Methyllycaconitine-sensitive neuronal nicotinic receptor-operated slow Ca2+ signal by local application or perfusion of ACh at the mouse neuromuscular junction

Local application of acetylcholine (ACh; 0.3 mM, 20 mu l) elicited bi-phasi c elevation of intracellular Ca2+ concentrations (contractile fast and non- contractile slow Ca2+ signal measured as aequorin luminescence) in diaphrag m muscle preparation. A neuronal nicotinic antagonist methyllycaconitine (M LA; 0.01-1 mu M), which did not affect the fast Ca2+ transients and twitch tension, concentration-dependently depressed only the slow Ca2+ component. Ca2+ channel blockers, Cd2+ (200 mu M), nitrendipine (1 mu M), verapamil(1 mu M) and diltiazem (1 mu M), or a Na+ channel blocker tetrodotoxin (TTX; 0 .1 mu M) failed to prevent the generation of slow Ca2+ response. Perfusion of ACh (1 mu M) to isolated single skeletal (flexor digitorum brevis) muscl e cells pretreated with TTX (0.1 mu M) also elicited a slow Ca2+ signal mea sured as confocal imaging with a fluorescent dye, fluo-3, at the endplate r egion. MLA (1 mu M) antagonized against the ACh perfusion-elicited slow Ca2 + signal. Perfusion of choline (1 mM), a neuronal nicotinic agonist, also e licited the MLA-sensitive slow Ca2+ signal. These results strongly suggest that the ACh-induced slow Ca2+ signal reflects Ca2+ entry through a postsyn aptic MLA-sensitive neuronal nicotinic ACh receptor subtype at the neuromus cular junction. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.