Expression and regulation of calpain in rat pancreatic acinar cells.
BACKGROUND
Calpains, cytosolic Ca(2+)-dependent cysteine proteases, are expressed in a variety of mammalian cells and have been found to participate in stimulus-secretion coupling in platelets and alveolar cells.


OBJECTIVE
In pancreatic acinar cells, expression of calpains and their role in the secretory process have not yet been elucidated. Both subjects, therefore, were examined in the current study.


METHODS
mu-calpain and m-calpain were detected immunochemically. Calpain activation was measured by fluorescence spectrophotometry and single-cell fluorometry using Suc-Leu-Leu-Val-Tyr-AMC as substrate. Amylase secretion and cell damage, characterized by lactate dehydrogenase release, were measured by colorimetric assays.


RESULTS
Immunochemistry revealed cytoplasmic localization of both calpain isoforms. Immediately after increasing the cytosolic Ca(2+) concentration with ionomycin, a marked dose-dependent protease activation and cellular damage were observed. Inhibition of ionomycin-mediated enzyme activation through preincubation of cells with Ca(2+)-free medium, BAPTA-AM, or Z-Leu-Leu-Tyr-CHN(2) significantly reduced cell injury. Cholecystokinin (100 pM) also induced proteolytic activity, preceding cholecystokinin-stimulated amylase secretion. Protease activity and amylase release were significantly inhibited by Z-Leu-Leu-Tyr-CHN(2 ) retreatment.


CONCLUSIONS
Calpains are expressed in pancreatic acinar cells and may participate in stimulus-secretion coupling. In addition, our study indicates that pathologic calpain activation may contribute to Ca(2+)-mediated acinar cell damage.
Weber
Heike
H
Institute of Clinical Chemistry and Pathobiochemistry, University of Rostock, Rostock, Germany. heike.wber@med.uni-rostock.de
Hopp
Hans-Heinrich
HH
Wagner
Andreas C C
AC
Noack
Thomas
T
Jonas
Ludwig
L
Lüthen
Frank
F
Schuff-Werner
Peter
P
D000681:Amylases / Q000032:analysis / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002154:Calpain / Q000032:analysis / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D002478:Cells, Cultured; D002766:Cholecystokinin; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation; D007150:Immunohistochemistry; D015759:Ionomycin; D007527:Isoenzymes / Q000096:biosynthesis; D009842:Oligopeptides / Q000494:pharmacology; D010179:Pancreas / Q000201:enzymology / Q000378:metabolism*; D051381:Rats
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009842:Oligopeptides; C062355:N-benzyloxycarbonyl-leucyl-valyl-glycine diazomethane; D015759:Ionomycin; D002766:Cholecystokinin; D000681:Amylases; D002154:Calpain; D002118:Calcium