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. 1997 Feb;63(2):440–447. doi: 10.1128/aem.63.2.440-447.1997

Interaction between Calcium Ions and Bacillus thuringiensis Toxin Activity against Sf9 Cells (Spodoptera frugiperda, Lepidoptera)

R Monette, L Potvin, D Baines, R Laprade, J L Schwartz
PMCID: PMC1389515  PMID: 16535509

Abstract

The effects of calcium ions and modulators of calcium movement on Bacillus thuringiensis insecticidal protein toxicity were investigated with Sf9 cells (Spodoptera frugiperda, fall armyworm) by a new B. thuringiensis toxicity assay based on measurement of fluorescence of ethidium homodimer, a high-affinity DNA stain. CryIC toxicity was substantially stimulated by extracellular calcium in a dose-dependent way (in the millimolar range), while toxicity enhancement could not be replicated when calcium was replaced by barium. This incremental toxicity was reduced by cobalt and lanthanum ions, two inorganic-calcium transport inhibitors. Methoxyverapamil, a voltage-dependent calcium channel blocker, and nifedipine, an inhibitor of dihydropyridine-sensitive L-type calcium channels, had no effect on CryIC toxin activity, but BAY K 8644, an L-type calcium channel activator, increased CryIC activity at high concentrations of extracellular calcium. While A23187, a calcium ionophore, and TMB-8, an inhibitor of intracellular-calcium mobilization, did not change CryIC-induced mortality, thapsigargin, an inhibitor of calcium uptake in intracellular stores, and more particularly trifluoperazine, which inhibits calcium-calmodulin-dependent processes, increased CryIC-mediated toxicity. The incremental effect of extracellular calcium on CryIC-induced toxicity was consistent with an increased concentration of intracellular calcium.

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Selected References

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  1. Anderberg E. K., Artursson P. Epithelial transport of drugs in cell culture. VIII: Effects of sodium dodecyl sulfate on cell membrane and tight junction permeability in human intestinal epithelial (Caco-2) cells. J Pharm Sci. 1993 Apr;82(4):392–398. doi: 10.1002/jps.2600820412. [DOI] [PubMed] [Google Scholar]
  2. Carafoli E. Intracellular calcium homeostasis. Annu Rev Biochem. 1987;56:395–433. doi: 10.1146/annurev.bi.56.070187.002143. [DOI] [PubMed] [Google Scholar]
  3. Chiou C. Y., Malagodi M. H. Studies on the mechanism of action of a new Ca-2+ antagonist, 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride in smooth and skeletal muscles. Br J Pharmacol. 1975 Feb;53(2):279–285. doi: 10.1111/j.1476-5381.1975.tb07359.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gaugain B., Barbet J., Capelle N., Roques B. P., Le Pecq J. B. DNA Bifunctional intercalators. 2. Fluorescence properties and DNA binding interaction of an ethidium homodimer and an acridine ethidium heterodimer. Biochemistry. 1978 Nov 28;17(24):5078–5088. doi: 10.1021/bi00617a002. [DOI] [PubMed] [Google Scholar]
  5. Gill S. S., Cowles E. A., Pietrantonio P. V. The mode of action of Bacillus thuringiensis endotoxins. Annu Rev Entomol. 1992;37:615–636. doi: 10.1146/annurev.en.37.010192.003151. [DOI] [PubMed] [Google Scholar]
  6. Glazer A. N., Rye H. S. Stable dye-DNA intercalation complexes as reagents for high-sensitivity fluorescence detection. Nature. 1992 Oct 29;359(6398):859–861. doi: 10.1038/359859a0. [DOI] [PubMed] [Google Scholar]
  7. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  8. Hess P. Calcium channels in vertebrate cells. Annu Rev Neurosci. 1990;13:337–356. doi: 10.1146/annurev.ne.13.030190.002005. [DOI] [PubMed] [Google Scholar]
  9. Hu Y., Rajan L., Schilling W. P. Ca2+ signaling in Sf9 insect cells and the functional expression of a rat brain M5 muscarinic receptor. Am J Physiol. 1994 Jun;266(6 Pt 1):C1736–C1743. doi: 10.1152/ajpcell.1994.266.6.C1736. [DOI] [PubMed] [Google Scholar]
  10. Hu Y., Vaca L., Zhu X., Birnbaumer L., Kunze D. L., Schilling W. P. Appearance of a novel Ca2+ influx pathway in Sf9 insect cells following expression of the transient receptor potential-like (trpl) protein of Drosophila. Biochem Biophys Res Commun. 1994 Jun 15;201(2):1050–1056. doi: 10.1006/bbrc.1994.1808. [DOI] [PubMed] [Google Scholar]
  11. Höfte H., Whiteley H. R. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev. 1989 Jun;53(2):242–255. doi: 10.1128/mr.53.2.242-255.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Johnson D. E. Cellular toxicities and membrane binding characteristics of insecticidal crystal proteins from Bacillus thuringiensis toward cultured insect cells. J Invertebr Pathol. 1994 Mar;63(2):123–129. doi: 10.1006/jipa.1994.1024. [DOI] [PubMed] [Google Scholar]
  13. Knight P. J., Knowles B. H., Ellar D. J. Molecular cloning of an insect aminopeptidase N that serves as a receptor for Bacillus thuringiensis CryIA(c) toxin. J Biol Chem. 1995 Jul 28;270(30):17765–17770. doi: 10.1074/jbc.270.30.17765. [DOI] [PubMed] [Google Scholar]
  14. Knowles B. H., Farndale R. W. Activation of insect cell adenylate cyclase by Bacillus thuringiensis delta-endotoxins and melittin. Toxicity is independent of cyclic AMP. Biochem J. 1988 Jul 1;253(1):235–241. doi: 10.1042/bj2530235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kolb H. A. Potassium channels in excitable and non-excitable cells. Rev Physiol Biochem Pharmacol. 1990;115:51–91. [PubMed] [Google Scholar]
  16. Lanciotti R. A., Bender P. K. Baculovirus-directed expression of the gamma-subunit of phosphorylase kinase: purification and calmodulin dependence. Biochem J. 1994 Apr 1;299(Pt 1):183–189. doi: 10.1042/bj2990183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Li J. D., Carroll J., Ellar D. J. Crystal structure of insecticidal delta-endotoxin from Bacillus thuringiensis at 2.5 A resolution. Nature. 1991 Oct 31;353(6347):815–821. doi: 10.1038/353815a0. [DOI] [PubMed] [Google Scholar]
  18. Markovits J., Roques B. P., Le Pecq J. B. Ethidium dimer: a new reagent for the fluorimetric determination of nucleic acids. Anal Biochem. 1979 Apr 15;94(2):259–264. doi: 10.1016/0003-2697(79)90357-9. [DOI] [PubMed] [Google Scholar]
  19. Martínez-Ramírez A. C., González-Nebauer S., Escriche B., Real M. D. Ligand blot identification of a Manduca sexta midgut binding protein specific to three Bacillus thuringiensis CryIA-type ICPs. Biochem Biophys Res Commun. 1994 Jun 15;201(2):782–787. doi: 10.1006/bbrc.1994.1769. [DOI] [PubMed] [Google Scholar]
  20. Morgan G. T. Identification in the human genome of mobile elements spread by DNA-mediated transposition. J Mol Biol. 1995 Nov 17;254(1):1–5. doi: 10.1006/jmbi.1995.0593. [DOI] [PubMed] [Google Scholar]
  21. Orrenius S., McConkey D. J., Bellomo G., Nicotera P. Role of Ca2+ in toxic cell killing. Trends Pharmacol Sci. 1989 Jul;10(7):281–285. doi: 10.1016/0165-6147(89)90029-1. [DOI] [PubMed] [Google Scholar]
  22. Pietrobon D., Di Virgilio F., Pozzan T. Structural and functional aspects of calcium homeostasis in eukaryotic cells. Eur J Biochem. 1990 Nov 13;193(3):599–622. doi: 10.1111/j.1432-1033.1990.tb19378.x. [DOI] [PubMed] [Google Scholar]
  23. Pressman B. C. Biological applications of ionophores. Annu Rev Biochem. 1976;45:501–530. doi: 10.1146/annurev.bi.45.070176.002441. [DOI] [PubMed] [Google Scholar]
  24. Sangadala S., Walters F. S., English L. H., Adang M. J. A mixture of Manduca sexta aminopeptidase and phosphatase enhances Bacillus thuringiensis insecticidal CryIA(c) toxin binding and 86Rb(+)-K+ efflux in vitro. J Biol Chem. 1994 Apr 1;269(13):10088–10092. [PubMed] [Google Scholar]
  25. Schilling W. P., Rajan L., Strobl-Jager E. Characterization of the bradykinin-stimulated calcium influx pathway of cultured vascular endothelial cells. Saturability, selectivity, and kinetics. J Biol Chem. 1989 Aug 5;264(22):12838–12848. [PubMed] [Google Scholar]
  26. Schwartz J. L., Garneau L., Masson L., Brousseau R. Early response of cultured lepidopteran cells to exposure to delta-endotoxin from Bacillus thuringiensis: involvement of calcium and anionic channels. Biochim Biophys Acta. 1991 Jun 18;1065(2):250–260. doi: 10.1016/0005-2736(91)90237-3. [DOI] [PubMed] [Google Scholar]
  27. Schwartz J. L., Garneau L., Savaria D., Masson L., Brousseau R., Rousseau E. Lepidopteran-specific crystal toxins from Bacillus thuringiensis form cation- and anion-selective channels in planar lipid bilayers. J Membr Biol. 1993 Feb;132(1):53–62. doi: 10.1007/BF00233051. [DOI] [PubMed] [Google Scholar]
  28. Thastrup O., Cullen P. J., Drøbak B. K., Hanley M. R., Dawson A. P. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2466–2470. doi: 10.1073/pnas.87.7.2466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Triggle D. J. Calcium, calcium channels, and calcium channel antagonists. Can J Physiol Pharmacol. 1990 Nov;68(11):1474–1481. doi: 10.1139/y90-224. [DOI] [PubMed] [Google Scholar]
  30. Tsien R. W., Tsien R. Y. Calcium channels, stores, and oscillations. Annu Rev Cell Biol. 1990;6:715–760. doi: 10.1146/annurev.cb.06.110190.003435. [DOI] [PubMed] [Google Scholar]
  31. Vachon V., Paradis M. J., Marsolais M., Schwartz J. L., Laprade R. Endogenous K+/H+ exchange activity in the Sf9 insect cell line. Biochemistry. 1995 Nov 21;34(46):15157–15164. doi: 10.1021/bi00046a023. [DOI] [PubMed] [Google Scholar]
  32. Vachon V., Paradis M. J., Marsolais M., Schwartz J. L., Laprade R. Ionic permeabilities induced by Bacillus thuringiensis in Sf9 cells. J Membr Biol. 1995 Nov;148(1):57–63. doi: 10.1007/BF00234156. [DOI] [PubMed] [Google Scholar]
  33. Vadlamudi R. K., Ji T. H., Bulla L. A., Jr A specific binding protein from Manduca sexta for the insecticidal toxin of Bacillus thuringiensis subsp. berliner. J Biol Chem. 1993 Jun 15;268(17):12334–12340. [PubMed] [Google Scholar]
  34. Weiss B., Levin R. M. Mechanism for selectively inhibiting the activation of cyclic nucleotide phosphodiesterase and adenylate cyclase by antipsychotic agents. Adv Cyclic Nucleotide Res. 1978;9:285–303. [PubMed] [Google Scholar]
  35. Wu D., Aronson A. I. Localized mutagenesis defines regions of the Bacillus thuringiensis delta-endotoxin involved in toxicity and specificity. J Biol Chem. 1992 Feb 5;267(4):2311–2317. [PubMed] [Google Scholar]

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