- Top of page
- Discussion and conclusions
- Conflict of interest
Monoterpenes, like camphor, borneol or menthol comprise a group of naturally occurring organic compounds derived from two isoprene units. Most of them are fragrant and form major constituents of many plant-derived essential oils. While most commonly used as antimicrobial agents, they also have a wide range of applications in pharmaceutical, medical and cosmetic fields. These uses range from anesthetic and analgesic (Galeotti et al., 2001, 2002; Xu et al., 2005) to anti-inflammatory (Santos and Rao, 2001) and antipruritic applications (Umezu et al., 2001; Anand, 2003).
Although the mechanisms of action are not completely understood for many of the described effects, different monoterpenes have been shown to activate, inactivate or modulate ion channels. Thus, borneol, thymol, α-thujone and menthol modulate γ-aminobutyric acid (GABA)A channels (Priestley et al., 2003; Hall et al., 2004; Granger et al., 2005); camphor and borneol are non-competitive inhibitors of nicotinic acetylcholine receptors (Park et al., 2003), and thymol affects calcium and potassium channels (Magyar et al., 2002; Szentandrassy et al., 2004). A number of monoterpenes have also been described as agonists or antagonists of different members of the transient receptor potential (TRP) channel family (Mckemy et al., 2002; Peier et al., 2002a, 2002b; Behrendt et al., 2004; Moqrich et al., 2005; Xu et al., 2005, 2006; Macpherson et al., 2006).
The TRP ion channel family contains several thermosensitive members, named thermoTRPs. ThermoTRPs are believed to serve as temperature sensors on the molecular level. While some members of the TRPV family activate on heating (TRPV1 and TRPV2 in the noxious range, at 42 and 52°C, respectively, TRPV3 at 39°C, and TRPV4 at 27–42°C), TRPM8 serves to detect innocuous cooling below 25°C (Smith et al., 2002; Xu et al., 2002; Patapoutian et al., 2003). TRPA1 has been described as being activated in the noxious cold temperature range below 17°C (Story et al., 2003), although the temperature sensitivity of this channel is controversial (Bautista et al., 2006).
In addition to temperature, thermoTRPs can be activated by other stimuli, such as osmolarity, pH or chemical agonists. Therefore, compounds such as (−)-menthol or capsaicin also cause changes in temperature perception through activation or inhibition of particular TRP channels. The ability of camphor, a naturally occurring monoterpene produced by the Camphor Laurel (Cinnamomum camphora), to modulate sensations of warmth in humans has been attributed to its ability to activate TRPV3 (Moqrich et al., 2005). TRPV3 is expressed in keratinocytes, the dorsal root ganglia, brain and spinal cord ([Peier et al., 2002a, 2002b; Xu et al., 2002). It has been implicated in hyperalgesia in inflamed tissues (Hu et al., 2006; Xu et al., 2006) and possibly skin sensitization (Xu et al., 2006).
TRPV3 is a sensitizing receptor (Peier et al., 2002b; Xu et al., 2002), such that upon prolonged or repeated stimulation, currents evoked by chemical or thermal stimuli increase in amplitude. The synthetic TRPV3 agonist, 2-aminoethoxy diphenyl borate (2-APB), which is structurally unrelated to camphor, has been shown to activate significant currents upon first application at concentrations of 100–300 μM. In contrast, camphor as a relatively weak agonist, activates sizeable currents only at concentrations of 10 mM or higher on first exposure. However, after preceding stimulation with higher doses, pronounced currents can already be observed with 2 mM camphor. Also, camphor's dose dependence is shifted to lower activating concentrations after prestimulating exposures to 2-APB before camphor treatment (Xu et al., 2005).
As still only few ligands are known for TRPV3, we aimed to investigate systematically the pharmacological profile of this receptor by testing compounds covering several important classes of terpenoids and thus establish a structure–function relationship. The most potent of the identified agonists were also tested on TRPM8 to determine whether similar structural requirements were relevant for activation of this channel and to assess the overlap of the receptor pharmacology.
Discussion and conclusions
- Top of page
- Discussion and conclusions
- Conflict of interest
The recent identification of thermosensitive TRP ion channels has been a major advance in our understanding of thermosensation. Interestingly, members of this ion channel family can also be activated or inhibited by chemical agonists. The ability of compounds to elicit warm, cool, hot or cold sensations is therefore currently attributed to the activation or inhibition of thermosensitive TRP channels. However, owing to the quite recent identification of most members of this ion channel family, relatively few agonists are known for most of them and little is known about the structural basis for activation.
While the general pharmacology of TRPM8 has been characterized in detail (Behrendt et al., 2004), chemical agonists for TRPV3 are only slowly emerging. The first chemical agonist identified for TRPV3 was the synthetic compound 2-APB (Hu et al., 2004). Shortly afterwards, the monoterpene camphor was found to be an agonist, albeit only at rather high concentrations (Moqrich et al., 2005). In addition, thymol, carvacrol and menthol have recently been described to activate TRPV3 (Xu et al., 2006; Macpherson et al., 2006), although no quantitative comparison was performed.
We therefore examined a number of terpenoid compounds in which different structural elements were systematically varied to identify features required for channel activation. Out of the 33 tested terpenes and related compounds, six agonists significantly stronger than camphor were identified. All of these activate TRPV3 with an EC50 substantially lower than that of camphor and the two most potent ones – 6-tert-butyl-m-cresol and carvacrol, also show a higher potency than the synthetic agonist 2-APB.
While camphor belongs to the group of the bicyclic monoterpenes, the best agonists were found among the monocyclic group. Interestingly, highly active agonists were encountered both among the aromatic and the non-aromatic compounds. Both tested acyclic monoterpenes activated the channel to a much lesser extent than camphor, which may indicate that a cyclic structure is required for activation.
It is striking that all of the six compounds more potent than camphor carry a secondary hydroxyl group. Oxidation to a carbonyl group reduced the activity of the substance drastically, arguing that a hydroxyl group is a structural requirement for efficient activation of TRPV3. In line with this, none of the compounds without an oxygen moiety (p-cymene and (+)-limonene) activated the channel to a significant extent.
While the position of the hydroxyl group on the ring does not appear to be critical for TRPV3 activation in aromatic substances, it is relevant for non-aromatic compounds. Here, much stronger activation is achieved with the hydroxyl group in the meta position to the isopropyl residue as in dihydrocarveol and (−)-carveol, rather than in the ortho position as in (−)-isopulegol and (−)-menthol. No significant activation can be seen by compounds in which the hydroxyl group is not located on the ring itself (as in terpineol, mugetanol or (−)-α-bisabolol).
A similar requirement for a ring-located hydroxyl group has also been described for the modulation of GABAA receptors by terpenoids (Mohammadi et al., 2001). Among the investigated compounds are several monoterpenes such as thymol and menthol, accounting for their ability to serve as anxiolytic or sedative agents (Priestley et al., 2003; Hall et al., 2004). Thymol was also identified as one of the most potent TRVP3 agonists in the presented work. In contrast, the high affinity GABAA ligand propofol did not activate TRPV3 to a significant extent, indicating differences in the structural requirements for activation and that the overlap between these unrelated receptors is probably confined to a small group of substances.
Like odorant receptors, thermoTRP channels are not highly specific for one given ligand, but are activated by a number of chemically similar agonists. Recent studies identified overlaps in the agonist profiles of different thermoTRPs, such as TRPV1, TRPV3, TRPM8 and TRPA1 (Macpherson et al., 2005, 2006), which complicates the prediction of the sensory effects elicited by a given compound. While the activation of TRPV1 and TRPA1, which both localize to nociceptors, by the same compounds offers a comprehensible explanation of the pungent sensation evoked by these substances, the concomitant activation of cool-activating TRPM8 and warm-activating TRPV3 offers more of a puzzle. In the case of (−)-menthol, activation of both receptors has been correlated with a paradoxical feeling of warmth at higher temperatures in addition to the well-known cooling sensation elicited by this compound when applied at lower temperatures (Macpherson et al., 2006).
To further investigate whether additional substances are shared agonists on these two receptors and whether structural requirements for activation are similar, we applied the most potent identified agonists of TRPV3 on TRPM8. As seen with TRPV3, monoterpenes without a substituent only weakly activated TRPM8. Also as for TRPV3, oxidation of the hydroxyl group in (−)-menthol to yield (−)-menthone drastically reduced M8 activation.
However, neither of the potent TRPV3 agonists could activate TRPM8 as effectively as (−)-menthol and even small changes in the structure of this ligand resulted in a much reduced activation: Both the introduction of a double bond in the isopropyl residue, as in (−)-isopulegol, or the change to an aromatic ring, as in thymol, reduced the activity by a factor of at least three. While a number of (−)-menthol derivatives are known to be potent TRPM8 activators, such as Frescolat ML, WS-3 or cooling- agent- 10, all of these compounds share the (−)-menthol structure and stereochemistry and carry different substituents at the position of the hydroxyl group only.
Interestingly, the non-terpenoid TRPV3-ligand 2-APB is known to inhibit TRPM8 (Hu et al., 2004) and thus has opposing effects on the two channels. Therefore, although both channels are activated by members of the monoterpene family, their pharmacological profiles appear to overlap only marginally.
It has been suggested that TRPV3 might be a molecular target for skin sensitizers (Xu et al., 2006). However, our data do not provide evidence for a correlation of TRPV3 agonism and skin sensitization. Thus, the potent TRPV3 agonists carvacrol and thymol are not sensitizers (Andersen, 2006), borneol is a weak skin sensitizer and allergic reactions to camphor are rare. In contrast, the known skin sensitizers, geraniol (Frosch et al., 1995) and carvone (Paulsen et al., 1993) are only poor TRPV3 agonists. While our study did not address the human receptor or test the behaviour of TRPV3 in the native system, a direct relationship between TRPV3 agonism and skin sensitization is not supported by our results, leading us to suggest that the role of TRPV3 in skin sensitization still needs to be clarified.
Terpenoids have been used in essential oils for centuries as medically and cosmetically relevant compounds, but still little is known about their mechanism of action. TRP channels as targets for terpenoids can potentially explain several of the described effects. For instance, the desensitization of nociceptive TRPV1 and/or the activation of TRPM8 have been suggested to underlie the analgesic effect of naturally occurring compounds like (−)-menthol or camphor (Xu et al., 2005; Macpherson et al., 2006; Proudfoot et al., 2006). Consequently, activation, desensitization or inhibition of particular TRP channels by natural or synthetic compounds is a promising tool in medicine and pharmacology. Moreover, a more comprehensive knowledge of the pharmacology of different thermoTRPs may offer the possibility to alter chemical structures to achieve activation or inhibition of these channels. In this context, the investigation of ligands for members of the TRP channel family is not only relevant for the understanding of this protein family, but also has implications for target-directed drug design.