Skip to main content
SpringerLink
Log in

Studies on the Cytosolic Phospholipase A2 in Immortalized Astrocytes (DITNC) Revealed New Properties of the Calcium Ionophore, A23187

  • Published:
Neurochemical Research Aims and scope Submit manuscript

Abstract

Besides playing an important role in the maintenance of cell membrane phospholipids, phospholipases A2 (PLA2) are responsible for the release of arachidonic acid (AA) which is a precursor for prostaglandin biosynthesis. The cytosolic PLA2 has been the focus of recent studies, probably due to its ability to respond to protein kinases and changes in intracellular calcium levels. In this study, we examined agents for stimulation of the cytosolic phospholipase A2 in immortalized astrocytes (DITNC). Incubation of DITNC cells with [14C]arachidonic acid (AA) resulted in a time-dependent uptake of the label into phospholipids (PL) and neutral glycerides. In prelabeled cells, release of labeled AA could be stimulated by calcium mobilizing agents such as calcium ionophore A23187 (4–20 μM) and thimerosal (100 μM), and by phorbol myristate acetate (PMA, 100 nM), an agent for activation of protein kinase C. The release of AA could also be stimulated by ATP (200 μM), probably through activation of the purinergic receptor but not by glutamate (1 mM). The stimulated release of AA was dependent on extracellular Ca2+ and was inhibited by mepacrine (50 μM), a non-specific PLA2 inhibitor. Western blot analysis further confirmed the presence of an 85 kDa cPLA2 in both membrane and cytosol fractions of these cells and stimulation by A23187 resulted in translocation of this protein to the membrane fraction. Besides labeled fatty acids, A23187 also stimulated the concomitant release of labeled PL into the culture medium and this event was accompanied by the increased release in lactate dehydrogenase (LDH). Results thus revealed that besides activation of cPLA2, the calcium ionophore A23187 is capable of perturbating cell membrane integrity.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

REFERENCES

  1. Azmi, S., Dhawan, D., and Singh, N. 1996. Calcium ionophore A23187 induces apoptotic cell death in rat thymocytes. Cancer Letters, 107:97–103.

    Google Scholar 

  2. Badiani, K., Lu, X., and Arthur, G. 1993. Effect of delta 9-tetrahydrocannabinol and merthiolate on acyltransferase activities in guinea pig liver microsomes. Lipids, 28:299–303.

    Google Scholar 

  3. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein dye binding. Anal. Biochem., 72:248–254.

    Google Scholar 

  4. Bruner, G., and Murphy, S. 1990. ATP-evoked arachidonic acid mobilization in astrocytes is via a P2Y-purinergic receptor. J. Neurochem., 55:1569–1575.

    Google Scholar 

  5. Channon, J. Y., and Leslie, C. C. 1990. A calcium-dependent mechanism for associating a soluble arachidonoyl-hydrolyzing phospholipase A2 with membrane in the macrophage cell line RAW 264.7. J. Biol. Chem., 265:5409–5413.

    Google Scholar 

  6. Chen, C. C., and Chen, W. C. 1996. ATP-evoked inositol phosphates formation through activation of P2U purinergic receptors in cultured astrocytes: Regulation by PKC subtypes α, δ, and ϑ. Glia. 17:63–71.

    Google Scholar 

  7. Pearce, G., Murphy, S., Jeremy, J., Morrow, C., and Dandona, P. (1989) ATP-evoked calcium mobilization and prostanoid release from astrocytes: P2-purinergic receptors linked to phosphoinositide hydrolysis. J. Neurochem., 52:971–977.

    Google Scholar 

  8. Clark, J. D., Lin, L., Kriz, R. W., Ramesha, C. S., Sultzman, L. A., Lin, A. Y., Milona, N., and Knopt, J. L. 1991. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca2+-dependent translocation domain with homology to PKC and GAP. Cell, 65:1043–1051.

    Google Scholar 

  9. Clark, J. D., Schievella, A. R., Nalefski, E. A., and Lin, L. L. 1995. Cytosolic phospholipase A2. J. Lipid Mediators Cell Signalling, 12:83–117.

    Google Scholar 

  10. Clemens, J. A., Stephenson, D. T., Smalstig, E. B., Roberts, E. F., Johnstone, E. M., Sharp, J. D., Little, S. P., and Kramer, R. M. 1996. Reactive glia express cytosolic phospholipase A2 after transient global forebrain ischemia in the rat. Stroke, 27:527–535.

    Google Scholar 

  11. Diez, E., Chilton, F. H., Stroup, G., Mayer, R. J., Winkler, J. D., and Fonteh, A. N. 1994. Fatty acid and phospholipid selectivity of different phospholipase A2 enzymes studied by using a mammalian membrane as substrate. Biochem. J., 301:721–726.

    Google Scholar 

  12. Farooqui, A. A., Yang, H. C., and Horrocks, L. A. 1997. Involvement of phospholipase A2 in neurodegeneration. Neurochem. Int., 30:517–522.

    Google Scholar 

  13. Joseph, R., Li, W., Han, E. 1993. Neuronal death, cytoplasmic calcium and internucleosomal DNA fragmentation: evidence for DNA fragments being released from cells. Mol. Brain Res., 17:70–76.

    Google Scholar 

  14. Kramer, R. M., Stephenson, D. T., Roberts, E. F., and Clemens, J. A. 1996. Cytosolic phospholipase A2 (cPLA2) and lipid mediator release in the brain. J. Lipid Mediators & Cell Signalling, 14:3–7.

    Google Scholar 

  15. LaDu, M. J., Gilligan, S. M., Lukens, J. R., Cabana, V. G., Reardon, C. A., Van Eldik, L. J., and Holtzman, D. M. 1998. Nascent astrocyte particles differ from lipoproteins in CSF. J. Neurochem., 70:2070–2081.

    Google Scholar 

  16. Matsubara, K., Kubota, M., Adachi, S., Kuwakado, K., Hirota, H., Wakazono, Y., Akiyama, Y., and Mikawa, H. 1994. Different mode of death induced by calcium ionophore in human leukemia cell lines: possible role of constitutive endonuclease. Experimental Cell Res., 210:19–25.

    Google Scholar 

  17. Molloy, G. Y., Rattray, M., Williams, R. J. 1998. Genes encoding multiple forms of phospholipase A2 are expressed in rat brain. Neurosci. Letters, 258: 139–142.

    Google Scholar 

  18. Moore, S. A., Yoder, E., Murphy, S., Dutton, G. R., and Spector A. A. 1991. Astrocytes, not neurons, produce docosahexaenoic acid (22:6W-3) and arachidonic acid (20:4W-6). J. Neurochem., 56:518–524.

    Google Scholar 

  19. Oka, S., and Arita, H. 1991. Inflammatory factors stimulate expression of group II PLA2 in rat cultured astrocytes. J. Biol., Chem. 266:9956–9960.

    Google Scholar 

  20. Pelassy, C., Brettmayer, J. P., Ticchioni, M., and Aussel, C. 1994. Effect of thimerosal on cytosolic calcium and phosphatidylserine synthesis in Jurkat T cells. Int. J. Biochem., 26:93–96.

    Google Scholar 

  21. Pintado, E., Baquero-leonis, D., Conde, M., and Sobrino, F. 1994. Effect of thimerosal and other sulfhydryl reagents on calcium permeability in thymus lymphocytes. Biochem. Pharmacol., 49:227–232.

    Google Scholar 

  22. Radany, E. H., Brenner, M., Besnard, F., Bigornia, V., Bishop, J. M., and Deschepper, C. F. 1992. Directed establishment of rat brain cell lines with the phenotypic characteristics of type 1 astrocytes. Proc. Natl. Acad. Sci. USA, 89:6467–6471.

    Google Scholar 

  23. Shao, Y., and McCarthy, K. D. 1994. Plasticity of astrocytes. GLIA. 11:147–155.

    Google Scholar 

  24. Shaposhnikova, V. V., Dobrovinskaya, O. R., Eidus, L. K., and Korystov, Y. N. 1994. Dependence of thymocyte apoptosis on protein kinase C and phospholipase A2. FEBS Letters 348:317–319.

    Google Scholar 

  25. Stephenson, D. T., Manetta, J. V., White, D. L., Chiou, X. G., Cox, L., Gitter, B., May, P. C., Sharp, J. D., Kramer, R. M., and Clemens, J. A. 1994. Calcium-sensitive cytosolic phospholipase A2 (cPLA2) is expressed in human astrocytes. Brain Res., 637:97–105.

    Google Scholar 

  26. Sun, G. Y. 1988. Preparation and analysis of acyl and alkenyl groups of glycerophospholipids from brain subcellular membranes. In: Neuromethods: Lipids and Related Compounds, (AA Boulton, R Baker and LA Horrocks, eds) Vol. 7, Humana Press, New Jersey, pp. 63–82.

    Google Scholar 

  27. Sun, G. Y., Aradottir, S., Gustavsson, L., and Alling, C. 1989. Ethanol alters the transfer of arachidonic acid to ethanolamine plasmalogens in C-6 glioma cells. J. Neurosci. Res., 24:268–275.

    Google Scholar 

  28. Takadera, T., and Ohyashiki, T. 1997. Apoptotic cell death and caspase 3 (CPP32) activation induced by calcium ionophore at low concentrations and their prevention by nerve growth factor in PC12 cells. European J. Biochem., 249:8–12.

    Google Scholar 

  29. Tong, W., Hu, Z. Y., and Sun, G. Y. (1995) Stimulation of group II phospholipase A2 mRNA expression and release in an immortalized astrocyte cell line (DI TNC) by LPS, TNFα, and IL-1β: interactive effects. Mol. Chem. Neuropath., 25:1–17.

    Google Scholar 

  30. Ui-Tei, K., Sato, S., Miyake, T., and Miyata, Y. 1996. Induction of apoptosis in a Drosophila neuronal cell line by calcium ionophore. Neuroscience Letters, 203:191–194.

    Google Scholar 

  31. Zanassi, P., Paolillo, M., and Schinelli, S. 1998. Coexpression of phospholipase A2 isoforms in rat striatal astrocytes. Neurosci. Letters, 247:83–86.

    Google Scholar 

  32. Zhang, J., Driscoll, T. A., Hannun, Y. A., and Obeid, L. M. 1998. Regulation of membrane release in apoptosis. Biochem. J., 334:479–485.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Biochemistry Department and Nutritional Sciences Program, University of Missouri, Columbia, MO, 65212

    Di Xue, Jianfeng Xu, Susan O. McGuire, David Devitre & Grace Y. Sun

Authors
  1. Di Xue
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jianfeng Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Susan O. McGuire
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David Devitre
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Grace Y. Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Grace Y. Sun.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Xue, D., Xu, J., McGuire, S.O. et al. Studies on the Cytosolic Phospholipase A2 in Immortalized Astrocytes (DITNC) Revealed New Properties of the Calcium Ionophore, A23187. Neurochem Res 24, 1285–1291 (1999). https://doi.org/10.1023/A:1020981224876

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1020981224876

Search

Navigation