Comparison of various calpain inhibitors in reduction of light scattering, protein precipitation and nuclear cataract in vitro.

ABSTRACT

PURPOSE: To compare effects of calpain inhibitors on in vitro light-scattering in rat lens soluble protein and calcium-ionophore (A23187)-induced cataract formation in cultured rat lenses. METHODS: Rat lens soluble protein was hydrolyzed for 24 hours by activation of endogenous lens calpain. Ten calpain inhibitors were tested in this model at 10 and 25 microM concentration. As an index of protein precipitation, light scattering was measured daily at 405 nm for 8 days. Lens proteins were analyzed by isoelectric-focussing. Subsequently, rat lenses were cultured for 5 days with 10 microM A23187. Calpain inhibitors (SJA6017, MDL28170, AK295 and PD150606), which inhibited light-scattering were tested at 100 microM concentration in this model. Cataract evaluation, isoelectric-focussing and calcium determinations were performed. RESULTS: At 25 microM concentration AK295, SJA6017, E-64, PD-150606 and MDL28170 produced greater than 25% inhibition of light-scattering. Isoelectric-focussing revealed that addition of Ca(2+) produced characteristic crystallin proteolysis and aggregation patterns. AK295, SJA6017, MDL28170 and E64c prevented these changes. Lenses cultured in A23187 exhibited nuclear cataract, elevated calcium and proteolysis and aggregation of crystallins. Co-culture with SJA6017, MDL28170 and E64c reduced A23187-induced nuclear opacities, proteolysis and aggregation of crystallins without affecting increased total calcium. CONCLUSIONS: Endogenous calpain-activation model and A23187-induced cataract model can be used sequentially to screen calpain inhibitors for potential anti-cataract activity. Proteolytic changes in lens cortex after exposure to A23187 are also due to calpain activation. AK295, SJA6017 and MDL28170 possess efficacy against calcium-induced models of rodent cataracts. Use of calpain inhibitors represents a promising approach to cataract therapy.