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

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, 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 1The abbreviations used are: GlyRglycine receptorGABAγ-aminobutyric acidGABAAGABA type AGABAARGABAA receptorGAginkgolide AGBginkgolide BGCginkgolide CGMginkgolide MGJginkgolide JBBbilobalidePTXpicrotoxininACSFartificial cerebrospinal fluid.1The abbreviations used are: GlyRglycine receptorGABAγ-aminobutyric acidGABAAGABA type AGABAARGABAA receptorGAginkgolide AGBginkgolide BGCginkgolide CGMginkgolide MGJginkgolide JBBbilobalidePTXpicrotoxininACSFartificial 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 (1.Ortells M.O. Lunt G.G. Trends Neurosci. 1995; 18: 121-127Abstract Full Text PDF PubMed Scopus (467) Google Scholar, 2.Leite J.F. Cascio M. Mol. Cell. Neurosci. 2001; 17: 777-792Crossref PubMed Scopus (36) Google Scholar). 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 (3.Flint A.C. Liu X. Kriegstein A.R. Neuron. 1998; 20: 43-53Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar) with α2 as the predominant subunit (4.Betz H. Kuhse J. Schmieden V. Laube B. Kirsch J. Harvey R.J. Ann. N. Y. Acad. Sci. 1999; 868: 667-676Crossref PubMed Scopus (115) Google Scholar). 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 (5.Betz H. Harvey R.J. Schloss P. Mohler, H. Handbook of Experimental Pharmacology. Springer-Verlag, Berlin2001: 150Google Scholar) but not the native α/β heteromers. GABAARs are functionally expressed very early in cortical development by proliferating precursor cells (6.LoTurco J.J. Owens D.F. Heath M.J. Davis M.B. Kriegstein A.R. Neuron. 1995; 15: 1287-1298Abstract Full Text PDF PubMed Scopus (917) Google Scholar), now known to be radial glial cells (7.Noctor S.C. Flint A.C. Weissman T.A. Dammerman R.S. Kriegstein A.R. Nature. 2001; 409: 714-720Crossref PubMed Scopus (1536) Google Scholar, 8.Malatesta P. Hartfuss E. Gotz M. Development. 2000; 127: 5253-5263Crossref PubMed Google Scholar), 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 (9.Owens D.F. Liu X. Kriegstein A.R. J. Neurophysiol. 1999; 82: 570-583Crossref PubMed Scopus (155) Google Scholar). GABAARs have been implicated not only in synaptic signaling but also in a number of developmental events including proliferation and migration (10.Owens D.F. Kriegstein A.R. Nat. Rev. Neurosci. 2002; 3: 715-727Crossref PubMed Scopus (915) Google Scholar). 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. glycine receptor γ-aminobutyric acid GABA type A GABAA receptor ginkgolide A ginkgolide B ginkgolide C ginkgolide M ginkgolide J bilobalide picrotoxinin artificial cerebrospinal fluid. glycine receptor γ-aminobutyric acid GABA type A GABAA receptor ginkgolide A ginkgolide B ginkgolide C ginkgolide M ginkgolide J bilobalide picrotoxinin artificial cerebrospinal fluid. 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 (11.DeFeudis F.V. Drieu K. Curr. Drug Targets. 2000; 1: 25-58Crossref PubMed Scopus (373) Google Scholar). 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 (11.DeFeudis F.V. Drieu K. Curr. Drug Targets. 2000; 1: 25-58Crossref PubMed Scopus (373) Google Scholar) and have been reported to improve memory, although the latter claim remains controversial (12.Solomon P.R. Adams F. Silver A. Zimmer J. DeVeaux R. JAMA. 2002; 288: 835-840Crossref PubMed Scopus (246) Google Scholar). 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 (13.Kondratskaya E.L. Lishko P.V. Chatterjee S.S. Krishtal O.A. Neurochem. Int. 2002; 40: 647-653Crossref PubMed Scopus (49) Google Scholar), and several reports have shown that BB interferes with the GABAergic system, although both enhancement and inhibition have been reported (14.Sasaki K. Hatta S. Haga M. Ohshika H. Eur. J. Pharmacol. 1999; 367: 165-173Crossref PubMed Scopus (64) Google Scholar, 15.Sasaki K. Hatta S. Wada K. Ohshika H. Haga M. Life Sci. 2000; 67: 709-715Crossref PubMed Scopus (23) Google Scholar, 16.Sasaki K. Oota I. Wada K. Inomata K. Ohshika H. Haga M. Comp. Biochem. Physiol. Part C Pharmacol. Toxicol. Endocrinol. 1999; 124: 315-321Crossref PubMed Scopus (24) Google Scholar). 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. 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 (17.Lichtblau D. Berger J.M. Nakanishi K. J. Nat. Prod. 2002; 65: 1501-1504Crossref PubMed Scopus (53) Google Scholar, 18.van Beek T.A. Lelyved G.P. J. Nat. Prod. 1997; 60: Scopus Google Scholar). M was from studies K. PubMed Scopus Google Scholar). The of these compounds was as by of bilobalide, and were by in to 10 mm and were at and γ-aminobutyric acid were as in and in ACSF to All were by NY). were performed with an Germany). were acquired with Germany). Corp., were with mm 2 mm CaCl2, 10 mm and mm at 25 mosmol/liter). were at calcium imaging were as a in fluorescence and was done by 10-15 cells for each and were as the ± between two was with a of were to be Molecular with and were with OR). molecular modeling were with at the with Mol. Scopus Google Scholar) including the GB and picrotoxinin were using a between and of GB and and of as in for 10 of the GB unique in the 10 were obtained from structural using and with the minimized The that were for GB and for and bilobalide, compounds as terpene trilactones, share several structural similarities, in particular and a The ginkgolides only by the number and of K. PubMed Scopus Google Scholar). GB, and GC are found in both leaves and of G. biloba, and are found only in the leaves and BB is to the but only and is found in the leaves K. J. Scopus Google Scholar). Glycine activation a chloride ion channel and a membrane in embryonic cortical because of a intracellular chloride in immature (3.Flint A.C. Liu X. Kriegstein A.R. Neuron. 1998; 20: 43-53Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). The leads to the activation of calcium channels and a of calcium We of this and used calcium imaging to the potency of various ginkgolides on glycine-induced responses in embryonic cortical neurons. of glycine μm) in an increase in intracellular which was by with GB μm) The effect was highly as of glycine and GB the glycine which by the was to The effect was as by the of glycine responses a with artificial cerebrospinal fluid The glycine responses were to and by of glycine and GB These are in with an report on the effect of GB on glycine receptors in from adult rats (13.Kondratskaya E.L. Lishko P.V. Chatterjee S.S. Krishtal O.A. Neurochem. Int. 2002; 40: 647-653Crossref PubMed Scopus (49) Google Scholar). We the ginkgolides and bilobalide also glycine responses because share a chemical and their has not been We GB, GC, and together with BB and at the low to the relative potency of these GB, GC, and were the most potent antagonists and by the glycine responses to of and the to a lesser of The BB effect on glycine-induced calcium in this at the effect on glycine responses in intracellular calcium only indirectly activation of GlyRs and on the of the calcium In of calcium both on the and on the of the intracellular calcium the effect of BB and the most potent GB and GC, on glycine-induced currents, we used A in GB followed by its with glycine μm) in potent inhibition of the glycine in cortical has been suggested as an for (3.Flint A.C. Liu X. Kriegstein A.R. Neuron. 1998; 20: 43-53Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, M. J. Physiol. 2002; Scopus Google Scholar), we also the effect of GB on responses The effect was with inhibition and was and BB about of the terpene trilactones, the major single component in G. biloba extracts, we glycine-induced BB μm) was potent GB but a of glycine μm) responses to the of GB BB not significantly with both BB and GB a more effect on glycine-induced currents, measured in with their effect on intracellular calcium and the that we not a in the glycine-induced calcium in the of BB most likely from an of glycine-induced by BB that was not to the intracellular calcium C an for the of Glycine is of the most potent glycine receptor antagonists present in G. biloba a of the effect of we used a recording and glycine μm) every s in the of 10 GC following two glycine a GC the glycine was ± and a The effect was as by a of the glycine a glycine were done every in the of The glycine responses a with open channels are for the we the effect of on the of We used a the glycine was a and during the glycine and GC were following a or GC In glycine was are as the between the and the glycine responses from the show that the of inhibition not on the of glycine and GC were responses were not only but the were an of glycine channels the of drug These a for open channels in glycine receptor and B GABAA receptor is the only functional GABA receptor type expressed in embryonic as by a of GABA-induced with (9.Owens D.F. Liu X. Kriegstein A.R. J. Neurophysiol. 1999; 82: 570-583Crossref PubMed Scopus (155) Google Scholar). performed on cortical of GABAA responses by both GB and with BB the more potent of the two A in the drug was followed by of the drug with GABA the responses to GABA that were by with GB or BB this GB and BB GABA responses to ± and ± the effect of GB on GABA responses was not as by in which the was further The for the inhibitory of GB and BB on GABA-induced μm) responses are in for the μm) that BB is about as potent as The for BB was significantly the obtained on recombinant expressed in M. K. Wada K. Eur. J. Pharmacol. PubMed Scopus Google Scholar) most likely a effect of BB on receptors with subunit B and to a structural for the effects of of GB, and to with We their molecular properties modeling F. J. Scopus Google Scholar) and by with the of GB G. P. C Google Scholar) and P.R. J. Scopus Google Scholar). The minimized were found to be to by GB and although were differences in the of The minimized of GB and similarity in several a the is composed of a or and the are a of The are of the between the and for GB and with a of for the of the two in both the of the is of a their in GB and in In to this similarity in the two compounds have important in The of GB is in the as the of although the is and the of GB very well with the of of the of also investigated differences in among the ginkgolides from a structural GB, GC, and the more potent glycine share a the potent and not The of the for the structure was investigated a performed on and GB as All of were found to have C and with very little in of GB was that the in is The in GB may form a with the to the two of with B and is to GB and in that but GB has only BB was with GB and not A of BB with GB that the important functional of GB and BB be but not at the important differences in GB and the minimized of BB and In of is a striking of or as was for GB and We have shown that the terpene constituents of G. biloba have at the two major of receptors mediating fast inhibitory synaptic transmission in the adult central nervous system, both of which have also been shown to be active during The in particular GB and GC, glycine responses in the embryonic in a as was suggested previously for GB in from adult rats (13.Kondratskaya E.L. Lishko P.V. Chatterjee S.S. Krishtal O.A. Neurochem. Int. 2002; 40: 647-653Crossref PubMed Scopus (49) Google Scholar). GB only at higher that GB is the selective of We have also investigated the relative potency of the ginkgolides at that GB, GC, and are significantly more potent and These differences be as GB, GC, and a and that the is important for of and GB we have shown that the relative of the is in and the in of and GB be because of a direct of with the or be that by the in GB in a that is more for receptor Additionally, we the major single component in G. biloba BB is to ginkgolides and several functional in particular and a and has been to have properties and to be as a F.V. Ginkgo Scholar). Recent studies indicated that BB might GABAergic as BB has been reported to the GABA by acid (14.Sasaki K. Hatta S. Haga M. Ohshika H. Eur. J. Pharmacol. 1999; 367: 165-173Crossref PubMed Scopus (64) Google Scholar, 15.Sasaki K. Hatta S. Wada K. Ohshika H. Haga M. Life Sci. 2000; 67: 709-715Crossref PubMed Scopus (23) Google Scholar), responses K. Oota I. Wada K. Inomata K. Ohshika H. Haga M. Comp. Biochem. Physiol. Part C Pharmacol. Toxicol. Endocrinol. 1999; 124: 315-321Crossref PubMed Scopus (24) Google Scholar), and the of GABA Chatterjee S.S. 2002; PubMed Scopus Google Scholar). BB has also been reported to recombinant M. K. Wada K. Eur. J. Pharmacol. PubMed Scopus Google Scholar). In the effect of BB on glycine-induced responses has not been we show that BB activation and also reduces glycine although at higher of both GABAA and receptors by BB was not the of GlyRs by GB and GC that BB has a of the ginkgolides at glycine receptors, also to its products containing such as and the and the have been suggested to to and GABAARs at the E. Y. 2002; PubMed Scopus Google Scholar, D.F. Mol. Pharmacol. Google Scholar). The terpene trilactones may also interact with this although this remains to be molecular modeling we have shown structural similarities between GB and Both form a structure of The and relative of the two are very is likely to be functionally as studies of have that the between and is for E. 1999; PubMed Scopus Google Scholar). In the of the of GB is to the of although the is more is that the of GB into a in as the of in The of this for at GABAARs is well as the of an in the component of leads to a in at E. 1999; PubMed Scopus Google Scholar). Finally, the of GB very well with the of The of is very important for of as of the of by leads to of E. 1999; PubMed Scopus Google Scholar). studies have suggested that with in the J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), is that of GB has a in light of evidence that open channels are for was suggested that GB an of adult GlyRs (13.Kondratskaya E.L. Lishko P.V. Chatterjee S.S. Krishtal O.A. Neurochem. Int. 2002; 40: 647-653Crossref PubMed Scopus (49) Google Scholar). experiments demonstrate that GC inhibition of embryonic GlyRs is but that the of inhibition not on the of the These are a for open channels in GC We show that GB and share several structural that may be important for their are also differences in their pharmacological i.e. not GlyRs containing β subunits, GB GlyRs containing both α and β subunits, as shown in adult (13.Kondratskaya E.L. Lishko P.V. Chatterjee S.S. Krishtal O.A. Neurochem. Int. 2002; 40: 647-653Crossref PubMed Scopus (49) Google Scholar). ginkgolides are equally potent in inhibition of GlyRs remains to be of inhibitory receptors in and GABAARs in are known the effect of BB and GB on GABAARs might have implications for G. biloba extract, with a for such as is suggested by a recent report of two with well 2 of G. biloba that of the 2001; PubMed Scopus Google Scholar). In the of GABAA and receptors in cortical development is also important to the effects of G. biloba studies are to not only the effect of terpene trilactones on the and adult nervous but also the effect of all In we have shown that the ginkgolides and bilobalide are antagonists of both and GABAA receptors. In GB and GC are selective and potent antagonists of the GlyRs highly pharmacological for the of this receptor. Molecular modeling several structural similarities between GB and a of with the and Finally, the inhibitory of these components of G. biloba extract, the of the major on the may have important implications for this We and Owens for on the We for with