Leminoprazole (an acid pump inhibitor) has a mucosal protective effect against various experimental gastric lesions, but the underlying mechanism remains unknown. We examined whether leminoprazole prevents indomethacin-induced damage to cultured gastric mucosal cells. The viability of rabbit gastric mucosal cells was assessed by the 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide and dye exclusion methods. [35S]Methionine-labeled proteins were detected by autoradiography after sodium dodecylsulfate-polyacrylamide gel electrophoresis. Western blot analysis was carried out using anti-heat shock protein (HSP)-70 and anti-HSP-72 antibodies. Exposure of gastric mucosal cells to indomethacin for 4 hr apparently reduced their viability in a dose-related manner. Pretreatment with leminoprazole for 4 hr significantly prevented the reduction in cell viability caused by 50 microM indomethacin, although omeprazole was not effective. However, such pretreatment did not prevent the severe damage induced by 500 microM indomethacin. 16,16-Dimethyl prostaglandin E2 significantly prevented the cell damage induced by indomethacin at both 50 and 500 microM. Leminoprazole alone did not affect cell viability. The cytoprotection by leminoprazole was expressed after a 2-hr lag period. Leminoprazole did not promote prostaglandin E2 synthesis by cells, but it apparently induced the synthesis of 83-kDa, 72-kDa, 52-kDa and 35-kDa proteins. Both the cytoprotection and the induction of such protein synthesis were abolished by cycloheximide and actinomycin D. The leminoprazole-induced 72-kDa protein did not react with the antibodies against HSP-70 and HSP-72. These results indicate that leminoprazole directly protects gastric mucosal cells against mild damage caused by indomethacin and that its cytoprotective effect might be mediated through de novo synthesized proteins. In addition, it is suggested that the leminoprazole-induced proteins might be unknown proteins related to cytoprotection, although the exact characters of the proteins are unclear.