Iizuka K, Kawaguchi H, Yasuda H, Kitabatake A
Department of Cardiovascular Medicine, Hokkaido University School of Medicine, Sapporo, Japan.
Jpn Heart J. 1992 Sep;33(5):707-15. doi: 10.1536/ihj.33.707.
The aim of this study was to investigate the correlation between hypoxic myocardial cell injury and intracellular protease activity. Cardiac myocytes were isolated from neonatal rat hearts and cultured in Eagle's modified minimum essential medium. Myocytes were incubated in hypoxic conditions for 6 hours. The cell death rate during hypoxia rose to 80% after 6 hours. Extracellular protease activity was elevated to 4 units during hypoxia, much higher than the 0.7 units in aerobic states at 6 hours. This extracellular protease activity in hypoxic conditions was markedly inhibited by leupeptin and EDTA, and weakly inhibited by the cysteine protease inhibitor, NCO-700, but phenylmethyl sulfonyl fluoride did not inhibit the protease activity. To identify the protease activated during hypoxia, calpain-specific inhibitors were added to the incubation mixture. Calpain inhibitor 1 and calpastatin, an endogenous selective calpain inhibitor, markedly inhibited extracellular protease activity during hypoxia. NCO-700 also inhibited intracellular protease activity. NCO-700 reduced hypoxic cell death to 30% after 6 hours of hypoxygenation. These observations indicate that calpain is activated during hypoxia and leads to irreversible cell membrane degradation after 6 hours of hypoxygenation.
本研究的目的是探讨缺氧心肌细胞损伤与细胞内蛋白酶活性之间的相关性。从新生大鼠心脏分离心肌细胞，并在伊格尔改良的最低必需培养基中培养。将心肌细胞置于缺氧条件下孵育6小时。缺氧6小时后，缺氧期间的细胞死亡率升至80%。缺氧期间细胞外蛋白酶活性升高至4个单位，远高于6小时有氧状态下的0.7个单位。缺氧条件下的这种细胞外蛋白酶活性被亮抑酶肽和乙二胺四乙酸显著抑制，被半胱氨酸蛋白酶抑制剂NCO - 700微弱抑制，但苯甲基磺酰氟不抑制蛋白酶活性。为了鉴定缺氧期间激活的蛋白酶，将钙蛋白酶特异性抑制剂添加到孵育混合物中。钙蛋白酶抑制剂1和钙蛋白酶抑制蛋白（一种内源性选择性钙蛋白酶抑制剂）显著抑制缺氧期间的细胞外蛋白酶活性。NCO - 700也抑制细胞内蛋白酶活性。在缺氧6小时后，NCO - 700将缺氧细胞死亡率降至30%。这些观察结果表明，钙蛋白酶在缺氧期间被激活，并在缺氧6小时后导致不可逆的细胞膜降解。
