豚鼠和兔培养晶状体中的核性白内障与光散射

Fukiage C, Azuma M, Nakamura Y, Tamada Y, Shearer T R

Department of Oral Molecular Biology and Ophthalmology, Oregon Health Sciences University, Portland, USA.

Curr Eye Res. 1998 Jun;17(6):623-35.

PURPOSE

To compare calcium ionophore-induced cataract formation and in vitro light scattering in cultured lenses from guinea pig and rabbit.

METHODS

Lenses from guinea pig and rabbit were cultured for 5 or 6 days with calcium ionophore A23187. To assess the involvement of calpain in cataract formation; SDS-PAGE, immunoblotting and calcium determinations were performed. For in vitro light scattering, lens soluble proteins from rabbit were hydrolyzed for 24 h by either endogenous lens calpain, or by addition of purified m-calpain and then further incubated for up to 10 days. Light scattering was measured daily at 405 nm.

RESULTS

Lenses from younger guinea pigs cultured in A23187 first developed outer cortical opacities followed by nuclear cataract. Total calcium was markedly increased by A23187 in lenses of all ages. Proteolysis of crystallins and alpha-spectrin were observed in nuclear cataract in younger guinea pigs. This was attenuated with age, in association with the attenuation of cataract formation with age. Calpain 80 kDa subunit in the lenses cultured with A23187 was also decreased. Co-culture with SJA6017 or E64d (reversible and irreversible inhibitors of calpain, respectively) reduced A23187-induced nuclear opacities, proteolysis of crystallins and alpha-spectrin, and loss of calpain without affecting increased total calcium. In contrast, rabbit lenses cultured in A23187 did not develop nuclear cataract, although biochemical changes in cultured rabbit lenses were similar to those in cultured guinea pig lenses. Furthermore, no appreciable in vitro light scattering occurred in soluble proteins from rabbit lenses after activation of endogenous m-calpain, or after addition of exogenous purified m-calpain, although crystallins were partially hydrolyzed by calpain.

CONCLUSIONS

Both rabbit and guinea pig lenses undergo calpain-induced proteolysis upon elevation of lenticular calcium. However, factors in intact guinea pig lenses may promote light scattering and insolubilization after proteolysis by calpain, but these factors were not functional in rabbit lenses. Discovery of the factors promoting light scatter and insolubilization after proteolysis will help to explain the role of certain crystallin polypeptides in cataract formation.

目的

比较钙离子载体诱导豚鼠和兔培养晶状体形成白内障及体外光散射的情况。

方法

将豚鼠和兔的晶状体用钙离子载体A23187培养5或6天。为评估钙蛋白酶在白内障形成中的作用；进行了SDS - PAGE、免疫印迹和钙测定。对于体外光散射，兔晶状体可溶性蛋白通过内源性晶状体钙蛋白酶或添加纯化的m - 钙蛋白酶水解24小时，然后进一步孵育长达10天。每天在405nm处测量光散射。

结果

在A23187中培养的幼年豚鼠晶状体首先出现外层皮质混浊，随后发展为核性白内障。所有年龄段晶状体中的总钙均因A23187而显著增加。在幼年豚鼠的核性白内障中观察到晶状体蛋白和α - 血影蛋白的蛋白水解。随着年龄增长，这种情况减弱，同时白内障形成也随年龄减弱。用A23187培养的晶状体中钙蛋白酶80kDa亚基也减少。与SJA6017或E64d（分别为钙蛋白酶的可逆和不可逆抑制剂）共培养可减少A23187诱导的核混浊、晶状体蛋白和α - 血影蛋白的蛋白水解以及钙蛋白酶的损失，而不影响总钙的增加。相比之下，在A23187中培养的兔晶状体未发展为核性白内障，尽管培养的兔晶状体中的生化变化与培养的豚鼠晶状体相似。此外，在内源性m - 钙蛋白酶激活后或添加外源性纯化的m - 钙蛋白酶后，兔晶状体可溶性蛋白中未出现明显的体外光散射，尽管晶状体蛋白被钙蛋白酶部分水解。