Proteolytic activity in intact sheets of polarized epithelial cells as determined by a cell-permeable fluorogenic substrate.
The purpose of the present investigation was to develop a system for continuous evaluation of extralysosomal proteolytic activity and its regulation in polarized epithelial cells. Filter inserts containing a tight monolayer of primary cultured pig thyrocytes were placed in a thermostated aluminium block. The cell-permeable, fluorogenic calpain and proteasome substrate succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin was added to the apical buffer and fluorescence changes were continuously measured via the fibre optics of a luminometer held at a fixed distance from the cell layer. Basal proteolytic activity was reduced by 60-70% by the proteasome inhibitor lactacystin. Proteolysis was increased within a few minutes after application of Ca(2+)-mobilizing agents (ionomycin, 4-bromo-A23187, thapsigargin and maitotoxin). Forskolin and staurosporine also enhanced the proteolytic activity. We conclude that Ca(2+)mobilization, and possibly also changes of protein kinase activity, rapidly increase non-lysosomal proteolysis in the intact thyroid epithelium.
Karlsson
J O
JO
Institute of Anatomy and Cell Biology, Göteborg University, Göteborg, SE-405 30, Sweden. janolof.karlsson@anatcell.gu.se
Lundquist
J
J
Skoglund
I
I
Nilsson
M
M
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000378:metabolism*; D016764:Cell Polarity; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D003374:Coumarins / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000201:enzymology*; D005453:Fluorescence; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D013552:Swine; D013961:Thyroid Gland / Q000166:cytology / Q000378:metabolism
D003374:Coumarins; D015853:Cysteine Proteinase Inhibitors; D009097:Multienzyme Complexes; C114703:N-succinyl-leucyl-leucyl-valyl-tyrosyl-7-amino-4-methylcoumarin; D009842:Oligopeptides; C067713:lactacystin; D005576:Colforsin; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D002154:Calpain; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D002118:Calcium; D000111:Acetylcysteine