The Differential Effects of Resveratrol and Trans-ε-viniferin on the Gaba-induced Current in Gabaa Receptor Subtypes Expressed in Xenopus Laevis Oocytes

Purpose: The natural products resveratrol and trans-ε-viniferin have been reported to have many beneficial effects, which include the enhancement of cognition and memory. There have been no studies which have reported the effects of these compounds on the different GABAA receptor subtypes and this study aimed to address this. Methods: The effects of both resveratrol, and its dimer, trans-ε-viniferin, have been investigated on different GABAA receptor subtypes expressed in Xenopus laevis oocytes, using the two-electrode voltage clamp technique. Results: Resveratrol induced a current of 22 ± 3.53 nA in the α1β2γ2L subtype of the GABAA receptor (but not in the α5β3γ2L and α2β2γ2L subtypes) when applied alone. It also positively modulated the GABA-induced current (IGABA) in α1β2γ2L receptors, in a dose-dependent manner (EC50 58.24 μM). The effects of resveratrol were not sensitive to the benzodiazepine antagonist flumazenil. trans-ε-Viniferin exhibited a different pattern of activity to resveratrol; it alone had no effect on any of the subtypes, but it did negatively modulate the GABA-induced current (IGABA) in all three subtypes. The greatest inhibition was found in the α1β2γ2L subtype (IC50 5.79 μM), with the inhibition in the α2β2γ2L (IC50 of 19.08 μM) and α5β3γ2L (IC50 of 21.05 μM) subtypes being similar. The effects of trans-ε-viniferin in α1β2γ2L and α2β2γ2L receptors were also not sensitive to the benzodiazepine antagonist flumazenil while, in the α5β3γ2L subtype the effect was not sensitive to the inverse agonist L-655,708, indicating different binding sites for this molecule. Conclusions: The results of the present study indicate that both resveratrol and trans-ε-viniferin modulate the GABA-induced current in different ways, and that trans-ε-viniferin may be a lead compound for the discovery of agents which selectively inhibit the GABA-induced current in α1-containing subtypes.


INTRODUCTION
GABAA receptors are membrane bound pentameric chloride selective ion channel composed of α, β, and γ subunits.There are 19 genes for GABAA receptors, which include 16 subunits (α1-6, β1-3, γ1-3, δ, ρ1-3, θ, π, ε) that are assembled as the different subtypes of GABAA receptors (1).These differences in the combinations of receptor subunits result in variations in the biophysical and pharmacological properties of the receptors.The distribution of these receptors in the body also differs, with GABAA receptors being widely distributed in the CNS.Most importantly, agonist affinity, receptor kinetics, and sensitivity to a variety of clinically important drugs (including benzodiazepines and general anaesthetics) are determined by the composition of the subunits (2,3).For example, receptors that are composed of α1-3, α5, γ2, and β2 or β3 are sensitive to the benzodiazepines, whereas receptors composed of α4 or α6, or δ instead of γ2, are not sensitive to this class of drugs (4).Simple changes in the receptor subunit combinations can lead to dramatically different activities; for example, receptors containing α1β2γ2 mediate the sedative and anticonvulsant effects of diazepam, α2βγ2-and α3βγ2-containing receptors are responsible for the anxiolytic and muscle relaxing effects of this drug, and α5βγ2-containing receptors may mediate learning and memory processes (5).The involvement of α5-containing GABAA receptors in cognition and memory is supported by both mutational and pharmacological studies on rats (6,7), so these receptors have become attractive targets for the development of memory enhancing drugs (8).
Vitis vinifera (common grape vine) belongs to the family Vitaceae and the extracts and pure compounds from this plant exhibit a variety of ________________________________________ biological activities, including effects on different neurological disorders.Resveratrol (3,5,4'trihydroxy-trans-stilbene), a phytoalexin from this plant, has been reported to improve scopolaminebut not mecamylamine-induced memory impairment in rats, in both passive avoidance and Morris water maze tests.The interaction of resveratrol with muscarinic cholinergic receptors has also been suggested by the same authors (9).The neuroprotective effects of resveratrol have been reported by a number of studies which include the protection of dopaminergic neurons from MPTP (1-methyl-4-phenyl-1,2,3,6tetrahydropyridine) induced toxicity in mice.It has also been reported to reduce the effect of acetylcholine esterase (AChE), with subsequent improvement of memory impairment in diabetic rats (10).The effect of resveratrol on the level of different neurotransmitters during ischemia/reperfusion in rats has been reported by Li et al., who found that it significantly increased the basal extracellular level of GABA (11).
The effect of resveratrol on different ligandgated ion channels has been studied by Lee et al. (15,16) and it has been found that it potentiates the 5-hydroxytryptamine (5-HT) induced current in the 5-HT3 receptor, with an EC50 value of 28.0 ± 2.4 µM.At the same time, it inhibits the GABAinduced current in the GABAC ρ receptor expressed in Xenopus laevis oocytes, with an IC50 value of 28.9 ± 2.8 µM.Resveratrol also reported to inhibit 1 μM GABA-induced current at human ρ1 GABAC receptors with an IC50 value of 72 μM (17).To date, however, no studies have been reported on the effects of resveratrol on the different subtypes of GABAA receptor.In the present study, the effects of both resveratrol and trans-ε-viniferin (Figure 1) have been examined in three different GABAA receptor subtypes.

Materials
Human α1, α5, β2, β3 and γ2L DNA in pcDM8 (Invitrogen, CA, USA) were a kind donation from Dr Paul Whiting (Merck, Sharpe and Dohme Research Labs, Harlow, UK).Xenopus laevis were obtained from NASCO, Fort Atkinson, Wisconsin, USA and housed in the Department of Veterinary Science, University of Sydney.DMSO, GABA, and zinc sulphate were purchased from Sigma Aldrich Chemical Co. Ltd. (St Louis, MO, USA).trans-ε-Viniferin was purchased from Cfm Oskar Tropitzsch GmbH, Germany and resveratrol was purchased from Sigma Aldrich, Australia.Flumazenil and L655,708 were purchased from Tocris Bioscience, Minneapolis, USA.The compounds used were dissolved in DMSO and any further dilution was made with ND96 (96 mM NaCl, 2 mM KCl, 1 mM MgCl2.6H2O, 1.8 mM CaCl2, 5 mM HEPES, 2.5 mM sodium pyruvate, 0.5 mM theophylline, 50 μg/mL gentamycin, pH 7.5) buffer before use (all drug solutions were standardised to contain 0.8% DMSO).

Oocyte preparation
After surgical removal, the ovarian lobes of female Xenopus laevis were rinsed with oocyte releasing buffer 2 (OR2; 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2.6H2O, 5 mM HEPES, pH 7.5), then suspended for 2 hours in collagenase (2 mg/ml in OR2, Bohringer Manheim, Germany) to allow the separation of oocytes from connective tissues and follicular cells.The separated oocytes were then washed several times with ND96 buffer solution.The oocytes were then sorted under a microscope in order to obtain mature and healthy cells with clear animal / vegetal pole divisions and without any spots or markings on the surface.Before injection, the oocytes were stored in a refrigerator at 2-8 0 C.

Oocyte recording
Two-three days after injection, the two-electrode voltage clamp technique was performed to measure the receptor activity with Digidata 1200, Geneclamp 500 amplifier (Axon Instruments, Foster City, CA, USA).Microelectrodes were made by pulling glass capillaries (0.94 mm I.D.x1.2 mm O.D.; Harvard Apparatus Ltd., Kent, UK) using an automated micropipette puller (PUL-100, World Precision Instruments, Inc.) filled with 3M potassium chloride solution.
Oocytes were placed in the oocyte bath chamber, impaled by electrodes with resistance of less than 10 MΩ (usually 0.5 to 2.0 MΩ).In the oocyte chamber, the cells were always perfused with ND96 buffer solution.The current traces elicited due to the application of drugs and / or GABA were recorded using a Mac Lab 2e recorder (ADInstruments, Sydney, NSW, Australia) and Chart Version 5.1 program.For all the electrophysiological experiments, the oocytes were clamped at a holding potential of -60 mV.

Data analysis
Data analysis was performed as described previously, with slight modifications (18).The analysis was performed on GraphPad Prism version 5; concentration-response curves were obtained from the currents recorded from the applied GABA concentrations (EC10 for potentiation and EC50 for inhibition) in the presence of range of resveratrol and trans-εviniferin concentrations.The data are expressed as a percentage of the averaged maximum current (Imax) and fitted by least squares non-linear regression with the empirical Hill equation.
where [A] is the agonist concentration, nH is the Hill coefficient and EC50 is the effective concentration that evoked a 50% of Imax response.Similarly, inhibition curves were assembled from the peak currents recorded from the range of εviniferin concentrations applied in the presence of a fixed concentration (EC50) of GABA.The data were expressed as a percentage of the peak current (Imax) obtained from the application of the GABA concentration alone.The concentration that inhibited 50% of Imax (IC50) was estimated from fitting the data with the Hill equation, where the concentration of the ε-viniferin is substituted for the agonist concentration.Unless otherwise stated, parameters were calculated from individual oocytes and then averaged.

Resveratrol
The addition of the maximal concentration of GABA (1 mM) induced a large inward current (IGABA) in all three subtypes of receptors, confirming the expression of the respective GABAA receptors by the oocytes.This current was not inhibited by either 10 or 100 μM solutions of zinc chloride, indicating the incorporation of the γ2L subunit (19).Resveratrol (Figure 1), at a concentration of 100 μM induced a slight current (22 ± 3.53 nA) (Figure 2B) at α1β2γ2L, but not at the α2β2γ2L and α5β3γ2L GABAA receptor subtypes.Resveratrol at 100 μM concentration did not modulate the GABA-induced current at the α2β2γ2L and α5β3γ2L subtypes of GABAA receptor but potentiated the EC10 (3 μM) GABA-induced current at α1β2γ2L by 126 ± 15 %.In a doseresponse study, when applied with a fixed dose of GABA (EC10, 3 μM), resveratrol positively modulated the GABA-induced current (62 ± 2.35 nA) in a concentration dependent manner, with an EC50 of 58.24 μM (Figure 2A).Moreover, the effect of resveratrol was not sensitive to the benzodiazepine antagonist, flumazenil (Figure 3), indicating that it does not interact with the high sensitivity benzodiazepine binding site, which is sensitive to flumazenil and is located at the interface of the α-γ subunits (20,21).

trans-ε-Vinferin
trans-ε-Viniferin (Figure 1), at a concentration of 100 μM, did not induce any current at all three subtypes of GABAA receptors when applied alone, but there was a small outward current for trans-εviniferin on the α5β3γ2L subtype (Figure 4F) of the GABAA receptor.However, it did negatively modulate the GABA-induced current (IGABA) at all three subtypes.In dose-response experiments, involving co-application with the EC50 GABA concentration, trans-ε-viniferin inhibited the GABA-induced current in a concentration dependent manner.The highest inhibitory potency was observed at the α1β2γ2L subtype, with an IC50 value of 5.79 μM Figure 4 (A-B),, followed by the α2β2γ2L (IC50 19.08 μM) Figure 4 (C-D), and then the α5β3γ2L (IC50 21.05 μM) (Figure 4 (E-F)).
Further studies showed that the effect of transε-viniferin on both the α1β1γ2L (Figure 5A), and α2β2γ2L subtypes is not affected by the benzodiazepine antagonist flumazenil (Figure 5B), indicating that it does not interact with the high affinity benzodiazepine binding site (which is sensitive to flumazenil).In addition, the effect of trans-ε-viniferin on the α5β3γ2L subtype is not sensitive to L-655,708, a preferential inverse agonist of this subtype of GABAA receptor (Figure 5C) (22).

DISCUSSION
γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system (CNS), and GABAergic neurons constitute 17-20% of all neurons in the brain (23).There are three different types of GABA receptors, which are classified as GABAA, GABAB, and GABAC (GABAρ) based upon their subunit composition, gating properties, and pharmacological profiles.GABAA and GABAC are ligand-gated ion channel receptors (LGICs), whereas GABAB are G-protein coupled receptors (24,25).GABAA receptors are an important target for anxiolytics, sedative, hypnotics, anticonvulsant and muscle relaxants (26) and, in spite of having a range of drugs for the treatment of anxiety, there is an increased demand for herbal preparations for the treatment of anxiety, depression, insomnia etc. (27).In the present study, we report the differential effects of resveratrol, and its dehydrodimer trans-ε-viniferin, which was originally obtained from plant, on different GABAA receptor subtypes expressed in Xenopus laevis oocytes.The effects of resveratrol on ligand gated ion channels have been investigated by many researchers and it has been reported that the neuroprotection by resveratrol in a cerebral ischaemia model is a result of its interaction with NMDA receptors (28).Resveratrol has been found to inhibit the acetylcholine-induced current in rat α3β4 nicotinic acetylcholine receptors (IC50 25.9 μM), inhibit the GABA-induced current in GABAC receptors, and to potentiate the 5-HT induced current in 5-HT3A receptors (15,16,29).It also inhibits the effect of GABA (1 μM) at the human ρ1 GABAC receptor as a non-competitive inhibitor with an IC50 of 72 μM (30).In the current study, resveratrol had no direct effect on the different subtypes of GABAA receptors, except α1β2γ2L, when applied alone.It did, however, positively modulate the GABA-induced current at the α1β2γ2L subtype (but not the α2β2γ2L and α5β3γ2L subtypes) in a dose-dependent manner.It appears, therefore, that the α1 subunit is essential for the modulatory effects of this compound on GABAA receptors.Moreover, the effect of resveratrol is not sensitive to the benzodiazepine antagonist flumazenil, indicating that its binding site is distinct from that of high affinity benzodiazepine binding site.In the previous studies, similar data has been obtained where resveratrol had no influence on the positive modulation of diazepam.At the same time, there was no significant effect on the effect of higher (40 μM) concentration of GABA at α1β2γ2L receptors (31).In the present study, resveratrol positively modulated the current induced by a lower (3 μM) GABA concentration.
Although both resveratrol and trans-εviniferin are present in comparable amount in grapes (32), the effects of trans-ε-viniferin have not been well studied (33), despite it having been found to be more active than resveratrol in a range of biological assays.For example, it is more active than resveratrol in inducing the relaxation of rat thoracic aorta preparations, has greater in vitro antioxidant activity, is a more potent inhibitor of platelet-derived growth factor-induced cell proliferation, and induces nitric oxide generation in vascular smooth muscle cells (VSMCs) (34)(35)(36).A number of reports on the modulatory effect of resveratrol on ion channel receptors have been published (15,16), however, to date, no reports on the modulatory effects of trans-ε-viniferin have been published.In the present study, trans-εviniferin, the dehydrodimer of resveratrol, has been shown to negatively modulate the GABAinduced current (IGABA) in all three subtypes of GABAA receptor in a dose-dependent manner.The effect of trans-ε-viniferin on the α1β2γ2L and α2β2γ2L subtypes is also not sensitive to benzodiazepine antagonist flumazenil, while the effects on the α5β3γ2L subtype are not sensitive to the inverse agonist L-655,708, indicating that this compound does not interact with the high affinity benzodiazepine binding site.The α5 subunit containing receptors are mainly located in the hippocampus, where they mediate a tonic chloride leak current and contribute a slow component to GABAergic inhibitory synaptic currents.The inhibitory effect of these receptors on the excitation of hippocampal neurons is thus partly responsible for their association with cognition, learning and memory.These receptors have thus become an important target for different pathological conditions including age related dementia, schizophrenia, and Down syndrome (37).Moreover, it has also been reported that the chronic treatment of TS mice (mouse model of Down syndrome) with an α5 negative allosteric modulator (NAM) reversed their deficit in spatial learning and memory (38).In the present study, trans-ε-viniferin negatively modulated the GABAinduced current at α5β3γ2L GABAA receptor with an IC50 of 21.05 μM, which indicate the potential of this molecule for the development of drug for the treatment age related dementia, Down syndrome and schizophrenia.
In conclusion, despite the structural similarity between resveratrol and trans-ε-viniferin, these compounds modulate the GABA-induced current in GABAA receptors in different ways.The effects of trans-ε-viniferin are subtype selective but, in order to increase the selectivity, particularly selectivity towards α5β3γ2L GABAA receptor, analogues of this compound should be designed and, in addition to being tested on GABAA receptors in vitro, should be tested in animal models.

Figure 2 .Figure 3 .
Figure 2. A. Dose-response curve for the effect of resveratrol on the GABA EC10 (3 μM) response at α1β2γ2L GABAA receptors.B. Typical traces for the positive modulation of the GABA EC10 (3 μM) induced current by different concentrations of resveratrol.

Figure 4 .
Figure 4. Dose-response curve and typical traces showing the effect of trans-ε-viniferin on the GABA-induced current in different subtypes of GABAA receptors; A-B α1β2γ2L , C-D α2β2γ2L, E-F α5β3γ2L.Data for all dose-response curves are the Mean ± SEM (n=3-4 oocytes).