Peripheral NMDA Receptors in Visceral Nociception

描述（由申请人提供）：肠道功能性和炎症性疾病的特征为异常性疼痛（将正常非疼痛刺激感知为疼痛）和痛觉过敏（疼痛感知增强）。内脏痛觉过敏及其潜在的中枢敏感化的发展关键地依赖于位于脊髓背角中的突触后神经元上的特定类型的谷氨酸受体（NMDA受体（NMDAR））的激活。最近，研究表明，外源性初级传入神经元（EPAN）中央末梢上的突触前NMDAR可以调节P物质向浅层背角的释放，从而促进中枢敏感化。可能在支配结肠的EPAN的DRG神经元中合成的NMDAR亚单位不仅被运输到中枢，而且被运输到外周神经末梢。在该基金的初始资助期间，我们已经确定了EPAN表达的NMDAR亚基，并评估了它们的功能特性。我们发现，激活NMDAR在结肠中的行为，以敏感某些传入终端机械扩张。在原代培养的DRG神经元中，我们发现NMDAR激活通过涉及蛋白激酶C的过程增强电压门控钙电流。这种机制是否能解释外周传入神经末梢对机械刺激的敏感性增强，以及NMDAR与蛋白激酶C（PKC）和其他下游靶点偶联的细胞内转导事件的性质将在更新申请中得到解决。此外，我们发现结肠炎症导致DRG神经元中NMDAR信号增强，这与NMDAR亚基之一的磷酸化有关。在这种竞争性更新应用中，使用体内（经验证的靶向KO小鼠）和体外技术（分子生物学，电生理学）的组合，我们提出了新的实验来解决以下3个假设：1）神经支配结肠的EPAN上的NMDAR通过调节神经元兴奋性和神经肽释放在外周致敏中发挥作用。2)EPAN末端的敏化作用由NMDAR通过PKC和PKD活化介导。3)结肠炎症通过非受体酪氨酸激酶磷酸化NMDAR使DRG神经元敏感。这些研究很可能对我们理解与胃肠道功能性和炎症性疾病相关的内脏痛增强的潜在机制做出重大贡献。

DESCRIPTION (provided by applicant): Functional and inflammatory disorders of the gut are characterized by allodynia (the perception of a normally non-painful stimulus as painful) and hyperalgesia (the enhanced perception of pain). The development of visceral hyperalgesia and its underlying central sensitization relies crucially on the activation of a particular type of glutamate receptor, the NMDA receptor (NMDAR), located on postsynaptic neurons in the dorsal horn of the spinal cord. Recently, a presynaptic NMDAR on the central terminals of extrinsic primary afferent neurons (EPANs) has been shown to regulate the release of substance P into the superficial dorsal horn, thereby contributing to central sensitization. It is likely that NMDAR subunits synthesized in DRG neurons of EPANs innervating the colon are transported not only to central, but also to peripheral nerve terminals. During the initial funding period of this grant, we have identified the NMDAR subunits expressed by EPANs and evaluated their functional properties. We found that activation of NMDARs in the colon acts to sensitize certain afferent terminals to mechanical distension. Using DRG neurons in primary culture, we showed that NMDAR activation enhances voltage gated calcium currents through a process that involves protein kinase C. Whether this mechanism accounts for the enhanced sensitivity of peripheral afferent nerve terminals to mechanical stimuli, and the nature of the intracellular transduction events coupling NMDAR to protein kinase C (PKC) and other downstream targets will be addressed in the renewal application. In addition, we showed that colon inflammation results in enhanced NMDAR signaling in DRG neurons, which is associated with phosphorylation of one of the NMDAR subunits. In this competitive renewal application, using a combination of in vivo (validated, targeted KO mice) and in vitro techniques (molecular biology, electrophysiology), we propose new experiments to address the following 3 hypotheses: 1) NMDARs on EPANs innervating the colon play a role in peripheral sensitization by regulating neuronal excitability and neuropeptide release. 2) Sensitization of EPAN terminals is mediated by the NMDAR through PKCepsilon and PKD activation. 3) Colon inflammation sensitizes DRG neurons via phosphorylation of NMDARs by non-receptor tyrosine kinases. These studies are likely to contribute significantly to our understanding of the mechanisms underlying enhanced visceral pain associated with functional and inflammatory disorders of the Gl tract.