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

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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