Kilpatrick-Smith L, Erecińska M
Circ Shock. 1983;11(2):85-99.
Intramitochondrial substrate metabolism was examined in cultured neuroblastoma NB41A3 cells exposed to endotoxin in order to elucidate possible causes for the changes in [ATP]/[ADP][Pi] and [NAD+]/[NADH] reported by us previously in these cells [1]. Flux through pyruvate dehydrogenase (PDH), measured with [1-14C]-pyruvate, was inhibited by 54% within 10 min in endotoxin-treated cells (0.99 nmol/min/mg dry wt vs 0.46 nmol/min/mg dry wt). In contrast, flux through 2-oxoglutarate dehydrogenase, measured with [1-14C]-glutamate was unaltered (0.79 nmol/min/mg dry wt). Dichloroacetate, an inhibitor of PDH kinase, restored flux through PDH to control levels. In endotoxin-treated cells, only 44% of the total PDH complex was in the active (nonphosphorylated) form as compared to 72% in control cells. Equilibrium uptake studies with 45Ca2+ and atomic absorption measurements showed that intracellular [Ca2+] in endotoxin-treated cells was about 20% lower than in control cells. It is postulated that binding of endotoxin to the plasma membrane triggers a sequence of events that lead to an initial decline in intracellular calcium concentration and that this latter event may be responsible for the inhibition of PDH phosphatase and consequent conversion of the complex to its inactive phosphorylated form.
为了阐明我们之前报道的这些细胞中[ATP]/[ADP][Pi]和[NAD+]/[NADH]变化的可能原因，我们检测了暴露于内毒素的培养神经母细胞瘤NB41A3细胞的线粒体内底物代谢情况。用[1-14C]-丙酮酸测量的丙酮酸脱氢酶（PDH）通量在内毒素处理的细胞中10分钟内被抑制了54%（0.99 nmol/分钟/毫克干重对比0.46 nmol/分钟/毫克干重）。相比之下，用[1-14C]-谷氨酸测量的2-氧代戊二酸脱氢酶通量未改变（0.79 nmol/分钟/毫克干重）。二氯乙酸，一种PDH激酶抑制剂，使PDH通量恢复到对照水平。在内毒素处理的细胞中，只有44%的总PDH复合物处于活性（非磷酸化）形式，而对照细胞中为72%。用45Ca2+进行的平衡摄取研究和原子吸收测量表明，内毒素处理细胞中的细胞内[Ca2+]比对照细胞低约20%。据推测，内毒素与质膜的结合引发了一系列事件，导致细胞内钙浓度最初下降，而后一事件可能是PDH磷酸酶受到抑制以及复合物随后转化为无活性磷酸化形式的原因。
