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Possible role of cGMP in excitatory amino acid induced cytotoxicity in cultured cerebral cortical neurons

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Abstract

Using cultured cerebral cortical neurons at mature stages (9 days in culture, d.i.c.) it was demonstrated that glutamate, NMDA (N-methyl-D-aspartate) and to a lesser extent KA (kainate) increase the intracellular cGMP concentration ([cGMP]i) whereas no such effect was observed after exposure of the cells of QA (quisqualate) and AMPA (2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate). No effect of glutamate, NMDA and KA was observed in immature neurons (2 d.i.c.). The pharmacology of these cGMP responses was investigated using the glutamate antagonists APV (2-amino-5-phosphonovalerate) with selectivity for NMDA receptors, CNQX (6-cyano-7-nitro-quinoxaline-2,3-dione) with selectivity for non-NMDA receptors and the novel KA selective antagonists AMOA (2-amino-3-[3-(carboxymethoxy)-5-methylisoxazol-4-yl]propionate) and AMNH (2-amino-3-[2-(3-hydroxy-5-methylisoxazol-4-yl)methyl-5-methyl-3-oxoisoxazolin-4-yl]propionate). In addition, the cytotoxicity of glutamate, NMDA and KA was studied and found to be enhanced by addition of the non-metabolizable cGMP analogue 8-Br-cGMP. On the contrary, the toxicity of QA and AMPA was not affected by 8-Br-cGMP. Pertussis toxin augmented the toxicity elicited by glutamate, NMDA, KA and QA but not that induced by AMPA. On the other hand, only glutamate and KA induced toxicity was potentiated by cholera toxin, which also enhanced the stimulatory effect of glutamate and NMDA but not that of KA on the cellular cGMP content. The toxicity as well as the effects on intracellular cGMP levels could be antagonized by the specific excitatory amino acid (EAA) antagonists. These results suggest that the mechanisms by which the various excitatory amino acids exert cytotoxicity are different, and that increased cGMP levels may participate in the mediation of glutamate, NMDA or KA induced toxicity but less likely in QA and AMPA mediated toxicity. Furthermore, G-proteins or other pertussis or cholera toxin sensitive entities seem to be involved in the cytotoxic action of all excitatory amino acids except AMPA.

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References

  1. Rothman, S. M., and Olney, J. W. 1986. Glutamate and the pathophysiology of hypoxic-ischemic brain damage. Ann. Neurol. 19:105–111.

    Google Scholar 

  2. Siesjö, B. K. 1990. Calcium in the brain under physiological and pathological conditions. Eur. Neurol. 30(S2):3–9.

    Google Scholar 

  3. Bouchelouche, P., Belhage, B., Frandsen, Aa., Drejer, J., and Schousboe, A. 1989. Glutamate receptor activation in cultured cerebellar granule cells increased cytosolic free Ca2+ by mobilization of cellular Ca2+ and activation of Ca2+ influx. Exp. Brain Res. 76:281–291.

    Google Scholar 

  4. Frandsen, Aa., and Schousboe, A. 1991. Dantrolene prevents glutamate cytotoxicity and Ca++ release from intracellular stores in cultured cerebral cortical neurons. J. Neurochem. 56:1075–1078.

    Google Scholar 

  5. Biggo, G., Brodie, B. B., Costa, E., and Guidotti, A. 1977. Mechanisms by which diazepam, muscimol, and other drugs change the content of cGMP in cerebellar cortex. Proc. Natl. Acad. Sci. USA 74:3592–3596.

    Google Scholar 

  6. Danysz, W., Wroblewski, J. T., Brooker, G., and Costa, E. 1989. Modulation of glutamate receptors by phencyclidine and glycine in the rat cerebellum: cGMP increase in vivo. Brain Res. 479:270–276.

    Google Scholar 

  7. Wroblewski, J. T., Nicoletti, F., and Costa, E. 1985. Different coupling of excitatory amino acid receptors with Ca++ channels in primary cultures of cerebellar granule cells. Neuropharmacology 24:919–921.

    Google Scholar 

  8. Garthwaite, G., and Garthwaite, J. 1988. Cyclic GMP and cell death in rat cerebellar slices. Neuroscience 26:321–326.

    Google Scholar 

  9. Garthwaite, J., and Garthwaite, G. 1987. Cellular origins of cyclic GMP responses to excitatory amino acid receptor agonists in rat cerebellum in vitro. J. Neurochem. 48:29–39.

    Google Scholar 

  10. Garthwaite, J., Southam, E., and Anderson, M. 1989. A kainate receptor linked to nitric oxide synthesis from arginine. J. Neurochem. 53:1952–1954.

    Google Scholar 

  11. Lolley, R. N., Farber, D. B., Rayborn, M. E., and Hollyfield, J. G. 1977. Cyclic GMP accumulation causes degeneration of photoreceptor cells: simulation of an inherited disease. Science 196:664–666.

    Google Scholar 

  12. Weill, C. L., and Greene, D. P. 1984. Prevention of natural motoneurone cell death by dibutyryl cyclic GMP. Nature 308:452–454.

    Google Scholar 

  13. Bowes, C., Li, T., Danciger, M., Baxter, L. C., Applebury, M. L., and Farber, D. B. 1990. Retinal degeneration in the rd mouse is caused by a defect in the beta subunit of rod cGMP-phosphodiesterase. Nature 347:677–681.

    Google Scholar 

  14. Schousboe, A., and Hertz, L. 1987. Primary cultures of GABAergic and glutamatergic neurons as model systems to study neurotransmitter function. II Developmental aspects. Pages 33–42,in Vernadakis, A., Privat, A., Lauder, J. M., Timiras, P., and Giacobini, E. (eds.), Model Systems of Development and Aging of the Nervous System. Martinus Nijhoff Publishing, New York.

    Google Scholar 

  15. Frandsen, Aa., and Schousboe, A. 1987. Time and concentration dependency of the toxicity of excitatory amino acids on cerebral neurons in primary culture. Neurochem. Int. 10:583–591.

    Google Scholar 

  16. Frandsen, Aa., Drejer, J., and Schousboe, A. 1989. Direct evidence that excitotoxicity in cultured neurons is mediated via N-methyl-D-aspartate (NMDA) as well as non-NMDA receptors. J. Neurochem. 53:297–299.

    Google Scholar 

  17. Frandsen, Aa., and Schousboe, A. 1990. Protection against excitotoxicity in cultured cerebral cortex neurones by inhibition of intracellular Ca++ release. Neurochem. Int. 16 S1:37–37.

    Google Scholar 

  18. Choi, D. W. 1988. Glutamate neurotoxicity and diseases of the nervous system. Neuron 1:623–634.

    Google Scholar 

  19. Larsson, O. M., Drejer, J., Kvamme, E., Svenneby, G., Hertz, L., and Schousboe, A. 1985. Ontogenetic development of glutamate and GABA metabolising enzymes in cultured cerebral cortex interneurons and in cerebral cortex in vivo. Int. J. Devl. Neurosci. 3:177–185.

    Google Scholar 

  20. Schousboe, A., Drejer, J., Hansen, G. H., and Meier, E. 1985. Cultured neurons as model systems for biochemical and pharmacological studies on receptors for neurotransmitter amino acids. Devl. Neurosci. 7:252–262.

    Google Scholar 

  21. Gilman, A. G. 1987. G proteins: Transducers of receptor-generated signals. Ann. Rev. Biochem. 56:615–649.

    Google Scholar 

  22. Sagi-Eisenberg, R. 1989. GTP-Binding proteins as possible targets for protein kinase C action. TIBS 14:355–357.

    Google Scholar 

  23. Martin, T. F. J. 1989. Lipid hydrolysis by phosphoinositidase C: Enzymology and regulation by receptors and guanine nucleotides. Pages 81–112,in Michell R. H., Drummond, A. H., and Downes, C. P. (eds.), Inositol Lipids in Cell Signalling. Academic Press, London.

    Google Scholar 

  24. Birnbaumer, L., Abrahamowitz, J., and Brown, A. M. 1990. Receptor-effector coupling by G proteins. Biochim. Biophys. Acta 1031:163–224.

    Google Scholar 

  25. Frandsen, Aa., and Schousboe, A. 1990. Development of excitatory amino acid induced cytotoxicity in cultured neurons. Int. J. Devl. Neurosci. 8:209–216.

    Google Scholar 

  26. Hertz, E., Yu, A. C. H., Hertz, L., Juurlink, B. H. J., and Schousboe, A. 1989. Preparation of primary cultures of mouse cortical neurons. Pages 183–186in Shahar, A., De Vellis, J., Vernadakis, A., and Haber, B. (eds.), A Dissection and Tissue Culture Manual of the Nervous System. Alan R. Liss, N.Y.

    Google Scholar 

  27. Dichter, M. A. 1978. Rat cortical neurons in cell culture: Culture methods, cell morphology, electrophysiology, and synapse formation. Brain Res. 149:279–293.

    Google Scholar 

  28. Drejer, J., Honoré, T., and Schousboe, A. 1987. Excitatory amino acid-induced release of3H-GABA from cultured mouse cerebral cortex interneurons. J. Neurosci. 7:2910–2916.

    Google Scholar 

  29. Scott, R. H., and Dolphin, A. C. 1988. The agonist effect of Bay K 8644 on neuronal calcium channel currents is promoted by G-protein activation. Neurosci. Lett. 89:170–175.

    Google Scholar 

  30. Lowry, O. H., Roseborough, N. J., Farr, A. L., and Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275.

    Google Scholar 

  31. Drejer, J., Larsson, O. M., and Schousboe, A. 1982. Characterization of L-glutamate uptake into and release from astrocytes and neurons cultured from different brain regions. Exp. Brain Res. 47:259–269.

    Google Scholar 

  32. Wahl, P., Honoré, T., Drejer, J., and Schousboe, A. 1991. Development of binding sites for excitatory amino acids in cultured cerebral cortex neurons. Int. J. Devl. Neurosci. 9:287–296.

    Google Scholar 

  33. Novelli, A., Nicoletti, F., Wroblewski, J. T., Alho, H., Costa, E., and Guidotti, A. 1987. Excitatory amino acid receptors coupled with guanylate cyclase in primary cultures of cerebellar granule cells. J. Neurosci. 7:40–47.

    Google Scholar 

  34. Davis, J., Francis, A. A., Jones, A. W., and Watkins, J. C. 1981. 2-Amino-5-phosphonovalerate (2APV), a potent and selective antagonist of amino acid-induced and synaptic transmission. Neurosci. Lett. 21:77–81.

    Google Scholar 

  35. Honoré, T., Davies, S. N., Drejer, J., Fletcher, E. J., Jacobsen, P., Lodge, D., and Nielsen, F. 1988. Quinoxalinediones: potent non-NMDA glutamate receptor antagonists. Science 241:701–703.

    Google Scholar 

  36. Frandsen, Aa., Krogsgaard-Larsen, P., and Schousboe, A. 1990. Novel glutamate antagonists selectively protect against kainic acid neurotoxicity in cultured cerebral cortex neurons. J. Neurochem. 55:1821–1823.

    Google Scholar 

  37. Krogsgaard-Larsen, P., Ferkany, J. W., Nielsen, E. Ø., Madsen, U., Ebert, B., Johansen, J. S., Diemer, N. H., Bruhn, T., Beattie, D., and Curtis, D. R. 1991. Novel class of amino acid antagonists at non-N-methyl-D-aspartic acid excitatory amino acid receptors. Synthesis, in vitro and in vivo pharmacology and neuroprotection. J. Med. Chem. 34:123–130.

    Google Scholar 

  38. Bunn, S. J., Garthwaite, J., and Wilkin, G. P. 1986. Guanylate cyclase activities in enriched preparations of neurones, astroglia and a synaptic complex isolated from rat cerebellum. Neurochem. Int. 8:179–185.

    Google Scholar 

  39. Milani, D., Facci, L., Guidolin, D., Leon, A., and Skaper, S. D. 1989. Activation of polyphosphoinositide metabolism as a signal transducing system coupled to excitatory amino acid receptors in astroglia cells. Glia 2:161–169.

    Google Scholar 

  40. McDonald, J. W., and Johnston, M. V. 1990. Physiological and pathophysiological roles of excitatory amino acids during central nervous system development. Brain Res. Rev. 15:41–70.

    Google Scholar 

  41. Novelli, A., and Henneberry, R. C. 1987. cGMP synthesis in cultured cerebellar neurons is stimulated by glutamate via a Ca2+-mediated, differentiation-dependent mechanism. Dev. Brain Res. 34:307–310.

    Google Scholar 

  42. Balázs, R., Hack, N., and Jørgensen, O. S. 1988. Stimulation of the N-methyl-D-aspartate receptor has a trophic effect on differentiating cerebellar granule cells. Neurosci. Lett. 87:80–86.

    Google Scholar 

  43. Pearce, I. A., Cambray-Deakin, M. A., and Burgoyne, R. D. 1987. Glutamate acting on NMDA receptors stimulates neurite outgrowth from cerebellar granule cells. FEBS Lett. 223:143–147.

    Google Scholar 

  44. Garthwaite, J., Charles, S. L., and Chess-Williams, R. 1988. Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as an intracellular messenger in the brain. Nature 336:385–388.

    Google Scholar 

  45. Miki, N., Kawabe, Y., and Kuriyami, K. 1977. Activation of cerebral guanylate cyclase by nitric oxide. Biochem. Biophys. Res. Commun. 75:851–856.

    Google Scholar 

  46. Palmer, R. M. J., Ferrige, A. G., and Moncada, S. 1987. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327:524–526.

    Google Scholar 

  47. Waldman, S. A., and Murad, F. 1987. Cyclic GMP synthesis and function. Pharmacol. Rev. 39:163–198.

    Google Scholar 

  48. Burch, R. M., Luini, A., and Axelrod, J. 1986. Phospholipase A2 and phospholipase C are activated by distinct GTP-binding proteins in response to alpha 1-adrenergic stimulation in FRTL5 thyroid cells. Proc. Natl. Acad. Sci. 83:7201–7205.

    Google Scholar 

  49. Kikuchi, A., Kozawa, O., Kaibuchi, K., Katada, T., Ui, M., and Takai, Y. 1986. Direct evidence for involvement of a guanine nucleotide-binding protein in chemotactic peptide-stimulated formation of inositol biphosphate and triphosphate in differentiated human leukemic (HL-60) cells. J. Biol. Chem. 261:11558–11562.

    Google Scholar 

  50. Boyer, J. L., Hepler, J. R., and Harden, T. K. 1989. Hormone and growth factor receptor-mediated regulation of phospholipase C activity. TIPS 10:360–364.

    Google Scholar 

  51. Cockcroft, S., and Comperts, B. D. 1985. Role of guanine nucleotide binding protein in the activation of polyphosphoinositide phosphodiesterase. Nature 314:534–536.

    Google Scholar 

  52. Kurachi, Y., Nakajima, T., and Sugimoto, T. 1986. On the mechanism of activation of muscarinic K+ channels by adenosine in isolated atrial cells: involvement of GTP-binding proteins. Pflugers Arch. 407:264–274.

    Google Scholar 

  53. Frandsen, Aa., Drejer, J., and Schousboe, A. 1989. Glutamate-induced45Ca+ uptake into immature cerebral cortex neurons shows a distinct pharmacological profile. J. Neurochem. 53:1959–1962.

    Google Scholar 

  54. Drejer, J., Honoré, T., Meier, E., and Schousboe, A. 1986. Pharmacologically distinct glutamate receptors on cerebellar granule cells. Life Sci. 38:2077–2085.

    Google Scholar 

  55. Balcar, V. J., Johnston, G. A. R., and Stephanson, A. L. 1976. Transport of L-proline by rat brain slices. Brain Res. 102:143–151.

    Google Scholar 

  56. Koh, J. Y., and Choi, D. W. 1987. Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay. J. Neurosci. Meth. 20:83–90.

    Google Scholar 

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  1. PharmaBiotec Research Center, The Neurobiology Unit, Department of Biological Sciences, The Royal Danish School of Pharmacy, Universitetsparken 2, DK 2100, Copenhagen, Denmark

    Aase Frandsen, Charlotte F. Andersen & Arne Schousboe

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  1. Aase Frandsen
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  2. Charlotte F. Andersen
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Frandsen, A., Andersen, C.F. & Schousboe, A. Possible role of cGMP in excitatory amino acid induced cytotoxicity in cultured cerebral cortical neurons. Neurochem Res 17, 35–43 (1992). https://doi.org/10.1007/BF00966863

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