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J.D. Morton, H.Y. Y. Lee, L.J. G. Robertson, J.D. McDermott; Ionomycin Causes Opacities in Cultured Sheep Lenses by Damaging the Outer Layers of the Cortex . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2895.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Cultured lenses treated with the calcium ionophore, ionomycin, have been used as model systems to test the efficacy of potential therapeutic agents for cataracts. The initial purpose of this experiment was to determine which parts of the lens were affected by ionomycin. The second objective was to look for specific evidence of calpain activation during opacification. Methods: Lamb eyes were dissected and the lenses were cultured in Eagles minimum essential medium (EMEM) for 48 hr. Lens opacity was induced in the test group by exposure to 1 or 2µM ionomycin for up to 5 days. Control lenses either remained in EMEM or EMEM plus ionomycin and EGTA. Following culture some lenses were fixed and the cell structure determined. The remaining lenses were dissected into epithelium, cortex, and nucleus. The calcium concentration, calpain activity and extent of proteolysis were determined in each fraction. Proteolysis of lens proteins was assessed by 2–dimensional electrophoresis (2–DE) and by Western blotting of spectrin and vimentin. Spots from 2–DE gels were identified by mass spectrometry and by comparison with previously mapped lens proteins. Results:Control lenses cultured in EMEM remained transparent over the 5–day period. Ionomycin initially caused swelling of cells and opacification at the lens equator which became more generalized. Calcium chelation with EGTA reduced opacification. Calpain 2 activation in the epithelium and cortex was associated with ionomycin treatment and extensive changes in the soluble protein profile. These included the disappearance of vimentin, breakdown of spectrin and changes in alpha and beta crystallins. No changes were seen in the nucleus. A liquid fraction formed between the lens capsule and the main body of the lens with ionomycin treatment. The proteins in this liquid most closely resembled the cortex fraction. Conclusions: The lens fraction most affected by ionomycin treatment was the epithelium and the outer cortex. Spectrin and vimentin proteolysis and changes in crystallins were prominent features of this opacification model. The formation of a liquid fraction between the lens epithelium and outer cortex following ionomycin treatment indicated protein degradation in this region. These changes were consistent with calpain activation. However the limited changes in the inner cortex and the absence of changes in the nuclear fraction implied that ionomycin had not penetrated the interior of the lens.
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