Opiod Receptor Function in the Enteric Nervous System

描述（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）的运输是很重要的，因为uOR介导许多阿片效应，包括镇痛、耐受性、呼吸抑制和肠道转运障碍。在之前的资助期内的主要发现是：1)配体诱导的uOR内吞作用降低了备用uOR受体失活的肠道神经肌肉制剂的神经原性反应；2)肠道神经元内吞作用是对内源性释放的阿片类药物的反应。目前应用的假设是激动剂诱导的uOR内吞作用作为一种调节机制来减弱神经元的反应性。该项目的长期目标是阐明uOR的转运机制和作用，以及uOR在肠道运动中的作用。这些研究将在器官型和原代细胞培养中使用转染细胞和肠神经元。豚鼠和小鼠将作为动物模型。特异性目的1将研究a)配体诱导的uOR内吞作用对长期治疗和未治疗的豚鼠纵向肌肌丛制剂的神经介导反应（由场刺激引起的胆碱能收缩和非肾上腺素能/非胆碱能松弛）的影响，以及b)不同能力的配体诱导受体内吞作用对慢性治疗神经元中uOR细胞分布的影响。特异性目的2将检查a) B型阻滞蛋白、动力蛋白和rab型GTPase在激动剂诱导的细胞内吞作用和转染细胞中uOR的转运中的作用，以及B)慢性阿片类药物治疗后肠神经元中B型阻滞蛋白、动力蛋白和rab型GTPase的细胞分布和表达。Specific Aim 3将使用uOR内吞观察内脏有害刺激释放的内源性阿片激活的肠道神经元回路，并验证内脏有害刺激诱导的阿片释放是由NMDA受体介导的假设，NMDA受体是谷氨酸离子门控受体。特异性目标4将a)通过使用核糖体失活蛋白皂苷偶联dermorphin（一种诱导受体内化的疼痛激动剂）对肠道运动的影响来确定消融性肠道疼痛的影响；b)研究内脏有害刺激对uOR敲除（-/-）和野生型（+1+）小鼠肠道运输的影响。这些研究将进一步加深我们对uOR在胃肠道功能的理解。

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.