Murata M, Monden M, Umeshita K, Nakano H, Kanai T, Gotoh M, Mori T
Department of Surgery II, Osaka University Medical School, Suita, Japan.
Hepatology. 1994 May;19(5):1223-8.
The mechanisms of hepatocyte injury caused by exogenous superoxide were investigated with the use of cultured rat hepatocytes. Cell viability, cytosolic free calcium concentration and cell surface structure were observed. Superoxide was produced by adding hypoxanthine and xanthine oxidase to the buffer. Cytosolic free calcium concentration was calculated by means of ratio imaging of fura 2 fluorescence with multiparameter digitized microscopy. In the buffer containing 1.27 mmol/L of calcium, lactate dehydrogenase release into the buffer began to increase at 1 hr and reached a plateau in 5 hr. Eighteen minutes after the addition of hypoxanthine and xanthine oxidase, small blebs were recognized on the cell surface with a scanning electron microscope; then a gradual rise in cytosolic free calcium concentration was observed. Thirty minutes after exposure to superoxide, large blebs were recognized with a phase-contrast microscope, when cytosolic free calcium concentration had risen to about 700 nmol/L. Depriving the buffer of calcium (< 10 mumol/L) significantly suppressed bleb formation and cell death, and cytosolic free calcium concentration was found to remain around the basal level (200 nmol/L). When ethylene glycol-bis (beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid was added to the buffer, bleb formation and cell death were suppressed more completely, and cytosolic free calcium concentration decreased. Superoxide dismutase combined with catalase or nifedipine allowed the hepatocytes to maintain their viability and suppressed cytosolic free calcium concentration elevation. Calpeptin, a Ca(2+)-dependent neutral protease inhibitor, did not affect the rise in cytosolic free calcium concentration but prevented cell injury.(ABSTRACT TRUNCATED AT 250 WORDS)
利用培养的大鼠肝细胞研究外源性超氧化物引起肝细胞损伤的机制。观察细胞活力、胞质游离钙浓度和细胞表面结构。通过向缓冲液中添加次黄嘌呤和黄嘌呤氧化酶产生超氧化物。利用多参数数字化显微镜通过fura 2荧光比率成像计算胞质游离钙浓度。在含有1.27 mmol/L钙的缓冲液中，乳酸脱氢酶释放到缓冲液中的量在1小时开始增加，并在5小时达到平台期。添加次黄嘌呤和黄嘌呤氧化酶18分钟后，用扫描电子显微镜在细胞表面观察到小泡；然后观察到胞质游离钙浓度逐渐升高。暴露于超氧化物30分钟后，当胞质游离钙浓度升至约700 nmol/L时，用相差显微镜观察到大气泡。去除缓冲液中的钙（<10 μmol/L）可显著抑制气泡形成和细胞死亡，且发现胞质游离钙浓度保持在基础水平（200 nmol/L）左右。当向缓冲液中添加乙二醇双（β-氨基乙醚）-N，N，N'，N'-四乙酸时，气泡形成和细胞死亡得到更完全的抑制，且胞质游离钙浓度降低。超氧化物歧化酶与过氧化氢酶或硝苯地平联合使用可使肝细胞维持其活力并抑制胞质游离钙浓度升高。钙肽素是一种钙依赖性中性蛋白酶抑制剂，它不影响胞质游离钙浓度的升高，但可防止细胞损伤。（摘要截短至250字）
