Effects of motilin on intracellular free calcium in cultured smooth muscle cells from the antrum of neonatal rats.

P Fang, L Dong, J Y Luo

Index: Acta Physiol. (Oxf.) 199(1) , 53-61, (2010)

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Abstract

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).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.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.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.