BACKGROUND After superficial mucosal injury, the disturbed gastric epithelial continuity is restored by cellular migration. Caspase-3 is an enzyme responsible for the execution of stress-induced apoptosis. Interestingly, heat shock proteins (Hsp) including Hsp60 are capable of modulating caspase-3 activity. Interestingly, we have demonstrated that heat shock preconditioning upregulates Hsp synthesis and inhibits restitution and cell proliferation via mechanisms related to de novo protein synthesis and eicosanoid pathways, both of which are crucial in the regulation of apoptosis and gastric mucosal defense systems. OBJECTIVE To assess whether caspase-3 activity is affected by heat shock preconditioning and associated pharmacological modulations after standard superficial injury to allow development of cytoprotective strategies. METHODS Guinea pig gastric mucosae were mounted and perfused in paired Ussing chambers. After heat shock (HS) preconditioning (42 degrees C) for 30 min, a superficial injury was induced (1.25 mol/l NaCl) followed by 3 h recovery. For mechanistic studies, the mucosa was exposed to 30 micromol/l arachidonic acid (AA) as a substrate for eicosanoid pathways, to 50 micromol/l quercetin (Q) to inhibit lipoxygenases, to 50 micromol/l indomethacin (In) to inhibit cyclo-oxygenases, or to 150 micromol/l cycloheximide (CHX) to inhibit de novo protein synthesis. After the experiment, the mucosa was prepared for analysis of caspase-3 activity. Hsp60 expression was analyzed to monitor the induction of heat shock response. RESULTS HS upregulated Hsp60 expression, indicating induction of the heat shock response without an effect on basal caspase-3 activity. Superficial injury itself did not affect caspase-3 activity nor Hsp60 synthesis. In all the experiments, exposure to CHX abolished caspase-3 activity and Hsp60 synthesis. AA+Q increased, Q decreased, while In+AA and In+AA+Q abolished caspase-3 activity independent of alterations in Hsp60 synthesis. Upon exposure to In+Q, HS decreased caspase-3 activity and upregulated Hsp60 synthesis. CONCLUSIONS Caspase-3 activity in isolated guinea pig gastric mucosa is regulated by mechanisms dependent on de novo protein synthesis and eicosanoid pathways but is not strictly related to Hsp synthesis. Upon modulation of the eicosanoid pathways, HS may be utilized to simultaneously decrease caspase-3 activity and increase Hsp synthesis. Modulations of the eicosanoid pathways may be utilized to reduce caspase-3 activity also upon normothermic conditions suggesting a novel mechanism by which caspase-3 is regulated in the gastric mucosa.