Keane W F, Welch R, Gekker G, Peterson P K
Am J Pathol. 1987 Feb;126(2):350-7.
Alpha-hemolysin (AH) is a 110,000-dalton protein secreted extracellularly by certain Escherichia coli. This protein is an acknowledged virulence factor for E coli and recently has been implicated as an important determinant in the pathogenesis of E coli pyelonephritis. Recombinant engineered strains of E coli were used that varied only in their ability to secrete AH extracellularly. The effect of AH on vital dye exclusion, oxygen consumption rate (QO2) adenosine triphosphate (ATP) levels, superoxide (O2-) and hydrogen peroxide (H2O2) production in preparations of isolated rat cortical renal tubular cells (RTCs) was assessed. Approximately 5-10 pg of AH dramatically stimulated QO2 by nearly 150%. This was associated with a marked increase in production of O2- and H2O2, to 13.9 +/- 1.7 and 13.2 +/- 2.1 nM/mg cell protein, respectively (P less than 0.05), as well as a 38% decrease in cellular ATP. These biochemical effects were all seen after a 30-minute exposure to AH and by 120 minutes were associated with 15.7% +/- 1.1% of RTCs that were unable to exclude vital dye. The effect of AH on QO2 and O2- formation was prevented by pretreatment of RTCs with ouabain, which indicates that the effect of AH on oxygen metabolism is linked to Na-K ATPase activity. However, when ouabain-treated RTCs were exposed to AH, ATP remained depressed despite the inhibition of QO2 and O2- production. In contrast, in ouabain-pretreated RTCs, cell membrane integrity was dramatically protected, because only 2.4% +/- 0.4% of RTCs were not unable to exclude vital dye. Thus, the data demonstrate that E coli AH provokes at least two biochemical events that may be injurious to RTC: increased oxygen intermediates (O2- and H2O2 and ATP depletion. These findings with ouabain suggest that the first mechanism of injury may be a more proximate cause of cell death. Moreover, the data suggest that endogenous production of reactive oxygen molecules may be critical modulators of RTC membrane injury.
α-溶血素（AH）是由某些大肠杆菌分泌到细胞外的一种110,000道尔顿的蛋白质。这种蛋白质是公认的大肠杆菌毒力因子，最近被认为是大肠杆菌肾盂肾炎发病机制中的一个重要决定因素。使用了仅在细胞外分泌AH能力上有所不同的重组工程化大肠杆菌菌株。评估了AH对分离的大鼠皮质肾小管细胞（RTC）制剂中活性染料排斥、氧消耗率（QO2）、三磷酸腺苷（ATP）水平、超氧化物（O2-）和过氧化氢（H2O2）产生的影响。约5 - 10皮克的AH使QO2显著增加近150%。这与O2-和H2O2的产生显著增加有关，分别达到13.9±1.7和13.2±2.1纳摩尔/毫克细胞蛋白（P小于0.05），同时细胞ATP减少38%。这些生化效应在暴露于AH 30分钟后均可见，到120分钟时，15.7%±1.1%的RTC无法排斥活性染料。用哇巴因预处理RTC可防止AH对QO2和O2-形成的影响，这表明AH对氧代谢的影响与钠钾ATP酶活性有关。然而，当用哇巴因处理的RTC暴露于AH时，尽管QO2和O2-产生受到抑制，但ATP仍保持降低。相反，在经哇巴因预处理的RTC中，细胞膜完整性得到显著保护，因为只有2.4%±0.4%的RTC无法排斥活性染料。因此，数据表明大肠杆菌AH引发了至少两种可能对RTC有害的生化事件：氧中间体增加（O2-和H2O2）和ATP耗竭。哇巴因的这些发现表明，第一种损伤机制可能是细胞死亡的更直接原因。此外，数据表明活性氧分子的内源性产生可能是RTC膜损伤的关键调节因子。
