Role of endonuclease activity and DNA fragmentation in Ca2+ ionophore A23187-mediated injury to rabbit isolated gastric mucosal cells.
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.
Tepperman
B L
BL
Department of Physiology, University of Western Ontario, London, Canada.
Lush
C W
CW
Soper
B D
BD
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001312:Aurintricarboxylic Acid / Q000494:pharmacology; D000001:Calcimycin / Q000633:toxicity*; D002118:Calcium / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D004396:Coloring Agents; D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D004720:Endonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005753:Gastric Mucosa / Q000187:drug effects* / Q000378:metabolism* / Q000473:pathology; D066298:In Vitro Techniques; D007476:Ionophores / Q000633:toxicity*; D010078:Oxazines; D011817:Rabbits; D014343:Trypan Blue; D014966:Xanthenes; D015032:Zinc / Q000494:pharmacology
D000975:Antioxidants; D004396:Coloring Agents; D007476:Ionophores; D010078:Oxazines; D014966:Xanthenes; C005843:resazurin; D000001:Calcimycin; D001312:Aurintricarboxylic Acid; D004247:DNA; D004720:Endonucleases; D014343:Trypan Blue; D015032:Zinc; D002118:Calcium