Tepperman B L, Lush C W, Soper B D
Department of Physiology, University of Western Ontario, London, Canada.
Dig Dis Sci. 1996 Jun;41(6):1172-80. doi: 10.1007/BF02088234.
In the current study, the role of endonuclease activity in calcium ionophore A23187-induced gastric mucosal cellular disruption was examined using rabbit gastric mucosal cells. Cell integrity was assessed using trypan blue dye exclusion and Alamar blue dye absorbance. Ionophore A23187 (1.6-25 microM) induced a concentration-dependent decrease in dye exclusion and cell metabolism in cells suspended in a medium containing Ca2+ (2 mM), while no such effect was observed in cells incubated in the absence of extracellular Ca2+. Cells that were pretreated with the endonuclease inhibitors aurintricarboxylic acid (ATCA; 0.2 or 0.5 mM or Zn2+; 0.01 and 0.1 mM) exhibited significant reduction in the total extent of cell injury when incubated with A23187 in the presence of Ca2+. DNA fragmentation as assessed by measurement of [3H]thymidine liberation or gel electrophoresis was increased in response to ionophore A23187 (12.5 or 25 microM) treatment. A minimal degree of fragmentation was observed when cells were suspended in a Ca(2+)-free medium or incubated in the presence of ATCA or Zn2+. Addition of ethanol (8% w/v) induced a significant increase in cell injury, which was not affected by either removal of extracellular Ca2+ or ATCA pretreatment. Furthermore, treatment with the antioxidants catalase (50 micrograms/ml) or 2',2'-dipyridyl (2 mM) reduced ionophore-induced cell injury but did not reduce the extent of DNA fragmentation. These data suggest that sustained increases in intracellular Ca2+ result in increased endonuclease activity in gastric mucosal cells, leading to extensive DNA lysis and cell damage. Ethanol-induced cell damage does not involve Ca2+ influx and therefore is not mediated by endonuclease activation. Furthermore, sustained increases in cellular Ca2+ may also mediate their effects via formation of reactive oxygen metabolites, but this mechanism of cell damage does not appear to involve DNA fragmentation.
在当前研究中，使用兔胃黏膜细胞检测了核酸内切酶活性在钙离子载体A23187诱导的胃黏膜细胞破坏中的作用。使用台盼蓝染料排斥法和阿拉玛蓝染料吸光度评估细胞完整性。离子载体A23187（1.6 - 25微摩尔）在含有Ca2 +（2毫摩尔）的培养基中悬浮的细胞中，诱导染料排斥和细胞代谢呈浓度依赖性降低，而在无细胞外Ca2 +的情况下孵育的细胞中未观察到这种作用。用核酸内切酶抑制剂金精三羧酸（ATCA；0.2或0.5毫摩尔）或Zn2 +（0.01和0.1毫摩尔）预处理的细胞，在Ca2 +存在下与A23187一起孵育时，细胞损伤的总程度显著降低。通过测量[3H]胸苷释放或凝胶电泳评估的DNA片段化在离子载体A23187（12.5或25微摩尔）处理后增加。当细胞悬浮在无Ca(2 +)的培养基中或在ATCA或Zn2 +存在下孵育时，观察到最小程度的片段化。添加乙醇（8% w/v）诱导细胞损伤显著增加，这不受细胞外Ca2 +去除或ATCA预处理的影响。此外，用抗氧化剂过氧化氢酶（50微克/毫升）或2',2'-联吡啶（2毫摩尔）处理可减少离子载体诱导的细胞损伤，但未降低DNA片段化程度。这些数据表明，细胞内Ca2 +的持续增加导致胃黏膜细胞中核酸内切酶活性增加，导致广泛的DNA裂解和细胞损伤。乙醇诱导的细胞损伤不涉及Ca2 +内流，因此不是由核酸内切酶激活介导的。此外，细胞内Ca2 +的持续增加也可能通过活性氧代谢产物的形成介导其作用，但这种细胞损伤机制似乎不涉及DNA片段化。
