An increase in intracelluar free calcium ions modulated by cholinergic receptors in rat facial nucleus.
BACKGROUND
Ca(2+) in the central nervous system plays important roles in brain physiology, including neuronal survival and regeneration in rats with injured facial motoneurons. The present research was to study the modulations of intracellular free Ca(2+) concentrations by cholinergic receptors in rat facial nucleus, and the mechanisms of the modulations.


METHODS
The fluorescence intensity of facial nucleus in Fluo-3 AM loaded acute brainstem slices was detected by applying intracellular free Ca(2+) measurement technique via confocal laser scanning microscope. The changes of fluorescence intensity of facial nucleus indicate the average changes of intracellular free Ca(2+) levels of the neurons.


RESULTS
Acetylcholine was effective at increasing the fluorescence intensity of facial nucleus. Muscarine chloride induced a marked increase of fluorescence intensity in a concentration dependent fashion. The enhancement of fluorescence intensity by muscarine chloride was significantly reduced by thapsigargin (depletor of intracellular Ca(2+) store; P < 0.01), rather than Ca(2+) free artifical cerebrospinal fluid or EGTA (free Ca(2+) chelator; P > 0.05). And the increase of fluorescence intensity was also significantly inhibited by pirenzepine (M(1) subtype selective antagonist; P < 0.01) and 4-DAMP (M(3) subtype selective antagonist; P < 0.01). In addition, fluorescence intensity was markedly increased by nicotine. The enhancement of fluorescence intensity by nicotine was significantly reduced by EGTA, nifedipine (L-type voltage-gated Ca(2+) channel blocker), dihydro-beta-erythroidine (alpha4beta2 subtype selective antagonist), and in Ca(2+) free artificial cerebrospinal fluid (P < 0.01), but not in the presence of mibefradil (M-type voltage-gated Ca(2+) channel blocker) or thapsigargin (P > 0.05).


CONCLUSIONS
The data provide the evidence that muscarinic receptors may induce the increase of intracellular free Ca(2+) levels through the Ca(2+) release of intracellular Ca(2+) stores, in a manner related to M(1) and M(3) subtypes of muscarinic receptors in rat facial nucleus. Nicotine may increase intracellular free Ca(2+) concentrations via the influx of extracellular Ca(2+)+ mainly across L-type voltage-gated Ca(2+) channels, in a manner related to the alpha4beta2 subtype of nicotinic receptors.
Sun
Da-wei
DW
Department of Otolaryngology, Affiliated Hospital of Medical College, Qingdao University, Qingdao, Shandong, China. sundw@163.com
Zhou
Rui
R
Li
Na
N
Zhang
Qiu-gui
QG
Zhu
Fu-gao
FG
D000109:Acetylcholine / Q000494:pharmacology; D000814:Aniline Compounds / Q000008:administration & dosage; D000818:Animals; D001933:Brain Stem / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D003959:Diamines / Q000494:pharmacology; D005154:Facial Nerve / Q000166:cytology*; D005260:Female; D005456:Fluorescent Dyes / Q000008:administration & dosage; D066298:In Vitro Techniques; D008297:Male; D018613:Microscopy, Confocal; D009046:Motor Neurons / Q000187:drug effects* / Q000378:metabolism*; D018721:Muscarinic Agonists / Q000494:pharmacology; D009538:Nicotine / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D010890:Pirenzepine / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011950:Receptors, Cholinergic / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000378:metabolism; D014331:Tropicamide / Q000494:pharmacology; D014966:Xanthenes / Q000008:administration & dosage
D000814:Aniline Compounds; D003959:Diamines; D005456:Fluorescent Dyes; D018721:Muscarinic Agonists; D018722:Nicotinic Agonists; D010880:Piperidines; D011950:Receptors, Cholinergic; D011976:Receptors, Muscarinic; D011978:Receptors, Nicotinic; D014966:Xanthenes; C059715:Fluo-3; D010890:Pirenzepine; D009538:Nicotine; C042375:4-diphenylacetoxy-1,1-dimethylpiperidinium; D014331:Tropicamide; D000109:Acetylcholine; C054938:methoctramine; D002118:Calcium