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

本研究通过将自主神经节或神经节细胞与平滑肌细胞共培养来重建神经肌肉接头，并利用电生理技术和细胞内Ca ~（2+）浓度的测量来阐明突触传递机制。作为共培养的准备，使用上级颈神经节和输精管平滑肌细胞的组合。取幼年大鼠上级颈神经节，切片进行器官培养。偶尔，单个神经节细胞分离酶分散从切片。用胶原酶从成年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，观察了交感神经末梢释放的化学递质去甲肾上腺素和ATP对新鲜分离的或原代培养的单个输精管平滑肌细胞膜离子电流的影响。 ...更多信息 几天去甲肾上腺素使钙电流（1_<Ca>）和钙依赖性钾电流（1_<K-Ca>）均降低。去甲肾上腺素使膜的兴奋性增加。NE降低I_2的机制可能<K-Ca>包括通过增加细胞内Ca ~（2+）浓度激活Ca ~（2+）通道，以及通过激活GTP结合蛋白直接抑制Ca ~（2+）通道活性。另一方面，NE引起的1_的减少<K-Ca>可能是通过NE引起的细胞内Ca动员的变化来介导的。然而，共培养成功的可能性很低，因为细菌污染经常发生在程序的后期。扫描电镜形态学观察发现交感神经元与平滑肌细胞间有突触形成。尚未获得表明功能可用的突触形成的确切证据，例如电刺激神经后平滑肌细胞中的收缩或突触后电位。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