Advertisement

Cellular and Molecular Neurobiology

, Volume 26, Issue 7–8, pp 1353–1363 | Cite as

Distribution of Secretory Pathway Ca 2+ ATPase (SPCA1) in Neuronal and Glial Cell Cultures

  • Radovan Murín
  • Stephan Verleysdonk
  • Luc Raeymaekers
  • Peter Kaplán
  • Ján Lehotský
Article

1. Secretory pathway Ca2+ ATPase type 1 (SPCA1) is a newly recognized Ca2+/Mn2+-transporting pump localized in membranes of the Golgi apparatus.

2. The expression level of SPCA1 in brain tissue is relatively high in comparison with other tissues.

3. With the aim to determine the expression of SPCA1 within the different types of neural cells, we investigated the distribution of SPCA1 in neuronal, astroglial, oligodendroglial, ependymal, and microglial cell cultures derived from rat brains.

4. Western Blot analysis with rabbit anti-SPCA1 antibodies revealed the presence of SPCA1 in homogenates derived from neuronal, astroglial, ependymal, and oligodendroglial, but not from microglial cells.

5. Cell cultures that gave rise to positive signal in the immunoblot analysis were also examined immunocytochemically.

6. Immunocytochemical double-labeling experiments with anti-SPCA1 serum in combination with antibodies against cell-type specific proteins showed a localization of the SPCA1signal within cells stained positively also for GFAP, α-tubulin or MBP.

7. These results definitely established the expression of SPCA1 in astroglial, ependymal, and oligodendroglial cells.

8. In addition, the evaluation of neuronal cultures for the presence of SPCA1 revealed an SPCA1-specific immunofluorescence signal in cells identified as neurons.

KEY WORDS:

Golgi apparatus secretory pathway Ca2+ ATPase SPCA1 neuron glial cell immunocytochemistry 

Notes

ACKNOWLEDGMENTS

The authors would like to thank Barbara Birk for her expert technical help in the preparation of the ependymal cell cultures. Part of this work was presented as an abstract on the 5th International Symposium on Experimental and Clinical Neurobiology, Tatranska Lomnica-Stara Lesna, Slovak Republic, September 2005. This study was supported by research grants: VEGA No. 1/0034/03, VEGA No. 3380/06, APVT No. 51-127404, and MVTS 39.

REFERENCES

  1. Calegari, F., Coco, S., Taverna, E., Bassetti, M., Verderio, C., Corradi, N., Matteoli, M., and Rosa, P. A. (1999). Regulated secretory pathway in cultured hippocampal astrocytes. J. Biol. Chem. 274:22539–22547.CrossRefPubMedGoogle Scholar
  2. Canaff, L., Brechler, V., Reudelhuber, T. L., and Thibault, G. (1996). Secretory granule targeting of atrial natriuretic peptide correlates with its calcium-mediated aggregation. Proc. Natl. Acad. Sci. U.S.A. 93:9483–9487.CrossRefPubMedGoogle Scholar
  3. Carnell, L., and Moore, H. P. (1994). Transport via the regulated secretory pathway in semi-intact PC12 cells: Role of intra-cisternal calcium and pH in the transport and sorting of secretogranin II. J. Cell Biol. 127:693–705.CrossRefPubMedGoogle Scholar
  4. Chandra, S., Kable, E. P., Morrison, G. H., and Webb, W. W. (1991). Calcium sequestration in the Golgi apparatus of cultured mammalian cells revealed by laser scanning confocal microscopy and ion microscopy. J. Cell Sci. 100:747–752.PubMedGoogle Scholar
  5. Chandra, S., Fewtrell, C., Millard, P. J., Sandison, D. R., Webb, W. W., and Morrison, G. H. (1994). Imaging of total intracellular calcium and calcium influx and efflux in individual resting and stimulated tumor mast cells using ion microscopy. J. Biol. Chem. 269:15186–15194.PubMedGoogle Scholar
  6. Evanko, D. S., Zhang, Q., Zorec, R., and Haydon, P. G. (2004). Defining pathways of loss and secretion of chemical messengers from astrocytes. Glia 47:233–240.CrossRefPubMedGoogle Scholar
  7. Giulian, D., and Baker, T. J. (1986). Characterization of ameboid microglia isolated from developing mammalian brain. J. Neurosci. 6:2163–2178.PubMedGoogle Scholar
  8. Hamprecht, B., and Loffler, F. (1985). Primary glial cultures as a model for studying hormone action. Methods Enzymol. 109:341–345.PubMedCrossRefGoogle Scholar
  9. Hirrlinger, J., Gutterer, J. M., Kussmaul, L., Hamprecht, B., and Dringen, R. (2000). Microglial cells in culture express a prominent glutathione system for the defense against reactive oxygen species. Dev. Neurosci. 22:384–392.CrossRefPubMedGoogle Scholar
  10. Hirrlinger, J., Resch, A., Gutterer, J. M., and Dringen, R. (2002). Oligodendroglial cells in culture effectively dispose of exogenous hydrogenperoxide: Comparison with cultured neurones, astroglial and microglial cells. J. Neurochem. 82:635–644.CrossRefPubMedGoogle Scholar
  11. Hu, Z., Bonifas, J. M., Beech, J., Bench, G., Shigihara, T., Ogawa, H., Ikeda, S., Mauro, T., and Epstein, E. H. Jr. (2000). Mutations in ATP2C1, encoding a calcium pump, cause Hailey-Hailey disease. Nat. Genet. 24:61–65.CrossRefPubMedGoogle Scholar
  12. Löffler, F., Lohmann, S. M., Walckhoff, B., Walter, U., and Hamprecht, B. (1986). Immunocytochemical characterization of neuron-rich primary cultures of embryonic rat brain cells by established neuronal and glial markers and by monospecific antisera against cyclic nucleotide-dependent protein kinases and the synaptic vesicle protein synapsin I. Brain Res. 363:205–221.CrossRefGoogle Scholar
  13. Michelangeli, F., Ogunbayo, O. A., and Wootton, L. L. (2005). A plethora of interacting organellar Ca2+ stores. Curr. Opin. Cell. Biol. 17:135–140.CrossRefPubMedGoogle Scholar
  14. Pezzati, R., Bossi, M., Podini, P., Meldolesi, J., and Grohovaz, F. (1997). High-resolution calcium mapping of the endoplasmic reticulum-Golgi-exocytic membrane system. Electron energy loss imaging analysis of quick frozen-freeze dried PC12 cells. Mol. Biol. Cell. 8:1501–1512.PubMedGoogle Scholar
  15. Pinton, P., Pozzan, T., and Rizzuto, R. (1998). The Golgi apparatus is an inositol 1,4,5-trisphosphate-sensitive Ca2+ store, with functional properties distinct from those of the endoplasmic reticulum. EMBO J. 17:5298–5308.CrossRefPubMedGoogle Scholar
  16. Prothmann, C., Wellard, J., Berger, J., Hamprecht, B., and Verleysdonk, S. (2001). Primary cultures as a model for studying ependymal functions: Glycogen metabolism in ependymal cells. Brain Res. 920:74–83.CrossRefPubMedGoogle Scholar
  17. Sudbrak, R., Brown, J., Dobson-Stone, C., Carter, S., Ramser, J., White, J., Healy, E., Dissanayake, M., Larregue, M., Perrussel, M., Lehrach, H., Munro, C. S., Strachan, T., Burge, S., Hovnanian, A., and Monaco, A. P. (2000). Hailey-Hailey disease is caused by mutations in ATP2C1 encoding a novel Ca(2+) pump. Hum. Mol. Genet. 9:1131–1140.CrossRefPubMedGoogle Scholar
  18. Surroca, A., and Wolff, D. (2000). Inositol 1,4,5-trisphosphate but not ryanodine-receptor agonists indu-ces calcium release from rat liver Golgi apparatus membrane vesicles. J. Membr. Biol. 177:243–249.CrossRefPubMedGoogle Scholar
  19. Sytnyk, V., Leshchyns’ka, I., Dityatev, A., and Schachner, M. (2004). Trans-Golgi network delivery of synaptic proteins in synaptogenesis. J. Cell. Sci. 117:381–388.CrossRefPubMedGoogle Scholar
  20. Ton, V. K., Mandal, D., Vahadji, C., and Rao, R. (2002). Functional expression in yeast of the human secretory pathway Ca(2+), Mn(2+)-ATPase defective in Hailey-Hailey disease. J. Biol. Chem. 277:6422–6427.CrossRefPubMedGoogle Scholar
  21. Van Baelen, K., Vanoevelen, J., Callewaert, G., Parys, J. B., De Smedt, H., Raeymaekers, L., Rizzuto, R., Missiaen, L., Wuytack, F. (2001). The contribution of the SPCA1 Ca2+ pump to the Ca2+ accumulation in the Golgi apparatus of HeLa cells assessed via RNA-mediated interference. Biochem. Biophys. Res. Commun. 306:430–436.Google Scholar
  22. Van Baelen, K., Dode, L., Vanoevelen, J., Callewaert, G., De Smedt, H., Missiaen, L., Parys, J. B., Raeymaekers, L., and Wuytack, F. (2004). The Ca2+/Mn2+ pumps in the Golgi apparatus. Biochim. Biophys. Acta 1742:103–112.CrossRefPubMedGoogle Scholar
  23. Vanoevelen, J., Dode, L., Van Baelen, K., Fairclough, R. J., Missiaen, L., Raeymaekers, L., and Wuytack, F. (2005). The secretory pathway Ca2+/Mn2+-ATPase 2 is a Golgi-localized pump with high affinity for Ca2+ ions. J. Biol. Chem. 280:22800–22808.CrossRefPubMedGoogle Scholar
  24. Volterra, A., and Meldolesi, J. (2005). Astrocytes, from brain glue to communication elements: The revolution continues. Nat. Rev. Neurosci. 8:626–640.CrossRefGoogle Scholar
  25. Wootton, L. L., Argent, C. C. A., Wheatley, M., and Michelangeli, F. (2004). The expression, activity and localisation of the secretory pathway Ca2+-ATPase (SPCA1) in different mammalian tissues. Biochim. Biophys. Acta 1664:189–197.CrossRefPubMedGoogle Scholar
  26. Wuytack, F., Raeymaekers, L., and Missiaen, L. (2002). Molecular physiology of the SERCA and SPCA pumps. Cell Calcium 32:279–305.CrossRefPubMedGoogle Scholar
  27. Xiang, M., Mohamalawari, D., and Rao, R. (2005). A novel isoform of the secretory pathway Ca2+, Mn(2+)-ATPase, hSPCA2, has unusual properties and is expressed in the brain. J. Biol. Chem. 280:11608–11614.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • Radovan Murín
    • 1
    • 2
  • Stephan Verleysdonk
    • 1
  • Luc Raeymaekers
    • 3
  • Peter Kaplán
    • 2
  • Ján Lehotský
    • 2
    • 4
  1. 1.Interfaculty Institute of BiochemistryUniversity of TuebingenTuebingenGermany
  2. 2.Department of Medical BiochemistryJessenius Faculty of Medicine, Comenius UniversityMartinSlovakia
  3. 3.Department of PhysiologyCatholic University Leuven, GasthuisbergLeuvenBelgium
  4. 4.Department of Medical BiochemistryJessenius Faculty of Medicine, Comenius UniversityMartinSlovakia

Personalised recommendations