Opioid tolerance and bowel dysfunction

描述（由申请人提供）：吗啡仍然是治疗中度至重度疼痛（包括癌症或手术引起的疼痛）最常用的处方药之一。然而，这种优秀的止痛药在男性中的长期使用受到副作用的限制，包括镇痛耐受性和阿片类药物引起的肠道功能障碍，便秘是最常见的和使人衰弱的症状。这项研究的长期目标是阐明导致对阿片类药物的许多影响产生耐受性的机制，包括减缓胃肠道转运，但不包括便秘。要测试的主要假设是，吗啡细胞信号特性的差异决定了回肠而不是结肠的耐受性的发展。初步数据表明，回肠的吗啡耐受与μ阿片受体与其下游信号蛋白的解偶联有关。与回肠不同，结肠是便秘的主要部位，它不会对反复使用吗啡产生耐受性。特异性目的1的主要目的是验证回肠中ß arrestin2下调与吗啡耐受性相关的假设。具体目标是表征β -抑制蛋白2下调和耐受性发展的浓度和时间关系。功能和生化研究将利用ß-arrestin2敲除小鼠来关联慢性吗啡在体外和体内的作用。特异性Aim 2将验证ß arrestin2作为支架蛋白调控下游信号通路的假设，包括MAP激酶、Src激酶、Akt和蛋白激酶c。初步研究结果表明，与吗啡诱导的抗痛觉耐受性不同，回肠中磷酸化erk的下调与耐受性的形成相关，这表明胃肠道与中枢神经系统的阿片类药物耐受性机制存在根本差异。这一目的也将检查β -阻滞蛋白2的下调是否通过改变泛素化介导。特异性目的3将探讨长期吗啡对成年小鼠肌丛离体肠神经元的影响。在这个目的中，将对来自结肠和回肠的单个肠神经元进行表征和测试，以确定吗啡诱导的电兴奋性变化以及对野生型和ß arrestin2敲除小鼠钠、钙和钾通道的影响。从这些研究中获得的信息将增加我们对胃肠道和大脑中阿片类药物耐受性和最终身体依赖机制的理解。

DESCRIPTION (provided by applicant): Morphine remains one of the most frequently prescribed drugs for the treatment of moderate to severe pain, including pain due to cancer or surgery. However, the long-term use of this excellent pain reliever in man is limited by side-effects that include analgesic tolerance and opioid-induced bowel dysfunction, with constipation being the most common and debilitating symptom. The long-term goals of this study are to elucidate the mechanisms that lead to tolerance to many of the effects of opioids, including slowing of gastrointestinal transit but not constipation. The main hypothesis to be tested is that differences in the cellular signaling properties of morphine determine the development of tolerance in the ileum but not the colon. Preliminary data suggest that morphine tolerance in the ileum is associated with an uncoupling of the μ opioid receptor from its downstream signaling proteins. Unlike the ileum, the colon which is the major site for constipation does not develop tolerance to repeated administration of morphine. The major objective of specific aim 1 is to test the hypothesis that down-regulation of ß arrestin2 is associated with morphine tolerance in the ileum. The specific goals are to characterize the concentration and temporal relationship for ß arrestin 2 downregulation and tolerance development. Functional and biochemical studies will be utilized to correlate the effect of chronic morphine in-vitro and in-vivo utilizing ß-arrestin2 knock-out mice. Specific Aim 2 will test the hypothesis that ß arrestin2 acts as a scaffolding protein to regulated downstream signaling including MAP kinase, Src kinase, Akt and protein kinase C. Preliminary findings suggest that unlike morphine induced antinociceptive tolerance, in the ileum downregulation of phospho-ERK correlates with tolerance development suggesting fundamental differences in the mechanism for opioid tolerance in the gastrointestinal tract from CNS. This aim will also examine if downregulation of ß arrestin 2 is mediated via altered ubiquitination. Specific Aim 3 will explore the effect of long-term morphine on isolated enteric neurons from the adult mouse myenteric plexus. In this aim, single enteric neurons from the colon and ileum will be characterized and tested to determine morphine- induced changes in electrical excitability and effects on sodium, calcium and potassium channels in wild-type and ß arrestin2 knock-out mice. The information obtained from these studies will increase our understanding of the mechanisms of opioid tolerance and ultimately physical dependence in the gastrointestinal tract and in the brain.