Calpain-mediated breakdown of cytoskeletal proteins contributes to cholecystokinin-induced damage of rat pancreatic acini.
The cytosolic cysteine protease calpain is implicated in a multitude of cellular functions but also plays a role in cell damage. Our previous results suggest that an activation of calpain accompanied by a decrease in its endogenous inhibitor calpastatin may contribute to pancreatic damage during cerulein-induced acute pancreatitis. The present study aimed at the time course of secretagogue-induced calpain activation and cellular substrates of the protease. Isolated rat pancreatic acini were incubated with a supramaximal concentration of cholecystokinin (0.1 microM CCK) for 30 min in the presence or absence of the calpain inhibitor Z-Val-Phe methyl ester (100 microM ZVP). The activation of calpain and the expression of calpastatin and the actin cytoskeleton-associated proteins alphaII-spectrin, E-cadherin and vinculin were studied by immunoblotting. The cell damage was assessed by lactate dehydrogenase release and ultrastructural analysis including fluorescence-labelled actin filaments. Immediately after administration, CCK led to activation of both calpain isoforms, mu- and m-calpain. The protease activation was accompanied by a decrease in the E-cadherin level and formation of calpain-specific breakdown products of alphaII-spectrin. A calpain-specific cleavage product of vinculin appeared concomitantly with changes in the actin filament organization. No effect of CCK on calpastatin was found. Inhibition of calpain by ZVP reduced CCK-induced damage of the actin-associated proteins and the cellular ultrastructure including the actin cytoskeleton. The results suggest that CCK-induced acinar cell damage requires activation of calpain and that the actin cytoskeleton belongs to the cellular targets of the protease.
Weber
Heike
H
Institute of Clinical Chemistry and Laboratory Medicine, University of Rostock, D-18057 Rostock, Germany. heike.weber@med.uni-rostock.de
Hühns
Saskia
S
Lüthen
Frank
F
Jonas
Ludwig
L
D000199:Actins / Q000032:analysis / Q000378:metabolism; D000208:Acute Disease; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D015820:Cadherins / Q000032:analysis / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D002108:Ceruletide; D002766:Cholecystokinin / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D003599:Cytoskeleton / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D004151:Dipeptides / Q000494:pharmacology*; D004789:Enzyme Activation; D005260:Female; D015870:Gene Expression; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D023421:Models, Animal; D009924:Organ Culture Techniques; D010179:Pancreas / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D010195:Pancreatitis / Q000201:enzymology* / Q000473:pathology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013049:Spectrin / Q000378:metabolism; D013268:Stimulation, Chemical; D013997:Time Factors; D016596:Vinculin / Q000378:metabolism
D000199:Actins; D015820:Cadherins; D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D003598:Cytoskeletal Proteins; D004151:Dipeptides; D012333:RNA, Messenger; D016596:Vinculin; D013049:Spectrin; C095174:N-benzyloxycarbonyl-valyl-phenylalanine methyl ester; C033668:calpastatin; D002108:Ceruletide; D002766:Cholecystokinin; D002154:Calpain