Digestive Diseases and Sciences

, Volume 41, Issue 6, pp 1172–1180 | Cite as

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

  • B. L. Tepperman
  • C. W. Lush
  • B. D. Soper
Esophageal, Gastric, And Duodenal Disorders

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.

Key words

calcium cell injury gastric mucosa endonuclease DNA fragmentation ethanol antioxidants 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Farber JL: The role of calcium in cell killing. Life Sci 29:1289–1295, 1981Google Scholar
  2. 2.
    Farber JL: The role of calcium of calcium ions in toxic cell injury. Environ Health Perspect 84:107–111, 1990Google Scholar
  3. 3.
    Leonard JP, Saltpeter MM: Calcium mediated myopathy at neuromuscular junctions of normal and dystrophic muscle. Exp Neurol 76:121–138, 1982Google Scholar
  4. 4.
    Pasternak CA: Effect of pore formers on intracellular calcium. Cell Calcium 7:387–397, 1986Google Scholar
  5. 5.
    Nicotera P, Hartzell P, Davis G, Orrenius S: The formation of plasma membrane blebs in hepatocytes exposed to agents that increase cytosolic Ca2+ is mediated by the activation of a non-lysosomal proteolytic system. FEBS Lett 209:1309–1344, 1986Google Scholar
  6. 6.
    Nicotera P, Hartzell P, Baldi C, Svenson S-A, Bellomo G, Orrenius S: Cystamine induces toxicity in hepatocytes through the elevation of cytosolic Ca2+. J Biol Chem 261:14628–14635, 1986Google Scholar
  7. 7.
    Mirabelli F, Salis A, Vairetti M, Bellomo G, Thor H, Orrenius S: Cytoskeletal alterations in human platelets exposed to oxidative stress are mediated by oxidative and Ca+-dependent mechanisms. Arch Biochem Biophys 270:478–488, 1989Google Scholar
  8. 8.
    Dziki AJ, Batzri S, Harmon JW, Molloy M: Cellular hypercalcemia is an early event in deoxycholate injury of rabbit mucosal cells. Am J Physiol 269(Gastrointest Liver Physiol 32):G287-G296, 1995Google Scholar
  9. 9.
    Tepperman BL, Soper BD: Calcium-mediated damage to rabbit gastric mucosal cells: Modulation by nitric oxide. Eur J Pharmacol 293:259–266, 1995Google Scholar
  10. 10.
    Ota S, Tsukahara H, Terano H, Hata Y, Hiraishi H, Mutoh H, Sugimoto T: Protective effects of tauroursodeoxycholate against chenodeoxycholate-induced damage to cultured rabbit gastric cells. Dig Dis Sci 36:409–416, 1991Google Scholar
  11. 11.
    Tepperman BL, Soper BD: Effect of extracellular Ca2+ on indomethacin-induced injury to rabbit dispersed gastric mucosal cells. Can J Physiol Pharmacol 72:63–69, 1994Google Scholar
  12. 12.
    McConkey DJ, Hartzell P, Duddy SK, Hakansson H, Orrenius S: 2,3,7,8-Tetrachlorodibenzo-p-dioxin kills immature thymocytes by Ca2+-mediated endonuclease activation. Science 242:256–295, 1989Google Scholar
  13. 13.
    Lohmann RD, Beyersmann D: Effects of zinc and calcium on apoptotic DNA fragmentation in isolated bovine liver nuclei. Environ Health Perspect 102(suppl 3):269–271, 1994Google Scholar
  14. 14.
    Zhivotovsky B, Cedervall B, Jiang S, Nicotera P, Orrenius S: Involvement of Ca2+ in the formation of high molecular weight DNA fragments in thymocyte apoptosis. Biochem Biophys Res Commun 202:120–127, 1994Google Scholar
  15. 15.
    Matsubara K, Kubota M, Adachi S, Kuwakado K, Hirota H, Wakazono Y, Akiyama Y, Mikawa H: Different mode of cell death induced by calcium ionophore in human leukemia cell lines: Possible role of constitutive endonuclease. Exp Cell Res 210:19–25, 1994Google Scholar
  16. 16.
    Valle VG, Fagian MM, Parentoni LS, Meinicke AR, Vercesi AE: The participation of reactive oxygen species and protein permeabilization by calcium plus proxidants. Arch Biochem Biophys 307:1–7, 1993Google Scholar
  17. 17.
    Wong HM, Tepperman BL: Reduced glutathione modulates Ca2+-mediated damage to rabbit isolated gastric mucosal cells. Am J Physiol 267(Gastrointest Liver Physiol 30):G1-G9, 1994Google Scholar
  18. 18.
    Ben-Yoseph O, Ross BD: Oxidation therapy: The use of a reactive oxygen species-generating enzyme system for tumor treatment. Br J Cancer 70:1131–1135, 1994Google Scholar
  19. 19.
    Kim H-R, Rho H-W, Park B-H, Park J-W, Kim J-S, Kim U-H, Chung M-Y: Role of Ca2+ in alloxan-induced pancreaticβ-cell damage. Biochim Biophys Acta 1227:87–91, 1994Google Scholar
  20. 20.
    Tepperman BL, Tan SY, Whittle BJR: Effects of calcium-modifying agents on integrity of rabbit gastric mucosal cells. Am J Physiol 261(Gastrointest Liver Physiol 24):G119-G127, 1994Google Scholar
  21. 21.
    McConkey DJ, Hartzell P, Nicotera P, Orrenius S: Calcium-activated DNA fragmentation kills immature thymocytes. FASEB J 3:1843–1849, 1989Google Scholar
  22. 22.
    Pagé B, Pagé M, Noel C: A new fluorometric assay for cytotoxicity measurementsin vitro. Int J Oncol 3:473–476, 1993Google Scholar
  23. 23.
    Philippe C, Philippe B, Fouqueray B, Perez J, Lebret M, Baud L: Protection from tumor necrosis factor-mediated cytolysis by platelets. Am J Pathol 143:1713–1723, 1993Google Scholar
  24. 24.
    Robaye B, Mosselmans R, Fiers W, Dumont JE, Galand F: Tumor necrosis factor induces apoptosis (programmed cell death) in normal endothelial cellsin vitro. Am J Pathol 138:447–453, 1991Google Scholar
  25. 25.
    Wong HM, Soper BD, Tepperman BL: Role of calcium in thromboxane B2-mediated injury to rabbit gastric mucosal cells. Dig Dis Sci 40:2022–2028, 1995Google Scholar
  26. 26.
    Mosmann T: Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity. J Immunol Methods 65:55–64, 1983Google Scholar
  27. 27.
    Nagy LS, Szabo S, Morales RE, Plebani M, Jenkins JM: Identification of subcellular targets and sensitive tests of ethanol-induced damage in isolated rat gastric mucosal cells. Gastroenterology 107:907–914, 1994Google Scholar
  28. 28.
    Jiang S, Chow SC, Nicotera P, Orrenius S: Intracellular Ca2+ signals activate apoptosis in thymocytes: Studies using the Ca2+-ATPase inhibitor thapsigargin. Exp Cell Res 212:84–92, 1994Google Scholar
  29. 29.
    Csernansky CA, Canzoniero LMT, Sensi SL, Yu SP, Choi DW: Delayed application of aurintricarboxylic acid reduces glutamate-induced cortical neuronal injury. J Neurosci Res 38:101–108, 1994Google Scholar
  30. 30.
    Kokileva L: Multi-step chromatin degradation in apoptosis. Int Arch Allergy Immunol 105:339–343, 1994Google Scholar
  31. 31.
    Walker PR, Weaver VM, Lach B, LeBlanc J, Sikorska M: Endonuclease activities associated with high molecular weight and internucleosomal DNA fragmentation in apoptosis. Exp Cell Res 213:100–106, 1994Google Scholar
  32. 32.
    Cain K, Inayat-Hussain SH, Wolfe JT, Cohen GM: DNA fragmentation into 200–250 and/or 30–50 kilobase pair fragments in rat liver nuclei is stimulated by Mg2+ alone and Ca2+/Mg2+ but not by Ca2+ alone. FEBS Lett 349:385–391, 1994Google Scholar
  33. 33.
    Cherner JA, Naik L, Tarnawski A, Brzozowski J, Stachura J, Singh G: Ability of prostaglandin to reduce ethanol injury to dispersed chief cells from guinea pig stomach. Am J Physiol 256(Gastrointest Liver Physiol 19):G704-G714, 1989Google Scholar
  34. 34.
    Duke RC, Chervenak R, Cohen JJ: Endogenous endonuclease-induced DNA fragmentation: An early event in cell-mediated cytolysis. Proc Natl Acad Sci USA 80:6361–6365, 1983Google Scholar
  35. 35.
    Patel T, Bronk SF, Gores GJ: Increases in intracellular magnesium promote glycodeoxycholate-induced apoptosis in rat hepatocytes. J Clin Invest 94:2183–2192, 1994Google Scholar

Copyright information

© Plenum Publishing Corporation 1996

Authors and Affiliations

  • B. L. Tepperman
    • 1
  • C. W. Lush
    • 1
  • B. D. Soper
    • 1
  1. 1.Department of Physiology, Faculty of MedicineUniversity of Western OntarioLondonCanada

Personalised recommendations