Calpain activation contributes to oxidative stress-induced pancreatic acinar cell injury.
Oxygen radicals have been implicated as mediators in the pathogenesis of pancreatic acinar cell necrosis. However, the sequence of events between the oxidative insult and cell damage remains unclear. In the current study, we investigated whether the Ca(2+)-regulated cytosolic cysteine protease calpain is activated by oxidative stress and contributes to oxidant-induced acinar cell damage. Isolated rat pancreatic acinar cells were exposed to hydrogen peroxide (H(2)O(2))-generated oxidative stress in the presence or absence of the Ca(2+) chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM) and different calpain inhibitors including benzyloxycarbonyl-valyl-phenylalanine methyl ester. Calpain activation was studied by fluorescence spectrophotometry and immunoblotting. Cell injury was assessed by lactate dehydrogenase (LDH) release and characterization of the cellular ultrastructure including fluorescence-labeled actin filaments. Exposure of acinar cells to H(2)O(2) provoked a time- and dose-dependent increase in calpain proteolytic activity involving the ubiquitous isoforms mu- and m-calpain. The activation of calpain reflected the time course of developing cytotoxicity as demonstrated by increased LDH release. Inhibition of oxidant-induced calpain activity by BAPTA-AM and various calpain inhibitors provoked a decline in oxidant-induced cell injury. In particular, changes in the actin filament organization characterized by an increase in the basolateral actin and by a detachment of actin from the cell membrane in the region of membrane blebs were clearly reduced. In summary, our findings suggest that acinar cell damage through oxidative stress requires activation of calpain and that the actin cytoskeleton belongs to the cellular targets of the protease. The results support the hypothesis that calpain activation may play a role in the development of acute pancreatitis.
Weber
H
H
Institute of Clinical Chemistry and Laboratory Medicine, University of Rostock, Germany. heike.weber@med.uni-rostock.de
Hühns
S
S
Lüthen
F
F
Jonas
L
L
Schuff-Werner
P
P
D000818:Animals; D015153:Blotting, Western; D002154:Calpain / Q000378:metabolism*; D004789:Enzyme Activation; D005260:Female; D005476:Fluphenazine / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008854:Microscopy, Electron; D018384:Oxidative Stress; D010179:Pancreas / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology / Q000648:ultrastructure; D051381:Rats; D011917:Rats, Inbred Lew
D006861:Hydrogen Peroxide; D007770:L-Lactate Dehydrogenase; D002154:Calpain; D005476:Fluphenazine