The effects of acetylcholine on intracellular calcium fluorescence in smooth muscle cells of human esophagogastric junction cultured in vitro.

Abstract

Most esophageal motility studies are based on animals. It is necessary to explore smooth muscle motility in the human esophagus. This study was undertaken to explore the feasibility of in vitro culture of smooth muscle cells (SMCs) from human esophagogastric junction (EGJ) and to determine changes of intracellular calcium (Ca2+ ) fluorescence ([Ca2+ ]i ) in SMCs stimulated by acetylcholine (ACh). Primary cells of EGJ (Clasp, Sling, esophageal circular muscle (ECM), and longitudinal muscle (ELM)) were obtained by enzymatic digestion (ED) and explant culture with tissues (EC-T) from 9 upper esophageal carcinoma patients. Cells were cultured in smooth muscle cell medium (SMCM) and DMEM/F-12medium containing 10% newborn bovine serum (10%-F12), respectively, and then identified by α-SMA staining. After incubation with 5μM Fluo-3/am, the effect of 10-6 mM ACh on [Ca2+ ]i in Ca2+ -containing and Ca2+ -free buffers was evaluated by confocal microscopy. Cultured cells from ED and EC-T were identified as SMCs by α-SMA with spindle surface and "hills and valleys" morphology. Cells cultured in 10%-F12showed better morphology. The main characteristic of [Ca2+ ]i in Clasp-, Sling- and ECM-SMCs was the release of intracellular Ca2+ stores; the main characteristic in ELM-SMCs was extracellular Ca2+ influx. However, these cells seemed not to rely on a unique Ca2+ activity, instead combining the two activities to maintain [Ca2+ ]i . It was feasible to culture human EGJ SMCs in vitro; moreover, Ach-induced changes of [Ca2+ ]i in EGJ SMCs represent a complex interaction of intracellular Ca2+ release and extracellular Ca2+ influx.