Allosteric positive interaction of thymol with the GABAA receptor in primary cultures of mouse cortical neurons
Introduction
GABAA 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 GABAA 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 GABAA 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 GABAA 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 GABAA receptors in synaptosomal membranes, suggesting a possible role for thymol as a GABAA receptor agonist/modulator.
Other phenolic compounds, among them the anaesthetic propofol (2,6-isopropyl-phenol), reportedly enhance GABA agonist activity and directly activate the GABAA 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 GABAA 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 GABAA 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 GABAA 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 GABAA receptors. Finally, we determined a lack of neurotoxic effects exerted by thymol at concentrations relevant to its neuroactive window.
Section snippets
Materials
Pregnant NMRI mice (16th gestational day) were obtained from Charles River, Iffa Credo (St. Germain-sur-l'Arbreste, France). Plastic culture multiwell plates were purchased from CoStar (Corning Science Products, Acton, MA, USA). Foetal calf serum was obtained from Gibco (Glasgow, UK) and Dulbecco's modified Minimum Essential Medium (DMEM) from Biochrom (Berlin, Germany). [3H]Flunitrazepam (88 Ci/mmol) and 36Cl− (0.1 Ci/mol) were procured from Amersham Life Sciences (Buckinghamshire, UK); [3
Parameters of [3H]flunitrazepam binding and 36Cl− uptake in intact cultured cortical neurons
In primary cultures of mice cortical neurons [3H]flunitrazepam binding had an apparent Kd of 7.4 ± 1.8 nM and Bmax of 731 ± 31 fmol/mg protein (Fig. 1A), in agreement with previous reports using this type of cultures (Mehta and Ticku, 1992, Vale et al., 1999). Fig. 1B shows the concentration–response curve for GABA-induced Cl− flux in intact cultured cortical neurons. The EC50 value was 8.4 μM (log EC50 = −5.1 ± 0.1), similar to that reported by Hu and Ticku (1994) and Vale et al. (1999). Because
Discussion
In the present work we provide clear evidence that thymol is a positive GABAA receptor modulator, as demonstrated by its ability to increase both 36Cl− influx and [3H]flunitrazepam binding, as well as by its tendency to increase [3H]muscimol binding. The inhibition of these effects by GABAA receptor antagonists further support this finding. The fact that thymol, at concentrations that elicited a chloride influx, did not inhibit [3H]muscimol binding precludes a direct agonist effect at the GABA
Acknowledgements
This work was supported by grant SAF 2003-4930 (Spanish Ministry of Science and Technology, cofinanced with FEDER funds). D.A.G. is a member of CONICET (Argentina) and during this work was the receipt of a postdoctoral fellowship from the Fundación Carolina (Spain).
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Present address: Department of Pharmacology, University of Santiago de Compostela, Lugo, Spain.