The involvement of calpains in opacification induced by Ca2+-overload in ovine lens culture.

Objective: To investigate biochemical changes accompanying Ca(2+)-induced lens opacification and the possible role of calpain activation in opacification within an ovine lens culture system.

Methods: Sheep lenses were cultured in minimal media. Lens opacification was induced by exposure to the Ca(2+) ionophore, ionomycin, and graded by digital image analysis. Cell viability was estimated by the release of lactate dehydrogenase into the culture medium. Opaque lenses were fixed and stained for a microscopic view of the lens structural changes. Ionic changes in the lens were measured by atomic absorption spectroscopy. Calpain activation was determined by zymography on casein gels and proteolysis was investigated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2DE) and Western blotting. The calpain inhibitor, SJA6017, was used to investigate the involvement of calpains in lens opacification.

Results: Treatment of cultured ovine lenses with ionomycin increased total lens Ca(2+) concentration and caused the cortical region of the lens to become opaque. Addition of the Ca(2+) chelator, EGTA, inhibited the ionomycin-induced changes. Progress of opacification correlated with the death of lens cells and lens swelling in differentiating fiber cells. Autolysis of calpain 2, following ionomycin treatment, suggested activation of this protease. 2DE revealed that the ionomycin did not result in substantial proteolysis of the crystallins. However, Western blotting revealed significant breakdown of the cytoskeletal proteins, spectrin and vimentin. The pattern of the breakdown products was consistent with calpain proteolytic activity. SJA6017 retarded the cortical opacity induced by Ca(2+)-overload in the ovine lens.

Conclusion: The ovine lens with Ca(2+)-induced opacification by ionomycin is associated with calpain activation and the subsequent proteolysis of cytoskeletal proteins. These events could be initial factors contributing to cell death and the loss of lens transparency which occurs in this ovine model of cataractogenesis. The ovine model supports the hypothesis that cytoskeletal proteins and Ca(2+) homeostasis play an important role in maintaining lens transparency.