Effect Of Resveratrol On Gaba_a-mediated Current In Cultured Rat Cortical Neurons

Ligand-gated ion channels permit cells to respond rapidly to changes in their external environment. They are particularly well known for mediating fast neurotrasmission in the nervous system.γ-Aminobutyric acid type-A receptors (GABA_AR) are the predominant inhibitory neurotransmitter receptors in the mammalian central nervous system (CNS). They belong to the ligand-gated channels superfamily. Activation of GABA_AR leads to an influx of Cl- and increases intracellular membrane potential and generates hyperpolarization,which inhibit neuronal firing. Therefore, the modulation of GABA_AR function would be expected to alter neuronal excitability.Resveratrol(trans-3,4',5-trihydroxy-stilbene), a natural polyphenols compounds, is present in grapes and wine, which was found to exert potent neuroprotective effects in different in vivo and in vitro models. It has been reported to have a variety of estrogenic, anti-inflammatory, anti-platelet, and anti-carcinogenic effects . But some another research examine the pharmacology function of resveratrol on the ion channel and receptor. For example, resveratrol increased the activity of large-conductance calcium-activated potassium channels in vascular endothelial cells . And resveratrol strengthen to activate the big electric conductivity of potassium channels. It can also inhibit the L-type calcium current of ventricles muscle cell, reduce the TTX sensitive and TTX drug-fast of Na⁺ current on the dorsal root ganglion cell, repress the voltage activate of potassium channels current on lymphoid cell and the hippocampal neurons, restain the EPSC of AMPA/NMDAR-mediated current in the acute rat hippocampal brain slice. However, at present, there is no report on the effect of resveratrol on the chlorine ion channels, particularly in GABAARs in CNS (central nervous system). Thus, in the present study, we decided to examine the modulatory effects of resveratrol on native GABAARs in cultured cortex by using the whole-cell patch–clamp recording technique.In this study, we found that Resveratrol reversibly and concentration-dependently depressed GABA induced current (IGABA), with IC₅₀ of 48.7±2.4μM. Resveratrol depressed maximum IGABA, and significant changed the EC₅₀ for GABA and Hill coefficient, suggesting that Resveratrol inhibited IGABA through a non-competitive mechanism.The time course of Resveratrol inhibition of GABA_AR was very rapid, and the response was completely reversed after washout for about 4 min. These effects are compatible with the fact that Resveratrol acts directly on GABA_ARs. Kinetic analysis indicated that Resveratrol accelerated the rates of desesnsitization. Enhancement of desensitization is one common mechanism underlying non-competitive inhibition. It is unlikely that Resveratrol acts as an open channel blocker because no rebound current was observed in all experiments after washout of GABA and Resveratrol.The rebound current is the direct evidence of an open channel blocker theory. Besides,the inhibition of Resveratrol on IGABA depended on different drug application mode, and the pre-perfusion of Resveratrol produced the maximal suppressive effect. When applied alone before the application of GABA, Resveratrol also inhibited IGABA, indicating that Resveratrol could bind to the recepter before the channel openning, eliminating the possibility that the acting site of Resveratrol is without the channel center,which further support the idea that the inhibition effect of Resveratrol is not an open channel blocker mechanism. Based on these results, we propose that Resveratrol might exert its action as an allosteric inhibitor. Therefore, Resveratrol is not likely to compete with GABA for its recognition site, but act somewhere else on GABA_ARs that could reduce the GABA's affinity of GABAARs through changing ion channel conformation.Taken together, our data clearly demonstrate that Resveratrol significantly inhibit the GABA_A receptor response on cultured cortical neuron through non-competitive mechanism. Resveratrol and its metabolin are able to pass blood brain barrier.So resveratrol could directly combine on GABA_ARs. Thus,resveratrol can modulate excitability ,inhibition and the blance between them of cortex nervous system. Our findings may be helpful to pile up the related receptor pharmacology data of resveratrol.