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Pharmacopsychiatry 2003; 36: 78-83
DOI: 10.1055/s-2003-40453
Original Paper
© Georg Thieme Verlag Stuttgart · New York

Excitotoxic Hippocampal Membrane Breakdown and its Inhibition by Bilobalide: Role of Chloride Fluxes

J. Klein1 , 2 , O. Weichel1 , M. Hilgert1 , J. Rupp1 , S. S. Chatterjee3 , H. Nawrath1
  • 1Department of Pharmacology, Johannes Gutenberg University of Mainz, Mainz, Germany
  • 2Department of Pharmaceutical Sciences, Texas Tech School of Pharmacy, Amarillo, TX, USA
  • 3Section of Pharmacology, Dr. Willmar Schwabe Arzneimittel, Karlsruhe, Germany
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Publication History

Publication Date:
07 July 2003 (online)

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We have previously shown that hypoxia and N-methyl-D-aspartate (NMDA) receptor activation induce breakdown of choline-containing phospholipids in rat hippocampus, a process which is mediated by calcium influx and phospholipase A2 activation. Bilobalide, a constituent of Ginkgo biloba, inhibited this process in a potent manner (Weichel et al., Naunyn-Schmiedeberg’s Arch. Pharmacol. 360, 609 - 615, 1999). In this study, we used fluorescence microscopy and radioactive flux measurements to show that bilobalide does not interfere with NMDA-induced calcium influx. Instead, bilobalide seems to inhibit NMDA-induced fluxes of chloride ions through ligand-operated chloride channels. In our experiments, substitution of chloride in the superfusion medium fully blocked the effect of NMDA on choline release from hippocampal slices, while the presence of chloride transport inhibitors (furosemide, DIDS) was partially antagonistic. The inhibitory effect of bilobalide and of HA-966, a glycine B receptor antagonist, on NMDA-induced choline release was attenuated in the presence of glycine. The inhibitory effect of bilobalide, but not that of HA-966, was also antagonized by GABA. The inhibitory effect of MK-801, an NMDA channel blocker, on choline release was insensitive to glycine. We conclude from our findings that bilobalide inhibits an NMDA-induced chloride flux through glycine/GABA-operated channels, thereby preventing NMDA-induced breakdown of membrane phospholipids. This effect is expected to contribute to the neuroprotective effects of ginkgo biloba extracts.

References

  • 1 Ahlemeyer B, Mowes A, Krieglstein J. Inhibition of serum deprivation- and staurosporine-induced neuronal apoptosis by Ginkgo biloba extract and some of its constituents.  Eur J Pharmacol. 1999;  367 423-430
    CrossrefPubMedGoogle Scholar
  • 2 Bazan NG J r. Effects of ischemia and electroconvulsic shock on free fatty acid pool in the brain.  Biochim Biophys Acta. 1970;  218 1-10
    PubMedGoogle Scholar
  • 3 Bonventre J V, Huang Z, Taheri M R, O’Leary E, Li E, Moskowitz M A, Sapirstein A. Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2 .  Nature. 1997;  390 622-625
    CrossrefPubMedGoogle Scholar
  • 4 Chandrasekaran K, Mehrabian Z, Spinnewyn B, Drieu K, Fiskum G. Neuroprotective effects of bilobalide, a component of the Ginkgo biloba extract (EGb 761®), in gerbil global brain ischemia.  Brain Res. 2001;  922 282-292
    CrossrefPubMedGoogle Scholar
  • 5 Choi D W. Ionic dependence of glutamate neurotoxicity.  J Neurosci. 1987;  7 369-379
    PubMedGoogle Scholar
  • 6 Crowder J M, Croucher M J, Bradford H F, Collins J F. Excitatory amino acid receptors and depolarization-induced Ca- influx into hippocampal slices.  J Neurochem. 1987;  48 1917-1924
    PubMedGoogle Scholar
  • 7 Danysz W, Parsons C G. Glycine and N-methyl-D-aspartate receptors: physiological significance and possible therapeutic applications.  Pharmacol Rev. 1998;  50 597-664
    PubMedGoogle Scholar
  • 8 DeFeudis F V, Drieu K. Ginkgo biloba extract (EGb 761®) and CNS functions: basic studies and clinical applications.  Curr Drug Targets. 2000;  1 25-58
    CrossrefPubMedGoogle Scholar
  • 9 Dumuis A, Sebben M, Haynes L, Pin J -P, Bockaert J. NMDA receptors activate the arachidonic acid cascade system in striatal neurons.  Nature. 1988;  336 68-70
    CrossrefPubMedGoogle Scholar
  • 10 Erdo S L, Michler A, Wolff J R. GABA accelerates excitotoxic cell death in cortical cultures: Protection by blockers of GABA-gated chloride channels.  Brain Res. 1991;  542 254-258
    CrossrefPubMedGoogle Scholar
  • 11 Flynn C J, Wecker L. Concomitant increases in the levels of choline and free fatty acids in rat brain: evidence supporting the seizure-induced hydrolysis of phosphatidylcholine.  J Neurochem. 1987;  48 1178-1184
    PubMedGoogle Scholar
  • 12 Grynkiewicz G, Poenie M, Tsien R Y. A new generation of Ca2- indicators with greatly improved fluorescence properties.  J Biol Chem. 1985;  260 3440-3450
    PubMedGoogle Scholar
  • 13 Hasbani M J, Hyrc K L, Faddis B T, Romano C, Goldberg M P. Distinct roles for sodium, chloride, and calcium in excitotoxic dendritic injury and recovery.  Exp Neurol. 1998;  154 241-258
    CrossrefPubMedGoogle Scholar
  • 14 Hollingsworth E B, McNeal E T, Burton J L, Williams R J, Daly J W, Creveling C R. Biochemical characterization of a filtered synaptoneurosome preparation from guinea pig cerebral cortex.  J Neurosci. 1985;  5 2240-2253
    PubMedGoogle Scholar
  • 15 Inglefield J R, Schwartz-Bloom R D. Activation of excitatory amino acid receptors in the rat hippocampal slice increases intracellular Cl- and cell volume.  J Neurochem. 1998;  71 1396-1404
    PubMedGoogle Scholar
  • 16 Israel M, Lesbats B. Application to mammalian tissues of the chemiluminescent method for detecting acetylcholine.  J Neurochem. 1982;  39 248-250
    PubMedGoogle Scholar
  • 17 Johnson M W, Chotiner J K, Watson J B. Isolation and characterization of synaptoneurosomes from single rat hippocampal slices.  J Neurosci Meth. 1997;  77 151-156
    PubMedGoogle Scholar
  • 18 Karcher L, Zagermann P, Krieglstein J. Effect of an extract of Ginkgo biloba on rat brain energy metabolism in hypoxia.  Naunyn-Schmiedeberg’s Arch Pharmacol. 1984;  327 31-35
    PubMedGoogle Scholar
  • 19 Katayama Y, Kawamata T, Maeda T, Ishikawa K, Tsubokawa T. Inhibition of the early phase of free fatty acid liberation during cerebral ischemia by excitatory amino acid antagonist administered by microdialysis.  Brain Res. 1994;  635 331-334
    CrossrefPubMedGoogle Scholar
  • 20 Katsura K -I, Rodriguez de Turco E B, Folbergrova J, Bazan N G, Siesjo B K. Coupling among energy failure, loss of ion homeostasis, and phospholipase A2 and C activation during ischemia.  J Neurochem. 1993;  61 1677-1684
    PubMedGoogle Scholar
  • 21 Klein J. Membrane breakdown in acute and chronic neurodegeneration: focus on choline-containing phospholipids.  J Neural Transm. 2000;  107 1027-1063
    CrossrefPubMedGoogle Scholar
  • 22 Klein J, Chatterjee S S, Loffelholz K. Phospholipid breakdown and choline release under hypoxic conditions: inhibition by bilobalide, a constituent of Ginkgo biloba.  Brain Res. 1997;  755 347-350
    CrossrefPubMedGoogle Scholar
  • 23 Krishtal O A, Osipchuk Y uV, Vrublevsky S V. Properties of glycine-activated conductances in rat brain neurons.  Neurosci Lett. 1998;  84 271-276
    PubMedGoogle Scholar
  • 24 Mori M, Gähwiler B H, Gerber U. β-Alanine and taurine as endogenous agonists at glycine receptors in rat hippocampus in vitro.  J Physiol. 2002;  539 191-200
    CrossrefPubMedGoogle Scholar
  • 25 Muir J K, Lobner D, Monyer H, Choi D W. GABAA receptor activation attenuates excitotoxicity but exacerbates oxygen-glucose deprivation-induced neuronal damage in vitro.  J Cereb Blood Flow Metab. 1996;  16 1211-1218
    PubMedGoogle Scholar
  • 26 Oberpichler H, Sauer D, Rossberg C, Mennel H D, Krieglstein J. PAF-antagonist ginkgolide B reduces postischemic neuronal damage in rat brain hippocampus.  J Cereb Blood Flow Metab. 1990;  10 133-135
    PubMedGoogle Scholar
  • 27 Oken B S, Storzbach D M, Kaye J A. The efficacy of Ginkgo biloba on cognitive function in Alzheimer disease.  Arch Neurol. 1998;  55 1409-1415
    CrossrefPubMedGoogle Scholar
  • 28 Olney J W, Price M T, Samson L, Labruyere J. The role of specific ions in glutamate neurotoxicity.  Neurosci Lett. 1986;  65 65-71
    CrossrefPubMedGoogle Scholar
  • 29 Pellerin L, Wolfe L S. Release of arachidonic acid by NMDA-receptor activation in the rat hippocampus.  Neurochem Res. 1991;  16 983-989
    CrossrefPubMedGoogle Scholar
  • 30 Raiteri M, Fontana G, Fedele E. Glycine stimulates [3H]-noradrenaline release by activating a strychnine-sensitive receptor present in rat hippocampus.  Br J Pharmacol. 1990;  184 239-250
    PubMedGoogle Scholar
  • 31 Rodriguez de Turco E B, Droy-Lefaix M T, Bazan N G. Decreased electroconvulsive-shock-induced diacylglycerol and free fatty acid accumulation in the rat brain by Ginkgo biloba extract (EGb 761): selective effect in hippocampus as compared with cerebral cortex.  J Neurochem. 1993;  61 1438-1444
    PubMedGoogle Scholar
  • 32 Rothman S M. The neurotoxicity of excitatory amino acids is produced by passive chloride influx.  J Neurosci. 1985;  5 1483-1489
    PubMedGoogle Scholar
  • 33 Rothman S M, Olney J W. Excitotoxicity and the NMDA receptor: still lethal after eight years.  Trends Neurosci. 1995;  18 299-302
    CrossrefPubMedGoogle Scholar
  • 34 Sasaki K, Oota I, Wada K, Inomata K, Ohshika H, Haga M. Effects of bilobalide, a sesquiterpene in Ginkgo biloba leaves, on population spikes in rat hippocampal slices.  Comp Biochem Physiol C. 1995;  124 315-321
    PubMedGoogle Scholar
  • 35 Suen P -C, Wu K, Levine E S, Mount H TJ, Xu J L, Lin S -Y, Black I E. Brain-derived neurotrophic factor rapidly enhances phosphorylation of the postsynaptic N-methyl-D-aspartate receptor subunit 1.  Proc Natl Acad Sci USA. 1997;  94 8191-8195
    CrossrefPubMedGoogle Scholar
  • 36 Sun D, Murali S G. Stimulation of Na+-K+-2Cl- cotransporter in neuronal cells by excitatory neurotransmitter glutamate.  Am J Physiol. 1998;  275 C772-C779
    PubMedGoogle Scholar
  • 37 Takahashi M, Lion S Y, Kunihara M. Ca# and Cl- dependent, NMDA receptor-mediated neuronal death induced by depolarization in rat hippocampal organotypic cultures.  Brain Res. 1995;  675 249-256
    CrossrefPubMedGoogle Scholar
  • 38 Weichel O, Hilgert M, Chatterjee S S, Lehr M, Klein J. Bilobalide, a constituent of Ginkgo biloba, inhibits NMDA-induced phospholipase A2 activation and phospholipid breakdown in rat hippocampus.  Naunyn-Schmiedeberg’s Arch Pharmacol. 1999;  360 609-615
    CrossrefPubMedGoogle Scholar
  • 39 Zhou L -J, Zhu X -Z. Reactive oxygen species-induced apoptosis in PC12 cells and protective effect of bilobalide.  J Pharmacol Exp Ther. 2000;  293 982-988
    PubMedGoogle Scholar

Jochen Klein, Ph. D.

Department of Pharmaceutical Sciences

Texas Tech School of Pharmacy

1300 Coulter Dr.

Amarillo, TX 79106

USA

Email: jklein@ama.ttuhsc.edu

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