Factors from glial cells regulate choline acetyltransferase and tyrosine hydroxylase activities in a hybrid-hybrid cell line.
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Biomedical subjects
Publications and source records attributed to B Hamprecht.
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Different cholinergic cell lines were fused with an adrenergic neuroblastoma cell line (N115-BU-8). Its fusion with a cholinergic neuroblastoma-glioma hybrid produced a "hybrid-hybrid" line containing cholinergic and adrenergic enzyme activities. Both activities were also present in subclones of this line. The presence of catecholamines in single cells was confirmed by microspectrofluorimetry. These results are discussed with respect to the possibility of a simultaneous synthesis of noradrenaline and acetylcholine in single cells. The cholinergic and adrenergic enzyme activities are influenced by cell density, by dexamethasone, and by conditioned medium.
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Electrical excitability is one of the various neuronal properties of neuroblastoma X glioma hybrid cells. At a Ca2+ concentration of 1.8 mM the action potential is inhibited by tetrodotoxin, suggesting that the inward current is carried by Na+ ions. In contrast, at a Ca2+ concentration of 20-36 mM and even in the absence of Na+, spikes (sometimes repetitive) with strong hyperpolarizing afterpotential occur, which are no longer affected by tetrodotoxin. They are, however, blocked by antagonists of Ca2+ like La3+, Co2+, Mn2+, and the synthetic compounds D-600 and BAY a-1040. This seems to indicate that at high concentrations of Ca2+, the inward current of the action potential is essentially carried by Ca2+. Sr2+, but not Mg2+ can effectively substitute for Ca2+. It slows down the time course of the action potential. Ba2+ depolarizes the membrane gradually. If Ca2+ is also present, Ba2+ causes a reduced depolarization and spontaneous action potentials with no hyperpolarizing after-potential are observed.
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The stability of a clonal mouse neuroblastoma x rat glioma hybrid cell line was examined. Cell volume and cellular content of DNA and protein were measured as functions of the passage number. They decreased with the number of serial subcultivations. Cellular volume was linearly related to cellular DNA and protein. Thus, measurements of cell volume can be used to monitor the loss of DNA from hybrid cells. After about 60 passages a stable population of hybrid cells arose, as judged by the constancy of cellular volume and by the decreased coefficient of variation of the cell volume distribution. A mathematical model for the kinetics of the simultaneous loss of cellular volume, DNA and protein is introduced. Several neuronal properties were investigated. The specific activity of the neurotransmitter enzyme choline acetyltransferase decreased by more than 50% during 56 passages. After 70 subcultivations, the hybrid cells were still capable of extending processes, action potentials could still be elicited electrically or by iontophoretic application of acetylcholine, and the cells still responded to prostaglandin E1 as they do at low passage number.
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A bromodeoxyuridine resistant mutant of rat gliom line C6 grown in the presence of N6,02'-dibutyryl adenosine-3':5'-cyclic monophosphate, although inexcitable by electrical current, responds to iontophoretic application of acetylcholine by slow hyperpolarization. The sensitivity to acetylcholine is strongly reduced by excessive amounts of acetylcholine. The hyperpolarization response is inhibited by atropine or alpha-bungarotoxin, but not by D-tubocurarine. In the absence of acetylcholine some cells display spontaneous hyperpolarizations at regular intervals. The membrane resting potential is decreased if the concentration of external K+ is raised.