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

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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