Calcium-Dependent Proteolysis in Rabbit Lens Epithelium after Oxidative Stress

The purpose of this study was to examine changes in calcium-dependent proteolytic activity in the lens epithelium from whole rabbit lenses exposed to long-term oxidative stress at near physiological levels. Rabbit lenses, incubated in 50 µM H₂O₂ for 1 or 24 h, were checked for clarity and morphological changes in the epithelium. Proteolytic activity was measured in the epithelium using a fluorogenic synthetic substrate; N-succinyl-Leu-Tyr-7-amino-4-methylcoumarin, both in the presence and the absence of calcium (1 mM Ca²⁺ and 5 mM EDTA respectively). The effect on transparency and morphology of the epithelium following a 1-hour incubation in 100 µM H₂O₂ was also studied. Lenses incubated in 50 µM H₂O₂ were clear even after 24 h. After a 1-hour incubation in 50 µM H₂O₂ the epithelium of the exposed lens appeared normal. However, after 24 h the epithelium cells appeared swollen and microscopical examination showed extensive intracellular and subepithelial vacuolization. Incubation in 100 µM H₂O₂ for 1 h caused loss of transparency; vacuole formation, globulization of the superficial lens fibers and death of the epithelial cells. There was a 55% increase in calcium-dependent proteolytic activity after 1 h in 50 µM H₂O₂, implying a role for the calcium-activated protease calpain in oxidatively induced cataract.