SEPSIS STIMULATES NONLYSOSOMAL, ENERGY-DEPENDENT PROTEOLYSIS AND INCREASES UBIQUITIN MESSENGER-RNA LEVELS IN RAT SKELETAL-MUSCLE
SEPSIS STIMULATES NONLYSOSOMAL, ENERGY-DEPENDENT PROTEOLYSIS AND INCREASES UBIQUITIN MESSENGER-RNA LEVELS IN RAT SKELETAL-MUSCLE
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DOI：
10.1172/jci117588
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发表时间：
1994-12-01
影响因子：
15.9
通讯作者：
HASSELGREN, PO
中科院分区：
文献类型：
--
作者：
TIAO, G;FAGAN, JM;HASSELGREN, PO
我们测试了不同细胞内蛋白水解途径在脓毒症诱导的肌肉蛋白水解中的作用。通过盲肠结扎和穿刺诱导大鼠败血症；控制是假操作的。在孵育的趾长伸肌中，分别测定作为酪氨酸和3-甲基组氨酸的释放的总蛋白水解和肌原纤维蛋白水解。使用促溶酶体试剂 NH4Cl、氯喹、亮肽素和甲胺评估溶酶体蛋白水解作用。 Ca2+ 依赖性蛋白水解作用是在存在或不存在 Ca2+ 的情况下测定的，或者通过阻断 Ca2+ 依赖性蛋白酶钙蛋白酶 I 和 II 来测定。在 2-脱氧葡萄糖和 2.4-二硝基苯酚耗尽 ATP 的肌肉中测定能量依赖性蛋白水解。分别通过 Northern 和 Western 印迹测定肌肉泛素 mRNA 以及游离和结合泛素的浓度，以评估 ATP 泛素依赖性蛋白水解途径的作用。败血症期间总蛋白分解和肌原纤维蛋白分解分别增加 50% 和 440%。对照组和脓毒症大鼠的溶酶体和 Ca2+ 依赖性蛋白水解相似。相比之下，脓毒症肌肉中能量依赖性总蛋白分解和肌原纤维蛋白分解分别增加了 172% 和四倍多。脓毒症肌肉中泛素 mRNA 增加数倍。结果表明，脓毒症期间肌肉蛋白水解的增加是由于非溶酶体能量依赖性蛋白质分解的增加，这可能涉及泛素系统。
We tested the role of different intracellular proteolytic pathways in sepsis-induced muscle proteolysis. Sepsis was induced in rats by cecal ligation and puncture; controls were sham operated. Total and myofibrillar proteolysis was determined in incubated extensor digitorum longus muscles as release of tyrosine and 3-methylhistidine, respectively. Lysosomal proteolysis was assessed by using the lysosomotropic agents NH4Cl, chloroquine, leupeptin, and methylamine. Ca2+-dependent proteolysis was determined in the absence or presence of Ca2+ or by blocking the Ca2+-dependent proteases calpain I and II. Energy-dependent proteolysis was determined in muscles depleted of ATP by 2-deoxyglucose and 2.4-dinitrophenol. Muscle ubiquitin mRNA and the concentrations of free and conjugated ubiquitin were determined by Northern and Western blots, respectively, to assess the role of the ATP-ubiquitin-dependent proteolytic pathway. Total and myofibrillar protein breakdown was increased during sepsis by 50 and 440%, respectively. Lysosomal and Ca2+-dependent proteolysis was similar in control and septic rats. In contrast, energy-dependent total and myofibrillar protein breakdown was increased by 172% and more than fourfold, respectively, in septic muscle. Ubiquitin mRNA was increased severalfold in septic muscle. The results suggest that the increase in muscle proteolysis during sepsis is due to an increase in nonlysosomal energy-dependent protein breakdown, which may involve the ubiquitin system.