N-methyl-D-aspartate Receptors in the Enteric Nervous System

描述(申请人提供)：每三个美国人中就有一个会在一生中的某个时刻遭受肠易激综合征(IBS)或炎症性肠病(IBD)等肠道疾病的困扰。这些疾病给社会造成了巨大的代价，每年的总成本约为300亿美元。这些患者的主要主诉是大便运动和疼痛的变化。目前的假说支持在肠病模型中出现的蠕动和内脏高敏感性的改变可能与N-甲基-D-天冬氨酸(NMDA)受体有关。我们先前报道了炎症前后大鼠肌间神经丛中NMDA受体NR1亚单位剪接变异体的表达。我们发现，在未经治疗的动物身上，存在NR1剪接变体NR1-001和NR1-000。炎症后NR1的表达明显增加。此外，炎症后第14天，剪接变异体NR1-011和NR1-111的表达也增加。我们推测，NMDA受体表达/活性的变化可能会影响结肠的收缩，从而导致疼痛。在这项建议中，我们计划使用2，4，6-三硝基苯磺酸(TNBS)诱导的结肠炎大鼠模型，利用多通道隔离组织浴系统来表征炎症和不同的NMDA配体对结肠收缩的影响。我们将使用针对不同NMDA受体亚型VIP和NOS的特异性抗体，采用多重标记免疫组织化学方法，评估炎症前后含抑制性VIP/NOS运动神经元上存在的肠道NMDA受体亚型。最后，我们将利用特异性的ELISA法和荧光分光光度法分析炎症前后NMDA受体激动剂和拮抗剂对血管活性肠肽(VIP)和一氧化氮(NO)释放的影响。我们预测，在TNBS诱导炎症后，肠内NMDA受体影响VIP和NO的释放，改变结肠的收缩能力，这可以解释在肠道疾病动物模型中发现的相关内脏高敏感性，以及患者报道的。由于蠕动障碍是影响肠病患者生活质量的主要因素，因此增加对肠道神经系统(ENS)中NMDA受体结构/功能变化的认识是至关重要的。对肠道神经系统中NMDA受体的识别可能会导致选择性调节肠道功能的药物的开发。

DESCRIPTION (provided by applicant): One out of three Americans will suffer a bowel disorder such as irritable bowel syndrome (IBS) or inflammatory bowel disease (IBD) at some point in their lives. These disorders result in significant costs to society, a combined total of approximately $30 billion annually. The major complaints in these patients are changes in bowel movements and pain. Current hypotheses support that changes in peristalsis and visceral hypersensitivity present in models of bowel disorders may be linked to the N-methyl-D-aspartate (NMDA) receptors. We previously reported the expression of the NMDA receptor NR1 subunit splice variants in the rat myenteric plexus before and after inflammation. We found that on untreated animals the NR1 splice variants present are NR1-001 and NR1-000. After inflammation the expression of NR1 was notably increased. Moreover, the expression of splice variants NR1-011 and NR1-111 was also increased 14 days after inflammation. We hypothesize that the changes in NMDA receptor expression/activity could affect colonic contractibility, which then induces pain. In this proposal we plan to use a rat model of colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) to characterize the effect of inflammation and different NMDA ligands in colon contractibility using a multi-channel isolated tissue bath system. We will evaluate the enteric NMDA receptor subtypes present on inhibitory VIP/NOS-containing motor neurons before and after inflammation with multiple-labeling immunohistochemistry using specific antibodies targeting the different NMDA receptors subtypes, VIP and NOS. Finally we will analyze the effect of NMDA receptors' agonists and antagonists in the release of vasoactive intestinal peptide (VIP) and nitric oxide (NO) using specific Elisa and fluorometric assays before and after inflammation. We predict that after the TNBS-induced inflammation the enteric NMDA receptors influence the release of VIP and NO, altering colonic contractibility which could explain the associated visceral hypersensitivity found in animal models of bowel disorders and reported by patients. Because disturbances in peristalsis are the major factors affecting the quality of life in patients with bowel disorders, it is critical that we increase our knowledge of NMDA receptor structural/functional changes in the enteric nervous system (ENS). The identification of the NMDA receptors in the enteric nervous system could lead to the development of drugs that selectively modulate bowel function.