Progress of Opacification Changes In Lens Proteins

lonomycin causes opacities in cultured sheep lenses by damaging the outer layers of the cortex:,:=.'!~=.=.::=:-...--Background Cortical r::ataracts have been assodmd wtth inclea$4!d levels of caldum'. A model has been developed for c;ata,.cts by Increasing the caldum levels In cultured sheep lenses using the calcium lonophore. lonomydn'. This model has been used to test the efficacy of potential therapeutic agents to r cataracts. parttcUlaT ~ calpaln Inhibitors'. The calcium-dependent calp.lns have been Implicated In cataract formation " and are known to cleave lens crystallins and c:ytoskeletlll proteins. Purpose The purpose of these experiments was to determine which parts of the lens were affected by ionomycin and to look for specific evidence of calpain activation during opacification. Methods Lamb Lenses Wi!I'I! dissected from whole eye globes and Qllhneci in Eagles minimum essential medium (EIo1E101) for 48 hr. Control Lenses (n-6) remained in ENEI<! for a further 4 days. Lens opacity was induced in the test: group (0-6) by exposure to 2~ ionomycin. The requirement forcalcium in the medium was tested using lenses tnsrted with ionomycin and 5mN EGTA (0-6). Remaining lenses WI!A! dissected into epitheUal. CDrt2x and nw::l.mrfradions. The calcium COIICI!IIIration in ead! frilction was determined by flame emission spectroscopy and the calpain artivity by casein zymography. Pruteolysis of lens proteins was assessed by two-dimensional electrophoresis (2-DE) and WI!5b!m blotting af the tytDskeletaL protl!ins spectrin and vimentin. Spots from 2-DE gels were identified by mass spectrometry and by comparison with previ ousJN mapped lens protl!ins