Opiod Receptor Function in the Enteric Nervous System

描述（由申请人提供）：配体诱导的受体内吞作用是调节受体介导的信号转导的关键步骤之一。了解阿片受体（uOR）的运输是非常重要的，因为uOR介导了许多阿片类药物的作用，包括镇痛，耐受，呼吸抑制和肠道运输障碍。上一个资助期的主要发现是：1）配体诱导的uOR内吞作用降低了肠神经肌肉制剂的神经原性反应，其中备用uOR受体已被灭活，2）肠神经元中发生uOR内吞作用，以响应内源性释放的阿片类药物。本申请的假设是激动剂诱导的uOR内吞作用用作减弱神经元响应性的调节机制。本项目的长期目标是阐明uOR转运机制和作用，以及uOR在肠动力中的作用。这些研究将在器官型和原代细胞培养物中使用转染的细胞和肠神经元。豚鼠和小鼠将作为动物模型。具体目标1将研究a）配体诱导的uOR内吞作用对神经介导的反应的影响（场刺激诱发的胆碱能收缩和非肾上腺素能/非胆碱能松弛），和B）急性诱导受体内吞作用的能力不同的配体对慢性治疗的小鼠中uOR的细胞分布的影响神经元具体目标2将检查a）B抑制蛋白、发动蛋白和rab GT3在转染细胞中激动剂诱导的内吞和uOR运输中的作用，和b）B抑制蛋白、发动蛋白和rab GT3在慢性阿片剂处理后肠神经元中的细胞分布和表达。具体目标3将使用uOR内吞作用来可视化由响应于内脏伤害性刺激而释放的内源性阿片样物质激活的肠神经元回路，并测试由内脏伤害性刺激诱导的阿片样物质释放由NMDA受体（谷氨酸盐离子门控受体）介导的假设。具体目标4将a）通过使用与皮吗啡肽（诱导受体内化的uOR激动剂）缀合的核糖体失活蛋白皂草素来确定肠uOR的消融对肠运动的影响;和B）检查内脏伤害性刺激对uOR敲除（-/-）和野生型（+1+）小鼠中肠转运的影响。这些研究将进一步了解uOR在胃肠道中的功能。

DESCRIPTION (provided by applicant): Ligand-induced receptor endocytosis is one of the key steps regulating receptor-mediated signal transduction. An understanding of opioid receptor (uOR) trafficking is of importance, since uOR mediates many opiate effects, including analgesia, tolerance, respiratory depression and impairment of intestinal transit. The major findings during the previous funding period were that: 1) Ligand-induced uOR endocytosis reduces the neurogenic response of intestinal neuromuscular preparations in which spare uOR receptors have been inactivated, and 2) uOR endocytosis occurs in enteric neurons in response to endogenously released opioids. The hypothesis of the present application is that agonist-induced uOR endocytosis serves as a regulatory mechanism to attenuate neuronal responsiveness. The long-term goals of this program are to elucidate uOR trafficking mechanisms and effects, and the role of uOR in intestinal motility. These studies will use both transfected cells and enteric neurons in organotypic and primary cell cultures. Guinea pigs and mice will serve as animal models. Specific Aim 1 will investigate a) the effect of ligand-induced uOR endocytosis on the nerve-mediated response (cholinergic contraction and non-adrenergic/non-cholinergic relaxation evoked by field stimulation) of longitudinal muscle-myenteric plexus preparations from chronically treated and untreated guinea pigs, and b) the effect of ligands that differ in their ability to induce receptor endocytosis acutely on the cellular distribution of uOR in chronically treated neurons. Specific Aim 2 will examine a) the role of B arrestins, dynamin and rab GTPase in agonist-induced endocytosis and trafficking of uOR in transfected cells, and b) the cellular distribution and expression of B arrestins, dynamin and rab GTPase in enteric neurons following chronic opiate treatment. Specific Aim 3 will use uOR endocytosis to visualize the enteric neuronal circuits activated by endogenous opioids released in response to visceral noxious stimuli and test the hypothesis that opioid release induced by visceral noxious stimuli is mediated by NMDA receptors, which are glutamate, ion-gated receptors. Specific Aim 4 will a) determine the effect of ablation of enteric uORs by using the ribosome-inactivating protein saporin conjugated to dermorphin (a uOR agonist that induces receptor internalization) on intestinal motility; and b) examine the effect of visceral noxious stimuli on intestinal transit in uOR knockout (-/-) and wild type (+1+) mice. These studies will further our understanding of uOR function in the gastrointestinal tract.