Peripheral NMDA Receptors in Visceral Nociception

描述(由申请人提供)：肠道的功能性和炎症性障碍的特征是痛觉异常(将通常不痛的刺激感觉为疼痛)和痛觉过敏(痛觉增强)。内脏痛觉过敏及其中枢敏感化的发生关键依赖于位于脊髓背角突触后神经元上的一种特殊类型的谷氨酸受体--NMDAR的激活。最近，外源性初级传入神经元(EPAN)中央终末上的突触前NMDAR被发现调节P物质向背角浅层的释放，从而促进中枢敏化。在支配结肠的EPAN的DRG神经元中合成的NMDAR亚单位可能不仅被运输到中枢，而且被运输到周围神经末梢。在这笔赠款的初始资助期，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，结肠中NMDAR的激活使某些传入终末对机械扩张敏感。利用原代培养的DRG神经元，我们发现NMDAR的激活通过涉及蛋白激酶C的过程来增强电压门控钙电流。这一机制是否解释了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应用中得到解决。此外，我们发现结肠炎导致DRG神经元中NMDAR信号的增强，这与NMDAR亚单位之一的磷酸化有关。在这种竞争性更新应用中，结合体内(有效的、靶向的KO小鼠)和体外技术(分子生物学、电生理学)，我们提出了新的实验来解决以下三个假设：1)支配结肠的EPAN上的NMDAR通过调节神经元的兴奋性和神经肽的释放而在外周敏化中发挥作用。2)NMDAR通过激活PKCepsilon和PKD介导EPAN末端的敏化。3)结肠炎通过非受体酪氨酸激酶使NMDAR磷酸化，使DRG神经元变得敏感。这些研究可能对我们理解内脏疼痛加剧的机制有很大帮助，这些内脏痛与胃肠道的功能和炎症障碍相关。

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.