Release of (3H)norepinephrine from a clonal line of pheochromocytoma cells (PC12) by nicotinic cholinergic stimulation

Abstract

Release experiments were carried out in vitro with a clonal line of rat pheochromocytoma cells (designated PC12) which synthesize and store catecholamines and which, after treatment with nerve growth factor (NGF), cease cell division and extend neuronal-like processes. In the present study, PC12 cells were exposed to [3H]norepinephrine (NE) which they took up and stored in reserpine-sensitive sites. Exposure of such cells to nicotinic cholinergic agonists resulted in release of [3H]NE into the external medium. Release terminated within 1 min and partially returned after 20 min in the absence of agonist. After 1 min of stimulation with nicotine, NGF-treated cells released 5-6% of their contents of [3H]NE while NGF-untreated cells released 1-2%. Release from both NGF-treated and -untreated PC12 cells was inhibited in the absence of Ca2+ or by elevated Mg2+ and was blocked by the nicotinic antagonists D-tubocurarine and mecamylamine (50% inhibition at 0.1 and 0.06 micrometer, respectively). Release was not affected by the presence of tetrodotoxin. Such findings suggest that release of [3H]NE from PC12 cells may be mediated via stimulation of nicotinic acetylcholine receptors and a consequent stimulation-secretion coupling mechanism.