Identification of a functional Ca2+-sensing receptor in normal human gastric mucous epithelial cells

The purpose of the present study was to determine whether human gastric muc ous epithelial cells express a functional Ca2+-sensing receptor (CaR). Huma n gastric mucous epithelial cells were isolated from surgical tissues and c ultured on glass coverslips, plastic dishes, or porous membrane filters. Ce ll growth was assessed by the MTT assay, CaR localization was detected by i mmunohistochemistry and confocal microscopy, CaR protein expression was ass essed by Western immunoblotting, and intracellular Ca2+ concentration( [Ca2 +](i)) was determined by fura 2 spectrofluorometry. In paraffin sections of whole stomach, we found strong CaR immunohistochemical staining at the bas olateral membrane, with weak CaR-staining at the apical membrane in mucous epithelial cells. Confocal microscopy of human gastric mucous epithelial ce ll cultures showed abundant CaR immunofluorescence at the basolateral membr ane and little to no CaR immunoreactivity at the apical membrane. Western i mmunoblot detection of CaR protein in cell culture lysates showed two signi ficant immunoreactive bands of 140 and 120 kDa. Addition of extracellular C a2+ to preconfluent cultures of human gastric mucous epithelial cells produ ced a significant proliferative response. Changes in [Ca2+](i) were also ob served in response to graded doses of extracellular Ca2+ and Gd3+. The phos pholipase C inhibitor U-73122 specifically inhibited Gd3+-induced changes i n [Ca2+](i) in the gastric mucous epithelial cell cultures. In conclusion, we have identified the localization of a functional CaR in human gastric mu cous epithelial cells.