Acetylcholine-induced calcium signaling associated with muscarinic receptor activation in cultured myenteric neurons.
BACKGROUND
Within the enteric nervous system, acetylcholine (ACh) is an important neurotransmitter. Experimental evidence has suggested that in myenteric neurons, calcium plays a key role in the coupling of cholinergic receptors to secretory responses.


METHODS
We investigated the effects of ACh on intracellular calcium concentration ([Ca2+]i) in individual myenteric neurons using fura-2 microspectrofluorometry.


RESULTS
Resting [Ca2+]i in myenteric neurons was 62.5 +/- 3 nM. Acetylcholine produced dose-dependent increases in [Ca2+]i in myenteric neurons. As the concentration of ACh was increased from 0.1 to 100 microM, both the peak [Ca2+]i response as well as the percentage of responding neurons progressively increased, with a maximal effect at 100 microM (347 +/- 31 nM, 95 percent of neurons). The effect of ACh was not sensitive to pertussis toxin (100 ng/mL). Calcium ion (Ca2+) responses to ACh were abolished by removal of extracellular Ca2+ as well as exposure to nifedipine (10 microM). Characterization of the specific muscarinic subtype(s) involved in ACh-mediated Ca2+ transients was performed using the specific antagonists pirenzepine (M1), gallamine (M2), and 4-DAMP (M3). Pirenzepine (1 microM) blocked increases in [Ca2+]i induced by ACh; gallamine (1 microM) and 4-DAMP (1 microM) had no significant effect. Intracellular Ca2+ responses to ACh were not affected by incubation with the phorbol ester tetradecanoylphorbol-13-acetate (1 microM).


CONCLUSIONS
These findings suggest that ACh induces increases in [Ca2+]i in myenteric neurons by promoting influx of extracellular Ca2+ through L-type voltage-dependent Ca2+ channels by activation of the M1 muscarinic receptor subtype. The Ca2+ response does not appear to involve a pertussis toxin-sensitive G protein.
Simeone
D M
DM
Department of Surgery, University of Michigan School of Medicine, Ann Arbor, USA.
Kimball
B C
BC
Mulholland
M W
MW
D000109:Acetylcholine / Q000502:physiology*; D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology*; D002478:Cells, Cultured; D019204:GTP-Binding Proteins / Q000502:physiology; D006168:Guinea Pigs; D008858:Microscopy, Phase-Contrast; D009197:Myenteric Plexus / Q000502:physiology*; D009474:Neurons / Q000502:physiology; D011976:Receptors, Muscarinic / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*
D015220:Calcium Channels; D011976:Receptors, Muscarinic; D019204:GTP-Binding Proteins; D000109:Acetylcholine; D002118:Calcium