title: Acetylcholine-induced calcium signaling associated with muscarinic receptor activation in cultured myenteric neurons.
abstract: 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.
authors.affiliation: Department of Surgery, University of Michigan School of Medicine, Ann Arbor, USA.
authors.forename: D M
authors.initials: DM
authors.lastname: Simeone
authors.forename: B C
authors.initials: BC
authors.lastname: Kimball
authors.forename: M W
authors.initials: MW
authors.lastname: Mulholland
mesh_terms: 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*
chemical_list: D015220:Calcium Channels; D011976:Receptors, Muscarinic; D019204:GTP-Binding Proteins; D000109:Acetylcholine; D002118:Calcium