Opiod Receptor Function in the Enteric Nervous System

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。 由于jaOR介导许多阿片类物质，因此了解jaOR的转运是非常重要的。 影响，包括镇痛、耐受性、呼吸抑制和肠道运输受损。主要 在上一个资助期的研究结果是：1）配体诱导的uOR内吞作用减少了 肠神经肌肉制剂的神经原性反应，其中备用uOR受体已被 失活，和2）uOR内吞作用发生在肠神经元中，响应于内源性释放的阿片样物质。 本申请的假设是激动剂诱导的uOR内吞作用充当调节剂。 调节机制，以减弱神经元的反应。这一长期目标 本研究旨在阐明uOR的转运机制和作用，以及uOR在 肠蠕动这些研究将使用转染的细胞和肠神经元， 原代细胞培养豚鼠和小鼠将作为动物模型。具体目标1将调查a） 配体诱导的uOR内吞作用对神经介导的反应（胆碱能收缩和非胆碱能收缩）的影响。 场刺激诱发的肾上腺素能/非胆碱能松弛 来自慢性处理和未处理的豚鼠的制剂，和B）在其作用上不同的配体的作用， 在慢性处理的神经元中，急性诱导受体内吞作用对uOR细胞分布的影响。 具体目标2将检查a）6种抑制蛋白、发动蛋白和rab GT3在激动剂诱导的细胞凋亡中的作用。 转染细胞中uOR的内吞和运输，和B）B的细胞分布和表达 抑制蛋白、发动蛋白和rab GT3在慢性阿片治疗后肠神经元中的表达。第3章将 使用uOR内吞作用来可视化肠神经元回路，肠神经元回路由肠内释放的内源性阿片样物质激活。 内脏伤害性刺激引起的阿片样物质释放的假设 刺激是由NMDA受体介导的，NMDA受体是谷氨酸离子门控受体。具体目标4 a） 通过使用核糖体失活蛋白皂草素缀合物测定肠uOR的消融效果， 皮吗啡肽（诱导受体内化的uOR激动剂）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（+/+）小鼠肠道传输的影响。这些研究将 进一步了解uOR在胃肠道中的功能。

Ligand-induced receptor endocytosis is one of the key steps regulating receptor-mediated signal transduction. An understanding of \i opioid receptor (uOR) trafficking is of importance, since jaOR 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 6 arrestins, dynamin and rab GTPase in agonist-induced endocytosis and trafficking of uOR in transfected cells, and b) the cellular distribution and expression ofB 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 conjugatedto 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 (+/+) mice. These studies will further our understanding of uOR function in the gastrointestinaltract.