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Pharmacology of peconstitued synapses in co-culture of autonomic neurons and smooth mescle cells

自主神经元和平滑肌细胞共培养中特构突触的药理学

基本信息

批准号：
04671363
负责人：
WATANABE Minoru
金额：
$ 1.34万
依托单位：
Nagoya City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

This project was undertaken to investigate molecular mechanisms of transmission between autonomic nerve and smooth muscle cell by means of electrophysiological technics and measuring intracellular Ca concentration if preparations where neuro-muscular interaction is reconstituted by co-culture of autonomic neurons and smooth muscle. Singles neuron and smooth muscle cells isolated fron superior cervical ganglia and vas deferens or iris of young or infant rats were co-cultured for up to 7 days. Cardiac myocytes isolated from infant rats were also used as the target cedds of the sympathetic innervation.Membrane currents recorded by whole-cell clamp were compared in freshly isolated cells and from cells cultured for 3-4 days. Major currents resolved by using specific blockers were not changed significantly during the culture. Since the success rate of synapse formation during co-culture was low, synaptic current could not be measured in innervated smooth muscle cells. Morphological changes … More in interaction between nerve endings and smooth muscle cells were observed under scanning electron microscopy. Although cardiac myocytes were co-cultured with sympathetic neurons to increase the success rate, electrical recordings form innervated myocytes were not succedful either.To investigate the functional changes in intracellular Ca storage setes during the primary clture, effects of cyclopiazonic acid. a novel inhibitor of Ca-pump in endo-or sarco-plasmic reticulum(ER/SR), were examined. The decrease in stored Ca in ER/SR resulted in the decrease in Ca activated membrane currents, especially Ca activated K current (I_<K-Ca>), in both sympathetic neurons and vas deferens smooth muscle cells. Since I_<K-Ca> is the major current responsible for action potential afterhyperpalarization if both cell types, the inhibition of ER/SR Ca-pump by CPA resulted in the potentiation of membrane excitability. Intracellular Ca mobikization was investigated using Ca-fluorescent indicator, Fluo 3-AM, and laser confocal fluorescent microscopy in these cells.although the reconstifution of synatic interaction by co-culture of freshly isolated sympathetic neurons and smooth muscle cells was not very successful, it is suggested that the ionic channels in co-cultured cells remained unchanged. Less

该项目旨在通过电生理学技术研究自主神经和平滑肌细胞之间传递的分子机制，并测量通过自主神经元和平滑肌共培养重建神经肌肉相互作用的制剂的细胞内Ca浓度。从幼鼠或幼鼠的颈上神经节和输精管或虹膜中分离出的单个神经元和平滑肌细胞共培养长达 7 天。从幼鼠身上分离的心肌细胞也被用作交感神经支配的目标细胞。通过全细胞钳记录的膜电流在新鲜分离的细胞和培养3-4天的细胞中进行比较。通过使用特定阻断剂解决的主要电流在培养过程中没有显着改变。由于共培养过程中突触形成的成功率较低，因此无法测量受神经支配的平滑肌细胞中的突触电流。在扫描电子显微镜下观察神经末梢和平滑肌细胞之间相互作用的形态变化。尽管将心肌细胞与交感神经元共培养以提高成功率，但神经支配的心肌细胞的电记录也不成功。为了研究原代培养过程中细胞内钙储存功能的变化，环吡嗪酸的影响。检测了一种新型的内质网或肌质网（ER/SR）钙泵抑制剂。 ER/SR 中储存的 Ca 减少导致交感神经元和输精管平滑肌细胞中 Ca 激活的膜电流，特别是 Ca 激活的 K 电流（I_<K-Ca>）减少。由于 I_<K-Ca> 是导致两种细胞类型超平衡后动作电位的主要电流，因此 CPA 对 ER/SR Ca 泵的抑制导致膜兴奋性增强。使用 Ca 荧光指示剂、Fluo 3-AM 和激光共聚焦荧光显微镜在这些细胞中研究了细胞内 Ca 动员。尽管通过共培养新鲜分离的交感神经元和平滑肌细胞来重建突触相互作用不是很成功，但这表明共培养细胞中的离子通道保持不变。较少的