Role of Glycine Receptors and Glycine Release for the Neuroprotective Activity of Bilobalide

BRAIN RESEARCH 1201 (2008) 143– 150

available at www.sciencedirect.com

www.elsevier.com/locate/brainres

Research Report

Role of glycine receptors and glycine release for the neuroprotective activity of bilobalide

Cornelia Kiewerta, Vikas Kumara, Oksana Hildmannb, Joachim Hartmanna, Markus Hillerta, 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, Johann Wolfgang Goethe University of Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt, Germany

ARTICLE INFO ABSTRACT

Article history: Bilobalide, a constituent of Ginkgo biloba, has neuroprotective properties. Its mechanism of Accepted 15 January 2008 action is unknown but it was recently found to interact with neuronal transmission Available online 31 January 2008 mediated by glutamate, γ-aminobutyric acid (GABA) and glycine. The goal of this study was to test the interaction of bilobalide with glycine in assays of neuroprotection. In rat Keywords: hippocampal slices exposed to N-methyl-D-aspartate (NMDA), release of choline indicates Edema formation breakdown of membrane phospholipids. NMDA-induced choline release was almost Excitotoxicity completely blocked in the presence of bilobalide (10 µM). Glycine (10–100 µM) antagonized Glycine receptor the inhibitory action of bilobalide in this assay. In a second assay of excitotoxicity, we Ginkgo biloba measured tissue water content as an indicator of cytotoxic edema formation in hippocampal Ischemia slices which were exposed to NMDA. In this assay, edema formation was suppressed by Oxygen-glucose deprivation bilobalide but bilobalide's action was attenuated in the presence of glycine and of D-serine (100 µM each). To investigate bilobalide's interaction with glycine receptors directly, we determined 36chloride flux in rat cortico-hippocampal synaptoneurosomes. Glycine (100 µM) was inactive in this assay indicating an absence of functional glycine-A receptors in this preparation. [3H]Glycine was used to assess binding at the glycine binding site of the NMDA receptor but bilobalide was found to be inactive in this assay. Finally, [3H]glycine release was monitored in hippocampal slices exposed to oxygen-glucose deprivation. In this model, glycine release was induced by ischemia, an effect that was strongly reduced by bilobalide. We conclude that bilobalide does not interact with glycine receptors in neurochemical assays but it significantly reduces the release of glycine under ischemic conditions. This effect likely contributes to bilobalide's neuroprotective effects in assays of excitotoxicity and ischemia. © 2008 Elsevier B.V. All rights reserved.

⁎ Corresponding author. Department of Pharmacology, Johann Wolfgang Goethe University of Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt, Germany. Fax: +49 69 798 29277. E-mail address: [email protected] (J. Klein). Abbreviations: AU, arbitrary units; AUC, area under the curve; DCKA, 5,7-dichlorokynurenic acid; DFP, di-isopropyl fluorophosphates; DIDS, 4,4′-di-isothiocyanostilbene-2,2′-disulfonic acid; GABA, γ-aminobutyric acid; HEPES, (4-(2-hydroxyethyl)-1-piperazine ethanesul- fonic acid; NMDA, N-methyl-D-aspartate; OGD, oxygen-glucose deprivation; TLC, thin-layer chromatography

Table 1 – Area under the curve (AUC) values from choline 1. Introduction efflux experiments Condition AUC Value⁎ N EGb761, a defined extract of the leaves of the Ginkgo tree (Ginkgo biloba), is a clinically useful medication for chronic neurodegen- NMDA 267±15.8 5 erative diseases such as Alzheimer's dementia (Oken et al., 1998; NMDA + Glycine (100 µM) 369±12.8a 4 b DeFeudis and Drieu, 2000). In experimental work, Ginkgo ex- NMDA + Bilobalide 26.5±10.3 5 NMDA + Bilo + Glycine (10 µM) 88.9±26.5b 5 tracts were found to interfere with amyloid formation, aggrega- NMDA + Bilo + Glycine (100 µM) 308±22.3c 6 tion and toxicity (Bastianetto et al., 2000; Luo et al., 2002; ⁎ Colciaghi et al., 2004), and this activity may underlie their anti- AUC values were calculated from the data shown in Fig. 1,for30min dementia actions. Recent publications have also demonstrated of choline release. They are given as arbitrary units (AU, formally anti-ischemic and anti-edema properties of Ginkgo extract and representing [%] min). Statistical evaluation (ANOVA with Tukey's Multiple Comparison Test): a, p<0.05 vs. NMDA. b, p<0.01 vs. NMDA. its constitutent, bilobalide, which represents 3% of EGb761 (De- c, p<0.01 vs. NMDA+Bilobalide. Feudis, 2002; Ahlemeyer and Krieglstein, 2003). Neuroprotection by bilobalide was observed in models of ischemia and excitotoxicity, both in vitro and in vivo (Krieglstein et al., 1995; Chan- ishvili et al., 2006). The mechanism of action of bilobalide in drasekaran et al., 2001). In our hands, bilobalide blocked cellular these models, however, remains elusive. toxicity and phospholipid breakdown in hippocampal slices A variety of cellular mechanisms were proposed for the neu- exposed to hypoxia or to N-methyl-D-aspartate (NMDA) acting roprotective effects of bilobalide, among them effects on nitric in the low micromolar range (Klein et al., 1997; Weichel et al., oxide and platelet-activating factor, on mitochondrial function 1999). Recently, we reported potent anti-edema effects of bilo- and apoptosis as well as genomic and proteomic effects (De- balide after middle cerebral artery occlusion in mice (Mdzinar- Feudis, 2002; Ahlemeyer and Krieglstein, 2003). Much recent evi- dence from our and other laboratories points to an interference of bilobalide with amino acidergic neurotransmission which is mediated by glutamate, γ-aminobutyricacid(GABA)and/orgly- cine. For instance, tissue levels of these transmitter amino acids in the brain were increased following chronic bilobalide administration (Sasaki et al., 2002). In hippocampal slices, bilobalide blocked glutamatergic, NMDA receptor-mediated actions both in electrophysiological and in neurochemical assays (Chatterjee et al., 2003; Klein et al., 2003). Bilobalide was also found to interfere with the release of glutamate under hypoxic/hypoglycemic conditions (Davies et al., 2003). Bilobalide was reported as a

potent noncompetitive antagonist at GABAA receptors (Huang et al., 2003; Ivic et al., 2003) and was also found to block glycine-A receptors, albeit at lower potency (Ivic et al., 2003; Hawthorne and Lynch, 2005). Moreover, it was reported that bilobalide structu- rally resembles picrotoxin as a ligand of GABA and glycine chan- nels (Hawthorne and Lynch, 2005). However, we have previously reported that interactions with GABAergic mechanisms cannot explain bilobalide's neuroprotective activities (Kiewertetal., 2007). In the present communication, we tested if bilobalide's neuroprotective activity can be explained by interactions with glycine. For this purpose, we tested bilobalide-glycine inter- Fig. 1 – Choline release from hippocampal slices induced by actions in two models of excitotoxicity. We also determined if NMDA receptor activation: effects of bilobalide and glycine. bilobalide directly interferes with glycine receptors or with — Hippocampal slices were superfused with glycine release. magnesium-free solution. N-methyl-D-aspartate (NMDA) 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”, 2. Results “NMDA + Gly” and “NMDA + Bilo + Gly” show the responses of NMDA in the presence of bilobalide (Bilo, 10 µM) or glycine 2.1. Choline release from hippocampal slices (10 or 100 µM), or both. Bilobalide and glycine, when present, were added together with NMDA. Bilobalide or glycine, when Membrane breakdown is a typical consequence of excitotoxicity given alone or together, did not affect basal choline efflux and can be monitored using the choline release assay which was (data not shown). Data are given as relative changes of the established in our laboratory (Weichel et al., 1999; Klein, 2000). In basal choline efflux (determined from three consecutive the present study, we superfused rat hippocampal slices with samples before addition of NMDA) and are means±S.E.M. of NMDA (100 µM) in magnesium-free Tyrode solution (Fig. 1). 4–6 experiments. Basal choline efflux from the slices (1.5±0.1 pmol/10 µl, N=24) BRAIN RESEARCH 1201 (2008) 143– 150 145 was unchanged in the absence or presence of magnesium (data not shown). Upon addition of NMDA (100 μM), we observed an immediate increase of choline release that lasted beyond the experimental period of observation (30 min). We used this model to investigate the effects of bilobalide and glycine on NMDA receptor-mediated excitotoxicity. For the statistical evaluation of the effects, we calculated the “areas under the curve” (AUC) values as a measure of the total amount of free choline that was released by NMDA receptor activation during 30 min of superfusion (Table 1). Bilobalide (10 µM) and glycine (100 µM), given alone, did not affect basal choline release (data not shown). Glycine (100 µM) modestly but significantly increased NMDA-induced release of choline (p<0.05). When given together with NMDA, bilobalide suppressed NMDA-induced choline release by 90% (p<0.01; Fig. 1). Importantly, glycine antagonized bilobalide's inhibitory Fig. 3 – Displacement of 3H-glycine binding by bilobalide. — effect on NMDA-induced choline release (Fig. 1). At 10 µM, The binding assay was carried out in rat glycine caused a minor increase of the AUC (Table 1) which did cortico-hippocampal membranes, as described in not reach statistical significance. At 100 µM, glycine counter- Experimental procedures. Data were from three different experiments, each averaged from incubations run in triplicates. Specific binding was calculated from incubations that contained unlabeled glycine (1 mM) and was 37–41% of total binding. Specific binding was tested in the presence of 3H-glycine (50 nM), 5,7-dichlorokynurenic acid (“DCKA”), bilobalide (“Bilo”, 0.1–100 µM) and strychnine (10 µM). **, p<0.01 vs control (“Specific binding”).

acted bilobalide's action almost completely (Fig. 1), and bilobalide's action on the effect of NMDA was no longer significant when glycine (100 µM) was present (Table 1).

2.2. Brain edema induced by excitotoxicity

As an alternative model of tissue damage induced by excitotoxicity, we exposed rat hippocampal slices to NMDA and measured tissue water contents to quantify cytotoxic edema formation (Fig. 2). In our hands, basal values of water contents showed some variation between experiments but relative changes of water contents after NMDA exposure were highly reproducible. Exposure of slices to NMDA caused a significant increase of the tissue water content, and this NMDA-induced edema was completely inhibited in the presence of bilobalide (Fig. 2). When glycine (100 µM) was added to the superfusion solution, bilobalide's protective action was attenuated (Fig. 2A). The same observation was made when D-serine, an agonist at the glycine binding site of the NMDA receptor, was used to Fig. 2 – Edema formation in hippocampal slices: effects of counteract bilobalide's action (Fig. 2B). Glycine and D-serine by bilobalide, glycine and D-serine. (A) Effects of bilobalide and themselves did not significantly affect tissue water contents glycine. (B) Effects of bilobalide and D-serine. Slices were (data not shown). superfused for 30 min with control (low-magnesium) buffer (“Ctr”) or with control buffer containing NMDA (300 µM). In 36 some experiments, bilobalide (“Bilo”, 10 µM) and/or glycine 2.3. Chloride flux assay (“Gly”, 100 µM) or D-serine (“D-Ser”, 100 µM) were present. When present, bilobalide was added 5 min before NMDA. To test the presence and functional relevance of glycine-A Water contents in the slices were determined at the end of receptors, we compared the GABA- and glycine-induced influx the superfusion procedure by differential weighing before of 36chloride in cortico-hippocampal synaptoneurosomes. and after drying the slices. Statistical significance was GABA (100 µM) significantly increased 36Cl uptake by 63% evaluated by paired ANOVA. **, p<0.01 vs Ctr. ##, p<0.01 vs. (p<0.01, N=10). Glycine (100 µM) was without significant effect NMDA. N=6 for (A), N=4 for (B). (−8%; pN0.3, N=5) (data not illustrated). 146 BRAIN RESEARCH 1201 (2008) 143– 150

2.4. [3H]glycine binding assay Table 2 – Area under the curve (AUC) values from 3H-glycine release experiments The glycine binding assay was used to test an interaction of Condition AUC value⁎ N bilobalide with the glycine B-receptor, i.e. the glycine binding Ctr 21.8±0.68 5 site on NMDA receptors. As shown in Fig. 3, specific binding of Bilobalide (10 µM) 23.5±2.07 5 3 [ H]glycine in cortico-hippocampal membranes was reduced OGD 46.3±4.18a 5 by 90% in the presence of 5,7-dichlorokynurenic acid (DCKA), a OGD + bilobalide 35.6±1.77a,b,c 5 well-known ligand at the glycine binding site of the NMDA OGD minus Ctr 24.5±4.52 receptor. In contrast, bilobalide was inactive in concentrations OGD + bilobalide minus Bilobalide 12.1±2.91d ⁎ between 0.1 and 100 µM. Strychnine, a blocker of glycine-A AUC values were calculated from the data shown in Fig. 4, for ten receptors, was also inactive. samples (50 min) following the switch to OGD. They are given as arbitrary units (AU, formally representing [%] min). Statistical 2.5. [3H]glycine release assay evaluation (ANOVA with Tukey's Multiple Comparison Test): a, p<0.01 vs. Ctr. b, p<0.05 vs. Bilobalide. c, p<0.05 vs. OGD. d, p<0.05 vs. “OGD ” The glycine release assay was established to investigate the minus Ctr . Ctr, control; OGD, oxygen-glucose deprivation. effect of bilobalide on glycine release under basal and ischemic conditions. Oxygen-glucose deprivation (OGD) was used as an in vitro-ischemia model and induced an immediate release of explanation of this effect is provided by the observation that tritium label from hippocampal slices that had been prelabeled bilobalide antagonizes excitotoxicity, i.e. toxic effects me- with [3H]glycine (Fig. 4). The identity of the measured efflux of diated by NMDA receptor activation (Weichel et al., 1999). The tritium as glycine was established using thin-layer chromato- molecular target of bilobalide for this action is, however, un- graphy (TLC) which revealed that N85% of all radioactivity that known. In the present study, we used two assays of excito- was released from the slices could be recovered as [3H]glycine toxicity to investigate a potential interaction of bilobalide with (not illustrated). In this assay, bilobalide (10 µM) did not affect glycine: the choline release assay and the brain slice edema basal release of [3H]glycine; however, it significantly reduced assay. the ischemia-induced release (Fig. 4). As estimated from AUC calculations, bilobalide reduced ischemia-induced release of 3.1. Antagonism of bilobalide by glycine in assays of glycine by 51% (p<0.05; Table 2). excitotoxicity

Exposure of hippocampal slices to NMDA leads to calcium 3. Discussion influx, activation of phospholipase A2, hydrolysis of phospha- tidylcholine and release of choline from membranes (Klein, Bilobalide has neuroprotective properties in vitro and in vivo 2000). The assay can be used to monitor NMDA receptor acti- (DeFeudis, 2002; Mdzinarishvili et al., 2006). A possible vation (Weichel et al., 1999; Klein et al., 2003). NMDA-induced choline release is potently inhibited by bilobalide (Weichel et al., 1999; Klein et al., 2003). Intriguingly, we found that bilobalide's action in this model is counteracted by glycine. A small glycine concentration (10 µM) reduced the effect of bilobalide while a larger concentration (100 µM) almost abolished it (Fig. 1 and Table 1). These glycine concentrations were selected because it is known that brain slices maintain micromolar concentrations of glycine in the extracellular space (Huettner, 1990). As a second model of excitotoxicity, we employed the brain slice edema assay. As reported previously (Kiewert et al., 2007), exposure of hippocampal slices to NMDA causes the formation of a cytotoxic edema which is reflected in an increase of tissue water. A similar effect was described during ischemia (oxygen-glucose deprivation; MacGregor et al., 2003). In the present study, the NMDA-induced increase of tissue water was completely inhibited in the presence of bilobalide (Fig. 2). Fig. 4 – Effects of bilobalide on ischemia-induced glycine Moreover, bilobalide's effect was attenuated in the presence of release. — Rat hippocampal slices were preloaded with glycine and D-serine, a selective agonist at the glycine binding 3H-glycine and then superfused in four lanes in the presence site of the NMDA receptor (Schell et al., 1995; see below). (“Bilo”) or absence (“Ctr”) of bilobalide (10 µM). At time zero, two lanes were switched to a buffer solution containing no 3.2. Interaction of bilobalide with glycine receptors glucose which was bubbled with nitrogen (oxygen-glucose deprivation, “OGD”). Efflux of 3H-glycine was monitored by Recent reports indicated an interference of bilobalide with scintillation counting and expressed as fractional release glycine-A receptors, i.e. inhibitory receptors that mediate chlo- (for details, see Experimental procedures). ride influx into neurons, in neonatal rat cortex (Ivic et al., 2003) BRAIN RESEARCH 1201 (2008) 143– 150 147 and in experiments using recombinant glycine receptors brain slice release assay will be required to monitor glycine expressed in HEK293 cells (Hawthorne and Lynch, 2005). levels in the immediate vicinity of NMDA receptors. Never- However, bilobalide's action on glycine-A receptors had low theless, the present data indicate that NMDA receptor activa- potency and could not be reproduced in hippocampal slices tion and excitotoxicity are attenuated when the release of obtained from 14 d old rats (Chatterjee et al., 2003). In our glycine is reduced. hands, glycine was inactive in a functional assay measuring 36chloride fluxes in synaptoneurosomes taken from adult rats. 3.4. Conclusion Thus, in agreement with the literature (Lynch, 2004), glycine does not seem to play a major role as an inhibitory neuro- Our data indicate that bilobalide has neuroprotective properties transmitter in adult hippocampus, and therefore the glycine-A that can be antagonized in vitro by the addition of exogenous receptor is unlikely to mediate the actions of glycine shown in glycine. Further analysis demonstrates that bilobalide does not Figs. 1 and 2. interact with glycine receptors or with glycine release under Glycine has a prominent high-affinity binding site on the basal conditions, but that it specifically inhibits the release of NMDA receptor complex which is sometimes called glycine-B glycine under ischemic conditions. An inhibitory effect on receptor and which is widely distributed in the forebrain (Danysz glycine release during excitotoxicity likely explains some of the and Parsons, 1998). Acting as a co-agonist in the NMDA receptor neuroprotective activity of bilobalide that was seen in the complex, glycine is required for NMDA receptor opening to choline release and tissue edema assays. In fact, the antagonism occur. Our data (Fig. 3), however, demonstrate that bilobalide is of bilobalide's action by D-serine suggests that bilobalide limits unable to compete with the binding of [3H]glycine in cortico- the availability of endogenous glycine to act as a co-agonist at hippocampal membranes. the NMDA receptor. Moreover, inhibition of glycine release under ischemic conditions may underlie some of bilobalide's 3.3. Interaction of bilobalide with glycine release neuroprotective effects in stroke (Krieglstein et al., 1995; Mdzi- narishvili et al., 2006). Bilobalide may act as a direct inhibitor of As bilobalide failed to interact directly with glycine receptors, glycine transport, which would be of interest in a variety of how can the interactions between bilobalide and glycine in therapeutic settings (Eulenburg et al., 2005); or it may interfere assays of neurotoxicity be explained? The sensible conclusion with the cellular energy status and the ionic gradients that go- seemed to be that bilobalide must somehow interact with the vern the release of amino acids in pathological situations (Dan- release of glycine from cellular sources. Therefore, we exposed bolt, 2001). These phenomena provide novel molecular targets hippocampal slices to oxygen-glucose deprivation (OGD), a for the investigation of bilobalide's neuroprotective effects. situation which mimics ischemia in vitro. In agreement with a previous report (Saransaari and Oja, 2001), glycine release was increased after onset of ischemia. Bilobalide did not affect 4. Experimental procedures glycine release under basal conditions but, importantly, reduced ischemia-induced glycine release to a significant extent 4.1. Materials (Fig. 4 and Table 2). Can the effect on glycine release explain neuroprotective Bilobalide was isolated in 99% purity from Ginkgo biloba leaves effects of bilobalide? Brain slices are long known to maintain a as described (Weinges and Bähr, 1969) and was made available microenvironment during superfusion that provides enough by Dr. Michael Nöldner (Dr. Willmar Schwabe Pharmaceuticals, glycine to activate the high-affinity glycine-B receptor; low Karlsruhe, Germany). Bilobalide stock solution was made in micromolar levels of glycine have been estimated in perfused DMSO and was diluted in buffer so that the final DMSO con- brain slices (Huettner, 1990; Wood, 1995). More recent work, centration in experiments was 0.1%. All incubation and super- however, demonstrated that the in situ levels of glycine are fusion buffers in Figs. 1–4 contained 0.1% DMSO. [3H]glycine kept at low levels through cellular uptake by glycine trans- (spec. activity: 0.05 Ci/mol; 1.85 GBq/mol) and 36chloride (0.6 Ci/ porters (Berger et al., 1998; Tsai et al., 2004). Accordingly, in our mol; 22.2 GBq/mol) as sodium chloride were from American hands, addition of exogenous glycine caused an increase of Radiolabeled Chemicals (St. Louis, MO). All other chemicals the NMDA-induced choline release per se (Fig. 1) and anta- were from Sigma at the highest purity available. gonized bilobalide's inhibitory action during NMDA receptor activation (Figs. 1 and 2). Release of amino acids (including 4.2. Animals glycine) is well characterized under ischemic conditions and involves the reversal of sodium-dependent uptake systems in Male Sprague–Dawley rats (250–350 g; Charles River) were kept glial cells (Danbolt, 2001; Saransaari and Oja, 2001). Bilobalide under standardised light/dark (12 h), temperature (22 °C) and inhibited this type of ischemia-induced release whereas it humidity (70%) conditions, with rat chow and water available left basal glycine release unchanged (Fig. 4). It is possible but ad libitum. Animal procedures were in accordance with NIH not conclusively known whether NMDA receptor activation regulations and were registered with the Institutional Animal causes glycine release in hippocampal tissue. In an earlier Care and Use Committee of TTUHSC (protocol #04003-02). work, NMDA-induced glycine release was reported in hippocampal slices from newborn rats but the effect disappeared 4.3. Choline release from rat hippocampal slices with maturation (Saransaari and Oja, 1994). Glutamatergic activation was found to release D-serine from glial cells (Schell Hippocampal slices (400 µm) were prepared from male rats as et al., 1995). More sophisticated methods than the present previously described (Klein et al., 1997; Weichel et al., 1999) 148 BRAIN RESEARCH 1201 (2008) 143– 150 and superfused (0.7 ml/min) at 35 °C with Tyrode solution of 30 °C in the presence of 100 µM DIDS and 10 µM furosemide the following composition: 142 mM NaCl, 2.7 mM KCl, 1.8 mM (Bloomquist and Soderlund, 1985). Aliquots of the incubations

CaCl2, 1.2 mM MgCl2, 11.9 mM NaHCO3, 5.6 mM glucose. All were then rapidly mixed with buffer B (composition identical superfusion solutions were continuously gassed with carbo- to buffer A, except that NaCl was 145 mM and glucose was 36 gen (95% O2,5%CO2). The slices were first incubated with 27 mM) containing chloride (1 µCi/ml; 37 kBq/ml). GABA and 0.1 mM di-isopropyl-fluorophosphate (DFP) for 30 min in order glycine (100 µM final concentration), when present, were added to prevent choline release from acetylcholine by the action of to buffer B. After 5 s of incubation, the assay tubes (0.4 ml) were acetylcholinesterase. Subsequently, the slices were washed mixed with a surplus (4 ml) of ice-cold buffer B and rapidly for 40 min with magnesium-free Tyrode solution, and basal filtered over GF/C Whatman filters in a vacuum filter unit. choline efflux was determined. Then, the superfusion solu- Filters were washed twice, dried and counted for radioactivity tions were switched to those containing NMDA (100 µM) and/ in a Beckman Coulter LS 6000 scintillation counter. or bilobalide (10 µM) or glycine (10 or 100 µM) in magnesium- 3 free Tyrode solution. The superfusates were collected at 5 or 4.6. [ H]glycine binding assay 10 min intervals and analyzed for choline content. Choline was determined by a chemoluminescence assay (Klein et al., Preparation of rat cortico-hippocampal membranes were car- 1997; Weichel et al., 1999). Briefly, 10 µl aliquots of the super- ried out as described (Vogel and Vogel, 2002). Aliquots of mem- fusates were given to a reaction mixture consisting of 20 mM branes were incubated in 25 mM Tris-maleate buffer (pH 7.4) 3 Tris buffer pH 8.6, 1 µg luminol, 10 µg peroxidase, and 1.25 U with [ H]glycine (10 nM) in the presence of bilobalide (0.1– choline oxidase, and the chemiluminescence resulting from 100 µM, in 0.1% DMSO), dichlorokynurenic acid or strychnine oxidation of choline to betaine was measured at 425 nm in a (see Fig. 3). Incubations with unlabeled glycine (1 mM) were used LKB-Wallac luminometer. The assay was linear from 1–5pmol to calculate specific binding which was 37–41% of total binding. choline. The data for choline efflux (Fig. 1) are expressed in % After incubation at 4 °C for 20 min, the assay was terminated of basal choline efflux which was 71.6±5.4 pmol/min/mg by centrifugation, followed by two washes with ice-cold buf- protein (N=24). Three values for choline efflux were taken at fer. Radioactivity on filters was counted in a Beckman Coulter 30, 20 and 10 min before addition of NMDA, and the average of LS 6000 scintillation counter. All experiments were done in these three values was set as 100% and used to calculate the triplicate. relative changes of choline efflux after addition of NMDA in 3 Fig. 1. Areas under the curve (AUC) values were calculated for 4.7. [ H]glycine release assay each individual experiment; they represent the amount of choline that was released by NMDA receptor activation during Rat hippocampal slices were prepared as described above (cf. 4.3) 30 min of superfusion (Table 1). and incubated at 37 °C for 15 min with [3H]glycine (5 µCi; 185 kBq) in Tyrode solution. Then the slices were randomly transferred to 4.4. Edema formation in hippocampal slices four lanes (A–D) of a superfusion chamber and were superfused with Tyrode solution. During the 40 min equilibration period, all Rat hippocampal slices were prepared and superfused with superfusion solutions were continuously gassed with carbogen

Tyrode solution as described above. During the equilibration (95% O2,5%CO2). Bilobalide, when used, was pre-incubated for period, all superfusion solutions were continuously gassed with 5 min before OGD. At time zero (cf. Fig. 4), lanes A and B were carbogen (95% O2,5%CO2). To induce cytotoxic edema formation, switched to Tyrode solutions containing 0.1% DMSO and 10 µM theslicesweresuperfusedwithNMDA(300µM),andthecon- bilobalide (in 0.1% DMSO), respectively. 5 min later, the slices in centration of magnesium chloride in the Tyrode solution was lanes C and D were switched to Tyrode solution which lacked lowered to 0.12 mM. Bilobalide, when used, was pre-incubated glucose and was gassed with nitrogen to remove oxygen from for 5 min before NMDA was added. Glycine and D-serine were the solution (oxygen-glucose deprivation, OGD); in addition, the added together with NMDA. Four lanes of slices were superfused slices received 0.1% DMSO (lane C) and 10 µM bilobalide (lane D), in parallel for 30 min. At the end of the superfusion period, slices respectively. The effluent from each lane was collected in 5 min from each lane were collected, superficially dried, transferred to intervals, and aliquots (1 ml) of the effluent were mixed with aluminum foil, and weighed (“wet weight”). They were then scintillation fluid and counted in a Beckman Coulter LS 6000 dried over night at 105 °C in a desiccating oven and weighed scintillation counter. At the end of the incubation, the slices were again (“dry weight”). Total tissue brain water was calculated collected and homogenized in Tyrode solution. Aliquots of the according to [(wet weight−dry weight)/wet weight]×100. homogenate were also counted for radioactivity. Data were expressed as [%] of radioactivity that was released during a time 36 4.5. Chloride flux assay in synaptoneurosomes period, relative to the total radioactivity present in the tissue at that time (the latter value was taken from the homogenized Synaptoneurosomes were prepared from rat cortico-hippo- tissue at the end of the incubation). Areas under the curve (AUC) campal tissue essentially as described (Hollingsworth et al., values were calculated for each individual experiment; they 1985). For 36chloride uptake assays, cortico-hippocampal ve- represent the amount of radioactivity that was released during sicles were suspended in buffer A (20 mM HEPES buffer pH 7.4 30 min of superfusion (Table 2). 3 with 54 mM glucose, 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, The identity of tritium release as [ H]glycine was confirmed

2.5 mM CaCl2), and chemicals (glycine or GABA) were added by TLC. Aliquots of the effluents and homogenates were and pre-incubated for 10 min at room temperature. Vials were spotted on silica gel TLC plates and developed in 90% ethanol/ then transferred to a heating block and incubated for 60 s at 10% acetic acid. After spraying with ninhydrin solution, the BRAIN RESEARCH 1201 (2008) 143– 150 149 spots corresponding to authentic glycine were collected and CNS functions: basic studies and clinical applications. Curr. – counted for radioactivity. Comparison of the spots with total Drug Targets 1, 25 58. Eulenburg, V., Armsen, W., Betz, H., Gomeza, J., 2005. Glycine radioactivity on the TLC plate revealed that [3H]glycine ac- transporters: essential regulators of neurotransmission. counted for N85% of total radioactivity in effluents and ho- Trends Biochem. Sci. 30, 325–333. mogenates (data not shown). Hawthorne, R., Lynch, J.W., 2005. A picrotoxin-specific conformational change in the glycine receptor M2–M3 loop. 4.8. Statistics J. Biol. Chem. 280, 35836–35843. Hollingsworth, E.B., McNeal, E.T., Burton, J.L., Williams, R.J., Statistical calculations were performed by GraphPad InStat 4.0 Daly, J.W., Creveling, C.R., 1985. Biochemical characterization program package, using analysis of variance (ANOVA) of paired of a filtered synaptoneurosome preparation from guinea pig cerebral cortex. J. Neurosci. 5, 2240–2253. or unpaired data as indicated in text and figure legends. AUC Huang, S.H., Duke, R.K., Chebib, M., Sasaki, K., Wada, K., Johnston, “ ” ( area under the curve ) was calculated as arbitrary units (AU) G.A.R., 2003. Bilobalide, a sesquiterpene trilactone from Ginkgo α from the data in Figs. 1 and 4 and was used to statistically biloba, is an antagonist at recombinant 1ß2g2L GABAA compare the effects of treatments (see Tables 1 and 2). receptors. Eur. J. Pharmacol. 464, 1–8. Huettner, J.E., 1990. Competitive antagonism of glycine at the N-methyl-D-aspartate (NMDA) receptor. Biochem. Pharmacol. 41, 9–16. Acknowledgments Ivic, L., Sands, T.T., Fishkin, N., Nakanishi, K., Kriegstein, A.R., Stromgaard, K., 2003. Terpene trilactones from Ginkgo biloba are

The authors are grateful to Drs. Shyam S. Chatterjee and Michael antagonists of cortical glycine and GABAA receptors. J. Biol. Nöldner, Dr. Willmar Schwabe Pharmaceuticals (Karlsruhe, Chem. 278, 49279–49285. Germany) for their helpful discussions and for supplying pure Kiewert, C., Kumar, V., Hildmann, O., Rueda, M., Hartmann, J., bilobalide; to Rachita Sumbria for the TLC experiments; and to Naik, R.S., Klein, J., 2007. Role of GABAergic antagonism in the neuroprotective effects of bilobalide. Brain Res. 1128, the National Center for Complementary and Alternative Med- 70–78. icine and to Texas Tech University Health Science Center (Car- Klein, J., 2000. Membrane breakdown in acute and chronic diovascular Seed Grant) for the financial support. neurodegeneration: focus on choline-containing phospholipids. J. Neural Transm. 107, 1027–1063. Klein, J., Chatterjee, S.S., Löffelholz, K., 1997. Phospholipid REFERENCES breakdown and choline release under hypoxic conditions: inhibition by bilobalide, a constituent of Ginkgo biloba. Brain Res. 755, 347–350. Ahlemeyer, B., Krieglstein, J., 2003. Neuroprotective effects of Klein, J., Weichel, O., Hilgert, M., Rupp, J., Chatterjee, S.S., Ginkgo biloba extract. Cell. Mol. Life Sci. 60, 1779–1792. Nawrath, H., 2003. Excitotoxic hippocampal membrane Bastianetto, S., Ramassamy, C., Dore, S., Christen, Y., Poirier, J., breakdown and its inhibition by bilobalide: role of chloride Quirion, R., 2000. The Ginkgo biloba extract (EGb 761) protects fluxes. Pharmacopsychiatry 36 (Suppl. 1), S78–S83. hippocampal neurons against cell death induced by ß-amyloid. Krieglstein, J., Ausmeier, F., El-Abhar, H., Lippert, K., Welsch, M., Eur. J. Neurosci. 12, 1882–1890. Ruppalla, K., Henrich-Noack, P., 1995. Neuroprotective effects Berger, A.J., Dieudonne, S., Ascher, P., 1998. Glycine uptake governs of Ginkgo biloba constituents. Eur. J. Pharm. Sci. 3, 39–48. glycine site occupancy at NMDA receptors of excitatory Luo, Y., Smith, J.V., Paramavisam, V., Burdick, A., Curry, K.J., synapses. J. Neurophysiol. 80, 3336–3340. Buford, J.P., Khan, I., Netzer, W.J., Xu, H., Butko, P., 2002. Bloomquist, J.R., Soderlund, D.M., 1985. Neurotoxic insecticides Inhibition of amyloid-ß aggregation and caspase-3 activation inhibit GABA-dependent chloride uptake by mouse brain by the Ginkgo biloba extract EGb761. Proc. Natl. Acad. Sci. U. S. A. vesicles. Biochem. Biophys. Res. Commun. 133, 37–43. 99, 12197–12202. Chandrasekaran, K., Mehrabian, Z., Spinnewyn, B., Drieu, K., Lynch, J.W., 2004. Molecular structure and function of the glycine Fiskum, G., 2001. Neuroprotective effects of bilobalide, a receptor chloride channel. Physiol. Rev. 84, 1051–1095. component of the Ginkgo biloba extract (EGb761), in gerbil global MacGregor, D.G., Avshalumov, M.V., Rice, M.E., 2003. Brain edema brain ischemia. Brain Res. 922, 282–292. induced by in vitro ischemia: causal factors and Chatterjee, S.S., Kondratskaya, E.L., Krishtal, O.A., 2003. neuroprotection. J. Neurochem. 85, 1402–1411. Structure-activity studies with Ginkgo biloba extract Mdzinarishvili, A., Kiewert, C., Kumar, V., Hillert, M., Klein, J., 2006. constituents as receptor-gated chloride channel blockers and Bilobalide prevents ischemia-induced edema formation in vitro modulators. Pharmacopsychiatry 36 (Suppl. 1), S68–S77. and in vivo. Neuroscience 144, 217–222. Colciaghi, F., Borroni, B., Zimmermann, M., Bellone, C., Longhi, A., Oken, B.S., Storzbach, D.M., Kaye, J.A., 1998. The efficacy of Ginkgo Padovani, A., Cattabeni, F., Christen, Y., Di Luca, M., 2004. biloba on cognitive function in Alzheimer disease. Arch. Neurol. Amyloid precursor protein metabolism is regulated toward 55, 1409–1415. alpha-secretase pathway by Ginkgo biloba extracts. Neurobiol. Saransaari, P., Oja, S.S., 1994. Glycine release from hippocampal Dis. 16, 454–460. slices in developing and ageing mice: modulation by Danbolt, N.C., 2001. Glutamate uptake. Prog. Neurobiol. 65, 1–105. glutamatergic receptors. Mech. Ageing Dev. 76, 113–124. Danysz, W., Parsons, C.G., 1998. Glycine and N-methyl-D-aspartate Saransaari, P., Oja, S.S., 2001. Characteristics of hippocampal receptors: physiological significance and possible glycine release in cell-damaging conditions in the adult and therapeutic applications. Pharmacol. Rev. 50, 597–659. developing mouse. Neurochem. Res. 26, 845–852. Davies, J.A., Johns, L., Jones, F.A., 2003. Effects of bilobalide on Sasaki, K., Hatta, S., Wada, K., Itoh, M., Yoshimura, T., Haga, M., cerebral amino acid neurotransmission. Pharmacopsychiatry 2002. Effects of chronic administration of bilobalide on amino 36 (Suppl. 1), S84–S88. acid levels in mouse brain. Cell. Mol. Biol. (Noisy-le-grand) 48, DeFeudis, F.V., 2002. Bilobalide and neuroprotection. Pharmacol. 681–684. Res. 46, 565–568. Schell, M.J., Molliver, M.E., Snyder, S.H., 1995. d-Serine, an DeFeudis, F.V., Drieu, K., 2000. Ginkgo biloba extract (EGb761) and endogenous synaptic modulator: localization to astrocytes and 150 BRAIN RESEARCH 1201 (2008) 143– 150

glutamate-stimulated release. Proc. Natl. Acad. Sci. U. S. A. 92, Weichel, O., Hilgert, M., Chatterjee, S.S., Lehr, M., Klein, J., 1999. 3948–3952. Bilobalide, a constituent of Ginkgo biloba, inhibits

Tsai, G., Ralph-Williams, R.J., Martina, M., Bergeron, R., NMDA-induced phospholipase A2 activation and phospholipid Berger-Sweeney, J., Dunham, K.S., Jiang, Z., Caine, S.B., breakdown in rat hippocampus. Naunyn-Schmiedeberg's Arch. Coyle, J.T., 2004. Gene knockout of glycine transporter 1: Pharmacol 360, 609–615. characterization of the behavioral phenotype. Proc. Natl. Acad. Weinges, K., Bähr, W., 1969. Kondensierte Ringsysteme II. Bilobalid, Sci. U. S. A. 101, 8485–8490. ein neues Sesquiterpen mit tertiärer Butylgruppe aus den Vogel, H.G., Vogel, W.H., 2002. Drug discovery and evaluation: Blättern von Ginkgo biloba L. Liebig's Ann. Chem. 724, 214–216. pharmacological assays. Springer electronic media, Wood, P.L., 1995. The co-agonist concept: is the NMDA-associated Heidelberg, Germany. glycine receptor saturated in vivo? Life Sci. 57, 301–310.