isolated segments of mouse and human colon, and nociceptive neurons in culture. Approaches will include: behavioral assessment of nociception, electrophysiological analysis of nociceptor activation, and biophysical and imaging approaches to assess PAR2 trafficking and signaling in nociceptors. Mice expressing fluorescent PAR2 will be used to study PAR2 trafficking. Mice with targeted deletion of PAR2 on nociceptors, and unique lipid-conjugated antagonists of endosomal PAR2, will be used to determine whether PAR2 in endosomes of nociceptors is a therapeutic target for persistent colonic pain. The studies will provide insights into the mechanisms and treatment of chronic pain. They have implications for therapeutic targeting of G protein-coupled receptors; this 1,000-member family of receptors is the target of 40% of drugs. Since many activated receptors internalize and continue to signal, effective therapy requires endosomally-targeted drugs.