Measurement of synaptic currents and changes in intracellular Ca concentration in smooth muscle cells co-cultured with autonomic nerve cells.

与自主神经细胞共培养的平滑肌细胞中突触电流和细胞内 Ca2+浓度变化的测量。

基本信息

批准号：
02671009
负责人：
WATANABE Minoru
金额：
$ 1.47万
依托单位：
Nagoya City University, Faculty of Pharmaceutical Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

The present study was undertaken to reconstitute neuromuscular junction by co-culture of autonomic ganglia or ganglion cells with smooth muscle cells and to elucidate the synaptic transmission mechanisms using electrophysiological techniques and measurement of intracellular Ca ion concentration. As preparations for co-culture, the combination of superior cervical ganglia and vas deferens smooth muscle cells were used. Superior cervical ganglia was removed from infant rat, sliced into several pieces and organ-cultured. Occasionally, single ganglion cells were isolated by enzymatical dispersion from the slices. Single smooth muscle cells were also obtained with coliagenase from vas deferentia of matured rats and guinea-pig.Before establishing co-culture methods, effects of norepinephrine and ATP, chemical transmitters released from sympathetic nerve endings, on membrane ionic currents-were examined in single smooth muscle cells freshly isolated from vas deferens or primary cultured for a … More few days. NE decreased both Ca current(1_<Ca>)and Ca-dependent K current(1_<K-Ca>). The decrease in the latter current was more prominent, NE increased the membrane excitability. It is suaaested that the mechanisms for 1_<K-Ca> decrease by NE includes Ca channel ind('Ftivation via an increase in intracellukar Ca concentration and, in addition, more direct inhibition of Ca channel activity by activation of GTP binding protein. On the other hand, it is suggested that the decrease in 1_<K-Ca> by Ne is mediated by changes in intracellular Ca mobilization by NE.The co-culture was performed by seeding single smooth muscle cells after neurocladism had been found in ganglion cells sprouting from ganglia slices. The probability of success of co-culture was, however, low since contamination with bacteria often happened in the later part of the procedure. The formation of synaps between sympathetic neuron and smooth muscle cells had been found in morphological survey with scanning electronmicroscope. Exact lines of evidence that indicate formation of functionally available synaps, such as contraction or post synaptic potentials in smooth muscle cells following electrical stimulation of the nerve, have not been obtained yet. Less

本研究通过自主神经节或神经节细胞与平滑肌细胞的共培养来重建神经肌肉接头，并使用电生理学技术和细胞内钙离子浓度的测量来阐明突触传递机制。使用颈上神经节和输精管平滑肌细胞，从幼鼠身上取出颈上神经节，切成几块并进行器官培养，有时也使用单个神经节细胞。通过酶法分散从切片中分离出单个平滑肌细胞，还用胶原酶从成熟大鼠和豚鼠的输精管中获得。在建立共培养方法之前，去甲肾上腺素和 ATP（从交感神经末梢释放的化学递质）对在从输精管新鲜分离或原代培养几天的单个平滑肌细胞中检查膜离子电流，NE 均降低了 Ca。 NE电流(1_<Ca>)和Ca依赖性K电流(1_<K-Ca>)的降低更为显着，说明NE增加了膜的兴奋性。 > NE 的减少包括 Ca 通道 ind（通过增加细胞内 Ca 浓度进行激活，此外，通过激活 GTP 结合蛋白更直接地抑制 Ca 通道活性。另一方面，表明Ne 的 1_<K-Ca> 是通过 NE 改变细胞内 Ca 动员来介导的。在从神经节切片中发芽的神经节细胞中发现神经分支后，通过接种单个平滑肌细胞进行共培养。共培养的成功概率。 -然而，由于细菌污染经常发生在该过程的后期部分，因此培养物较低。在用扫描电子显微镜进行形态学观察时发现了交感神经元和平滑肌细胞之间突触的形成。尚未获得表明功能可用突触形成的证据，例如神经电刺激后平滑肌细胞的收缩或突触后电位。