Bioimaging analysis of hypoxic cell death and its cellular defense mechanisms.

在分离的成年大鼠单个心肌细胞中，我们利用新设计的荧光成像系统研究了细胞内氧浓度、线粒体氧化代谢、细胞内ATP浓度和线粒体膜电位的变化。在生理氧浓度(3%-5%)下，氧化磷酸化解偶联剂增加线粒体的氧通量可促进坏死细胞死亡。由于这些细胞线粒体膜电位的变化很小，氧通量的增加似乎对细胞在低氧中的生存产生了至关重要的影响。肌酸激酶(CK)抑制后，观察到的细胞死亡明显加快。在CK抑制的氧通量升高的心肌细胞中，细胞核心的膜电位几乎被取消，这与我们之前关于单个心肌细胞(缺氧细胞核心)内氧浓度的径向梯度的发现一致。线粒体膜电位…的破坏缺氧核中的L越多，F_1F_0-ATP合成酶(复合体V)就会转化为ATPase。如果发生这种情况，不仅氧化的ATP生产将无法进行，而且在缺氧核心的线粒体中将爆发大量的ATP消耗。这种ATP的缺乏，虽然局限于细胞核心，但会迅速扰乱依赖ATPase的细胞内钙调节，最终导致细胞死亡。然而，在正常心肌细胞中，PCR-CK系统可能通过扩散将高能磷酸盐从细胞表面(那里氧气充足，氧化ATP的产生不受阻碍)供应到缺氧的细胞核心。因此，最初在细胞表面附近的线粒体产生的聚合酶链式反应会向缺氧细胞核心的线粒体补充三磷酸腺苷，从而保持线粒体膜电位，而不需要呼吸链中的电子传递。这一机制可显著延缓线粒体的功能性解体和随后的钙超载。因此，PCR-CK系统可能是保护呼吸心肌细胞免受缺氧性死亡的内在机制。较少

In single cardiomyocytes isolated from the adult rat, we investigated changes in intracellular oxygen concentration, mitochondrial oxidative metabolism, intracellular ATP concentration, and mitochondrial membrane potential using newly devised fluorescence imaging system. At physiological oxygen concentration (3%-5%),elevating oxygen flux to mitochondria by an uncoupler of oxidative phosphorylation promoted necrotic cell death. Because changes in the mitochondrial membrane potential in these cells were minimum, increases in the oxygen flux appeared to critically affect cell survival in hypoxia. Observed cell death was significantly accelerated after inhibition of creatine kinase (CK). In CK-inhibited cardiomyocytes with elevated oxygen flux, membrane potential was almost abolished in the cell core that is consistent with our previous findings regarding radial gradients of oxygen concentration within a single cardiomyocyte (anoxic cell core). Disruption of mitochondrial membrane potentia … More l in the anoxic core would turn the F_1F_0-ATP synthase (complex V) to an ATPase. If these happen, not only oxidative ATP production would be unable to proceed, but also massive consumption of ATP should break out in mitochondria in the anoxic core. This deficiency of ATP, albeit restricted in the cell core, quickly disturbs ATPase-dependent intracellular regulations of Ca^<2+>, and Ca^<2+> overload leading to cell death would finally commence. However, in the normal cardiomyocyte, the PCr-CK system may supply high energy phosphate from the cell surface (where oxygen is abundant and oxidative ATP production is not hampered) to the oxygen deficit cell core by diffusion. Thus, PCr originally produced in mitochondria near the cell surface would supplement ATP to the mitochondria in the anoxic cell core, so that mitochondrial membrane potential is maintained without electron transport in the respiratory chain. Functional disintegration of the mitochondria and following Ca^<2+> overload could be significantly retarded by this mechanism. Hence, the PCr-CK system may be an intrinsic mechanism that protects respiring cardiomyocytes against hypoxic death. Less