Proteolysis in human lens epithelium determined by a cell-permeable substrate

PURPOSE. To develop a system for continuous evaluation of proteolytic activ ity in human lens epithelium and to characterize factors of importance for the regulation of proteolytic activity in lens epithelial cells. METHODS. Human lens epithelial cells were obtained during cataract surgery. Capsule epithelium specimens consisted of the central parts of the anterio r capsule and the underlying lens epithelium. The sample, with the cell-per meable substrate Suc-Leu-Leu-VaI-Tyr-7-amino-4-methylcoumarin, was placed i n a chamber, which was placed in a thermostat-controlled aluminum block. Fl uorescence changes were continuously measured by the fiber optics of the lu minometer, which was placed 5 mm above the buffer surface. RESULTS. After administration of substrate to the medium overlying the cell s, the substrate was degraded at a relatively slow rate. Approximately 10 p icomoles of amino-4-methylcoumarin were formed per minute. A significant in crease of proteolytic activity could be observed after application of 1 mu M ionomycin or 2 mu M thapsigargin. No leakage of lactate dehydrogenase fro m the cells was observed during these procedures. Basal proteolytic activit y was totally inhibited by the proteasome inhibitor lactacystin. Lactacysti n also attenuated the response to ionomycin and thapsigargin. CONCLUSIONS. Human lens epithelium responds to increased Ca levels from ext ernal or internal stores with an increased proteolytic activity that may be mediated by calpain, by the proteasome, or by both. This calcium-dependent change in proteolytic activity may be of importance in the development of cataract.