Heat Shock Preconditioning and Eicosanoid Pathways Modulate Caspase 3-Like Activity in Superficially Injured Isolated Guinea Pig Gastric Mucosa

Abstract

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. Aims: 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°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 µmol/l arachidonic acid (AA) as a substrate for eicosanoid pathways, to 50 µmol/l quercetin (Q) to inhibit lipoxygenases, to 50 µmol/l indomethacin (In) to inhibit cyclo-oxygenases, or to 150 µmol/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. Conclusion: 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.

Heat Shock Protein 60 or 70 Activates Nitric-oxide Synthase (NOS) I- and Inhibits NOS II-associated Signaling and Depresses the Mitochondrial Apoptotic Cascade during Brain Stem Death

“…Superimposed on the augmentation of HSP60 expression in the RVLM during both phases of Mev intoxication was a progressive decline in mitochondrial or elevation in cytosolic HSP60 in the ventrolateral medulla. We reason that this re-distribution of HSP60 may arise from a higher cytosolic presence of the newly synthesized HSP60 (73) or as a result of mitochondrial to cytosolic translocation (31). More importantly, our novel results implicate that the re-distributed HSP60 in the RVLM exerts its anti-apoptotic action by an enhanced interaction between HSP60 and mitochondrial or cytosolic Bax or Bcl-2.…”

Essential Role for Zinc-Triggered p75NTRActivation in Preconditioning Neuroprotection

“…Among the steps shown to be involved in the mechanism of ischemic PC are poly(ADP-ribose) polymerase (PARP) inhibition (Liaudet et al, 2001), caspase-3 activation (McLaughlin et al, 2003), and PARP-1 cleavage by caspase-3 (Garnier et al, 2003). In addition, heat shock protein 70 (HSP70), which regulates apoptotic cell death (Beere et al, 2000;Saleh et al, 2000;Steel et al, 2004;Stankiewicz et al, 2005), may be involved in ischemic PC (McLaughlin et al, 2003;Oksala et al, 2004).…”

Heat Shock Preconditioning Induces Protein Carbonylation and Alters Antioxidant Protection in Superficially Injured Guinea Pig Gastric Mucosa In Vitro

“…HSPs protect against ethanol-induced deep, ulcerative gastric injury [4][5][6]. Previously in in vitro models, we have shown that heat shock preconditioning leads to accumulation of HSPs without histologic injury or induction of apoptosis and is accompanied by a reversible increase of electrophysiologic resistance [7][8][9][10]. Interestingly, we have demonstrated that heat shock preconditioning exerts a general inhibition on restitution and proliferation after superficial injury by a mechanism that involves eicosanoid pathways and protein synthesis [7][8][9].…”

“…The experiments were performed as described previously [7][8][9][10]. Two halves of fundic mucosae from guinea pigs (n = 50) were mounted in Ussing-chambers and perfused (37 • C) in vitro.…”

“…Interestingly, we have demonstrated that heat shock preconditioning exerts a general inhibition on restitution and proliferation after superficial injury by a mechanism that involves eicosanoid pathways and protein synthesis [7][8][9]. These pathways also regulate mucosal caspase-3 activity, the major executor of apoptosis [10,11].…”