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

本研究通过自主神经节或神经节细胞与平滑肌细胞共培养重建神经肌肉连接，并利用电生理技术和细胞内钙离子浓度测量阐明突触传递机制。采用颈上神经节与输精管平滑肌细胞联合培养作为共培养的准备物。取幼鼠颈上神经节，切成若干块，进行器官培养。偶尔，单个神经节细胞被酶分散从切片中分离。用胶原酶从成熟大鼠和豚鼠的输精管中获得单个平滑肌细胞。在建立共培养方法之前，在刚从输精管分离或原代培养的单个平滑肌细胞中，研究了从交感神经末梢释放的化学递质去甲肾上腺素和ATP对膜离子电流的影响。NE降低了Ca电流（1_<Ca>）和Ca依赖的K电流（1_<K-Ca>）。后一种电流的降低更为明显，NE增加了膜的兴奋性。推测NE降低1_<K-Ca>的机制包括通过增加细胞内Ca浓度激活Ca通道，以及通过激活GTP结合蛋白更直接地抑制Ca通道活性。另一方面，Ne对1_<K-Ca>的降低可能是由Ne对细胞内Ca动员的改变所介导的。在从神经节切片中萌发的神经节细胞中发现神经分支病后，通过播撒单个平滑肌细胞进行共培养。然而，由于细菌污染经常发生在过程的后期，因此共培养成功的可能性很低。扫描电镜观察发现交感神经元与平滑肌细胞间有突触形成。表明功能性突触形成的确切证据线，如神经电刺激后平滑肌细胞的收缩或突触后电位，尚未获得。少

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