Kimura M, Goto S, Wada A, Yahiro K, Niidome T, Hatakeyama T, Aoyagi H, Hirayama T, Kondo T
Department of Applied Chemistry, Faculty of Engineering, Nagasaki, 852, Japan.
Microb Pathog. 1999 Jan;26(1):45-52. doi: 10.1006/mpat.1998.0241.
We investigated the effects of vacuolating cytotoxin (VacA) prepared from Helicobacter pylori on the metabolism of gastric epithelial cells, AZ-521. VacA caused the ATP levels to decrease in a time-dependent manner; by approximately 20% in 6 h, 35% in 12 h and 50% in 24 h, at a concentration of 120 nM. This decrease was also dependent on the concentration of VacA. To evaluate the impairment of mitochondria by VacA, mitochondrial membrane potential was estimated by flow cytometric analysis using 3, 3'-dihexyloxacarbocyanine iodide as a substrate. VacA decreased membrane potential with the relative fluorescence intensity of AZ-521 cells in 6 h from 52+/-3 to 24+/-1. Treatment of the cells with bafilomycin A1, a specific inhibitor of vacuolar ATPase proton pump, showed no apparent effect on these changes in the levels of ATP and the mitochondrial membrane potential. Secondly, we estimated the effect of VacA on oxygen consumption. VacA inhibited oxygen consumption in AZ-521 cells: the levels of PO 2 in the medium of control cells decreased by 73% in 3 h and 37% in 6 h, whereas those in VacA-treated cells were 84% in 3 h and 59% in 6 h. Flow cytometric analysis showed the number of cells in the G0/G1 phase was increased by VacA. Taken together, VacA induced an inactivation of energy metabolism followed by mitochondrial damage, leading to impairment of the cell cycle in gastric epithelial cells.
我们研究了从幽门螺杆菌制备的空泡毒素（VacA）对胃上皮细胞AZ-521代谢的影响。VacA导致ATP水平呈时间依赖性下降；在浓度为120 nM时，6小时内下降约20%，12小时内下降35%，24小时内下降50%。这种下降也依赖于VacA的浓度。为了评估VacA对线粒体的损害，使用3,3'-二己基氧杂羰花青碘化物作为底物，通过流式细胞术分析来估计线粒体膜电位。VacA在6小时内使AZ-521细胞的膜电位下降，相对荧光强度从52±3降至24±1。用液泡ATP酶质子泵的特异性抑制剂巴弗洛霉素A1处理细胞，对ATP水平和线粒体膜电位的这些变化没有明显影响。其次，我们估计了VacA对氧气消耗的影响。VacA抑制了AZ-521细胞的氧气消耗：对照细胞培养基中的PO₂水平在3小时内下降73%，在6小时内下降37%，而VacA处理细胞的PO₂水平在3小时内为84%，在6小时内为59%。流式细胞术分析显示，VacA使处于G0/G1期的细胞数量增加。综上所述，VacA诱导能量代谢失活，随后导致线粒体损伤，进而损害胃上皮细胞的细胞周期。
