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Terpene Trilactones from Ginkgo biloba Are Antagonists of Cortical Glycine and GABAA Receptors*

Open AccessPublished:September 22, 2003DOI:https://doi.org/10.1074/jbc.M304034200
      Glycine and γ-aminobutyric acid, type A (GABAA) receptors are members of the ligand-gated ion channel superfamily that mediate inhibitory synaptic transmission in the adult central nervous system. During development, the activation of these receptors leads to membrane depolarization. Ligands for the two receptors have important implications both in disease therapy and as pharmacological tools. Terpene trilactones (ginkgolides and bilobalide) are unique constituents of Ginkgo biloba extracts that have various effects on the central nervous system. We have investigated the relative potency of these compounds on glycine and GABAA receptors. We find that most of the ginkgolides are selective and potent antagonists of the glycine receptor. Bilobalide, the single major component in G. biloba extracts, also reduces glycine-induced currents, although to a lesser extent. Both ginkgolides and bilobalide inhibit GABAA receptors, with bilobalide demonstrating a more potent effect. Additionally, we provide evidence that open channels are required for glycine receptor inhibition by ginkgolides. Finally, we employ molecular modeling to elucidate the similarities and differences in the structure of the terpene trilactones to account for the pharmacological properties of these compounds and demonstrate a striking similarity between ginkgolides and picrotoxinin, a GABAA and recombinant glycine α-homomeric receptor antagonist.
      Glycine and γ-aminobutyric acid receptors (GlyRs
      The abbreviations used are: GlyR
      glycine receptor
      GABA
      γ-aminobutyric acid
      GABAA
      GABA type A
      GABAAR
      GABAA receptor
      GA
      ginkgolide A
      GB
      ginkgolide B
      GC
      ginkgolide C
      GM
      ginkgolide M
      GJ
      ginkgolide J
      BB
      bilobalide
      PTX
      picrotoxinin
      ACSF
      artificial cerebrospinal fluid.
      1The abbreviations used are: GlyR
      glycine receptor
      GABA
      γ-aminobutyric acid
      GABAA
      GABA type A
      GABAAR
      GABAA receptor
      GA
      ginkgolide A
      GB
      ginkgolide B
      GC
      ginkgolide C
      GM
      ginkgolide M
      GJ
      ginkgolide J
      BB
      bilobalide
      PTX
      picrotoxinin
      ACSF
      artificial cerebrospinal fluid.
      and GABAARs) are anion-selective ligand-gated ion channels, which together with the cation-selective nicotinic acetylcholine and serotonin receptors constitute a superfamily of membrane receptors that mediate fast chemical synaptic transmission in the nervous system. These receptors share several structural similarities, i.e. a pentameric arrangement of subunits, each composed of four transmembrane domains (M1-M4) and an extracellular 15-residue Cys-loop motif-bearing N terminus (
      • Ortells M.O.
      • Lunt G.G.
      ,
      • Leite J.F.
      • Cascio M.
      ). In the adult, GlyRs consisting of α1-α4 and β subunits are found primarily in spinal cord and brain stem but are also present in higher brain regions such as hippocampus. During embryonic development, functional GlyRs are also expressed in the neocortex (
      • Flint A.C.
      • Liu X.
      • Kriegstein A.R.
      ) with α2 as the predominant subunit (
      • Betz H.
      • Kuhse J.
      • Schmieden V.
      • Laube B.
      • Kirsch J.
      • Harvey R.J.
      ). Few antagonists for GlyRs are known; the classical example is strychnine, which is a competitive antagonist. Also, the plant alkaloids picrotin and picrotoxinin (components of picrotoxin) are non-use-dependent antagonists that are equally efficacious in blocking recombinant homo-oligomeric GlyRs containing α subunits (
      • Betz H.
      • Harvey R.J.
      • Schloss P.
      ) but not the native α/β heteromers. GABAARs are functionally expressed very early in cortical development by proliferating precursor cells (
      • LoTurco J.J.
      • Owens D.F.
      • Heath M.J.
      • Davis M.B.
      • Kriegstein A.R.
      ), now known to be radial glial cells (
      • Noctor S.C.
      • Flint A.C.
      • Weissman T.A.
      • Dammerman R.S.
      • Kriegstein A.R.
      ,
      • Malatesta P.
      • Hartfuss E.
      • Gotz M.
      ), and by immature cortical neurons. The subunit composition of GABAARs changes significantly during development, and this is reflected in the pharmacological properties of the GABA-induced responses (
      • Owens D.F.
      • Liu X.
      • Kriegstein A.R.
      ). GABAARs have been implicated not only in synaptic signaling but also in a number of developmental events including proliferation and migration (
      • Owens D.F.
      • Kriegstein A.R.
      ). It is also likely that GlyRs and GABAARs have overlapping pathways during development because both receptors activate chloride channels and indirectly induce an increase in intracellular calcium in embryonic cortical neurons, thereby mediating a variety of cellular processes.
      Plant and animal products traditionally provide a rich source of drug candidates and pharmacological tools. The tree Ginkgo biloba has long been believed to have medicinal properties, and its extracts are among the most widely sold herbal supplements in the world. G. biloba extracts are standardized according to their content of flavonoids (22-24%) and terpene trilactones (6-8%), which are believed to be the active components. Although the bioavailability of flavonoids is limited, terpene trilactones, in particular ginkgolides A and B (GA and GB), and bilobalide (BB) are highly bioavailable (
      • DeFeudis F.V.
      • Drieu K.
      ).
      Extracts of G. biloba have been shown to be effective in symptomatic treatment of mild to moderate dementia of Alzheimer disease type as well as dementia of cerebrovascular origin (
      • DeFeudis F.V.
      • Drieu K.
      ) and have been reported to improve memory, although the latter claim remains controversial (
      • Solomon P.R.
      • Adams F.
      • Silver A.
      • Zimmer J.
      • DeVeaux R.
      ). Although a number of studies have shown various effects of G. biloba extract, very little is known about the direct effect of the individual components on the central nervous system. Recent work has indicated that ginkgolides and BB might interact with inhibitory signaling in the brain. A recent report suggested that GB inhibits glycine receptors in the hippocampus (
      • Kondratskaya E.L.
      • Lishko P.V.
      • Chatterjee S.S.
      • Krishtal O.A.
      ), and several reports have shown that BB interferes with the GABAergic system, although both enhancement and inhibition have been reported (
      • Sasaki K.
      • Hatta S.
      • Haga M.
      • Ohshika H.
      ,
      • Sasaki K.
      • Hatta S.
      • Wada K.
      • Ohshika H.
      • Haga M.
      ,
      • Sasaki K.
      • Oota I.
      • Wada K.
      • Inomata K.
      • Ohshika H.
      • Haga M.
      ).
      For these reasons we have addressed exactly how the unique components of G. biloba, ginkgolides and bilobalide, may exert their effect in the central nervous system. We show that the ginkgolides and bilobalide are selective antagonists of glycine and GABAA receptors, we demonstrate that GC inhibition of embryonic cortical GlyRs requires open channels, and that GB has several structural similarities to the GABAA receptor antagonist, picrotoxinin.

      EXPERIMENTAL PROCEDURES

      All experiments followed National Institutes of Health guidelines and were done in full compliance with the Columbia University Institutional Animal Care and Use Committee.
      Brain Slice Preparation—Timed pregnant Sprague-Dawley rats (Taconic, New York) at embryonic day E20 were used for all experiments. Rats were anesthetized (ketamine, 90 mg/kg, and xylazine, 10 mg/kg), and embryos were removed for further dissection. Following rapid decapitation, the brain was removed in chilled artificial cerebrospinal fluid (ACSF; 125 mm NaCl, 5 mm KCl, 1.25 mm NaH2PO4, 1 mm MgSO4, 2 mm CaCl2, 25 mm NaHCO3, and 25 mm glucose, pH 7.4, 310 mosmol/liter). The dissected brain was placed in 4% low melting agarose (Fisher Scientific) in ACSF. Agarose was cooled on ice and allowed to solidify, and the embedded brain was sliced into coronal sections (400 μm) in ice-cold ACSF using a Leica VT100S vibrating blade microtome (Nussloch, Germany). Slices were allowed to recover at room temperature in oxygenated (95% O2 and 5% CO2) ACSF. They were subsequently used for electrophysiological recording or loaded for calcium imaging.
      Calcium Imaging—Brain slices were bath-loaded with the acetoxymethyl ester form of the calcium indicator dye fluo-3 (fluo-3 AM, 10-15 μm, Molecular Probes, Eugene, OR). Loading was performed in the dark, at room temperature for 1-3 h. Loaded slices were placed in an imaging chamber on the stage of an upright compound microscope (Olympus BX50-WI, Tokyo, Japan) and continuously perfused with oxygenated ACSF. Epifluorescence imaging of fluo-3 was performed using a 100-watt mercury light source and a low light charge-coupled device camera (Dage-MTI 300-T, Michigan City, IN). For fluo-3 imaging, we used following fluorescence filters (Chroma Technology, Brattleboro, VT): excitation filter, 480 ± 20 nm; dichroic mirror, 505-nm long-pass; and emission filter, 535 ± 25 nm. Cells were imaged using 10× water immersion objective, and the photobleaching was minimized by controlling a shutter positioned in the excitation light path (Uniblitz S25, Vincent Associates, Rochester, NY). Time-lapse images were acquired every 2 s using the Scion Image program on a Macintosh G3 computer equipped with video frame grabber (Scion LG-3, Scion Corp., Frederick, MD). Fluorescence changes were measured in selected cells using Scion Image.
      Pharmacological Agents—Ginkgolides A, B, C, and J (GA, GB, GC, and GJ) and bilobalide were obtained by extraction of leaves from G. biloba, purification by column chromatography, and recrystallization, as previously described (
      • Lichtblau D.
      • Berger J.M.
      • Nakanishi K.
      ,
      • van Beek T.A.
      • Lelyved G.P.
      ). Ginkgolide M (GM) was from previous studies (
      • Nakanishi K.
      ). The purity of these compounds was >99%, as determined by 1H NMR. Stock solutions of ginkgolides, bilobalide, and picrotoxin (RBI, Natick, MA) were prepared by dissolving in Me2SO to 10 mm and were stored at -20 °C until use. Glycine, taurine (Sigma), and γ-aminobutyric acid (RBI, Natick, MA) were prepared as stock solutions in distilled deionized H2O and diluted in ACSF to final concentration. All drugs were applied focally by DAD-12 Superfusion System (ALA Scientific Instruments, Westbury, NY).
      Electrophysiology—Whole-cell patch-clamp recordings were performed with an EPC-9 patch-clamp amplifier (HEKA Electronic, Lambrecht, Germany). Data were acquired with HEKA Pulse v. 8.0 software (HEKA Electronic, Lambrecht, Germany). Borosilicate glass (Warner Instrument Corp., Hamden, CT) electrode pipettes (5-8 megohms) were filled with 130 mm CsCl, 2 mm CaCl2, 10 mm HEPES, and 11 mm EGTA (pH 7.4 at 25 °C, 265-275 mosmol/liter). Unless otherwise indicated, neurons were voltage-clamped at -60 mV.
      Data Analysis—All calcium imaging data were presented as a change in fluorescence over base-line fluorescence, ΔF/F0 = (F - F0)/F0, and plotted over time. Analysis was done by averaging 10-15 cells for each condition, and data were plotted as the mean ± S.E. Statistical significance between two groups was evaluated with a Student's t test. Probability (p) values of less than 0.01 were considered to be statistically significant.
      Molecular Modeling—Molecular mechanics calculations with MM2* force field and Monte Carlo conformational searches were executed with MacroModel 6.0 (Schrödinger, Inc., Portland, OR). Non-empirical molecular modeling calculations were executed with Jaguar 4.1 (Schrödinger, Inc., Portland, OR) at the B3LYP level with 6-31 G basis set (
      • Hariharan P.C.
      • Pople J.A.
      ) including the geometry optimization. GB and picrotoxinin (PTX) were overlaid using a rigid body fit between C-11, C-15, O-6, and O-10 of GB and C-15, C-14, O-5, and O-3 of PTX as seen in Fig. 5a. Monte Carlo simulations revealed nine conformers for GA within 10 kcal/mol of the global minimum, whereas GB had 32 unique conformations in the 10 kcal/mol range. X-ray crystallographic structures were obtained from Cambridge structural data base using ConQuest 1.1 (CCDC, Cambridge, UK) and compared with the minimized conformations. The comparison showed that root mean square values were 0.4 Å for GB and 0.6 Å for PTX.
      Figure thumbnail gr5
      Fig. 5Comparison of GB with PTX by molecular modeling.a, the structures of GB (blue) and PTX (orange) were minimized in MacroModel using density functional theory parameters and overlaid. GB and PTX are both cave molecules and have a highly similar position of important functional groups that all can be overlaid. Red, carbonyl. b, structures of GB and PTX indicating the similar distances between the carbonyl oxygen and similar position of the lipophilic tert-butyl and isoprenyl groups, respectively.

      RESULTS

      Ginkgolides and bilobalide, compounds classified as terpene trilactones, share several structural similarities, in particular three lactones and a tert-butyl group. The five ginkgolides differ only by the number and placement of hydroxyl groups (Fig. 1a) (
      • Nakanishi K.
      ). GA, GB, and GC are found in both leaves and root bark of G. biloba, whereas GJ and GM are found only in the leaves and root bark, respectively. BB is chemically related to the ginkgolides, having three lactones but only one carbocycle, and is found in the leaves (Fig. 1a) (
      • Nakanishi K.
      ).
      Figure thumbnail gr1
      Fig. 1Effects of terpene trilactones and picrotoxin on GlyR.a, the ginkgolides are diterpenoids with a unique structure comprising a highly oxygenated carbon skeleton, including three lactone rings and a tert-butyl group. Bilobalide is chemically related to the ginkgolides, in that it is a sesquiterpenoid trilactone with a tert-butyl group, but in contrast to the ginkgolides it has only one carbocycle making it smaller and more compact. b, Gly (200 μm) leads to an increase in intracellular Ca2+ in pyramidal neurons in cortical slices of embryonic rat (E20) brains. Incubation of the slices with GB (10 μm) for 1 min followed by co-application of Gly (200 μm) and GB (10 μm) reduces the response. The application was followed by a 5-min washing with GB, and the incubation and co-application were repeated a total of three times. c, consecutive co-application of glycine and GB reduced the glycine responses to 67, 48, and 30% of the control. d, GA, GC, GJ, GM, BB, and picrotoxin (PC) were investigated as described for GB, showing that GB was the most potent inhibitor. Asterisks indicate a statistically significant reduction of glycine-induced responses. For GB, GC, and GM, p < 0.001 (**); for GA and GJ, p < 0.01 (*).
      Ginkgolides Antagonize Glycine Receptors—GlyR activation opens a chloride ion channel and induces a membrane depolarization in embryonic cortical neurons because of a high intracellular chloride concentration in immature neurons (
      • Flint A.C.
      • Liu X.
      • Kriegstein A.R.
      ). The resulting depolarization leads to the activation of voltage-gated calcium channels and a subsequent influx of calcium ions. We took advantage of this mechanism and used calcium imaging to compare the potency of various ginkgolides on glycine-induced responses in embryonic cortical neurons.
      Application of glycine (200 μm) resulted in an increase in intracellular calcium, which was reduced by co-application with GB (10 μm) (Fig. 1b). The effect was highly use-dependent as repeated applications of glycine and GB gradually decreased the glycine response, which by the third application was reduced to 30% (Fig. 1b). The effect was reversible, as seen by the recovery of glycine responses after a 20-min wash with artificial cerebrospinal fluid (Fig. 1c). The glycine responses were reduced to 67, 48, and 30%, respectively, by three consecutive co-applications of glycine and GB (Fig. 1c). These findings are in agreement with an initial report on the effect of GB on glycine receptors in hippocampal pyramidal neurons isolated from adult rats (
      • Kondratskaya E.L.
      • Lishko P.V.
      • Chatterjee S.S.
      • Krishtal O.A.
      ).
      We wondered whether the other ginkgolides and bilobalide also antagonized glycine responses because they share a similar chemical structure, and their affect has not yet been reported. We tested GA, GB, GC, GJ, and GM, together with BB and picrotoxin at the same low concentration (10 μm), to determine the relative inhibiting potency of these compounds. GB, GC, and GM were the most potent antagonists and by the third application decreased glycine responses to 30-40% of control, whereas GA and GJ reduced the response to a lesser extent (70-80% of control, Fig. 1d). The sesquiterpenoid BB had no effect on glycine-induced calcium increases in this assay at the concentration tested. Picrotoxin had no effect on glycine responses (Fig. 1d).
      Changes in intracellular calcium only indirectly reflect activation of GlyRs and depend on the kinetics of the voltage-gated calcium channels. In addition, restoration of base-line calcium levels depends both on the GlyR gating and on the recovery of the resting intracellular calcium concentration. To directly observe the effect of BB and the most potent ginkgolides, GB and GC, on glycine-induced currents, we used whole-cell patchclamp recordings.
      A short (1-min) preincubation in GB (10 μm), followed by its co-application with glycine (500 μm) resulted in potent inhibition of the glycine response in cortical neurons (Fig. 2a). Because taurine has been suggested as an endogenous ligand for GlyR (
      • Flint A.C.
      • Liu X.
      • Kriegstein A.R.
      ,
      • Mori M.
      • Gahwiler B.H.
      • Gerber U.
      ), we also tested the effect of GB on taurine (5 mm)-induced responses (Fig. 2b). The effect was consistent with GlyR inhibition and was use-dependent and reversible.
      Figure thumbnail gr2
      Fig. 2Whole-cell recordings demonstrate effects of GB and BB on glycine receptors.a, application of Gly (500 μm) leads to an inward current that was reduced by repeated preincubation with GB (10 μm). The blocking effect of GB was reversible, as the Gly response recovered after a 20-min wash with ACSF. b, taurine (Tau, 5 mm) responses were affected in a similar fashion. c, BB (10 μm) reduced Gly (200 μm) response to 62.99 ± 5.17% of the control values. GB at the same concentration (10 μm) almost completely abolished glycine responses (8.67 ± 3.40%).
      Because BB represents about 50% of the terpene trilactones, being the major single component in G. biloba extracts, we wondered if it would reduce glycine-induced currents. BB (10 μm) was much less potent than GB (10 μm), but it still induced a significant decrease of glycine (200 μm) responses (Fig. 2c). Contrary to the pattern of GB inhibition, BB antagonism did not change significantly with repeated application. Interestingly, both BB and GB showed a more pronounced effect on glycine-induced currents, measured in whole-cell patch-clamp recordings, compared with their effect on intracellular calcium increases (compare Figs. 1d and 2c). Therefore, the fact that we did not observe a significant decrease in the glycine-induced calcium response in the presence of BB most likely results from an incomplete block of glycine-induced currents by BB that was not sufficient to alter the intracellular calcium change.
      Ginkgolide C Requires an Open Channel for the Inhibition of Embryonic Glycine Receptors—GC is one of the three most potent glycine receptor antagonists present in G. biloba extracts. To get a better understanding of the use-dependent effect characteristic of ginkgolides, we used a whole-cell patchclamp recording and applied glycine (500 μm) every 60 s in the presence of 10 μm GC (Fig. 3a) following two initial control glycine applications. Even after a short 1-min GC preincubation, the glycine response was dramatically reduced (to 32.7 ± 4.5%) and quickly (after 3-4 applications) reached a plateau. The effect was partially reversible as demonstrated by a slow recovery of the glycine response after a washout period. Control glycine applications were done every minute in the absence of GC. The glycine responses showed a slight desensitization with repeated application. To determine whether open channels are necessary for the use-dependent effect, we examined the effect of prolonged preincubation on the degree of inhibition. We used a double application protocol, wherein the first glycine application was a control, and during the second, glycine and GC were co-applied following a 1- or 10-min-long GC preincubation (Fig. 3b). In control experiments, glycine was applied twice without GC. Results are presented as the ratio between the second and the first glycine responses recorded from the same cell. Our data show that the degree of inhibition did not depend on the length of preincubation. Moreover, when glycine and GC were co-applied without any preincubation, responses were not only reduced, but the response kinetics were dramatically changed reflecting an abrupt blockade of glycine channels even before the end of drug application (Fig. 3c). These data, taken together, suggest a requirement for open channels in glycine receptor inhibition.
      Figure thumbnail gr3
      Fig. 3GC inhibition of embryonic glycine receptors requires open channels.a, after the two initial 5-s-long control glycine (500 μm) applications (Ctrl), we applied GC (10 μm) for 60 s before co-applying the drug with glycine (filled circles, n = 12). Glycine was co-applied with GC every 60 s until it reached a plateau (applications 1-4). The effect of GC was partially reversible after washout with ACSF (Rec1-Rec4). Control glycine application (empty circles) was done following the same protocol in the absence of GC (n = 5). Glycine-induced currents showed a slight desensitization with repeated application. Data points represent the ratio of the response to the initial glycine response. b, longer GC preincubation did not increase the degree of inhibition. Neurons were preincubated in GC for 1 or 10 min following the initial glycine stimulation, and then glycine and GC were co-applied (n = 5). The degree of GC inhibition is presented as the ratio of the glycine response after GC preincubation to a control glycine response from the same cell. In the absence of GC there is no significant decrease in glycine-induced current (n = 5). For a and b data were presented as mean values ± S.E. c, glycine responses decreased when co-applied with GC even without preincubation in GC (n = 6). In addition, the response kinetics changed. Glycine and GC (Gly+GC) were co-applied 1 min after the control glycine application (Gly).
      Bilobalide and Ginkgolide B Antagonize GABAAReceptors—The GABAA receptor subtype is the only functional ionotropic GABA receptor type expressed in embryonic pyramidal neurons as demonstrated by a complete block of GABA-induced current with 50 μm bicuculine (
      • Owens D.F.
      • Liu X.
      • Kriegstein A.R.
      ). Whole-cell recordings performed on cortical neurons demonstrated attenuation of GABAA responses by both GB and BB, with BB proving the more potent of the two compounds. A short (1-min) incubation in the drug was followed by co-application of the drug with GABA (30 μm). Fig. 4 illustrates the typical responses to 30 μm GABA that were reduced by co-application with 50 μm GB (Fig. 4a) or BB (Fig. 4b). At this concentration, GB and BB reduced GABA responses to 63.2 ± 0.3% (n = 5) and 46.8 ± 0.3% (n = 5), respectively. Interestingly, the effect of GB on GABA responses was not use-dependent, as tested by three successive applications in which the response was reduced no further (not shown). The dose-response relationships for the inhibitory action of GB and BB on GABA-induced (30 μm) responses are illustrated in Fig. 4c. Calculated values for the IC50 (GB, IC50 = 73 μm; BB, IC50 = 46 μm) demonstrated that BB is about 1.6 times as potent as GB. The IC50 value for BB was significantly smaller than the one obtained on recombinant α1β2γ2L GABAAR expressed in Xenopus oocytes (
      • Huang S.H.
      • Duke R.K.
      • Chebib M.
      • Sasaki K.
      • Wada K.
      • Johnston G.A.
      ) most likely reflecting a different effect of BB on receptors with different subunit composition.
      Figure thumbnail gr4
      Fig. 4Effects of GB and BB on GABAAR.a and b, whole-cell recordings from E20 cortical neurons demonstrate typical GABA (30 μm) responses, which were then partially blocked by co-application with GB or BB following a 1-min preincubation. The final trace is the response after a 1-min washout (wash). GB and BB (at 50 μm) reduced GABA responses to 63.2 ± 0.3 and 46.8 ± 0.3%, respectively. c, inhibition response curves for GB and BB obtained for GABA (30 μm) responses. I/I0 values are plotted against the log of the drug concentration. The error bars represent S.D. IC50 values are calculated to be about 73 and 46 μm for GB and BB, respectively.
      Ginkgolide B and Picrotoxinin Show Structural Similarity—These findings led us to investigate a structural rationalization for the effects of ginkgolides, particularly of GB, and to compare them with PTX. We analyzed their molecular properties through modeling (
      • Mohamadi F.
      ) and validated our approach by comparison with the x-ray crystallographic structures of GB (
      • Dupont L.
      • Dideberg O.
      • Germain G.
      • Braquet P.
      ) and PTX (
      • Andrews P.R.
      ). The minimized structures were found to be similar to those determined by x-ray (root mean square deviations: GB = 0.4 Å and PTX = 0.6 Å), although there were some differences in the extent of intramolecular hydrogen bonding.
      The minimized structures of GB and PTX showed remarkable similarity in several respects. Each molecule forms a cave structure, where the bottom is composed of a five- or sixmembered carbocycle, and the walls are a pair of lactones (Fig. 5). The caves are of similar size, the distance between the lactone carbonyl oxygen molecules being 5.8 and 5.2 Å for GB and PTX, respectively, with a root mean square deviation of 0.4 Å for the rigid body fit of the two lactone rings in both molecules (Fig. 5). Moreover, the orientation of the lactone pairs is of a similar magnitude, their projection angles being -26° in GB and -24° in PTX. In addition to this similarity in core structure, the two compounds have important moieties located in analogous positions. The tert-butyl group of GB is located in the same region as the isoprenyl group of PTX, although the tert-butyl group is bulkier, and the 1-OH of GB overlays very well with the 6-OH of PTX (Fig. 5).
      Structural Investigation of the Relative Activities of Ginkgolides—We also investigated differences in activity among the ginkgolides from a structural perspective. GB, GC, and GM, the more potent glycine antagonists, share a 1-OH, whereas the less potent GA and GJ do not (Fig. 1a). The importance of the 1-OH for the core structure was investigated via a Monte Carlo conformational search performed on GA and GB as representative compounds. All conformers of GA were found to have larger distance-spanning lactone rings C and F compared with GB. Moreover, very little flexibility in either lactone ring of GB was seen, suggesting that the larger lactone distance in GA is significant. The 1-OH in GB may form a hydrogen bond with the 10-OH to hold the two lactones together.
      Comparison of Bilobalide with Ginkgolide B and Picrotoxinin—BB is structurally related to GB and PTX in that it contains three lactone groups but unlike GB has only one carbocycle. BB was overlaid with GB and PTX, respectively (data not shown). A comparison of BB with GB showed that the proposed important functional groups of GB and BB can be overlaid individually but not at the same time, underlining important differences in GB and BB. Comparing the minimized structures of BB and PTX showed much less similarity. In neither of those cases is there a striking overlap of lactones, tert-butyl/isoprenyl groups, or hydroxyl functionalities as was observed for GB and PTX (Fig. 5).

      DISCUSSION

      We have shown that the terpene trilactone constituents of G. biloba have antagonistic activity at the two major types of ionotropic receptors mediating fast inhibitory synaptic transmission in the adult mammalian central nervous system, both of which have also been shown to be active during development. The ginkgolides, in particular GB and GC, antagonize glycine responses in the embryonic rat cortex in a use-dependent manner as was suggested previously for GB in hippocampal neurons isolated from adult rats (
      • Kondratskaya E.L.
      • Lishko P.V.
      • Chatterjee S.S.
      • Krishtal O.A.
      ). Moreover, GB affects GABAARs, only at much higher concentrations, thus suggesting that GB is the first selective non-competitive antagonist of GlyRs. We have also investigated the relative potency of the ginkgolides at GlyRs, showing that GB, GC, and GM are significantly more potent than GA and GJ. These differences can be explained structurally as GB, GC, and GM possess a 1-OH group, whereas GA and GJ do not, thus suggesting that the 1-OH group is important for activity. However, when performing Monte Carlo simulations of GA and GB we have shown that the relative position of the lactone groups is different in GA and GB. Thus, the difference in activity of GA and GB could be because of a direct interaction of 1-OH with the receptor, or it could be that 1-OH, by hydrogen-bonding, keeps the lactone groups in GB in a position that is more favorable for receptor interaction.
      Additionally, we studied BB, the major single component in G. biloba extract constituting 3-4%. BB is structurally related to ginkgolides and shares several functional groups, in particular three lactone groups and a tert-butyl group, and has been postulated to have anxiolytic properties and possibly to be useful as a neuroprotective agent (
      • DeFeudis F.V.
      ). Recent studies indicated that BB might modulate GABAergic neurotransmission, as BB has been reported to elevate the GABA levels, probably by enhancing glutamic acid decarboxylase activity (
      • Sasaki K.
      • Hatta S.
      • Haga M.
      • Ohshika H.
      ,
      • Sasaki K.
      • Hatta S.
      • Wada K.
      • Ohshika H.
      • Haga M.
      ), reduce muscimol responses (
      • Sasaki K.
      • Oota I.
      • Wada K.
      • Inomata K.
      • Ohshika H.
      • Haga M.
      ), and decrease the frequency of GABA uptake inhibitor-induced depolarizations (
      • Jones F.A.
      • Chatterjee S.S.
      • Davies J.A.
      ). BB has also been reported to antagonize recombinant α1β2γ2L GABAAR (
      • Huang S.H.
      • Duke R.K.
      • Chebib M.
      • Sasaki K.
      • Wada K.
      • Johnston G.A.
      ). In contrast, the effect of BB on glycine-induced responses has not been demonstrated. Here we show that BB antagonizes GABAAR activation and also reduces glycine responses, although at higher concentrations than GB. Interestingly, antagonism of both GABAA and Gly receptors by BB was not use-dependent, whereas the blockade of GlyRs by GB and GC was. This suggests that BB has a different mode of action than the ginkgolides at glycine receptors, perhaps also relating to its lower potency. Several terpenoid natural products containing lactones, such as anisatin and the picrodendrins, and the γ-butyrolactones have been suggested to bind to and antagonize GABAARs at the same site (
      • Kuriyama T.
      • Schmidt T.J.
      • Okuyama E.
      • Ozoe Y.
      ,
      • Klunk W.E.
      • Kalman B.L.
      • Ferrendelli J.A.
      • Covey D.F.
      ). The terpene trilactones may also interact with this site, although this remains to be investigated.
      Using molecular modeling we have shown structural similarities between GB and PTX. Both molecules form a cave structure of similar size. The absolute and relative positions of the two lactone groups are very similar. This is likely to be functionally relevant, as structure-activity relationship studies of PTX have revealed that the lactone fused between C-3 and C-5 is essential for activity (
      • Schmidt T.J.
      • Okuyama E.
      • Fronczek F.R.
      ). In addition, the projected position of the tert-butyl group of GB is similar to the isoprenyl group of PTX, although the former is much more bulky. Hence, it is possible that the tert-butyl of GB fits into a lipophilic pocket in GlyRs, as does the isoprenyl group of PTX in GABAARs. The importance of this isoprenyl group for activity at GABAARs is well established, as the presence of an isopropyl alcohol moiety in picrotin, the other component of picrotoxin, leads to a 30-50-fold decrease in activity at GABAAR (
      • Schmidt T.J.
      • Okuyama E.
      • Fronczek F.R.
      ). Finally, the 1-OH of GB overlaid very well with the 6-OH of PTX. The 6-OH of PTX is very important for antagonism of GABAAR, as masking of the 6-OH of PTX by acetylation leads to loss of activity (
      • Schmidt T.J.
      • Okuyama E.
      • Fronczek F.R.
      ). Because docking studies have suggested that 6-OH forms hydrogen bonds with threonine residues in the M2 pore segment (
      • Zhorov B.S.
      • Bregestovski P.D.
      ), it is possible that 1-OH of GB has a similar function, particularly in light of evidence that open channels are necessary for inhibition. Previously it was suggested that GB caused an open-channel block of adult GlyRs (
      • Kondratskaya E.L.
      • Lishko P.V.
      • Chatterjee S.S.
      • Krishtal O.A.
      ). Our experiments demonstrate that GC inhibition of embryonic GlyRs is use-dependent but that the degree of inhibition does not depend on the length of the preincubation. These results are a consistent requirement for open channels in GC inhibition.
      We show here that GB and PTX share several structural features that may be important for their antagonistic effects. However, there are also distinct differences in their pharmacological action, i.e. PTX does not antagonize GlyRs containing β subunits, whereas GB does antagonize GlyRs containing both α and β subunits, as shown in adult rat hippocampal neurons (
      • Kondratskaya E.L.
      • Lishko P.V.
      • Chatterjee S.S.
      • Krishtal O.A.
      ). Whether ginkgolides are equally potent in inhibition of homomeric GlyRs remains to be investigated.
      Antagonists of inhibitory receptors in general, and GABAARs in particular, are known convulsive agents; hence the antagonizing effect of BB and GB on GABAARs might have wide ranging implications for individuals taking G. biloba extract, especially those with a lower threshold for seizures, such as epileptic patients. This risk is suggested by a recent report of two patients with well controlled epilepsy who had recurrent seizures within 2 weeks of commencing G. biloba extract that ceased after discontinuation of the supplement (
      • Granger A.S.
      ).
      In considering the role of GABAA and Gly receptors in cortical development it is also important to evaluate the potential teratogenic effects of G. biloba extract. Further studies are necessary to establish not only the effect of terpene trilactones on the developing and adult nervous system but also the combinatorial effect of all extract constituents.
      In conclusion, we have shown that the ginkgolides and bilobalide are antagonists of both Gly and GABAA receptors. In particular, GB and GC are selective and potent antagonists of the GlyRs thus being highly promising pharmacological tools for the study of this receptor. Molecular modeling reveals several structural similarities between GB and PTX, suggesting a similar mode of interaction with the GlyR and GABAAR, respectively. Finally, the inhibitory activity of these components of G. biloba extract, especially the action of the major constituent, BB, on the GABAAR may have important implications for people taking this supplement.

      Acknowledgments

      We thank Drs. Abhay Kini and David Owens for valuable suggestions on the manuscript. We thank Dr. Stephen Noctor for help with preparing figures.

      References

        • Ortells M.O.
        • Lunt G.G.
        Trends Neurosci. 1995; 18: 121-127
        • Leite J.F.
        • Cascio M.
        Mol. Cell. Neurosci. 2001; 17: 777-792
        • Flint A.C.
        • Liu X.
        • Kriegstein A.R.
        Neuron. 1998; 20: 43-53
        • Betz H.
        • Kuhse J.
        • Schmieden V.
        • Laube B.
        • Kirsch J.
        • Harvey R.J.
        Ann. N. Y. Acad. Sci. 1999; 868: 667-676
        • Betz H.
        • Harvey R.J.
        • Schloss P.
        Mohler, H. Handbook of Experimental Pharmacology. Springer-Verlag, Berlin2001: 150
        • LoTurco J.J.
        • Owens D.F.
        • Heath M.J.
        • Davis M.B.
        • Kriegstein A.R.
        Neuron. 1995; 15: 1287-1298
        • Noctor S.C.
        • Flint A.C.
        • Weissman T.A.
        • Dammerman R.S.
        • Kriegstein A.R.
        Nature. 2001; 409: 714-720
        • Malatesta P.
        • Hartfuss E.
        • Gotz M.
        Development. 2000; 127: 5253-5263
        • Owens D.F.
        • Liu X.
        • Kriegstein A.R.
        J. Neurophysiol. 1999; 82: 570-583
        • Owens D.F.
        • Kriegstein A.R.
        Nat. Rev. Neurosci. 2002; 3: 715-727
        • DeFeudis F.V.
        • Drieu K.
        Curr. Drug Targets. 2000; 1: 25-58
        • Solomon P.R.
        • Adams F.
        • Silver A.
        • Zimmer J.
        • DeVeaux R.
        JAMA. 2002; 288: 835-840
        • Kondratskaya E.L.
        • Lishko P.V.
        • Chatterjee S.S.
        • Krishtal O.A.
        Neurochem. Int. 2002; 40: 647-653
        • Sasaki K.
        • Hatta S.
        • Haga M.
        • Ohshika H.
        Eur. J. Pharmacol. 1999; 367: 165-173
        • Sasaki K.
        • Hatta S.
        • Wada K.
        • Ohshika H.
        • Haga M.
        Life Sci. 2000; 67: 709-715
        • Sasaki K.
        • Oota I.
        • Wada K.
        • Inomata K.
        • Ohshika H.
        • Haga M.
        Comp. Biochem. Physiol. Part C Pharmacol. Toxicol. Endocrinol. 1999; 124: 315-321
        • Lichtblau D.
        • Berger J.M.
        • Nakanishi K.
        J. Nat. Prod. 2002; 65: 1501-1504
        • van Beek T.A.
        • Lelyved G.P.
        J. Nat. Prod. 1997; 60: 735-738
        • Nakanishi K.
        Pure Appl. Chem. 1967; 14: 89-113
        • Hariharan P.C.
        • Pople J.A.
        Mol. Phys. 1974; 27: 209-214
        • Nakanishi K.
        J. Am. Chem. Soc. 1971; 93: 3544-3546
        • Mori M.
        • Gahwiler B.H.
        • Gerber U.
        J. Physiol. (Lond.). 2002; 539: 191-200
        • Huang S.H.
        • Duke R.K.
        • Chebib M.
        • Sasaki K.
        • Wada K.
        • Johnston G.A.
        Eur. J. Pharmacol. 2003; 464: 1-8
        • Mohamadi F.
        J. Comput. Chem. 1990; 11: 440-467
        • Dupont L.
        • Dideberg O.
        • Germain G.
        • Braquet P.
        Acta Crystallogr. Sect. C Cryst. Struct. Commun. 1986; 42: 1759-1762
        • Andrews P.R.
        Aust. J. Chem. 1983; 36: 2219-2225
        • DeFeudis F.V.
        Ginkgo Biloba Extract (EGb 761). Ullstein Medical, Wiesbaden1998 (From Chemistry to Clinic)
        • Jones F.A.
        • Chatterjee S.S.
        • Davies J.A.
        Amino Acids (Vienna). 2002; 22: 369-379
        • Kuriyama T.
        • Schmidt T.J.
        • Okuyama E.
        • Ozoe Y.
        Bioorg. Med. Chem. 2002; 10: 1873-1881
        • Klunk W.E.
        • Kalman B.L.
        • Ferrendelli J.A.
        • Covey D.F.
        Mol. Pharmacol. 1983; 23: 511-518
        • Schmidt T.J.
        • Okuyama E.
        • Fronczek F.R.
        Bioorg. Med. Chem. 1999; 7: 2857-2865
        • Zhorov B.S.
        • Bregestovski P.D.
        Biophys. J. 2000; 78: 1786-1803
        • Granger A.S.
        Age Ageing. 2001; 30: 523-525