Role of Gabaergic Antagonism in the Neuroprotective Effects of Bilobalide

BRAIN RESEARCH 1128 (2007) 70– 78 available at www.sciencedirect.com www.elsevier.com/locate/brainres Research Report Role of GABAergic antagonism in the neuroprotective effects of bilobalide Cornelia Kiewerta, Vikas Kumara, Oksana Hildmannb, Misty Ruedaa, Joachim Hartmanna, Runa S. Naika, Jochen Kleina,b,⁎ aDepartment of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Science Center, 1300 Coulter Dr., Amarillo, TX 79106, USA bDepartment of Pharmacology, School of Medicine, Johannes Gutenberg University of Mainz, Obere Zahlbacher Str. 67, D-55101 Mainz, Germany ARTICLE INFO ABSTRACT Article history: Bilobalide, a constituent of Ginkgo biloba, has neuroprotective properties. Its mechanism of Accepted 20 October 2006 action is unknown but it was recently found to block GABAA receptors. The goal of this study Available online 28 November 2006 was to test the potential role of a GABAergic mechanism for the neuroprotective activity of bilobalide. In rat hippocampal slices exposed to NMDA, release of choline indicates Keywords: breakdown of membrane phospholipids. NMDA-induced choline release was almost Bicuculline completely blocked in the presence of bilobalide (10 μM) and under low-chloride μ Edema formation conditions. Bicuculline (100 M), a competitive antagonist at GABAA receptors, reduced GABAA receptor NMDA-induced choline release to a small extent (−23%). GABA (100 μM) partially Ginkgo biloba antagonized the inhibitory action of bilobalide. Exposure of hippocampal slices to NMDA Low-chloride condition also caused edema formation as measured by increases of tissue water content. NMDA- Water content induced edema formation was suppressed by bilobalide and by low-chloride conditions. Bicuculline exerted partial protection (by 30%) while GABA reduced bilobalide's effect by Abbreviations: about one third. To investigate bilobalide's interaction with GABAA receptors directly, we AU, arbitrary units measured binding of [35S]-TBPS to rat cortical membranes. TBPS binding was competitively μ AUC, area under the curve inhibited by bilobalide in the low micromolar range (IC50 =3.7 M). As a functional test, we GABA, γ-aminobutyric acid determined 36chloride flux in rat corticohippocampal synaptoneurosomes. GABA (100 μM) NMDA, N-methyl-D-aspartate significantly increased 36chloride flux (+65%), and this increase was blocked by bilobalide, μ TBPS, t-butylbicyclo- but with low potency (IC50:39 M). We conclude that, while antagonism of GABAA receptors phosphorothionate may contribute to bilobalide's neuroprotective effects, additional mechanisms must be postulated to fully explain bilobalide's actions. © 2006 Elsevier B.V. All rights reserved. 1. Introduction Alzheimer's disease (Oken et al., 1998; DeFeudis and Drieu, 2000). Experimental work during the last ten years has shown Ginkgo biloba extracts such as EGb 761 are widely used for the that Ginkgo extracts and their constituents, such as ginkgo- treatment of chronic neurodegenerative disorders such as lides and bilobalide, also exert beneficial effects in animal ⁎ Corresponding author. Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Science Center, 1300 Coulter Dr., Amarillo, TX 79106, USA. Fax: +1 806 356 4034. E-mail address: [email protected] (J. Klein). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.10.042 BRAIN RESEARCH 1128 (2007) 70– 78 71 models of acute neurodegeneration, e.g. in cerebral hypoxia and ischemia (Krieglstein et al., 1995; Chandrasekaran et al., 2001). In an experimental model of hypoxia-induced phos- pholipid breakdown, we found that bilobalide, a sesquiterpene lactone which constitutes ca. 3% of EGb761, was the active constituent of the extract (Klein et al., 1997) acting in the μ submicromolar range (IC50 =0.38 M). In further work, we described the antagonistic effect of bilobalide on NMDA receptor-induced choline release in the low micromolar μ range (IC50 =2.3 M) (Weichel et al., 1999). Other groups reported neuroprotection by bilobalide in neuronal cell cultures where it counteracted apoptotic cell death induced by amyloid (Luo et al., 2002) and serum deprivation (Ahle- meyer et al., 1999). The neuroprotective properties of bilobalide have recently been reviewed (DeFeudis, 2002; Ahlemeyer and Krieglstein, 2003). While evidence for neuroprotective properties of bilobalide is available from a variety of models, its mechanism of action is elusive. Bilobalide has been found to interfere with glutamatergic transmission, with mitochondrial function and apoptosis and it has genomic as well as proteomic effects (DeFeudis, 2002; Ahlemeyer and Krieglstein, 2003). The best available evidence, however, points to an interference with GABAergic neurotransmission. Electro-physio- logical data in hippocampal slices (Sasaki et al., 1995a,b)as well as antagonistic effects of bilobalide on barbital-induced sleeping time (Brochet et al., 1999) gave the first indirect evidence for an interference of bilobalide with GABAergic actions. Further data on GABAergic actions of bilobalide were reported by us in neurochemical models (Klein et al., 2003) and by others in electrophysiological studies (Chatterjee et al., 2003). Subsequently, it was demonstrated in careful Fig. 1 – Choline release from hippocampal slices induced by patch-clamp studies that bilobalide is a noncompetitive NMDA receptor activation: effects of low chloride and — antagonist (open channel blocker) at GABAA receptors in bicuculline Hippocampal slices were superfused with a embryonic cortical slices (Ivic et al., 2003) and at recombi- magnesium-free solution, and the superfusion fluid was nant GABAA receptors and GABAC receptors (Huang et al., switched to one containing N-methyl-D-aspartate (NMDA, 2003, 2006). Moreover, it was reported that bilobalide 100 μM) at time zero. Choline efflux was measured using a structurally resembles picrotoxin as a ligand of GABA and chemoluminescence assay. (A) Effect of NMDA in solutions glycine channels (Hawthorne and Lynch, 2005). with varying concentrations of chloride. In these “ ” Several groups have reported that blockade of GABAA experiments, NaCl (142 mM in controls, NaCl ) was partially “ ” “ ” receptors can be neuroprotective in certain situations (Erdö ( 50% Cl ) or completely ( Low Cl ) replaced by Na2SO4 in an et al., 1991; Muir et al., 1996; Chen et al., 1999). It remains isoosmotic manner. For the zero-chloride (“Zero Cl”) unclear, however, if the recently described GABAergic antag- condition, KCl and CaCl2 were additionally replaced by their onism contributes to the neuroprotective properties of bilo- nitrate salts. (B) Effect of bicuculline (100 μM), a competitive balide. In the present communication, we tested the potential antagonist at GABAA receptors. In these experiments, choline of GABAergic antagonism in two models of excitotoxicity, and efflux was recorded under basal conditions (“Ctr”) or in the we determined if bilobalide's neuroprotective effects may be presence of bicuculline (“Bicuculline”). When NMDA was “ ” due to interactions with the GABAA receptor. present, it was added at time zero in the absence ( NMDA )or presence (“NMDA+Bicu”) of bicuculline. Data are given as relative changes of the basal choline efflux (determined from 2. Results three consecutive samples before addition of NMDA) and are means±SEM of 4–6 experiments. 2.1. Choline release from hippocampal slices The model of NMDA-induced choline release was established mental period (30 min; Fig. 1). Basal choline efflux was in our laboratory and can be used to follow NMDA receptor- unchanged in the absence or presence of magnesium (data induced excitotoxicity and membrane breakdown (Weichel et not shown). Upon addition of NMDA (100 μM) in magnesium- al., 1999; Klein, 2000). In the present study, basal efflux of free solution, we observed an immediate, approximately two- choline from rat hippocampal slices was 1.5±0.1 pmol/10 μl fold increase of choline release that lasted beyond the (N=24); it increased slightly (by 15–20%) during the experi- experimental period of observation (30 min; Figs. 1 and 2). 72 BRAIN RESEARCH 1128 (2007) 70– 78 choline release (data not shown), but it suppressed NMDA- induced choline release by more than 90% (Fig. 2)(p<0.01; Table 1). GABA (100 μM) slightly increased choline release per se (p<0.05; Table 1) but did not significantly affect NMDA- induced release of choline (Fig. 2; Table 1). However, GABA partially counteracted bilobalide's effect (Fig. 2). Based on AUC values, the NMDA effect in the presence of GABA was 1474 AU (1844−370); in the presence of GABA, bilobalide reduced the NMDA effect by 781 AU (1844−1063) which is equivalent to a 47% reduction of this response. In two experiments, this GABA effect was apparently blocked in the presence of picrotoxin, a well-characterized GABAA channel blocker (data not shown); however, picrotoxin interfered with the choline oxidase-based detection of choline in this assay, and further experiments with picrotoxin were not performed. Fig. 2 – Choline release from hippocampal slices induced by NMDA receptor activation: effects of bilobalide and GABA—Hippocampal slices were superfused with a magnesium-free solution. When N-methyl-D-aspartate (NMDA) was present, it was added at time zero. Choline efflux was measured using a chemoluminescence assay. Data are as follows: “NMDA 100 μM” indicates the effect of NMDA alone. “NMDA+Bilo”, “NMDA+GABA,” and “NMDA+Bilo+GABA” show the responses of

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