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

描述（由申请人提供）：三分之一的美国人在他们生命中的某个阶段会患有肠道疾病，如肠易激综合征（IBS）或炎症性肠病（IBD）。这些疾病给社会带来了巨大的成本，每年总计约300亿美元。这些病人的主要主诉是排便改变和疼痛。目前的假设支持肠疾病模型中出现的蠕动和内脏超敏反应的变化可能与n -甲基- d -天冬氨酸（NMDA）受体有关。我们之前报道了NMDA受体NR1亚基剪接变异体在大鼠肌丛炎症前后的表达。我们发现，在未经处理的动物中，NR1剪接变体为NR1-001和NR1-000。炎症后NR1表达明显升高。此外，NR1-011和NR1-111剪接变异体的表达在炎症后14天也有所增加。我们假设NMDA受体表达/活性的变化可能影响结肠收缩性，从而引起疼痛。在这项提议中，我们计划使用2,4,6-三硝基苯磺酸（TNBS）诱导的大鼠结肠炎模型，利用多通道分离组织浴系统表征炎症和不同NMDA配体对结肠收缩性的影响。我们将利用针对不同NMDA受体亚型VIP和NOS的特异性抗体，采用多标记免疫组织化学方法评估炎症前后含抑制性VIP/NOS运动神经元肠道内NMDA受体亚型的存在。最后，我们将利用特异性Elisa和荧光法分析NMDA受体激动剂和拮抗剂对炎症前后血管活性肠肽（VIP）和一氧化氮（NO）释放的影响。我们预测，在tnbs诱导的炎症后，肠道NMDA受体影响VIP和NO的释放，改变结肠收缩性，这可以解释肠道疾病动物模型中发现的相关内脏超敏反应，并由患者报告。由于肠蠕动障碍是影响肠道疾病患者生活质量的主要因素，因此增加我们对肠神经系统（ENS）中NMDA受体结构/功能变化的了解至关重要。肠道神经系统中NMDA受体的鉴定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.