Effects of motilin on intracellular free calcium in cultured smooth muscle cells from the antrum of neonatal rats.
OBJECTIVE
The aim of this study was to determine the effects of motilin on [Ca(2+)](i) regulation and its underlying molecular mechanism in cultured antral smooth muscle cells (ASMCs).


METHODS
Antral cells were isolated and cultured from neonatal rats, and then the [Ca(2+)](i) in these cells was evaluated by calcium fluorescent probe Fluo-3/AM on a laser scanning confocal microscope.


RESULTS
We show that motilin dose-dependently increased [Ca(2+)](i) concentration in cultured ASMCs. Pre-incubation of cells with either the calcium antagonist verapamil (10(-5) mol L(-1)) or the calcium chelator Egtazic (EGTA, 0.1 mmol L(-1)) significantly suppressed motilin (10(-6) mol L(-1)) induced [Ca(2+)](i) increase as indicated by fluorescent intensity. Interestingly, after mixing with the non-selective intracellular calcium release blocker TMB-8 (10(-5) mol L(-1)), guanosine triphosphate regulatory protein antagonist NEM (10(-5) mol L(-1)), phospholipase C (PLC) inhibitor compound 48/80 (1.2 microg mL(-1)) and ryanodine at high concentration (10(-5) mol L(-1)), the motilin-induced [Ca(2+)](i) increase was only partially blocked. The protein kinase C inhibitor d-sphingosine (10(-6) mol L(-1)), however, did not show any inhibitory effect on motilin-induced [Ca(2+)](i) elevation.


CONCLUSIONS
Our study suggests that motilin-stimulated [Ca(2+)](i) elevation in ASMCs is probably due to sustained extracellular Ca(2+) influx and Ca(2+) release from Ca(2+) stores via inositol tris-phosphate receptors and ryanodine receptors. Specifically, motilin-induced [Ca(2+)](i) release is accompanied with guanosine triphosphate-binding protein-coupled receptor-PLC-inositol tris-phosphate signalling cascades.
Fang
P
P
Department of Internal Medicine, The Second Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi, China.
Dong
L
L
Luo
J Y
JY
D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism; D005707:Gallic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005765:Gastrointestinal Agents / Q000494:pharmacology*; D008297:Male; D009037:Motilin / Q000494:pharmacology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D011706:Pyloric Antrum / Q000166:cytology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012433:Ryanodine / Q000494:pharmacology; D013110:Sphingosine / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology; D014966:Xanthenes / Q000378:metabolism; D003189:p-Methoxy-N-methylphenethylamine / Q000494:pharmacology
D000814:Aniline Compounds; D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D005765:Gastrointestinal Agents; D014966:Xanthenes; D012433:Ryanodine; C059715:Fluo-3; D003189:p-Methoxy-N-methylphenethylamine; D009037:Motilin; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D005707:Gallic Acid; D014700:Verapamil; D013110:Sphingosine; D005033:Ethylmaleimide; D002118:Calcium