Effect of ebrotidine on gastric mucosal calcium channel activity

Ebrotidine is a new H2-receptor antagonist also known for its gastroprotective effect against ethanol-induced mucosal injury. In this study, we investigated the effect of ebrotidine on the activity of the gastric mucosal calcium channels. The channel complex was isolated from the solubilized gastric epithelial cell membranes by affinity chromatography on wheat germ agglutinin. After being labeled with [3H]PN200-110, the complex was reconstituted into phosphatidylcholine vesicles which exhibited active 45Ca2+ uptake into intravesicular space and responded in a concentration-dependent manner to calcium channel activator, BAY K8644, as well as to calcium channel antagonist, PN200-110. The 45Ca2+ uptake was inhibited by ebrotidine. Maximum inhibitory effect was attained at 50 micrograms/ml ebrotidine, at which point a 54.9% decrease in uptake occurred. The gastric mucosal calcium channels, on epidermal growth factor binding (EGF) in the presence of ATP, responded by an increase in tyrosine phosphorylation of 55 and 170 kDa proteins, and the vesicles containing the phosphorylated channels displayed a 48% greater 45Ca2+ uptake. This phosphorylation process was inhibited by ebrotidine. Furthermore, ebrotidine also interfered with the binding of EGF to calcium channel protein. The results point toward the importance of EGF in the maintenance of gastric mucosal calcium homeostasis, and suggest that ebrotidine has the ability to protect the cellular integrity from calcium imbalance by modulating the EGF-stimulated gastric mucosal calcium channel phosphorylation.