The Effect of Oxidative Damage on the Calpain Proteases in Cultured Ovine Lens

Abstract

Abstract: : Purpose: A line of sheep at Lincoln University inherits cataracts and provides a unique opportunity to investigate the role of calpain in cataract formation. 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 to test the efficacy of calpain inhibitors in preventing cataracts. The purpose of this experiment was to determine which calpain isoforms were present in the cultured lenses and whether oxidative damage led to changes in the amount and type of calpain isoforms. Methods: Eyes were obtained from 11-month-old sheep. Lenses were dissected out and cultured in EMEM for 3 days. Lenses were cultured in the presence of hydrogen peroxide (0.5mM, 1mM, 2mM) for 24 hours, then were returned to control EMEM for a further 48 hours. At 4 hours, 24 hours and 72 hours after addition of H₂O₂ lenses were removed from each treatment and stored at -80°C. Lens transparency was monitored daily. Frozen lenses were homogenised and an aliquot analysed for calpain activity by casein gel zymography. Another aliquot was separated by ion exchange chromatography on DEAE Sepharose and the activity of calpain II was determined. Results: Lenses cultured under control conditions remained transparent over the 10-day period of the experiment. Addition of H₂O₂ caused a generalised cortical opacity of the lenses after 4 hours. By 24 hours the lenses treated with 0.5mM H₂O₂ had recovered, with the opacity restricted to the area of the suture lines and the equatorial cortex. The lenses exposed to 2mM H₂O₂ retained a more generalised opacity although it was still most notable in the equatorial regions. Calpain II was the only isoform revealed by casein zymography and significant amounts of this enzyme were still present at the 72-hour time point in lenses challenged with 2mM H₂O₂. Separating the calpain and assaying it directly confirmed these results. Conclusions: Sheep lenses can be maintained in culture and remain transparent for at least 10 days. Incubation with hydrogen peroxide causes opacification in these lenses. Calpain II has been shown to be the only isoform present at significant levels. The lens culture system will allow investigation of other cataractogenic stimuli on the transparency of the lens and the role of calpain in the opacification process.

Keywords: cataract • oxidation/oxidative or free radical damage • proteolysis