Resveratrol Inhibits Glycine Receptor-Mediated Ion Currents

Abstract

Resveratrol is found in grapes, red wine, and berries. Resveratrol has been known to have many beneficial health effects, such as anti-cancer, neuroprotective, anti-nociceptive, and life-prolonging effects. However, the single cellular mechanisms by which resveratrol acts are relatively unknown, especially in terms of possible regulation of receptors involved in synaptic transmission. The glycine receptor is an inhibitory ligand-gated ion channel involved in fast synaptic transmission in spinal cord. In the present study, we investigated the effect of resveratrol on human glycine receptor channel activity. Glycine α1 receptors were expressed in Xenopus oocytes and glycine receptor channel activity was measured using a two-electrode voltage clamp technique. Treatment with resveratrol alone had no effect on oocytes injected with H₂O or on oocytes injected with glycine α1 receptor cRNA. In the oocytes injected with glycine α1 receptor cRNA, co- or pre-treatment of resveratrol with glycine inhibited the glycine-induced inward peak current (I_Gly) in a reversible manner. The inhibitory effect of resveratrol on I_Gly was also concentration dependent, voltage independent, and non-competitive. These results indicate that resveratrol regulates glycine receptor channel activity and that resveratrol-mediated regulation of glycine receptor channel activity is one of several cellular action mechanisms of resveratrol for pain regulation.

Resveratrol is a phytoalexin found in grapes, red wine, and other berries (Fig. 1A) and is also produced as an anti-fungal chemical by plants.¹⁾ The concentration of resveratrol in red wine is as high as 0.2–5.8 mg/L.²⁾ Resveratrol exhibits diverse physiological and pharmacological activities, such as anti-cancer, chemopreventive, antiviral, cardio-protective, anti-aging, anti-nociceptive, and life-prolonging effects.^2–4) Resveratrol exhibits beneficial effects on the nervous system. It attenuates neurodegenerative disorders such as Alzheimer’s disease.⁵⁾ It also attenuates neuronal cell death from in vitro or in vivo brain hypoxia or ischemic conditions.^6,7) Although accumulating evidence indicates that resveratrol has diverse beneficial properties, including protective effects on the nervous system, relatively little is known about its cellular action, especially with respect to possible regulation of the receptors involved in synaptic transmission.

Fig. 1. Chemical Structure of Resveratrol and Effect of Resveratrol

(A) Chemical structure of resveratrol. (B) Effects of resveratrol (Res) (100 µM) on glycine receptor channel activity. Resveratrol alone had no effect on oocytes expressing glycine receptors.

The glycine receptor belongs to the superfamily of ligand-gated ion channel receptors that are structurally similar to nicotinic acetylcholine, 5-HT₃, and γ-aminobutyric acid A (GABA_A) receptors.^8,9) Glycine receptors are dominantly expressed in the spinal cord.¹⁰⁾ The physiological roles of glycine receptors in these regions mediate post-synaptic inhibition for reflex responses, voluntary motor control, and the processing of sensory signals,¹⁰⁾ because the glycine receptor forms a chloride-permeable and -selective transmembrane channel. Glycine receptors are responsible for fast inhibitory synaptic transmission in spinal cord.¹¹⁾ Inhibition of the glycine receptor causes the convulsion, while enhancement of the glycine receptor causes central nervous system depression and muscle relaxation.¹⁰⁾ Several studies showed that resveratrol regulate voltage- or ligand-gated ion channel such as potassium channel, sodium channel, 5-HT₃ receptor and GABAρ receptor.^12–15) However, information regarding how resveratrol regulates glycine receptors has not been reported in cellular level.

In this study, we first investigated the possible regulation of glycine receptor channel activity by resveratrol expressed in Xenopus oocytes. For this study, we expressed human glycine receptor cRNAs into Xenopus laevis oocytes and examined the effect of resveratrol on glycine-elicited inward peak currents (I_Gly). The reason we used this system was that (1) Xenopus oocytes have been widely used as a tool to express membrane proteins encoded by exogenously administered cDNAs or cRNAs, including receptors, ion channels, and transporters,¹⁶⁾ and that (2) glycine receptor channels expressed in Xenopus oocytes by injection of glycine receptor cRNA subunits are well studied and characterized.¹⁷⁾ We report that co-treatment of resveratrol with glycine inhibits I_Gly in a concentration-dependent, voltage-independent, and non-competitive manner. We further discuss the pharmacological role of resveratrol through the regulations of glycine receptor in nervous systems.

MATERIALS AND METHODS

Materials

Figure 1A shows the structure of resveratrol. Resveratrol used in this study was dissolved in dimethyl sulfoxide (DMSO) as previously reported.¹⁸⁾ and was diluted with bath medium before use. Because resveratrol is sensitive to light, it was kept in the dark and a fresh stock solution was prepared for every experiment. The final DMSO concentration was less than 0.1%. Resveratrol and other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.).

Oocyte Preparation

Xenopus laevis frogs were purchased from Xenopus I (Ann Arbor, MI, U.S.A.). Animal care and handling were in accordance with the highest standards of Konkuk University guidelines. Frogs underwent surgery only twice, separated by an interval of at least 3 weeks. To isolate oocytes, frogs were anesthetized with an aerated solution of 3-aminobenzoic acid ethyl ester. Oocytes were separated by treatment with collagenase, with gentle shaking for 2 h in a CaCl₂-free medium containing 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2.5 mM sodium pyruvate, 100 units/mL penicillin, and 100 µg/mL streptomycin. Only stage 5 or 6 oocytes were collected and were maintained at 18°C with continuous gentle shaking in ND96 (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, and 5 mM HEPES; pH 7.5) supplemented with 0.5 mM theophylline and 50 µg/mL gentamycin. All solutions were changed daily. All experiments were performed within 2–4 d following isolation of the oocytes.¹⁹⁾

Oocyte Recording

A single oocyte was placed in a small Plexiglas net chamber (0.5 mL) and was constantly superfused with ND96 medium in the absence or presence of glycine or resveratrol during recording. The microelectrodes were filled with 3 M KCl and had a resistance of 0.2–0.7 MΩ. Two-electrode voltage-clamp recordings were performed at room temperature using Oocyte Clamp (OC-725C, Warner Instrument, Hamden, CT, U.S.A.) with Digidata 1200 A. For most of the electrophysiological experiments, the oocytes were clamped at a holding potential of −80 mV. For the current–voltage relationship, voltage ramps were applied from −100 to +40 mV for 300 ms.

cRNA Preparation of Glycine α1 Receptor and Microinjection

A recombinant plasmid containing a human glycine α1 cDNA insert was linearized by digestion with appropriate restriction enzymes. The cRNAs from linearized templates were obtained by using an in vitro transcription kit (mMessage mMachine; Ambion, Austin, TX, U.S.A.) with a T3 polymerase. The RNA was dissolved in RNase-free water at 1 µg/µL, divided into aliquots, and stored at −80°C until use. Oocytes were injected with H₂O or human glycine α1 receptor cRNAs (5–10 ng) by using a Nanoject Automatic Oocyte Injector (Drummond Scientific, Broomall, PA, U.S.A.). The injection pipette was pulled from the glass capillary tubing used for the recording electrodes and the tip was broken to ca. 20 µm outer diameter (OD).¹⁹⁾ The final cRNA products were resuspended with RNase-free water at a concentration of 1 µg/µL and stored at −80°C.

Data Analysis

To obtain the concentration-response curve for glycine-induced current in the presence of resveratrol, the observed peak amplitudes were normalized and plotted, and then fitted to the Hill equation below using Origin software (Northampton, MA, U.S.A.): y/y_max=[A]ⁿ/([A]ⁿ+[IC₅₀]ⁿ), where y represents percent (%) inhibition at a given concentration of resveratrol, y_max represents percent (%) maximal inhibition, IC₅₀ is the concentration of resveratrol producing half-maximum inhibition of the control response to glycine, [A] is the concentration of resveratrol, and n is the interaction coefficient. All values are presented as the mean±S.E.M. The differences between means of control and resveratrol treatment data were analyzed using Student’s t-test. A value of p<0.05 was considered statistically significant.

RESULTS AND DISCUSSION

As shown in previous studies,²⁰⁾ the addition of glycine to the bathing solution induced a large inward current (I_Gly) in oocytes injected with glycine α1 receptor cRNA (Fig. 1B). I_Gly was completely blocked by glycine receptor antagonist, strychnine (0.5 µM), and in H₂O-injected control oocytes, treatment of glycine did not induce any inward current as previously shown (data not shown). Resveratrol (100 µM) alone had no effect on oocytes expressing the glycine receptor at a holding potential of −80 mV (Fig. 1B). However, co- or pre-treatment of resveratrol (100 µM) with glycine (100 µM) for 30 s inhibited I_Gly in oocytes expressing the glycine receptor (Fig. 2A, n=10–12 from 3 different frogs). Thus, pre- and co-treatment of resveratrol with glycine induced the inhibition of I_Gly by 52.6±3.4% and 56.3±5.7%, respectively, indicating that resveratrol effect on I_Gly was not affected by pre-treatment (Fig. 2B). In concentration-dependent experiments using resveratrol, co-treatment with resveratrol for 30 s inhibited I_Gly in a concentration-dependent manner in oocytes expressing glycine receptors (Figs. 3A, B). Thus, resveratrol inhibited I_Gly by 5.2±1.0%, 20.8±3.9%, 56.6±3.7%, and 72.4±7.4% at 10, 30, 100, and 300 µM in oocytes expressing glycine receptors, respectively. The IC₅₀ of resveratrol on I_Gly was 54.0±1.7 µM in oocytes expressing glycine receptor (n=9–12, from 3 different frogs for each point) (Fig. 3B).

Fig. 2. Effect of Resveratrol on I_Gly in Oocytes Expressing Glycine Receptors

(A) The representative traces showing treatment of glycine (100 µM) for 30 s induced inward current (I_Gly) in a reversible manner, and both pre- and co-treatment of resveratrol with glycine inhibited I_Gly. Glycine (100 µM) and resveratrol (Res) (100 µM) were pre- or co-treated for 30 s in oocytes expressing glycine receptor. (B) Summary of the inhibition induced by co- and pre-treatment of resveratrol. Each value represents the mean±S.E.M. (n=10–12 oocytes).

Fig. 3. Concentration-Dependent Effect of Resveratrol on I_Gly in Oocytes Expressing Glycine Receptors

(A) The representative traces showing concentration-dependent effect of resveratrol on I_Gly in oocytes expressing glycine receptor. (B) Percent inhibition by resveratrol of I_Gly in glycine receptors was calculated from the average of the peak inward current elicited by glycine and the peak inward current elicited by glycine after co-treatment of resveratrol with glycine for 30 s. Oocytes were voltage-clamped at a holding potential of −80 mV. Each value represents the mean±S.E.M. (n=9–12 oocytes).

In experiments exploring the current–voltage relationship, the membrane potential was held at −80 mV and a voltage ramp was applied from −100 to +40 mV for 300 ms. In the absence of glycine, the inward current at −100 mV was <0.1 µA and the outward current at +40 mV was 0.1–0.4 µA (data not shown). The addition of glycine to the bathing medium mainly induced an inward current at negative voltages and an outward current at positive voltages. Co-treatment of resveratrol with glycine inhibited both inward and outward currents. The reversal potential was near −20 mV in both glycine and glycine plus resveratrol conditions. This suggests that glycine induces the anion current.²¹⁾ Moreover, co-treatment of resveratrol with glycine did not affect the glycine receptor channel property because resveratrol did not change the reversal potential of the glycine receptor (Fig. 4A). The inhibitory effect of resveratrol on I_Gly in oocytes expressing glycine receptor was independent of the membrane holding potential (Fig. 4B). Co-treatment of resveratrol inhibited I_Gly by 54.2±3.6%, 51.7±4.7%, 51.5±5.1%, 53.3±6.9% and 55.6±4.5% at −90, −60, −30, 0 and +30 mV membrane holding potentials in oocytes expressing glycine receptor, respectively (n=8–11, from 3 different frogs).

Fig. 4. Current–Voltage Relationship and Voltage-Independent Inhibition by Resveratrol (Res) on I_Gly in Oocytes Expressing Glycine Receptors

(A) The representative current–voltage relationship was obtained using voltage ramps from −100 to +40 mV for 300 ms. Voltage steps were applied before and after application of 100 µM glycine in the absence or presence of 100 µM resveratrol. (B) Summary of the inhibition induced by resveratrol at the indicated holding membrane potentials in oocytes expressing glycine receptors. Oocytes were voltage-clamped at a holding potential of −80 mV. Each point represents the mean±S.E.M. (n=8–11 oocytes).

To further investigate the mechanism by which resveratrol inhibits I_Gly in oocytes expressing glycine receptor, we analyzed the effect of 100 µM resveratrol on I_Gly evoked by different glycine concentrations in oocytes expressing glycine receptor (Fig. 5A). Co-treatment of resveratrol for 30 s with various concentrations of glycine did not greatly shift the concentration-response curve of glycine to the right (EC₅₀, from 156.1±5.0 to 158.6±5.6 µM, p>0.05 and Hill coefficient, from 2.6 to 3.1) in oocytes expressing glycine receptor, although co-treatment of resveratrol significantly inhibited I_Gly induced by concentrations of 100, 300, and 1000 µM glycine (Fig. 5B) (* p<0.05; ** p<0.005, compared to treatment with glycine alone). These results indicate that resveratrol inhibits I_Gly in a non-competitive manner.

Fig. 5. Concentration–Response Relationships between Glycine and Glycine+Resveratrol (Res) on I_Gly in Oocytes Expressing Glycine Receptors

(A) The representative trace of the concentration–response relationship for glycine and glycine +100 µM resveratrol in oocytes expressing glycine receptors. I_Gly in oocytes expressing glycine receptors was measured with the indicated concentration of glycine in the absence or the presence of 100 µM resveratrol. Oocytes were exposed to glycine alone or to glycine and resveratrol for 30 s. (B) Concentration–response curves for the effect of 100 µM resveratrol on oocytes expressing glycine receptors. * p<0.05; ** p<0.005, compared to treatment with glycine alone. The solid lines fit the Hill equation. Each point represents the mean±S.E.M. (n=10–12 oocytes).

In the present study, we demonstrated that (1) pre- or co-treatment of resveratrol with glycine induced an inhibition of I_Gly in oocytes expressing glycine receptors in a reversible and concentration-dependent manner; and (2) the inhibition of I_Gly by resveratrol co-treatment with glycine did not shift the glycine concentration–response curve and was voltage independent.

From the present results, however, we still have not been able to elucidate how resveratrol acts to inhibit I_Gly in oocytes expressing glycine receptors. One possibility is that resveratrol may work as a novel agent that inhibits the binding of glycine to its binding site(s) in the glycine receptor. In competition experiments, we observed that the presence of resveratrol did not shift the concentration curve of glycine to the right in oocytes expressing glycine receptor without significantly changing the Hill coefficient (Fig. 5B), suggesting that resveratrol regulates glycine receptor channel activity in a non-competitive manner. Another possibility is that resveratrol may act as an open channel blocker of the glycine receptor, although this seems unlikely because the inhibitory effect of resveratrol on I_Gly in oocytes expressing glycine receptors was not voltage-dependent. It is known that open-channel blockers, such as local anesthetics or hexamethonium, are strongly voltage-dependent because of the charge that they carry in the transmembrane electrical field.^22–24)

Previous studies have shown that the effects of resveratrol on nervous systems might be related to ligand-gated ion channels. For example, resveratrol-mediated neuroprotection against brain ischemia is inhibited by N-methyl-D-aspartate (NMDA) receptor antagonist.²⁵⁾ Resveratrol also attenuates kainite-induced epilepsy.²⁶⁾ In addition, resveratrol suppresses catecholamine secretion by inhibiting the α3β4 nicotinic acetylcholine receptor in adrenal medullar cells.²⁷⁾ On the other hand, resveratrol potentiates 5-HT_3A receptor channel activity.¹⁴⁾ Thus, although resveratrol shows differential effects related with excitatory receptor activities, there is still only a rudimentary understanding of how resveratrol regulates receptor or ion channel activities at the cellular level. The present findings that resveratrol inhibits I_Gly in oocytes expressing glycine receptor indicates the possibility that resveratrol might be also involved in glycine receptor-mediated physiological or pharmacological activity.

On the other hand, blockage of the glycine receptor by glycine receptor antagonist such as strychnine causes the convulsion.¹⁰⁾ However, it is unlikely that resveratrol-induced inhibition of I_Gly induces the convulsion like strychnine, since resveratrol is not a full glycine receptor antagonist compared to alkaloid strychnine (Fig. 2A) and there is no report that resveratrol induces a convulsion. However, resveratrol-mediated inhibition of I_Gly might be coupled to depolarization in neurons that express glycine receptors and affect the synaptic transmissions. In spinal dorsal horn glycinergic receptors are involved in the delivery of pain transmission to brain.²⁸⁾ Thus, one possible pharmacological role of resveratrol through glycine receptor regulation by resveratrol in nervous systems is association with pain regulations. For example, intrathecal or intracerebral ventricle administration of resveratrol elicits analgesic effects.^29–31) Currently, we do not know whether resveratrol-mediated inhibition of I_Gly, as observed in the present study, is directly or indirectly linked to regulation of spinal cord or brain pain transmission via glycinergic receptors. Further studies will be required to determine how in vitro resveratrol-mediated inhibition of I_Gly is coupled to glycine receptor-related beneficial effects including anti-nociception in central nervous systems.

In conclusion, we found that resveratrol, an active ingredient found in grapes, inhibits glycine receptor-mediated ion currents by interacting with other sites that are different from the glycine-binding site expressed in Xenopus oocytes, and these results further indicate that this regulation of the glycine receptor channel activity by resveratrol might be the basis of its cellular action.

Acknowledgment

This paper was supported by Konkuk University in 2013.

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