期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 275, 期 13, 页码 9452-9460出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.275.13.9452
关键词
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资金
- NIAMS NIH HHS [R01 AR44066] Funding Source: Medline
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.
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