PROTEOLYTIC ACTIVITY IN INTACT SHEETS OF POLARIZED EPITHELIAL CELLS AS DETERMINED BY A CELL-PERMEABLE FLUOROGENIC SUBSTRATE
✍ Scribed by Jan-Olof Karlsson; Jenny Lundquist; Ida Skoglund; Mikael Nilsson
- Publisher
- Elsevier Science
- Year
- 2000
- Tongue
- English
- Weight
- 184 KB
- Volume
- 24
- Category
- Article
- ISSN
- 1065-6995
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✦ Synopsis
Abstract
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.