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仍不清楚。 部分 这种缺乏明确性的原因是， 阿片肽必须通过其扩散到达细胞外空间 他们的受体。 由于种种原因，现在看来， 突触的经典概念不是一个合适的模型， 探索阿片类药物的生理作用。 最重要的是， 突触前神经末梢的活动区（ 通过胞吐作用从其中释放“经典”递质）可以 释放阿片类药物的地方 这项建议的目的 是在细胞和亚细胞水平上确定 阿片肽从轴突释放出来。 经典的小分子神经递质储存在小突触中， 囊泡;阿片类药物和其他神经肽优先储存 形成大颗粒囊泡。 存在独立的机制， 负责这两种蛋白的细胞内运输和胞吐作用 囊泡的数量 在这个提议中，提出了实验 它将直接决定分子的空间分布 参与大颗粒囊泡的释放。 以下 将进行以下研究： 1. N型和L型钙通道的空间分布 模型神经元系统中的阿片样物质能神经纤维的膜将 使用荧光标记的欧米茄毒素、尼索地平和 漏斗网蜘蛛毒素ω-Aga-IIIA 2.大鼠脑L型α 1亚单位的空间分布 阿片类神经纤维膜内钙通道的变化 小鼠输精管和豚鼠肠神经系统 使用对肽序列具有选择性的抗体测定回肠 这个subunit。 3.钙调蛋白的空间出现，假定的对接蛋白， 大颗粒囊泡，将相对于阿片类药物定位 模型神经系统中的神经纤维和终末。 4.某些小的GTP结合蛋白将相对于 阿片样物质神经纤维，因为它可能是至少一个成员， 这个家族在大颗粒囊泡的最后阶段至关重要 融合与释放