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 microl) elicited bi-phasic elevation of intracellular Ca2+ concentrations (contractile fast and non-contractile slow Ca2- signal measured as aequorin luminescence) in diaphragm muscle preparation. A neuronal nicotinic antagonist methyllycaconitine (MLA; 0.01-1 microM), which did not affect the fast Ca2+ transients and twitch tension, concentration-dependently depressed only the slow Ca2+ component. Ca2+ channel blockers, Cd2+ (200 microM), nitrendipine (1 microM), verapamil (1 microM) and diltiazem (1 microM), or a Na+ channel blocker tetrodotoxin (TTX; 0.1 microM) failed to prevent the generation of slow Ca2+ response. Perfusion of ACh (1 microM) to isolated single skeletal (flexor digitorum brevis) muscle cells pretreated with TTX (0.1 microM) also elicited a slow Ca2+ signal measured as confocal imaging with a fluorescent dye, fluo-3, at the endplate region. MLA (1 microM) antagonized against the ACh perfusion-elicited slow Ca2+ signal. Perfusion of choline (1 mM), a neuronal nicotinic agonist, also elicited the MLA-sensitive slow Ca2+ signal. These results strongly suggest that the ACh-induced slow Ca2+ signal reflects Ca2+ entry through a postsynaptic MLA-sensitive neuronal nicotinic ACh receptor subtype at the neuromuscular junction.
Dezaki
K
K
Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Japan.
Tsuneki
H
H
Kimura
I
I
D000109:Acetylcholine / Q000008:administration & dosage / Q000494:pharmacology*; D000157:Aconitine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000287:Administration, Topical; D000818:Animals; D015220:Calcium Channels / Q000502:physiology; D020013:Calcium Signaling / Q000187:drug effects*; D004594:Electrophysiology; D008297:Male; D051379:Mice; D009045:Motor Endplate / Q000187:drug effects / Q000502:physiology; D009469:Neuromuscular Junction / Q000187:drug effects / Q000502:physiology*; D009474:Neurons / Q000378:metabolism*; D018722:Nicotinic Agonists / Q000494:pharmacology; D018733:Nicotinic Antagonists / Q000494:pharmacology*; D010477:Perfusion; D011978:Receptors, Nicotinic / Q000502:physiology*; D013779:Tetrodotoxin / Q000494:pharmacology
D015220:Calcium Channels; D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; C054634:methyllycaconitine; D013779:Tetrodotoxin; D000109:Acetylcholine; D000157:Aconitine