Calpain, not caspase, is the causative protease for hypoxic damage in cultured monkey retinal cells

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 mon...

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Bibliographic Details
Published in:Investigative ophthalmology & visual science 2011-09, Vol.52 (10), p.7059-7067
Main Authors: Nakajima, Emi, Hammond, Katherine B, Rosales, Jennifer L, Shearer, Thomas R, Azuma, Mitsuyoshi
Format: Article
Language:English
Subjects:
Animals
Apoptosis
Biomarkers - metabolism
Calpain - antagonists & inhibitors
Calpain - physiology
Carbamates - pharmacology
Caspase 3 - physiology
Caspase Inhibitors
Cell Count
Cell Proliferation
Cells, Cultured
Fluorescent Antibody Technique, Indirect
Hypoxia - metabolism
Immunoblotting
In Situ Nick-End Labeling
Macaca mulatta
Neuroglia - pathology
Oligopeptides - pharmacology
Photoreceptor Cells, Vertebrate - pathology
Recoverin - metabolism
Reperfusion Injury - metabolism
Reperfusion Injury - pathology
Rhodopsin - metabolism
Spectrin - metabolism
Vimentin - metabolism
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Summary: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. 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. 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. Calpain, not caspase-3, was involved in hypoxic damage in cultured monkey retinal cells.
ISSN:1552-5783
0146-0404
1552-5783
DOI:10.1167/iovs.11-7497