calcimycin and resazurin

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.

Topics: Animals; Antioxidants; Aurintricarboxylic Acid; Calcimycin; Calcium; Cell Survival; Coloring Agents; DNA; Endonucleases; Gastric Mucosa; In Vitro Techniques; Ionophores; Oxazines; Rabbits; Trypan Blue; Xanthenes; Zinc