Calpeptin provides functional neuroprotection to rat retinal ganglion cells following Ca2+ influx
Calpeptin provides functional neuroprotection to rat retinal ganglion cells following Ca2+ influx
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DOI：
10.1016/j.brainres.2006.02.051
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发表时间：
2006-04-21
期刊：
影响因子：
2.9
通讯作者：
Ray, Swapan K.
中科院分区：
文献类型：
--
作者：
Das, Arabinda;Garner, Dena P.;Ray, Swapan K.
视网膜神经节细胞凋亡损害青光眼患者的视力。RGCs在多发性硬化症（MS）中也会退化，导致MS患者的视觉感知丧失。我们研究了钙蛋白酶和半胱天冬酶级联反应在大鼠视网膜神经节细胞系RGC-S细胞凋亡中的作用，250 nM离子霉素（IMN）或300单位/ml干扰素-γ（IFN-γ）暴露24小时后，然后用2 μ M钙蛋白酶肽（CP，钙蛋白酶特异性抑制剂）评价功能性神经保护作用。IMN或IFN-γ作用于RGC-5细胞后，可观察到细胞凋亡的形态学和生化特征。Fura-2测定确定暴露于IMN或IFN-γ后细胞内游离[Ca 2 +]显著增加。CP预处理1 h可抑制RGC-5细胞内Ca ~（2+）、内流、蛋白水解活性和凋亡。Western blot分析显示，在细胞凋亡过程中，钙蛋白酶和caspase-12的活性增加，Bax：Bcl-2的比例上调，细胞色素c从线粒体中释放，caspase-9和caspase-3的活性增加。增加的半胱天冬酶-3活性也通过比色测定证实。在暴露于IFN-γ后，RGC-5细胞中caspase-8的活化和Bid至tBid的裂解表明细胞凋亡的外在和内在途径之间的合作。膜片钳记录显示，预处理与CP衰减凋亡和维持正常的全细胞膜电位，表明功能性神经保护。综上所述，我们的结果表明，Ca 2+超载可能是负责激活钙蛋白酶和caspase级联反应，导致RGC-S细胞的凋亡死亡和CP提供功能性神经保护。(c)2006 Elsevier B. V.保留所有权利。
Apoptosis of retinal ganglion cells (RGCs) impairs vision in glaucoma patients. RGCs are also degenerated in multiple sclerosis (MS), resulting in loss of visual perception in MS patients. We examined the involvement of calpain and caspase cascades in apoptosis of the rat retinal ganglion cell line RGC-S following 24 h of exposure to 250 nM ionomycin (IMN) or 300 units/ml interferon-gamma (IFN-gamma) and then evaluated functional neuroprotection with 2 mu M calpeptin (CP, a calpain-specific inhibitor). Morphological and biochemical features of apoptosis were detected in RGC-5 cells following exposure to IMN or IFN-gamma. Fura-2 assay determined significant increases in intracellular free [Ca2+] following exposure to IMN or IFN-gamma. Pretreatment with CP for 1 h prevented Ca2+, influx, proteolytic activities, and apoptosis in RGC-5 cells. Western blot analyses showed an increase in activities of calpain and caspase-12, upregulation of Bax:Bcl-2 ratio, release of cytochrome c from mitochondria, and increase in caspase-9 and caspase-3 activities during apoptosis. Increased caspase-3 activity was also confirmed by a colorimetric assay. Activation of caspase-8 and cleavage of Bid to tBid in RGC-5 cells following exposure to IFN-gamma indicated co-operation between extrinsic and intrinsic pathways of apoptosis. Patch-clamp recordings showed that pretreatment with CP attenuated apoptosis and maintained normal whole-cell membrane potential, indicating functional neuroprotection. Taken together, our results demonstrated that Ca2+ overload could be responsible for activation of calpain and caspase cascades leading to apoptotic death of RGC-S cells and CP provided functional neuroprotection. (c) 2006 Elsevier B.V. All rights reserved.