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

Purpose. To compare effects of calpain inhibitors on in vitro light-scatter ing in rat lens soluble protein and calcium-ionophore (A23187)-induced cata ract 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 mod el at 10 and 25 muM concentration. As an index of protein precipitation, li ght scattering was measured daily at 405 nm for 8 days. Lens proteins were analyzed by isoelectric-focussing. Subsequently, rat lenses were cultured f or 5 days with 10 muM A23187. Calpain inhibitors (SJA6017, MDL28170, AK295 and PD150606), which inhibited light-scattering were tested at 100 muM conc entration in this model. Cataract evaluation, isoelectric-focussing and cal cium determinations were performed. Results. At 25 muM concentration AK295, SJA6017, E-64, PD-150606 and MDL281 70 produced greater than 25% inhibition of light-scattering. Isoelectric-fo cussing revealed that addition of Ca2+ produced characteristic crystallin p roteolysis and aggregation patterns. AK295, SJA6017, MDL28170 and E64c prev ented 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 tota l calcium. Conclusions. Endogenous calpain-activation model and A23187-induced catarac t 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 p ossess efficacy against calcium-induced models of rodent cataracts. Use of calpain inhibitors represents a promising approach to cataract therapy.