Agonist-selective endocytosis of mu opioid receptor by neurons in vivo
Agonist-selective endocytosis of mu opioid receptor by neurons in vivo
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DOI：
10.1073/pnas.93.17.9241
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发表时间：
1996-08-20
影响因子：
11.1
通讯作者：
Brecha, NC
中科院分区：
文献类型：
--
作者：
Sternini, C;Spann, M;Brecha, NC
阿片类生物碱是一种有效的镇痛药，通过激活G蛋白偶联受体在神经系统中发挥多种药理作用。刺激后的受体内化对于脱敏和再敏化可能是重要的，其影响细胞对配体的反应性。在此，我们通过使用豚鼠回肠作为模型系统和使用针对大鼠莫尔C末端的亲和纯化抗体的免疫组织化学来研究激动剂诱导的μ阿片受体（莫尔）在体内的内化。抗体特异性通过用表位标记的莫尔cDNA转染的人胚肾293细胞的阳性染色来证实，通过用δ或κ受体cDNA转染的细胞缺乏染色，以及通过当用莫尔肽片段预吸附莫尔抗体时染色的消除，丰富的莫尔免疫反应性（MOR-IR）定位于肌间神经元的细胞体、树突和轴突突起，免疫染色主要局限于细胞体和突起的质膜。在腹腔注射阿片受体激动剂埃托啡后15 min内，囊泡样结构中出现强烈的莫尔免疫反应，共聚焦显微镜下鉴定为内体。30 min时，MOR免疫反应遍及胞质和核周囊泡，120 min时MOR免疫反应仍被内化。有趣的是，吗啡，一种高亲和力的莫尔激动剂，没有引起可检测的内化，但它部分抑制埃托啡诱导的莫尔内吞作用。这些结果表明，发生激动剂选择性莫尔内吞作用的神经元自然表达这种受体在体内，并建议存在不同的机制调节细胞对配体的反应。
Opiate alkaloids are potent analgesics that exert multiple pharmacological effects in the nervous system by activating G protein-coupled receptors. Receptor internalization upon stimulation may be important for desensitization and resensitization, which affect cellular responsiveness to ligands. Here, we investigated the agonist-induced internalization of the mu opioid receptor (MOR) in vivo by using the guinea pig ileum as a model system and immunohistochemistry with an affinity-purified antibody to the C terminus of rat MOR, Antibody specificity was confirmed by the positive staining of human embryonic kidney 293 cells transfected with epitope-tagged MOR cDNA, by the lack of staining of cells transfected with the delta or kappa receptor cDNA, and by the abolition of staining when the MOR antibody was preadsorbed with the MOR peptide fragment, Abundant MOR immunoreactivity (MOR-IR) was localized to the cell body, dendrites, and axonal processes of myenteric neurons, Immunostaining was primarily confined to the plasma membrane of cell bodies and processes. Within 15 min of an intraperitoneal injection of the opiate agonist etorphine, intense MOR IR was present in vesiclelike structures, which were identified as endosomes by confocal microscopy, At 30 min, MOR-IR was throughout the cytoplasm and in perinuclear vesicles, MOR-IR was still internalized at 120 min, Agonist-induced endocytosis was completely inhibited by the opiate antagonist naloxone. Interestingly, morphine, a high-affinity MOR agonist, did not cause detectable internalization, but it partially inhibited the etorphine-induced MOR endocytosis. These results demonstrate the occurrence of agonist-selective MOR endocytosis in neurons naturally expressing this receptor in vivo and suggest the existence of different mechanisms regulating cellular responsiveness to ligands.