title: Effects of motilin on intracellular free calcium in cultured smooth muscle cells from the antrum of neonatal rats.
abstract: 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.
authors.affiliation: Department of Internal Medicine, The Second Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi, China.
authors.forename: P
authors.initials: P
authors.lastname: Fang
authors.forename: L
authors.initials: L
authors.lastname: Dong
authors.forename: J Y
authors.initials: JY
authors.lastname: Luo
mesh_terms: 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
chemical_list: 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