Rapid increase in cytosolic calcium ion concentration mediated by acetylcholine receptors in cultured retinal neurons and Müller cells.
OBJECTIVE
This study was conducted to detect the presence of muscarinic or nicotinic receptors in cultured retinal neurons and Müller cells.


METHODS
Pure Müller cell cultures and cocultures of retinal neurons and Müller cells were used; the former, obtained from adult rabbit retinas, and the latter, retinal neurons from neonatal rats, were cocultured with Müller cells. Intracellular calcium ion concentration ([Ca2+]i) following the administration of acetylcholine, a cholinesterase inhibitor (trichlorfon), nicotine or muscarinic agonist with or without a receptor antagonist was monitored using the calcium ion indicator, fura-2.


RESULTS
Acetylcholine and trichlorfon induced rapid increase in [Ca2+]i in half of either cell type. Trichlorfon induced positive response in coculture but not in the pure Müller cell cultures. This positive response was blocked only partially in the presence of atropine. Approximately 30-40% of neurons responded to nicotine at 5 microM, which was significantly blocked by alpha-bungarotoxin at 50 nM. No response to nicotine could be detected in Müller cells. Approximately 50% of neurons responded to muscarine at 50 microM, but 500 microM was required for the formation of calcium transients in 50% of Müller cells. The muscarine inducement of rapid increase in [Ca2+]i was blocked by atropine. The agonist of M1 (a muscarinic receptor subtype), McN-A-343, at 0.5 microM induced the most significant and rapid increase in [Ca2+]i both in neurons and Müller cells. McN-A-343 administration at 0.05 microM induced positive response in half the neurons, but only in approximately 10% of Müller cells. Such positive response was not observed following preincubation with the M1 antagonist, pirenzepine, at 50 microM.


CONCLUSIONS
Cocultured retinal neurons enhance the release of acetylcholine following anticholinesterase administration, and approximately half the neurons were found to possess muscarinic and nicotinic receptors. However, Müller cells appeared to possess only the less sensitive muscarinic receptor. Muscarinic receptor subtypes on either type of cell contained at least M1.
Wakakura
M
M
Department of Ophthalmology, Kitasato University School of Medicine, Sagamihara, Japan. wakakura@medcc.kitasato-u.ac.jp
Utsunomiya-Kawasaki
I
I
Ishikawa
S
S
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured / Q000187:drug effects; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes; D016257:Fura-2; D008858:Microscopy, Phase-Contrast; D009116:Muscarine / Q000494:pharmacology; D018721:Muscarinic Agonists / Q000494:pharmacology; D009457:Neuroglia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009538:Nicotine / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D011950:Receptors, Cholinergic / Q000378:metabolism*; D012165:Retinal Ganglion Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D014236:Trichlorfon / Q000494:pharmacology
D002800:Cholinesterase Inhibitors; D005456:Fluorescent Dyes; D018721:Muscarinic Agonists; D011950:Receptors, Cholinergic; D009538:Nicotine; D009116:Muscarine; D014236:Trichlorfon; D000109:Acetylcholine; D002118:Calcium; D016257:Fura-2