Menthol shares general anesthetic activity and sites of action on the GABAA receptor with the intravenous agent, propofol

https://doi.org/10.1016/j.ejphar.2008.06.003Get rights and content

Abstract

Menthol and related compounds were investigated for modulation of recombinant human γ-aminobutyric acid type A (GABAA, α1β2γ2s) receptor currents expressed in Xenopus oocytes. Sub-maximal (EC20) GABA currents were typically enhanced by co-applications of 3–300 μM (+)-menthol (e.g. by ~ 2-fold at 50 μM) > isopulegol > isomenthol> α-terpineol >> cyclohexanol. We studied menthol's actions on GABAA receptors compared to sedatives (benzodiazepines) and intravenous anesthetics (barbiturates, steroids, etomidate and propofol). Flumazenil (a benzodiazepine antagonist) did not inhibit menthol enhancements while currents directly activated by 50 μM propofol were significantly inhibited (by 26 ± 3%) by 50 μM (+)-menthol. GABAA receptors containing β2 subunits with either a point mutation in a methionine residue to a tryptophan at the 286 position (in transmembrane domain 3, TM-3) or a tyrosine to a tryptophan at the 444 position (TM-4) are insensitive to modulation by propofol. Enhancements of GABA EC20 currents by menthol were equally abolished in GABAA α1β2(M286W)γ2s and α1β2(Y444W)γ2s receptors while positive modulations by benzodiazepines, barbiturates and steroids were unaffected. Menthol may therefore exert its actions on GABAA receptors via sites distinct from benzodiazepines, steroids and barbiturates, and via sites important for modulation by propofol. Finally, using an in vivo tadpole assay, addition of (+)-menthol resulted in a loss of righting reflex with an EC50 of 23.5 ± 4.7 μM (~ 10-fold less potent anesthesia than propofol). Thus, menthol and analogs share general anesthetic action with propofol, possibly via action at similar sites on the GABAA receptor.

Introduction

Menthol is a fragrant monoterpenoid alcohol derived from peppermint (Mentha x piperita) oil and has been used for centuries as a food additive, a local anesthetic, a topical analgesic (Galeotti et al., 2002), an antipruritic and a gastric sedative agent. While menthol has long been recognized for its narcotic properties (Overton, 1901), it was recently shown to have profound effects on rodent behavior when administered intravenously (Umezu et al., 2001) and to act as a general anesthetic in fish (Kasai, 2004). Investigations of this monoterpenoid alcohol's neuroactive properties have revealed effects on neuronal intracellular signaling and modulating neurotransmitter-gated currents. For instance, menthol is an activator of the cold- and menthol-sensitive receptor-1 (CMR1) or transient receptor potential (TRPM8) channel, inducing Ca2+ release from intracellular stores and enhancing neurotransmission at sensory synapses (Tsuzuki et al., 2004). A previous study described menthol isomers as potent positive modulators of human γ-aminobutyric acid type A (GABAA) and glycine receptors (Hall et al., 2004b). This study also revealed a number of related agents (e.g. borneol, camphor, menthone and carvone) as positive modulators of GABAA receptors following reports of receptor modulation by other monoterpenes including citronellol and pinene (Aoshima and Hamamoto, 1999), thujone (Hold et al., 2000), thymol (Priestley et al., 2003), and borneol isomers (Granger et al., 2005).

GABAA receptors are the predominant ionotropic receptors for fast inhibitory neurotransmission in the mammalian central nervous system (CNS). Their pentameric structure is composed of different subunits (α1–6, β1–4, γ1–3, δ, ε, π, and θ) forming membrane-spanning chloride-selective ion channel complexes that are activated through the binding of GABA (Barnard et al., 1998). In mammalian neurons, the predominant GABAA receptor combination appears to be α1β2γ2 (McKernan and Whiting, 1996). These receptors are recognized as important targets for modulation by sedative, anxiolytic and general anesthetic agents (Franks and Lieb, 1994, Krasowski and Harrison, 1999). For instance, at clinically relevant concentrations the common intravenous anesthetic propofol is a potent enhancer of neuronal GABAergic currents and directly activates GABA currents at supraclinical concentrations (Hales and Lambert, 1991). More recently, specific residues on the GABAA receptor have been postulated as important binding sites for propofol including a methionine in the third transmembrane domain (M286, Krasowski et al., 2001b) and a tyrosine within the fourth transmembrane domain of the β2 subunit (Richardson et al., 2007). Since propofol and menthol have some structural similarities, including adjacency of an isopropyl group to the hydroxyl group on their respective rings (see Fig. 2), we hypothesized that they may share sites for modulatory action at the GABAA receptor.

In the following study, we used the Xenopus oocyte expression system to investigate modulation of recombinant human wild-type and mutant GABAA receptors (α1β2γ2s) by (+)-menthol to determine whether this monoterpenoid shares sites of action with common sedative and anesthetic agents. Our results indicated that (+)-menthol may indeed share sites on the receptor with the intravenous anesthetic propofol. We tested a number of menthol analogs for enhancement of GABA currents and describe structure–activity relationships for receptor modulation. Finally, we explored whether menthol can act as a general anesthetic in an established loss of tadpole righting reflex assay (Downes and Couragen, 1996). In this assay menthol acted as a general anesthetic with a potency that corresponded with its ability to enhance GABA currents compared to the other analogs tested.

Section snippets

Xenopus oocyte expression

cDNAs encoding for the α1, β2, γ2s subunits of human GABAA receptors were kindly provided by Dr. Paul J. Whiting (Merck, Sharp & Dohme Research Laboratories, UK). The GABAA receptor subunit cDNAs were prepared in pcDNA3.1+ vector and stored at − 20 °C prior to injection as described previously (Hall et al., 2004a). Mutations were introduced into the GABAA receptor β2 cDNA using the QuikChange Method (Stratagene, La Jolla, CA) by mixing together 6 µg of the GABAAR-β2-pcDNA3.1+ clone, synthetic

Menthol is a positive modulator of GABAA receptor currents via action at a different site to benzodiazepines

Oocytes were routinely screened for wild-type α1β2γ2s receptor expression by applications of 30 μM GABA that evoked ~ EC20 currents (effective concentration that evoked 20% of maximal current, see Hall et al., 2004a). As reported previously (see Hall et al., 2004b), co-applications of (+)-menthol produced dose-dependent potentiations of the GABA responses with 50 μM (+)-menthol typically resulting in ~ 2-fold enhancement of the EC20 GABA current (Fig. 1A). Pre-exposure to (+)-menthol did not

Discussion

GABAA receptors are the main ionotropic receptors responsible for fast inhibitory neurotransmission throughout the mammalian nervous system. Modulation of these receptors can produce profound influences on neuronal activity which may produce sedation and anesthesia in vivo. The major findings of the this study are that (1) the monoterpenoid menthol positively modulates GABAA receptor currents through sites of action other than those required for benzodiazepine, neurosteroid or barbiturate

Acknowledgements

We thank Dr. Lale Burk, Department of Chemistry, Smith College for helpful comments on the manuscript. This work was supported by grants from the Arthur Vining Davis Foundation to Smith College Neuroscience Program, from the Howard Hughes Medical Institute and the Beckman Foundation for funding BAB, from Merck/AAAS and Tomlinson Funding for EEW, from Blakeslee funding to ACH.

References (36)

  • UmezuT. et al.

    Ambulation-promoting effect of peppermint oil and identification of its active constituents

    Pharm. Biochem. Behav.

    (2001)
  • AoshimaH. et al.

    Potentiation of GABA-A receptors expressed in Xenopus oocytes by perfume and phytoncid

    Biosci. Biotechnol. Biochem.

    (1999)
  • BarnardE.A. et al.

    Subtypes of γ-aminobutyric acidA receptors: classification on the basis of subunit structure and receptor function

    Pharmacol. Rev.

    (1998)
  • DownesH. et al.

    Contrasting effects of anesthetics in tadpole bioassays

    J. Pharmacol. Exp. Ther.

    (1996)
  • FranksN.P. et al.

    Molecular and cellular mechanisms of general anesthesia

    Nature

    (1994)
  • GaleottiN. et al.

    Local anesthetic activity of (+)- and (−)-menthol

    Planta Med.

    (2001)
  • HaeselerG. et al.

    Voltage-dependent block of neuronal and skeletal muscle sodium channels by thymol and menthol

    Eur. J. Anaesth.

    (2002)
  • HalesT.G. et al.

    The actions of propofol on inhibitory amino acid receptors of bovine adrenomedullary chromaffin cells and rodent central neurons

    Br. J. Pharmacol.

    (1991)
  • Cited by (0)

    View full text