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Abstract #17098 Published in IGR 9-1
Calpain-specific proteolysis in primate retina: Contribution of calpains in cell death
Nakajima E; David LL; Bystrom C; Shearer TR; Azuma MInvestigative Ophthalmology and Visual Science 2006; 47: 5469-5475
See also comment(s) by Cynthia Grosskreutz •
PURPOSE: One of the leading causes of blindness is retinal damage caused by the high intraocular pressure (IOP) in glaucoma. Previous studies in rats have suggested that the proteolytic enzyme calpain (EC 3.4.22.17) is involved in retinal cell death during ischemia and in acute high IOP. Ubiquitous, calcium-activated calpain-1 and -2 from monkey retina are highly homologous to rat calpains, although expression patterns in variants of tissue-specific calpain-3 are different between monkey and rodent retinas. Thus, the purpose of the present study was to investigate the involvement of calpain-induced proteolysis in retinal cell death in primates. METHODS: Calpain involvement in a simulated pathologic condition was examined by incubating monkey retinas in hypoxic conditions (95% N2 and 5% CO2 ) in RPMI medium without glucose. Endogenous tissue calpains were also directly activated in monkey and human retinal soluble proteins by incubating with 2.5 mM calcium. The resultant proteolysis of monkey retinal proteins was assessed by 2D electrophoresis (2-DE). RESULTS: In hypoxic retina, leakage of lactate dehydrogenase (LDH) from retinas into the medium increased, indicating cell death. LDH leakage was partially inhibited by the calpain inhibitor SJA6017. Calpain autolysis was observed, and the calpain-preferred substrate α-spectrin was proteolyzed. In retinal soluble proteins incubated with calcium, a total of 15 spots from 2-DE of retinal soluble proteins were identified by mass spectrometry. Proteolysis of major proteins, vimentin, β-tubulin, α-enolase, and Hsp70 were confirmed by immunoblot analysis. Activation of calpains and proteolysis of these substrates were inhibited by the calpain-specific inhibitor SJA6017. CONCLUSIONS: Taken together, these results suggested that calpain activation in primate retinas could play an important role in cell death during hypoxia caused by elevated IOP from glaucoma.
Dr. E. Nakajima, Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Co., Ltd., Kobe, Japan
Classification:
3.12 Proteomics (Part of: 3 Laboratory methods)
2.13 Retina and retinal nerve fibre layer (Part of: 2 Anatomical structures in glaucoma)
3.6 Cellular biology (Part of: 3 Laboratory methods)
