Qian T, Nieminen A L, Herman B, Lemasters J J
Department of Cell Biology and Anatomy, School of Medicine, University of North Carolina at Chapel Hill 27599-7090, USA.
Am J Physiol. 1997 Dec;273(6):C1783-92. doi: 10.1152/ajpcell.1997.273.6.C1783.
To simulate ischemia and reperfusion, cultured rat hepatocytes were incubated in anoxic buffer at pH 6.2 for 4 h and reoxygenated at pH 7.4. During anoxia, intracellular pH (pHi) decreased to 6.3, mitochondria depolarized, and ATP decreased to < 1% of basal values, but the mitochondrial permeability transition (MPT) did not occur as assessed by confocal microscopy from the redistribution of cytosolic calcein into mitochondria. Moreover, cell viability remained > 90%. After reperfusion at pH 7.4, pHi returned to pH 7.2, the MPT occurred, and most hepatocytes lost viability. In contrast, after reperfusion at pH 6.2 or with Na(+)-free buffer at pH 7.4, pHi did not rise and cell viability remained > 80%. After acidotic reperfusion, the MPT did not occur. When hepatocytes were reperfused with cyclosporin A (0.5-1 microM) at pH 7.4, the MPT was prevented and cell viability remained > 80%, although pHi increased to 7.2. Reperfusion with glycine (5 mM) also prevented cell killing but did not block recovery of pHi or the MPT. Retention of cell viability was associated with recovery of 30-40% of ATP. In conclusion, preventing the rise of pHi after reperfusion blocked the MPT, improved ATP recovery, and prevented cell death. Cyclosporin A also prevented cell killing by blocking the MPT without blocking recovery of pHi. Glycine prevented cell killing but did not inhibit recovery of pHi or the MPT.
为模拟缺血和再灌注过程，将培养的大鼠肝细胞置于pH 6.2的缺氧缓冲液中孵育4小时，然后在pH 7.4条件下复氧。缺氧期间，细胞内pH（pHi）降至6.3，线粒体去极化，ATP降至基础值的<1%，但通过共聚焦显微镜观察胞质钙黄绿素向线粒体的重新分布评估，线粒体通透性转换（MPT）未发生。此外，细胞活力保持>90%。在pH 7.4复氧后，pHi恢复至pH 7.2，MPT发生，大多数肝细胞失去活力。相反，在pH 6.2复氧或在pH 7.4的无钠缓冲液中复氧后，pHi未升高，细胞活力保持>80%。酸中毒复氧后，MPT未发生。当肝细胞在pH 7.4用环孢素A（0.5 - 1 microM）复氧时，MPT被阻止，细胞活力保持>80%，尽管pHi升至7.2。用甘氨酸（5 mM）复氧也可防止细胞死亡，但不阻断pHi的恢复或MPT。细胞活力的保留与30 - 40%的ATP恢复相关。总之，防止复氧后pHi升高可阻断MPT，改善ATP恢复，并防止细胞死亡。环孢素A也通过阻断MPT防止细胞死亡，而不阻断pHi的恢复。甘氨酸可防止细胞死亡，但不抑制pHi的恢复或MPT。
