SUBCELLULAR LOCALIZATION OF NEURONAL SITES OF OPIOID PEPTIDE RELEASE

阿片类神经传递的生理作用尚不清楚。零件 这种不明确性的原因是关于 阿片肽必须扩散才能到达的细胞外空间 它们的受体。由于各种原因，现在看起来 经典的突触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，它非常 不太可能突触前神经末梢的活动区(该部位 其中，通过胞吐作用释放出“经典”递质)可以 阿片类药物被释放的地方。这项建议的目的是 是在细胞和亚细胞水平上确定 阿片肽是从轴突释放出来的。 经典的小分子神经递质储存在小突触中 囊泡；阿片类物质和其他神经肽优先储存 在大的颗粒状小泡中。存在独立的机制，它们是 负责这两种物质的细胞内运输和胞吐 囊泡的种群。在这项提案中，提出了实验 这将直接决定分子的空间产状 参与大颗粒囊泡的释放。以下是 我们会进行以下研究： 1.N型和L型钙通道在体内的空间分布 模型神经系统中阿片能神经纤维膜将 用荧光标记的欧米茄毒素、尼索地平和 漏斗网蜘蛛毒素Omega-Aga-IIIA。 2.L型大鼠脑内α1亚单位的空间分布 阿片能神经纤维膜上钙通道的变化 小鼠输精管与豚鼠肠道神经系统 回肠将使用对多肽序列具有选择性的抗体来确定 这个亚单位的。 3.钙化肌动蛋白的空间分布 大颗粒囊泡，将定位于阿片能 模型神经系统中的神经纤维和神经末梢。 4.某些小的GTP结合蛋白将定位于 阿片能神经纤维，因为很可能至少有一个成员 这个家族在大颗粒小泡的最后阶段是至关重要的。 融合和释放。

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.