Calcium influx through calcium leak channels is responsible for the elevated levels of calcium-dependent proteolysis in dystrophic myotubes
Calcium influx through calcium leak channels is responsible for the elevated levels of calcium-dependent proteolysis in dystrophic myotubes
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DOI：
10.1074/jbc.275.13.9452
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发表时间：
2000-03-31
影响因子：
4.8
通讯作者：
Steinhardt, RA
中科院分区：
文献类型：
--
作者：
Alderton, JM;Steinhardt, RA
为了评估钙蛋白酶的蛋白分解，我们测量了正常和营养不良的MDX小鼠肌管培养中一种荧光性钙蛋白酶底物的水解率。成肌细胞和肌管排列和融合过程中水解率较高，排列和融合停止后，水解率下降。对于正常的肌管，在收缩活动发展后，水解率仍然很低。相反，在收缩活动发展后，营养不良的MDX肌管具有异常高水平的依赖于外部钙的水解性，这种水解性可以被Calain的抑制剂CalPeptin所消除。我们通过添加河豚毒素消除了水解性测量中收缩的直接影响，溶酶体或蛋白体底物的水解性分别用NH4Cl和clasto-lactacystinβ-内酯来控制。成熟的MDX肌管中钙激活的蛋白水解酶活性增加与钙特异性泄漏通道的异常活性有关，因为这些通道的拮抗剂将营养不良肌管中较高水平的水解度降低到接近正常的水平。这些通道的异常活动与更脆弱的MDX肌管(1，2)中瞬时肌膜破坏的频率增加有关。用甲基强的松龙前药治疗MDX肌管也可将钙蛋白酶底物的水解度降低到接近正常的水平。然而，这种抑制只需要2.5h的预处理，这不足以发挥已知的强的松龙对钙稳态的作用。
To estimate calpain proteolysis, we measured the hydrolysis rate of a fluorogenic calpain substrate in individual resting normal and dystrophic mdx mouse myotubes in culture. Hydrolysis rates were high during myoblast and myotube alignment and fusion, After alignment and fusion ceased, hydrolysis rates declined. For normal myotubes, hydrolysis remained low after the development of contractile activity. In contrast, after the development of contractile activity, dystrophic mdx myotubes had abnormally high levels of hydrolysis that were dependent on external calcium and that could be abolished by calpeptin, an inhibitor of calpain, We eliminated the direct effects of contraction during measurements of hydrolysis by the addition of tetrodotoxin, Substrate hydrolysis by lysosomes or proteosomes was controlled for using NH4Cl and clasto-lactacystin beta-lactone, respectively. Increased activity of the calcium-activated protease in mature mdx myotubes was linked to the abnormal activity of calcium-specific leak channels because an antagonist of these channels reduced the higher levels of hydrolysis in dystrophic myotubes to nearly normal levels. The abnormal activity of these channels is linked to an increased frequency of transient sarcolemmal disruptions in the more fragile mdx myotubes (1, 2). Treatment of mdx myotubes with a prodrug of methylprednisolone also reduced calpain substrate hydrolysis to nearly normal levels. However, this inhibition only required 2.5 h of pretreatment, which was not long enough to act by the known effects of prednisolone on calcium homeostasis.