Allosteric positive interaction of thymol with the GABA_A receptor in primary cultures of mouse cortical neurons

Abstract

Thymol is a naturally occurring phenolic monoterpene known for its anti-microbial and anti-oxidant properties. It is used in dental practice and in anaesthetic halothane preparations. Recent studies have reported enhanced GABA_A receptor-operated chloride channel activity and increased binding affinity of [³H]flunitrazepam in the presence of thymol. In the present work, we more closely examined the pharmacological action of thymol on the native GABA_A receptor by using primary cultures of cortical neurons. Thymol enhanced GABA-induced (5 μM) chloride influx at concentrations lower than those exhibiting direct activity in the absence of GABA (EC₅₀ = 12 μM and 135 μM, respectively). This direct effect was inhibited by competitive and non-competitive GABA_A receptor antagonists. Thymol increased [³H]flunitrazepam binding (EC₅₀ = 131 μM) and showed a tendency to increase [³H]muscimol binding. These results confirm that thymol is a positive allosteric modulator of the GABA_A receptor. The thymol structural analogues menthol and cymene, which lack an aromatic ring or a hydroxyl group, did not affect [³H]flunitrazepam binding. Using a pharmacophoric model that includes a hydrogen bond donor group as well as an aromatic ring with two aliphatic substituents, we propose to demonstrate the molecular essential features of these compounds to interact with GABA_A receptors. Thymol (0–1 mM) did not affect cellular viability.

Introduction

GABA_A receptors are ligand-gated ion channels that mediate rapid synaptic inhibition in the central nervous system. They are structured in an arrangement of five different individual subunits from seven families with multiple isoforms. The GABA_A receptor possesses binding sites for drugs other than the neurotransmitter GABA, including benzodiazepines, barbiturates, and the convulsant picrotoxinin, which behave as allosteric modulators or channel blockers. A wide spectrum of drugs, toxic agents and metals modify GABA_A receptor function by directly interacting either with these binding sites or with other as yet not well-described sites present in the receptor complex. Known allosteric modulations include the enhanced binding of benzodiazepine agonists by GABA, the enhanced GABA-induced chloride flux by benzodiazepines and barbiturates, and the different modifications of [³⁵S]t-butylbicyclophosphorothionate (³⁵S-TBPS, ligand of choice for the picrotoxinin recognition site) binding induced by GABA, benzodiazepines and barbiturates (for review see Macdonald and Olsen, 1994; Sieghart, 1995, Sieghart, 2000, Korpi et al., 2002, Smith and Simpson, 2003, Rudolph and Mohler, 2004).

Thymol is a naturally occurring phenolic monoterpene which is found as a component of many essential oils used extensively in fragrances, flavour additives, or scenting products. This compound is a particularly well-known anti-microbial agent commonly incorporated in mouthwashes and used in dental practices for its bactericidal properties against oral bacteria (Shapiro and Guggenheim, 1995, Burt, 2004). As with other phenolic compounds, thymol possesses anti-oxidant properties, which explains its use as anaesthetic stabiliser in halothane preparations (MacPherson, 2001). A thymol-supplemented diet has been reported to increase anti-oxidant status and to safeguard polyunsaturated fatty acid levels in the aging rat brain (Youdim and Deans, 1999, Youdim and Deans, 2000). In addition to its anti-oxidant action in the brain, thymol has been shown to specifically interact with synaptic neural functions. Studies have reported blocking action on neuronal Na⁺ channels (Haeseler et al., 2002) as well as enhanced chloride channel activity in oocytes and cell lines expressing GABA_A receptor subunits (Mohammadi et al., 2001, Priestley et al., 2003). Recently, Sánchez et al. (2004) described the ability of thymol to incorporate itself in artificial membranes and to increase the binding affinity of [³H]flunitrazepam to GABA_A receptors in synaptosomal membranes, suggesting a possible role for thymol as a GABA_A receptor agonist/modulator.

Other phenolic compounds, among them the anaesthetic propofol (2,6-isopropyl-phenol), reportedly enhance GABA agonist activity and directly activate the GABA_A receptor (Squires et al., 1999, Trapani et al., 2000, Mohammadi et al., 2001 and references therein). Krasowski et al. (2002), by means of a QSAR analysis of several propofol analogues, have described two main structural conditions for a molecule to exhibit activity on this receptor: (i) an OH group as a hydrogen bond donor and (ii) two hydrophobic groups close to the hydroxyl. Based on this finding, and considering that thymol partially meets such structural requirements, we examined whether the action of thymol on the GABA_A receptor is mimicked by its structural analogues menthol and cymene. These compounds share with thymol the presence of either the OH group (menthol) or the aromatic ring (cymene) (see Fig. 2). Furthermore, both thymol and menthol have been reported to block sodium channels (Haeseler et al., 2002).

In the current study, we more closely examined the pharmacological action of thymol on native GABA_A receptors by determining its effects on GABA and benzodiazepine recognition sites, as well as on chloride influx by using primary cultures of cortical neurons, which express functional GABA_A receptors (Pomés et al., 1994, Vale et al., 1999 and references therein). Moreover, we propose a pharmacophoric model, based on that proposed by Krasowski et al. (2002), addressing the action of thymol and its analogue phenolic compound, propofol, on GABA_A receptors. Finally, we determined a lack of neurotoxic effects exerted by thymol at concentrations relevant to its neuroactive window.