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Role of endonuclease activity and DNA fragmentation in Ca2+ ionophore A23187-mediated injury to rabbit isolated gastric mucosal cells

  • Esophageal, Gastric, And Duodenal Disorders
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Abstract

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 µM) 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 µM) treatment. A minimal degree of fragmentation was observed when cells were suspended in a Ca2+-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 µg/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.

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Authors and Affiliations

  1. Department of Physiology, Faculty of Medicine, University of Western Ontario, N6A 5C1, London, Ontario, Canada

    B. L. Tepperman PhD, C. W. Lush BSc & B. D. Soper MSc

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  1. B. L. Tepperman PhD
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  2. C. W. Lush BSc
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  3. B. D. Soper MSc
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This work was supported by a grant from the Medical Research Council of Canada (MT 6426).

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Tepperman, B.L., Lush, C.W. & Soper, B.D. Role of endonuclease activity and DNA fragmentation in Ca2+ ionophore A23187-mediated injury to rabbit isolated gastric mucosal cells. Digest Dis Sci 41, 1172–1180 (1996). https://doi.org/10.1007/BF02088234

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  • DOI: https://doi.org/10.1007/BF02088234

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