Comparison of various calpain inhibitors in reduction of light scattering, protein precipitation and nuclear cataract in vitro
Comparison of various calpain inhibitors in reduction of light scattering, protein precipitation and nuclear cataract in vitro
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DOI：
10.1076/ceyr.21.6.926.6990
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发表时间：
2000-01-01
影响因子：
2
通讯作者：
Vajpayee, RB
中科院分区：
文献类型：
--
作者：
Mathur, P;Gupta, SK;Vajpayee, RB
目的.比较钙蛋白酶抑制剂对体外培养的大鼠透镜中可溶性蛋白的光散射和钙离子载体（A23187）诱导的大鼠晶状体白内障形成的影响。通过激活内源性透镜钙蛋白酶水解大鼠透镜可溶性蛋白24小时。在该模型中以10和25 μ M浓度测试了10种钙蛋白酶抑制剂。作为蛋白质沉淀的指标，每天在405 nm下测量光散射，持续8天。通过等电聚焦分析透镜蛋白。随后，用10 μ M A23187培养大鼠晶状体5天。在该模型中以100 μ M浓度测试抑制光散射的钙蛋白酶抑制剂（SJA 6017、MDL 28170、AK 295和PD 150606）。进行白内障评估、等电聚焦和钙测定。在25 μ M浓度下，AK 295、SJA 6017、E-64、PD-150606和MDL 28170产生大于25%的光散射抑制。等电聚焦显示，除了Ca 2+产生的特征晶体蛋白的蛋白水解和聚集模式。AK 295、SJA 6017、MDL 28170和E64 c阻止了这些变化。在A23187中培养的晶状体表现出核性白内障、钙和蛋白水解升高以及晶体蛋白聚集。与SJA 6017、MDL 28170和E64 c共培养可减少A23187诱导的核混浊、蛋白水解和晶体蛋白聚集，而不影响总钙的增加。内源性钙蛋白酶激活模型和A23187诱导的白内障模型可用于筛选钙蛋白酶抑制剂的潜在抗白内障活性。暴露于A23187后透镜皮质中的蛋白水解变化也是由于钙蛋白酶激活。AK 295、SJA 6017和MDL 28170对钙诱导的啮齿动物白内障模型具有功效。钙蛋白酶抑制剂的使用代表了白内障治疗的一种有前途的方法。
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 muM 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 muM A23187. Calpain inhibitors (SJA6017, MDL28170, AK295 and PD150606), which inhibited light-scattering were tested at 100 muM concentration in this model. Cataract evaluation, isoelectric-focussing and calcium determinations were performed.Results. At 25 muM concentration AK295, SJA6017, E-64, PD-150606 and MDL28170 produced greater than 25% inhibition of light-scattering. Isoelectric-focussing revealed that addition of Ca2+ 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.