title: Proteolysis in human lens epithelium determined by a cell-permeable substrate.
abstract: OBJECTIVE
  To develop a system for continuous evaluation of proteolytic activity 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 anterior capsule and the underlying lens epithelium. The sample, with the cell-permeable substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, was placed in a chamber, which was placed in a thermostat-controlled aluminum block. Fluorescence changes were continuously measured by the fiber optics of the luminometer, which was placed 5 mm above the buffer surface.
  
  
  RESULTS
  After administration of substrate to the medium overlying the cells, the substrate was degraded at a relatively slow rate. Approximately 10 picomoles of amino-4-methylcoumarin were formed per minute. A significant increase of proteolytic activity could be observed after application of 1 microM ionomycin or 2 microM thapsigargin. No leakage of lactate dehydrogenase from the cells was observed during these procedures. Basal proteolytic activity was totally inhibited by the proteasome inhibitor lactacystin. Lactacystin also attenuated the response to ionomycin and thapsigargin.
  
  
  CONCLUSIONS
  Human lens epithelium responds to increased Ca levels from external 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.
authors.affiliation: Institute of Anatomy and Cell Biology, Göteborg University, Sweden.
authors.forename: J O
authors.initials: JO
authors.lastname: Karlsson
authors.forename: M
authors.initials: M
authors.lastname: Andersson
authors.forename: A
authors.initials: A
authors.lastname: Kling-Petersen
authors.forename: J
authors.initials: J
authors.lastname: Sjöstrand
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002470:Cell Survival; D003374:Coumarins / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004848:Epithelium / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D007908:Lens, Crystalline / Q000187:drug effects / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D013379:Substrate Specificity; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: D003374:Coumarins; D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; C114703:N-succinyl-leucyl-leucyl-valyl-tyrosyl-7-amino-4-methylcoumarin; D009842:Oligopeptides; C067713:lactacystin; D015759:Ionomycin; D019284:Thapsigargin; D007770:L-Lactate Dehydrogenase; D002154:Calpain; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D002118:Calcium; D000111:Acetylcysteine