Calcium-Dependent Proteolysis in Rabbit Lens Epithelium after Oxidative Stress

Abstract

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.

Hepatitis C Virus NS5A and Subgenomic Replicon Activate NF-κB via Tyrosine Phosphorylation of IκBα and Its Degradation by Calpain Protease

“…The mechanism(s) of HCV replicon and NS5A-induced degradation of IB␣ by calpain is under further investigation. While studies relating to the mechanism(s) of calpain activation are currently under intense study, Ca 2ϩ release after exposure to hydrogen peroxide together with calpain activation under oxidative stress conditions is well documented (55,56). Several groups have reported that the PEST sequence is essential for oxidative stress mediated degradation of IB␣ (57)(58)(59).…”

Crucial Role of the Amino-Terminal Tyrosine Residue 42 and the Carboxyl-Terminal PEST Domain of IκBα in NF-κB Activation by an Oxidative Stress

“…Exposure to agents that produce hydroperoxides or the addition of exogenous hydroperoxides also causes elevation of intracellular Ca 2ϩ in some cells (60). Because Ca 2ϩ release after exposure to H 2 O 2 is well documented together with calpain activation under oxidative stress conditions (62,63), it is clear why I B␣ phosphorylation in the PEST sequence becomes a substrate for degradation by calpains.…”

Differential Inhibition of Three Peptidase Activities of the Proteasome in Human Lens Epithelium by Heat and Oxidation

“…Oxidative inhibition was induced by incubating the lens epithelium cytoplasm solution with 0n5 m H # O # for 1 or 24 h. For each experiment, fresh H # O # was prepared from a 30 % stock solution (Merck, Darmstadt, Germany). The concentration of H # O # was almost constant during the incubation period, as measured by the method of Guilbault, Brignac and Zimmer (1968) as previously described (Andersson et al, 1998b). During the longer exposure period, some samples were also incubated with α-crystallin or with heat shock protein 90 (final concentrations in the assay 1 and 0n1 mg ml −" respectively).…”