SUBCELLULAR LOCALIZATION OF NEURONAL SITES OF OPIOID PEPTIDE RELEASE

阿片肽释放神经元位点的亚细胞定位

• 批准号: 6237946
• 负责人: ROBERT P ELDE
• 金额: $ 8.62万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-03-10 至 1998-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6237946
• 关键词: Anura; alternatives to animals in research; animal poison; animal tissue; annexins; axon; biotin; calcium channel; calcium channel blockers; confocal scanning microscopy; dihydropyridines; endogenous opioid; epididymis; fluorescent dye /probe; guanine nucleotide binding protein; guinea pigs; ileum; immunocytochemistry; intracellular; laboratory mouse; laboratory rabbit; laboratory rat; membrane fusion; molecular site; neuronal transport; neurotoxins; neurotransmitter transport; opioid receptor; parasympathetic nervous system; pons; preganglionic fiber; protein structure; reticulospinal tract; synapses; vesicle /vacuole; western blottings

The physiological role of opioid neurotransmission remains unclear. Part of this lack of clarity is due to uncertainties concerning the volume of extracellular space through which opioid peptides must diffuse to reach their receptors. For a variety of reasons it now seems that the classical concept of the synapse is not an appropriate model in which to explore the physiological role of opioids. Most importantly, it is very unlikely that the active zone of presynaptic nerve terminals (the site from which "classical" transmitters are released by exocytosis) can be the site from which opioids are released. The purpose of this proposal is to determine at the cellular and subcellular level the sites at which opioid peptides are released from axons. Classical, small molecule neurotransmitters are stored in small synaptic vesicles; the opioids and other neuropeptides are preferentially stored in large granular vesicles. Independent mechanisms exist which are responsible for intracellular trafficking and exocytosis of these two populations of vesicles. In this proposal, experiments are proposed which will determine the spatial occurrence of molecules directly involved in the release of large granular vesicles. The following studies will be conducted: 1. The spatial occurrence of N- and L-types of calcium channels within the membranes of opioidergic nerve fibers in model neuronal systems will be determined using fluorescently-labeled omegaconotoxin, nisoldipine and funnel web spider toxin omega-Aga-IIIA. 2. The spatial occurrence of the alpha1 subunit of the rat brain L-type of calcium channel within the membranes of opioidergic nerve fibers in the mouse vas deferens and the enteric nervous system of the guinea pig ileum will be determined using antibodies selective for peptide sequences of this subunit. 3. The spatial occurrence of calpactin, the putative docking protein for large granular vesicles, will be localized with respect to opioidergic nerve fibers and terminals in the model neural systems. 4. Certain small GTP-binding proteins will be localized with respect to opioidergic nerve fibers, since it is likely that at least one member of this family is crucial in the final stages of large granular vesicle fusion and release.

阿片类神经传递的生理作用尚不清楚。 部分 缺乏明确性是由于关于数量的不确定性 阿片类肽必须扩散到达到达的细胞外空间 他们的受体。 由于各种原因，现在似乎 突触的经典概念不是适当的模型 探索阿片类药物的生理作用。 最重要的是，这是非常 突触前神经终端的活性区不可能（该部位 通过胞吞作用释放“经典”发射器）可以是 释放阿片类药物的网站。 该提议的目的 是确定在细胞和亚细胞水平的位置 阿片类肽从轴突释放。 经典的小分子神经递质存储在小突触中 囊泡；阿片类药物和其他神经肽优先存储 在大的颗粒囊泡中。 存在独立机制 负责这两个的细胞内贩运和胞吐作用 囊泡种群。 在此建议中，提出了实验 这将直接确定分子的空间发生 参与大型颗粒囊泡的释放。 下列 将进行研究： 1。钙通道的N型和L型的空间发生 模型神经元系统中阿片类神经纤维的膜 使用荧光标记的omegaconotoxin，nisoldipine和 漏斗网络蜘蛛毒素omega-aga-iiia。 2。大鼠脑L型的α1亚基的空间发生 阿片神经纤维膜内的钙通道的 小鼠VAS延迟和豚鼠的肠神经系统 回肠将使用选择性肽序列的抗体确定 这个亚基。 3。卡尔帕辛的空间发生，calpactin，推定的对接蛋白 大型颗粒囊泡将与阿片类囊泡进行定位 模型神经系统中的神经纤维和终端。 4。某些小的GTP结合蛋白将与 阿片类神经纤维，因为很可能至少有一个成员 这个家庭在大颗粒囊泡的最后阶段至关重要 融合和释放。