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

The aim of this study was to determine the effects of motilin on [Ca²⁺]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 [Ca²⁺]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 [Ca²⁺]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 [Ca²⁺]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 μg mL⁻¹) and ryanodine at high concentration (10⁻⁵ mol L⁻¹), the motilin-induced [Ca²⁺]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 [Ca²⁺]i elevation. Our study suggests that motilin-stimulated [Ca²⁺]i elevation in ASMCs is probably due to sustained extracellular Ca²⁺ influx and Ca²⁺ release from Ca²⁺ stores via inositol tris-phosphate receptors and ryanodine receptors. Specifically, motilin-induced [Ca²⁺]i release is accompanied with guanosine triphosphate-binding protein-coupled receptor-PLC-inositol tris-phosphate signalling cascades.