Mechanisms of oxidant-mediated cell injury. The glycolytic and mitochondrial pathways of ADP phosphorylation are major intracellular targets inactivated by hydrogen peroxide.
Mechanisms of oxidant-mediated cell injury. The glycolytic and mitochondrial pathways of ADP phosphorylation are major intracellular targets inactivated by hydrogen peroxide.
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DOI：
10.1016/s0021-9258(19)77928-9
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发表时间：
1988-02
期刊：
影响因子：
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通讯作者：
P. Hyslop;Daniel;Hinshawz;Wayne A. Halsey;Ingrid;Schraufstatter;Richard D. Sauerhebery;Roger G. Spraggj;Janis H. Jackson;C G Cochrane
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文献类型：
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作者：
P. Hyslop;Daniel;Hinshawz;Wayne A. Halsey;Ingrid;Schraufstatter;Richard D. Sauerhebery;Roger G. Spraggj;Janis H. Jackson;C G Cochrane
在P388 D1细胞暴露于H2 O2的抑制ADP磷酸化的糖酵解和线粒体。净葡萄糖摄取和乳酸产生受到氧化剂暴露的抑制（ED 50 = 50-100 μ M）。糖酵解在3-磷酸甘油醛脱氢酶步骤中通过三种独立的机制特异性失活：（a）细胞内酶的直接失活（ED 50约等于100 μ M）;（b）其烟酰胺辅因子的细胞内浓度和氧化还原电位的降低;和（B）胞浆pH值进一步偏离酶最佳值。与在甘油醛-3-磷酸脱氢酶步骤抑制糖酵解一致，观察到甘油醛-3-磷酸、二羟丙酮磷酸和果糖1，6-二磷酸的细胞内浓度升高。计算的甘油醛-3-磷酸脱氢酶活性的联合抑制可以合理地与糖酵解通量速率的抑制相关。线粒体对总细胞内ATP池的稳态贡献是通过使用各种代谢抑制剂间接确定的，并发现在暴露于300-800 μ M H2 O2后迅速下降。ADP磷酸化的抑制似乎更多地与ATP酶合酶复合物的直接抑制有关，而不是与呼吸链偶联电子传递能力的降低有关。通过糖酵解和线粒体的ADP磷酸化的估计速率和通过正在进行的代谢的ATP水解的估计速率被用于模拟在15分钟暴露于各种H2 O2浓度时预期的细胞内ATP的近似下降。理论计算和测量的细胞内ATP状态符合良好。氧化剂暴露15分钟导致细胞的剂量依赖性杀伤（ED 50 = 500 μ M），表明H2 O2介导的细胞内ATP损失与细胞活力之间密切相关。氧化剂介导的损伤过程中的细胞功能障碍和死亡的能量稳态受损的可能贡献进行了讨论。
Inhibition of ADP phosphorylation by both glycolysis and mitochondria in P388D1 cells exposed to H2O2 is described. Net glucose uptake and lactate production were inhibited by oxidant exposure (ED50 = 50-100 microM). Glycolysis was specifically inactivated at the glyceraldehyde-3-phosphate dehydrogenase step by three independent mechanisms: (a) direct inactivation of the intracellular enzyme (ED50 approximately equal to 100 microM); (b) reduction of the intracellular concentration and redox potential of its nicotinamide cofactors; and (c) a cytosolic pH shift further from the enzyme optima. Consistent with inhibition of glycolysis at the glyceraldehyde-3-phosphate dehydrogenase step, a rise in the intracellular concentration of glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, and fructose 1,6-bisphosphate was observed. The calculated combined inhibition of glyceraldehyde-3-phosphate dehydrogenase activity could be reasonably correlated with the depression in glycolytic flux rate with the appropriate modeling. The steady-state contribution by mitochondria to the total intracellular ATP pool was indirectly determined by the use of various metabolic inhibitors and was found to rapidly decline following exposure to 300-800 microM H2O2. The inhibition of ADP phosphorylation appeared to be related more to the direct inhibition of the ATPase-synthase complex rather than to the diminished capacity of the respiratory chain for coupled electron transport. Both the estimated rates of ADP phosphorylation by glycolysis and mitochondria and the estimated rate of ATP hydrolysis by ongoing metabolism were utilized to model the approximate decline in intracellular ATP expected at 15-min exposure to various H2O2 concentrations. Theoretical calculations and the measured intracellular ATP status were in good agreement. Oxidant exposure for 15 min resulted in dose-dependent killing of the cells (ED50 = 500 microM), indicating a close correlation between H2O2-mediated loss of intracellular ATP and cell viability. The possible contribution of impaired energy homeostasis during oxidant-mediated injury to the process of cell dysfunction and death is discussed.