title: Calpain, not caspase, is the causative protease for hypoxic damage in cultured monkey retinal cells.
abstract: OBJECTIVE
  Cell death occurring in human retina during AMD, high IOP, and diabetic retinopathy could be caused by activation of calpain or caspase proteolytic enzymes. The purpose of the present study was to determine whether calpains and/or caspase-3 were involved in cell death during retinal hypoxia in a monkey model.
  
  
  METHODS
  Dissociated monkey retinal cells were cultured for two weeks and subjected to 24-hour hypoxia/24-hour reoxygenation. TUNEL staining and immunostaining for Müller and photoreceptor markers were used to detect which retinal cell types were damaged.
  
  
  RESULTS
  Culturing dissociated monkey retina cells for two weeks resulted in proliferation of Müller cells and maintenance of some rod and cone photoreceptor cells, as identified by vimentin, recoverin, and rhodopsin immunocytochemical staining. Hypoxia/reoxygenation increased the number of cells staining positive for TUNEL. Immunoblotting showed that the calpain-specific 145 kDa α-spectrin breakdown product (SBDP) increased in hypoxic cells, but no caspase-specific 120 kDa α-spectrin breakdown product was detected. TUNEL staining and proteolysis were significantly reduced in the retinal cells treated with 10 and 100 μM calpain inhibitor SNJ-1945. Caspase inhibitor, z-VAD, did not inhibit cell damage from hypoxia/reoxygenation. Intact pro-caspase-3 was in fact cleaved by activated calpain during hypoxia/reoxygenation to pre 29 kDa caspase-3 and 24 kDa inactive fragments. No 17 and 12 kDa fragments, which form the active caspase-3 hetero-dimer, were detected. Calpain-induced cleavage of caspase was inhibited by SNJ-1945.
  
  
  CONCLUSIONS
  Calpain, not caspase-3, was involved in hypoxic damage in cultured monkey retinal cells.
authors.affiliation: Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Corporation Limited, Beaverton, Oregon 97006, USA.
authors.forename: Emi
authors.initials: E
authors.lastname: Nakajima
authors.forename: Katherine B
authors.initials: KB
authors.lastname: Hammond
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Rosales
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Shearer
authors.forename: Mitsuyoshi
authors.initials: M
authors.lastname: Azuma
mesh_terms: D000818:Animals; D017209:Apoptosis; D015415:Biomarkers / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors / Q000502:physiology*; D002219:Carbamates / Q000494:pharmacology; D053148:Caspase 3 / Q000502:physiology*; D061945:Caspase Inhibitors; D002452:Cell Count; D049109:Cell Proliferation; D002478:Cells, Cultured; D019084:Fluorescent Antibody Technique, Indirect; D000860:Hypoxia / Q000378:metabolism*; D015151:Immunoblotting; D020287:In Situ Nick-End Labeling; D008253:Macaca mulatta; D009457:Neuroglia / Q000473:pathology*; D009842:Oligopeptides / Q000494:pharmacology; D020419:Photoreceptor Cells, Vertebrate / Q000473:pathology*; D051603:Recoverin / Q000378:metabolism; D015427:Reperfusion Injury / Q000378:metabolism* / Q000473:pathology; D012243:Rhodopsin / Q000378:metabolism; D013049:Spectrin / Q000378:metabolism; D014746:Vimentin / 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; D015415:Biomarkers; D002219:Carbamates; D061945:Caspase Inhibitors; D009842:Oligopeptides; D014746:Vimentin; C517629:benzyloxycarbonyl-valyl-alanyl-aspartic acid; D013049:Spectrin; D051603:Recoverin; D012243:Rhodopsin; D002154:Calpain; D053148:Caspase 3