title: Involvement of calpain 2 in ionomycin-induced cell death in cultured mouse lens epithelial cells.
abstract: OBJECTIVE
  Calpains are calcium-activated, intracellular, non-lysosomal, cysteine proteases that hydrolyze lens crystallins and cytoskeletal proteins. Elevated calcium is a frequent finding in both rodent and human cataracts, and calpain 2 is present in lenses of both species. Lens epithelium forms a critical barrier to influx of calcium, but the role of calpain 2 in lens epithelium is poorly characterized. Thus, the purpose of the present experiment was to determine the role of calpain 2 in lens epithelial cell death.
  
  
  METHODS
  Mouse lens epithelial cells (α-TN4) were cultured with the calcium ionophore ionomycin to promote calcium influx. Release of LDH into the culture medium was measured as a general marker of cell death, while necrosis and apoptosis were detected by staining with ethidium homodimer III (EtD-III) or FITC-annexin V. Calpain activity was determined by zymography and immunoblotting for activation-associated, fragments of calpain. Breakdown products of calpain substrate α-spectrin were also detected by immunoblotting as additional markers of calpain activation.
  
  
  RESULTS
  Calpain 2 was found to be the major calpain isozyme in α-TN4 cells. Ionomycin caused leakage of LDH into the medium, activation of calpain 2, proteolysis of α-spectrin, and changes in α-TN4 cell morphology and staining characteristic of necrotic cell death. Calpain inhibitor SNJ-1945 significantly inhibited these changes.
  
  
  CONCLUSIONS
  The ability of mouse lens epithelium to maintain lens transparency would be compromised by activation of calpain 2 and associated necrotic cell death. Since calpain 2 is ubiquitously present in all animal lenses so far observed, the current results may predict the pathological consequences of calpain 2 activation in animal lenses including those of man.
authors.affiliation: Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Co., Ltd., Kobe, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Nakajima
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Shearer
authors.forename: Mitsuyoshi
authors.initials: M
authors.lastname: Azuma
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D061207:Calcium Ionophores / Q000633:toxicity*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002219:Carbamates / Q000494:pharmacology; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D015759:Ionomycin / Q000633:toxicity*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D007908:Lens, Crystalline / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D051379:Mice; D008822:Mice, Transgenic; D009336:Necrosis; D013049:Spectrin / Q000378:metabolism
chemical_list: C515164:((1S)-1-((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-methylbutyl)carbamic acid 5-methoxy-3-oxapentyl ester; D061207:Calcium Ionophores; D002219:Carbamates; D013049:Spectrin; D015759:Ionomycin; D007770:L-Lactate Dehydrogenase; D002154:Calpain; D002118:Calcium