Acetylcholine sensitivity of biphasic Ca 2+ mobilization induced by nicotinic receptor activation at the mouse skeletal muscle endplate

Acetylcholine (ACh) was locally applied onto the endplate region in a mouse phrenic nerve‐diaphragm muscle preparation to measure intracellular free calcium ([Ca 2+ ] i ) entry through nicotinic ACh receptors (AChRs) by use of Ca 2+ ‐aequorin luminescence. ACh (0.1–3 m m , 20 μl) elicited biphasic elevation of [Ca 2+ ] i (fast and slow Ca 2+ mobilization) in muscle cells. The peak amplitude of the slow Ca 2+ mobilization (not accompanied by twitch tension) was concentration‐dependently increased by ACh, whereas that of the fast component (accompanied by twitch tension) reached a maximum response at a lower concentration (0.1 m m ) of applied ACh. A pulse of nicotinic agonists, (−)‐nicotine (10 m m ) and 1,1‐dimethyl‐4‐phenyl‐piperazinium (10 m m ), but not a muscarinic agonist pilocarpine (10 m m ), also elicited a biphasic Ca 2+ signal. Even though ACh release from motor nerve endings was blocked by botulinum toxin (5 μg, bolus i.p. before isolation of the tissue), the generation of both a fast and slow Ca 2+ component caused by ACh application was observed. These results strongly suggest that ACh locally applied onto the endplate region of skeletal muscle induces a slow Ca 2+ signal reflecting Ca 2+ entry through a postsynaptic nicotinic AChR, which has a low sensitivity to transmitter ACh.British Journal of Pharmacology (1998) 123 , 1418–1424; doi: 10.1038/sj.bjp.0701725