Abstract
Abstract: :
Purpose: The sheep lens culture system has been developed as an alternative, controlled model of cataract formation allowing the measurement of early changes in cataractogenesis and the ability to test the efficacy of calpain inhibitors in preventing cataracts. The purpose of this experiment was to confirm a causal link between calpain activation and lens opacification. Methods: Eyes were obtained from 11–month–old lambs. Eyes were dissected and whole lenses cultured in five groups in Eagles minimum essential medium (EMEM) for 48 hr prior to treatment. After 48 hr two groups of lenses were pre–incubated in EMEM containing different inhibitors for 24 hr, while three groups remained in EMEM only. The experiment began at 72 hr with lenses under the following conditions: lenses were cultured in EMEM (control), EMEM + ionomycin (a calcium ionophore), EMEM + EGTA (a calcium chelator), or EMEM + ionomycin + inhibitor for 48 hr. A concentration range from 0.1 to 5.0 µM calcium ionophore was applied to induce lens opacity. Calpain specific inhibitors were tested for their ability to prevent ionomycin–induced opacity. Lens transparency was monitored daily. Following culture, frozen lenses were homogenised and analysed for calpain activity and calpain substrate breakdown by zymography, immunoblotting, SDS–PAGE and 2D–PAGE. Total calcium levels in the lenses were measured by a colorimetric calcium assay. Results: Lenses cultured under control conditions remained transparent over the 5–day period of the experiment. Addition of ionomycin caused generalised cortical opacities in the lenses, while lenses treated with EGTA remained transparent. Total calcium levels in the ionomycin–treated lenses were also much higher. The ionomycin–treated lenses had lower levels of active calpain II. Breakdown of the cytoskeletal protein, spectrin, was detected and consistent with calpain II proteolysis. Calpain inhibitors reduced opacification of the treated lenses. Conclusions:The development of opacities in lenses treated with ionomycin, while control and EDTA treated lenses remained transparent, supports the action of calcium–dependent proteases on cataractogenesis. Application of calpain specific inhibitors to ionomycin–treated lenses reduced opacification, suggesting a causal link between ionomycin–induced calpain activation and cultured lens opacification. Calpain II is the major isoform present in these sheep lenses and its specific cleavage of spectrin was associated with lens opacification.
Keywords: calcium • cataract • proteolysis