Neurochemical Research

, Volume 14, Issue 7, pp 677–682 | Cite as

Lack of excitatory amino acid-induced effects on calcium fluxes measured with45Ca2+ in rat cerebral cortex synaptosomes

  • Michele Simonato
  • Richard S. Jope
  • Clementina Bianchi
  • Lorenzo Beani
Original Articles


Ca2+ uptake was measured in purified rat cerebral cortex synaptosomes (P3 pellets) using45Ca2+ as a tracer. Ca2+ influx increased in time, and with an increase in external K+ concentration and temperature. The net (external K+-induced, depolarization-dependent) uptake follows a two-component course. The exponential term, due to the opening of voltage-operated calcium channels (VOC), has a rate constant which increases with an increase in the depolarization level (1.04 versus 0.54 nmol/s/mg protein for 50 mM—versus 15 mM [K+]-dependent net influx). The linear term, due to the Na+/Ca2+ exchange system, has a similar rate constant at all depolarization levels (0.16+/−0.05 and 0.11+/−0.02 nmol/s/mg protein). Excitatory amino acids (glutamate, kainate and n-methyl-d-aspartate-NMDA-) were tested on this preparation at doses ranging between 5×10−5 M and 5×10−3M and at multiple incubation times, under resting conditions and under two depolarizing conditions (partial depolarization: 15 mM external K+ and maximal depolarization: 50 mM external K+). NMDA was also tested in the absence of Mg2+. No effect was detectable under any of these experimental conditions. Hypotheses to interpret these data are discussed. Further studies on other preparations are needed in order to directly investigate the presynaptic effects of excitatory amino acids.

Key words

Synaptosomes calcium fluxes glutamate kainate NMDA 


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  1. 1.
    Fagg, G. E., Foster, A. C., and Ganong A. H. 1986. Excitatory amino acid synaptic mechanisms and neurological function. Trends Pharmacol. Sci. 7:357–363.Google Scholar
  2. 2.
    Dingledine, R. 1986. NMDA receptors: what do they do? Trends Neurosci. 9:47–49.Google Scholar
  3. 3.
    Collingridge, G. L., and Bliss, T. V. P. 1987. NMDA receptorstheir role in long-term potentiation. Trends Neurosci. 10:278–293.Google Scholar
  4. 4.
    Rothman, S. M., and Olney, J. W. 1987. Excitotoxicity and the NMDA receptor. Trends Neurosci. 10:299–302.Google Scholar
  5. 5.
    Cull-Candy, S. G., and Usowicz, M. M. 1987. Multiple-conductance channels activated by excitatory amino acids in cerebellar neurons. Nature 325:525–527.Google Scholar
  6. 6.
    Jahr, C. E., and Stevens, C. F. Glutamate activates multiple single channel conductances in hippocampal neurons. Nature 325:522–527.Google Scholar
  7. 7.
    Cull-Candy, S. G., and Usowicz, M. M. 1987. Patch clamp recording from single glutamate-receptor channels. Trends Pharmacol. Sci. 8:218–224.Google Scholar
  8. 8.
    Sladeczek, F., Recasens, M. and Bockaert, J. 1988. A new mechanism for glutamate receptor action: phosphoinositide hydrolysis. Trends Neurosci. 12:545–549.Google Scholar
  9. 9.
    Foster, A. C., Mena, E. E., Fagg, G. E., and Cotman, C. W. 1981. Glutamate and aspartate binding sites are enriched in synaptic junctions isolated from rat brain. J. Neurosci. 1:620–625.Google Scholar
  10. 10.
    Migani, P., Virgili, M., Contestabile, A., Poli, A., Villani, L., and Bernabei, O. 1985. [3H]Kainic acid binding sites in the synaptosomal-mitochondrial (P2) fraction from goldfish brain. Brain Res. 361:36–45.Google Scholar
  11. 11.
    Miwa, A., and Kawai, N. 1986. Presynaptic glutamate receptorpossible involvement of a K+ channel. Brain Res. 385:161–164.Google Scholar
  12. 12.
    Miller, R. J. 1987. Multiple calcium channels and neuronal function. Science 235:46–52.Google Scholar
  13. 13.
    Augustine, G. J., Charlton, M. P., and Smith, S. J. 1987. Calcium action in synaptic transmitter release. Ann. Rev. Neurosci. 10:633–693.Google Scholar
  14. 14.
    Tsien, R. W., Lipscombe, D., Madison, D. V., Bley, K. R., and Fox, A. P. 1988. Multiple types of neuronal calcium channels and their selective modulation. Trends Neurosci. 11:431–438.Google Scholar
  15. 15.
    Ashley, R. H., Brammer, M. J., and Marchbanks, R. 1984. Measurement of intrasynaptosomal free calcium by using the fluorescent indicator quin-2. Biochem. J. 219:149–158.Google Scholar
  16. 16.
    Blaustein, M. P. 1975. Effects of potassium, veratridine and scorpion venom on calcium accumulation and transmitter release by nerve terminals in vitro. J. Physiol. 247:617–655.Google Scholar
  17. 17.
    Nachshen, D. A., and Blaustein, M. P. 1980. Some properties of potassium-stimulated calcium influx in presynaptic nerve endings. J. Gen. Physiol. 76:709–727.Google Scholar
  18. 18.
    Nachshen, D. A., and Blaustein, M. P. 1980. Influx of calcium, strontium, and barium in presynaptic nerve endings. J. Gen. Physiol. 79:1065–1087.Google Scholar
  19. 19.
    Turner, T. J., and Goldin, S. M. 1985. Calcium channels in rat brain synaptosomes: identification and pharmacological characterization. J. Neurosci. 5:841–849.Google Scholar
  20. 20.
    Suszkiw, J. B., O'Leary, M. E., Murawsky, M. M., and Wang, T. 1986. Presynaptic calcium channels in rat cortical synaptosomes: fast-kinetics of phasic calcium influx, channel inactivation, and relationship to nitrendipine receptors. J. Neurosci. 6:1349–1357.Google Scholar
  21. 21.
    Harris, R. A. 1985. Effects of excitatory amino acids on calcium transport by brain membranes. Brain Res. 337:167–170.Google Scholar
  22. 22.
    Lazarewicz, J. W., Lehmann, A., Hagberg, H., and Hamberger, A. 1986. Effects of kainic acid on brain calcium fluxes studied in vivo and in vitro. J. Neurochem. 46:494–498.Google Scholar
  23. 23.
    Booth, R. F. G., and Clark, J. B. 1978. A rapid method for the preparation of relatively pure, metabolically competent synaptosomes from rat brain. Biochem. J. 176:365–370.Google Scholar
  24. 24.
    Koenig, M. L., and Jope, R. S. 1987. Aluminum inhibits the fast phase of voltage-dependent calcium influx into synaptosomes. J. Neurochem. 49:316–320.Google Scholar
  25. 25.
    Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, A. J. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:259–265.Google Scholar
  26. 26.
    Cotman, C. W., Monaghan, D. T., Ottersen, O. P., and Storm-Mathisen, J. 1987. Anatomical organization of excitatory amino acid receptors and their pathways. Trend Neutosci. 10:263–270.Google Scholar
  27. 27.
    O'Shaughnessy, C. T., and Lodge, D. 1988. N-methyl-D-aspartate receptor-mediated increase in intracellular calcium is reduced by ketamine and phencyclidine. Eur. J. Pharmacol. 153:201–209.Google Scholar
  28. 28.
    Nicoletti, F. 1988. Receptor-operated calcium channels activated by excitatory amino acids. Neurosci. Lett. Suppl. 33:5136.Google Scholar

Copyright information

© Plenum Publishing Corporation 1989

Authors and Affiliations

  • Michele Simonato
    • 1
  • Richard S. Jope
    • 2
  • Clementina Bianchi
    • 1
  • Lorenzo Beani
    • 1
  1. 1.Institute of PharmacologyUniversity of FerraraFerraraItaly
  2. 2.Department of Pharmacology and Neuropsychiatry Research ProgramUniversity of Alabama at BirminghamBirminghamUSA

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