Mu opioid receptor function in the enteric nervous system

描述(申请人提供)：配体诱导的G蛋白偶联受体的运输及其与相关蛋白的相互作用是调节受体介导的信号转导和细胞反应的关键步骤。？阿片受体(？or)介导阿片类药物的止痛作用及其副作用，包括阿片类肠道综合征(严重便秘和腹痛)和耐受性。我们先前的研究表明：a)内源性阿片类药物和大多数阿片类药物对肠神经元胞内吞饮的反应，而不是吗啡；b)慢性激活使吗啡具有触发？或内吞作用的能力，并诱导对受体内化很重要的细胞内蛋白质Dynamin的过度表达和转位；c)高效阿片类药物诱导的内吞作用的大小在阿片类药物耐受的情况下不会改变；d)？或内化激动剂激活？或内化激动剂可刺激培养的肌间神经元中丝裂原活化蛋白激酶(MAPK)的磷酸化。这一应用将检验一种假说，即长时间的激活会导致调节激动剂诱导的肠神经元运输和受体后信号的细胞内机制发生变化，这可能是导致耐受发展的机制。具体目的1将通过神经细胞转染表达OR的天然细胞，确定在体外急性和慢性激活后，哪些细胞内蛋白调节？或转运，并将它们的表达与体内受体内吞作用和肠道耐受状态相关联。具体目标2将确定急性和慢性刺激对肠神经元MAPK激活的影响，MAPK抑制剂对激动剂诱导的或脱敏和内化的影响，以及慢性阿片类药物治疗导致肠道耐受状态是否诱导完整回肠段MAPK持续激活。这些研究将更好地了解阿片类药物诱导的肠道神经元自然表达的适应，或者，这将增加我们对阿片类肠道综合征和耐受性的了解，阿片类肠道综合征是一种影响接受慢性阿片类药物止痛的患者的疾病，而耐受性是阻碍这些药物使用的因素。

DESCRIPTION (provided by applicant): Ligand-induced trafficking of G protein-coupled receptors and their interaction with associated proteins are key steps regulating receptor-mediated signal transduction and cellular responsiveness. ? opioid receptor (?OR) mediates the analgesic effects of opiates, potent drugs used for pain control, and their side effects, including opioid bowel syndrome (profound constipation and abdominal pain), and tolerance. We previously showed that a) ?OR endocytoses in enteric neurons in response to endogenously opioids and to most opiates, but not to morphine, b) chronic ?OR activation confers morphine the ability to trigger ?OR endocytosis and induces over expression and translocation of dynamin, an intracellular protein that is important for receptor internalization, c) the magnitude of ?OR endocytosis induced by high efficacy opiates does not change in a condition of opioid tolerance, d) activation of ?OR with an internalizing agonist stimulates mitogen-activated protein kinase (MAPK) phosphorylation in cultured myenteric neurons. This application will test the hypothesis that prolonged ?OR activation induces alterations in the intracellular machinery that regulates agonist-induced ?OR trafficking and post-receptor signaling in enteric neurons, which are possible mechanisms underlying the development of tolerance. Specific Aim 1 will establish which intracellular proteins regulate ?OR trafficking following acute and chronic activation in vitro using neuronal transfection of native cells expressing ?OR and correlate their expression with receptor endocytosis and state of gut tolerance in vivo. Specific Aim 2 will determine the effects of acute and chronic ?OR stimulation on MAPK activation in enteric neurons, the effects of MAPK inhibitors on agonist- induced ?OR desensitization and internalization, and whether chronic opiate treatment resulting in a state of tolerance in the gut induces sustained MAPK activation in intact segments of the ileum. These studies will provide a better knowledge of opiate-induced adaptations in enteric neurons naturally expressing ?OR, which will increase our understanding of opioid bowel syndrome, a condition affecting patients receiving chronic opiates for pain, and tolerance, which hamper the use of these drugs.