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Keywords:

  • proteolysis;
  • epithelial cells;
  • calpain;
  • proteasome

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 Ca2+-mobilizing agents (ionomycin, 4-bromo-A23187, thapsigargin and maitotoxin). Forskolin and staurosporine also enhanced the proteolytic activity. We conclude that Ca2+mobilization, and possibly also changes of protein kinase activity, rapidly increase non-lysosomal proteolysis in the intact thyroid epithelium.