Inhibitory effects of linalool, an essential oil component of lavender, on nociceptive TRPA1 and voltage-gated Ca²⁺ channels in mouse sensory neurons

Abstract

Linalool, an essential oil component of lavender is commonly used in fragrances. It is known that linalool has anxiolytic, sedative, and analgesic actions. However, the mechanism of its analgesic action has not yet been fully clarified. Pain signals elicited by the activation of nociceptors on peripheral neurons are transmitted to the central nervous system. In the present study, we investigated the effects of linalool on transient receptor potential (TRP) channels and voltage-gated channels, both of which are important for pain signaling via nociceptors in somatosensory neurons. For detection of channel activity, the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured using a Ca²⁺-imaging system, and membrane currents were recorded using the whole-cell patch-clamp technique. Analgesic actions were also examined in vivo.

In mouse sensory neurons linalool at concentrations that did not induce [Ca²⁺]_i increases did not affect [Ca²⁺]_i responses to capsaicin and acids, TRPV1 agonists, but suppressed those induced by allyl isothiocyanate (AITC) and carvacrol, TRPA1 agonists. Similar inhibitory effects of linalool were observed in cells that heterologously expressed TRPA1. Linalool attenuated the [Ca²⁺]_i increases induced by KCl and voltage-gated Ca²⁺ currents but only slightly suppressed voltage-gated Na^＋currents in mouse sensory neurons. Linalool diminished TRPA1-mediated nociceptive behaviors.

The present data suggest that linalool exerts an analgesic action via the suppression of nociceptive TRPA1 and voltage-gated Ca²⁺ channels.

Highlights

• •Linalool inhibited TRPA1 activation but not TRPV1 in native and heterologously expressed cells.
• •[Ca²⁺]_i responses to KCl and Cav currents were suppressed by linalool in mouse sensory neurons.
• •Linalool decreased TRPA1-mediated nociceptive behaviors.

1. Introduction

Various chemical compounds extracted from plants are useful resources for providing low-cost, safe substances useful for druggability. Linalool is an acyclic monoterpene alcohol included in essential oils of bergamot, rosewood, and lavender [1]. These are commonly used as fragrances in soaps, cosmetics, and perfumes [2]. Essential oils including linalool possess analgesic effects. For example, bergamot oil and linalool inhibit formalin-induced nociceptive behaviors in mice [3]. Lavender essential oil alleviates mechanical allodynia in a mouse model of neuropathic pain [4]. However, the molecular targets involved in the analgesic action of linalool have not been fully elucidated.

Transient receptor potential (TRP) channels consist of a large family with more than 20 subtypes [5]. TRP ankyrin 1 (TRPA1) and TRP vanilloid 1 (TRPV1) channels, non-selective cation channels, are well-known as nociceptors in peripheral nerves and play important roles in transmitting pain signaling to the central nervous system [6]. Therefore, the inhibition of TRPA1 and TRPV1 activation is an attractive pharmacological strategy for developing analgesics [7]. TRPA1 is activated by various pungent chemicals such as allyl isothiocyanate (AITC), and acts as a cold sensor [8,9], but the temperature sensitivity of mammalian TRPA1 remains controversial [10]. TRPV1 is activated by various stimuli, including high temperature (>43 °C), acids and capsaicin from hot chili peppers [11,12]. The inhibitory effects of monoterpenes on TRPA1 and TRPV1 have been reported. For example, borneol and eucalyptol suppress the activation of TRPA1 [13]. Limonene inhibits oxidative stress-induced TRPA1 activation [14]. Camphor activates and desensitizes TRPV1 [15].

Voltage-gated Na⁺ (Nav) and voltage-gated Ca²⁺ (Cav) channels are involved in peripheral pain signals [16,17]. Nav channels play a pivotal role for conduction of action potentials elicited at peripheral nociceptors. Nav 1.8 TTX-resistant Nav channels expressed in unmyelinated C-fibers and their cell bodies are important for perceiving pain sensation [18]. N-type Cav channels expressed in presynaptic nerve terminals and sensory neurons play a critical role in pain transmission, and secretion of inflammatory mediators [17]. Therefore, Nav and Cav are the principal targets for pain-relieving drugs.

As mentioned above, the effects of some monoterpenes and essential oil components on various ion channels related to pain signaling have been investigated [19], but there is little information on the analgesic effects of linalool specifically focusing on nociceptive channels. Therefore, we investigated the effects of linalool on nociceptive TRP channels as well as Nav and Cav channels in mouse sensory neurons and heterogeneously expressed TRP channels. We found that linalool suppressed the increases of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) induced by TRPA1 agonists and Cav currents. Furthermore, linalool reduced endogenous TRPA1 agonist-induced nociceptive behaviors. Our present data suggest that the analgesic effect of linalool is mediated via the blockade of TRPA1 and Cav channels in sensory neurons.

2. Materials and methods

Animals: We used adult C57BL/6 wild type and TRPA1-gene deficient (A1KO) mice (male or female, 8–24 weeks old). A1KO mice were kindly provided by Dr. Julius, University of California. To isolate sensory neurons the animals were euthanized by CO₂ gas inhalation. All experimental protocols were approved by the Committee on Animal Experimentation of Tottori University. We made efforts to use a minimal number of animals.

Culture of sensory neurons: Mouse sensory neurons were isolated from dorsal root ganglia (DRG) and cultured as previously described [20]. Briefly, isolated DRG were put into phosphate-buffered saline (PBS: in mM, 137 NaCl, 10 Na₂HPO₄, 1.8 KH₂PO₄, 2.7 KCl) and enzymatically digested for 30 min at 37 °C in PBS containing collagenase (1 mg/ml, type II, Worthington, Ranklin, OH) and DNase I (1 mg/ml, Roche, Basel, Switzerland). Next, the digested ganglia were immersed in PBS containing trypsin (10 mg/ml, Sigma, St. Louis, MO) and DNase I (1 mg/ml) at 37 °C for 15 min. The ganglia were subsequently washed with the culture medium (Dulbecco's modified Eagle's medium [DMEM], Sigma) supplemented with 10% fetal bovine serum (Sigma), penicillin G (100 U/ml), and streptomycin (100 μg/ml). After gentle trituration with a fire-polished Pasteur pipette, the DRG cell suspension was centrifuged (800 rpm, 2 min, room temperature) and the pellet was suspended again with the culture medium. Aliquots were placed onto glass cover slips coated with poly-D/l-lysine (Sigma) and cultured in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C. Cells were used within 24 h after the isolation.

Heterologous expression: HEK 293 cells were cultured in the culture medium described above. The cells were transfected with the expression vectors (mouse TRPA1 and mouse TRPV1) [21] by using a transfection reagent (Lipofectamine 2000, Invitrogen, Thermo Fisher Scientific, Waltham, MA) and used within 24 h after the transfection.

Ca²⁺imaging: Measurement of [Ca²⁺]_i in each cell was performed using a fluorescent imaging system (Aqua Cosmos, Hamamatsu Photonics, Hamamatsu, Japan) [22]. The cells were loaded with 10 μM fura-2 AM (Thermo Fisher Scientific) for 40 min at 37 °C in HEPES-buffered solution contained (in mM) 134 NaCl, 6 KCl, 1.2 MgCl₂, 2.5 CaCl₂, 10 glucose, and 10 HEPES (adjusted with NaOH to pH 7.4). A cover slip with fura-2-loaded cells was placed in an experimental chamber mounted on the stage of an inverted microscope (Olympus IX71, Tokyo, Japan) equipped with an image acquisition and analysis system. The cells were illuminated every 5 s with lights at 340 and 380 nm, and the respective fluorescent signals at 500 nm were detected. [Ca²⁺]_i was calculated from the ratio of fluorescent signals (F340/F380) as reported previously [23]. Cells were continuously superfused with an external solution via gravity through a tube placed close to the cells at a flow rate of ∼2.5 ml/min and drugs were applied, placing the same perfusion tube into the solution with each drug. For the preparation of acid solution (pH 5.0), MES was used as a pH buffer instead of HEPES for the external solution. The composition of 80 mM KCl was (in mM) 60 NaCl, 80 KCl, 1.2 MgCl₂, 2.5 CaCl₂, and 10 HEPES (pH 7.4 adjusted with NaOH). A 40 mM KCl solution was prepared by diluting 80 mM KCl solution with HEPES-buffered solution. For the experiments using DRG cells, high KCl responding ones were identified as neurons. All experiments were carried out at room temperature (22–25 °C).

Whole-cell current recording: Membrane currents were recorded using the conventional whole-cell configuration of the patch-clamp technique. Neurons with small diameters (<20 μm) were selected for current recording. The average membrane capacitance of neurons used was 15.4 ± 0.86 pF (n = 15). A patch pipette (4–5 MΩ resistance) was filled with an internal solution (in mM: 120 KCl, 20 CsOH, 10 HEPES, 10 EGTA, 1.2 MgATP, pH 7.3 adjusted with CsOH). For measurement of Nav currents, cells were superfused with HEPES-buffered solution as for Ca²⁺ imaging experiments. For measurement of Cav currents, the external solution was (in mM) 134 TEA-Cl, 5 CsCl, 5 BaCl_2, 0.5 MgCl₂, 10 HEPES, and 10 glucose (pH 7.4 adjusted with CsOH). Membrane potential was clamped at −60 mV and brief depolarization pulses to 0 mV (50 ms in duration for Cav currents, 10 ms in duration for Nav currents) were applied every 5 s. The whole-cell currents were sampled at 5 kHz and filtered at 2 kHz using a patch-clamp amplifier (Axopatch 200 B; Molecular Devices, Sunnyvale, CA) in conjunction with an A/D converter (Digidata 1322 A; Molecular Devices) using data acquisition software (pClamp 9, Molecular Devices). Data were analyzed using Clampfit 9.2 software (Molecular Devices). In neurons with diminished current amplitude without any treatment, no further analysis was performed. Experiments were performed at room temperature (22–25 °C).

Nociceptive behavioral test: Male wild-type and A1KO mice were used. The wild-type mice were divided into two groups to examine the effect of linalool on nociceptive behaviors (biting and lifting of an injected hind paw) induced by PGJ₂. All mice were acclimated to the environment for 30 min before the tests. Mice were injected on the plantar surface of the right paw with 10 μl of linalool or vehicle (5% DMSO). Ten minutes after the injection of linalool or vehicle, we injected 10 μl of PGJ₂ (10 nmol) into the ipsilateral hind paw and observed nociceptive behaviors. To confirm that the behavioral responses to PGJ₂ was due to TRPA1 activation, we also observed PGJ₂-induced nociceptive behaviors of A1KO mice.

Chemicals: The following drugs were used (vehicle and concentration for stock solution). AITC (dimethyl sulfoxide [DMSO], 1 M) was from Nakarai (Tokyo, Japan). Capsaicin (ethanol, 1 mM), capsazepine (DMSO, 0.1 M) and A967079 (DMSO, 0.1 M) were from Sigma. Carvacrol (DMSO, 1 M) and linalool (DMSO, 1 M) were from Tokyo Chemical Industry (Tokyo, Japan). 15-deoxy-Δ^12,14-PGJ₂ (PGJ₂) (DMSO, 0.02 M) was from Cayman chemical (Ann Arbor, MI, USA).

Data analysis: The data are indicated as the mean ± SEM (n = number of cells). To compare two groups, data were analyzed using the unpaired Student's t-test, and to compare multiple groups, one-way ANOVA followed by the Tukey-Kramer test was used. A P-value of less than 0.05 was considered a significant difference.

3. Results

3.1. Effects of linalool on the [Ca²⁺]_i responses to TRPV1 and TRPA1 agonists in mouse sensory neurons

Linalool has two enantiomeric forms, S-(+)- and R-(−)-linalool, which are included in various essential oils [1]. We used (±)-linalool in this study. First, we examined whether linalool changed the resting [Ca²⁺]_i in mouse sensory neurons. We defined cells showing [Ca²⁺]_i responses of over 20 nM as positively responding neurons. Linalool (≤1 mM) did not affect the resting [Ca²⁺]_i but it increased [Ca²⁺]_i at 3 mM and intense [Ca²⁺]_i responses were observed at 10 mM (Fig. 1a). The percentage of responding neurons increased with increasing concentrations of linalool and almost all neurons responded at 10 mM (Fig. 1b). These results indicate that linalool at high concentrations elicits nonspecific [Ca²⁺]_i responses in mouse sensory neurons. In the following experiments, therefore, we used linalool at 1 mM or less.

Fig. 1.

Effects of linalool on the [Ca²⁺]_i responses to TRPV1 and TRPA1 agonists in mouse sensory neurons. (a) [Ca²⁺]_i responses to linalool in mouse sensory neurons. Representative averaged traces of [Ca²⁺]_i responses to linalool (1, 3, and 10 mM). Vertical lines show mean ± SEM (n = 27). (b) The relationship of the amplitudes of [Ca²⁺]_i increases and the percentages of neurons responding to linalool against the concentration of linalool. Symbols and columns with vertical lines show mean ± SEM (n = 177, from 5 mice). Some small parts of the vertical lines are hidden by the symbols. **P < 0.01 vs. 1 mM linalool. (c) Representative averaged traces of [Ca²⁺]_i responses to 0.1 μM capsaicin alone and together with 1 mM linalool. Vertical lines show mean ± SEM (left; n = 23, right; n = 23). The agonist was applied for 5 min. Linalool was applied 2 min before and at the first 1 min during the stimulation with each agonist. After washout of linalool, capsaicin was applied alone and together with 10 μM capsazepine. (d) Summarized amplitudes of [Ca²⁺]_i responses to TRPV1 agonists (lines, capsaicin left, pH 5.0 right) and % of responding cells (columns) in the absence and presence of linalool. ns; not significant vs. 0 mM linalool. (e) Representative averaged traces of [Ca²⁺]_i responses to 50 μM AITC alone and together with 1 mM linalool. Vertical lines show mean ± SEM (left; n = 36, right; n = 23). The protocol for agonist and linalool application was the same as shown in c. After washout of linalool, AITC was applied alone and together with 10 μM A967079. (f) Summarized amplitudes of [Ca²⁺]_i responses to TRPA1 agonists (lines, AITC left, carvacrol right) and % of responding cells (columns) in the absence and presence of linalool. Symbols and columns with vertical lines indicate mean ± SEM (a; n = 81–110 from 4 mice, b; n = 60–75, from 3 mice). **P < 0.01 vs. 0 mM linalool.

Since TRPV1 and TRPA1 channels are involved in the pain sensation as nociceptors [6], we examined the effects of linalool on the [Ca²⁺]_i responses to agonists of TRPV1 and TRPA1 in mouse sensory neurons. Capsaicin (0.1 μM, a TRPV1 agonist) or AITC (50 μM, a TRPA1 agonist) evoked [Ca²⁺]_i increases that were sustained in its presence. Considering channel desensitization due to repetitive application of agonists, cells were stimulated only once by each agonist with or without linalool (Fig. 1c and e). Linalool (1 mM) did not affect the magnitude of [Ca²⁺]_i increases or the percentages of neurons responding to capsaicin and acids (pH 5.0) (Fig. 1d). The capsaicin responses were inhibited by capsazepine, a TRPV1 antagonist. In contrast, AITC-induced [Ca²⁺]_i increases and the percentages of responding neurons were greatly suppressed by linalool (Fig. 1f). After washout of linalool, AITC responses appeared and were suppressed by A967079, a TRPA1 antagonist. Linalool also inhibited the [Ca²⁺]_i response to carvacrol (0.1 mM, another TRPA1 agonist). The IC₅₀ values for AITC- and carvacrol-induced responses by linalool were 0.37 ± 0.06 mM and 0.32 ± 0.04 mM, respectively. These results suggest that linalool suppress the activation of TRPA1 but not TRPV1 in mouse sensory neurons.

3.2. Effects of linalool on the voltage-gated Ca²⁺ and Na ⁺ channels in mouse sensory neurons

Since voltage-gated Ca²⁺ (Cav) channels are related to neural transmission through synapses [17] and secretion of inflammatory substances such as calcitonin gene-related peptide and substance P [24], we examined the effects of linalool on the [Ca²⁺]_i increases induced by KCl in mouse sensory neurons. Linalool reversibly inhibited KCl-induced [Ca²⁺]_i increases in a dose-dependent manner (Fig. 2a). The concentration-inhibition relation of linalool on KCl-induced responses is shown in Fig. 2b. The IC₅₀ was 0.38 ± 0.06 mM. These results suggest that linalool suppresses Cav channels in mouse sensory neurons.

Fig. 2.

Effects of linalool on the voltage-gated Ca²⁺ (Cav) and voltage-gated Na⁺ (Nav) channels in mouse sensory neurons. (a) Effects of linalool on the [Ca²⁺]_i responses to KCl in mouse sensory neurons. Representative averaged traces of [Ca²⁺]_i responses to KCl (40 mM). Vertical lines show mean ± SEM (n = 58). Neurons were repetitively stimulated with KCl for 1 min with intervals of 10 min in the absence or presence of linalool. Linalool was applied 1 min before and during the KCl stimulation. (b) Summarized responses to KCl in the presence of various concentrations of linalool. Symbols with vertical lines show mean ± SEM (n = 108, from 3 mice). *P < 0.05, **P < 0.01 vs. 0 mM linalool. Some parts of the vertical line are hidden by the symbols. (c) Time courses of changes in peak amplitude of inward Cav currents repetitively evoked every 5 s by depolarizing pulses to 0 mV for 50 ms from a holding potential of −60 mV. An increasing concentration of linalool was applied for the period shown by the horizontal bars with arrows. Inset shows superimposed current records corresponding to the numbered data points. (d) Current-voltage relationships in the absence (Control; open circles) and presence of 1 mM linalool (+Linalool; closed circles). (e) Time course of changes in peak amplitudes of inward Nav currents repetitively evoked every 5 s by depolarizing pulses to 0 mV for 10 ms from a holding potential of −60 mV. An increasing concentration of linalool was continuously applied. Inset shows actual current records corresponding to the numbered data points. (f) Concentration-inhibition relationships of linalool. Relative amplitudes of Cav and Nav currents in the presence of various concentrations of linalool, which are expressed as the normalized values of that obtained prior to its application, are plotted against concentrations of linalool. Symbols with vertical lines show mean ± SEM (Cav; n = 8, Nav; n = 7). *P < 0.05, **P < 0.01 vs. 0 mM linalool. Some parts of the vertical line are hidden by the symbols.

To obtain direct evidence for the linalool-induced Cav channel inhibition, we next examined the effect of linalool on the Cav currents by using the whole-cell patch-clamp technique. In cells dialyzed with Cs⁺-containing internal solution, a depolarizing pulse to 0 mV from a holding potential of −60 mV evoked inward currents. These currents were greatly suppressed by Cd²⁺ (0.1 mM) (data not shown). After stable currents were obtained, various concentrations of linalool were applied. Inward Cav currents were decreased by the application of linalool in increasing concentrations (Fig. 2c). The current-voltage relationships in the absence and presence of linalool (1 mM) are shown in Fig. 2d.

We also examined the effects of linalool on voltage-gated Na⁺ (Nav) currents. Contrary to Cav currents, the inward Nav currents were only slightly inhibited by linalool, even at 1 mM (Fig. 2e). Fig. 2f shows the concentration-inhibition relations of linalool on Cav and Nav currents in mouse sensory neurons. The Cav currents were dose-dependently inhibited by linalool (IC₅₀: 0.20 ± 0.04 mM) much more than the Nav ones. These results indicate that linalool suppress Cav channels but has only a slight inhibitory effect on Nav channels.

3.3. The effects of linalool on the [Ca²⁺]_i responses to TRPV1 and TRPA1 agonists in HEK293 cells expressing TRP channels

To confirm the inhibitory action of linalool on TRPA1 and its lesser one on TRPV1, we investigated the effect of linalool in each heterologously expressed channel. Before evaluating the action of linalool on TRP channels, we examined whether linalool itself affected the [Ca²⁺]_i in transfected naïve HEK293 cells. Linalool (1 mM) did not change the resting [Ca²⁺]_i in naïve HEK293 cells. Moreover, it failed to change the [Ca²⁺]_i in TRPV1-expressing cells (Fig. 3a). In contrast, linalool elicited [Ca²⁺]_i increases in about half of the TRPA1-expressing cells (0.3 mM; 52.7%, 1 mM; 44.6%), but the amplitude of the [Ca²⁺]_i responses to it (0.3 mM, 38.1 ± 4.3 nM) was smaller than that to AITC (50 μM, 90.0 ± 5.7 nM) (Fig. 3b). These [Ca²⁺]_i responses were abolished by 10 μM A967079, a TRPA1 blocker, indicating that linalool had a partial agonistic effect on heterogeneously expressed TRPA1 channels.

Fig. 3.

Effects of linalool on the [Ca²⁺]_i responses to TRPV1 and TRPA1 agonists in HEK293 cells expressing TRP channels. (a, b) Concentration-response relationships of linalool in HEK293 cells expressing TRPV1 (TRPV1-HEK) and TRPA1 (TRPA1-HEK) with and without A967079 (10 μM). Lines with vertical lines show mean ± SEM (a; n = 11–24 from 1 to 3 transfections, b; n = 53–112, from 5 transfections). (c, d) Representative averaged traces of [Ca²⁺]_i responses to 0.1 μM capsaicin and 50 μM AITC alone (left) and together with 1 mM linalool (right) in cells expressing each TRP channel. Vertical lines show mean ± SEM (c; left; n = 5, right; n = 4, d; left; n = 4, right; n = 4). Linalool was applied 2 min before and at the first 1 min during the stimulation with each agonist. After washout of linalool, capsaicin or AITC was applied alone and together with 10 μM capsazepine or 10 μM A967079. (e, f) Summarized [Ca²⁺]_i responses to the TRPV1 and TRPA1 agonists in the absence and presence of linalool. Lines with vertical lines show mean ± SEM (0.1 μM capsaicin; n = 25–29 from 3 transfections, pH 5.0; n = 28–29 from 4 transfections, AITC; n = 26–60 from 4 transfections, carvacrol; n = 29–44 from 4 transfections). ns; no significant vs. 0 mM linalool. **P < 0.01 vs. 0 mM linalool.

We evaluated the inhibitory actions of linalool on heterogeneously expressed TRP channels. It did not affect the [Ca²⁺]_i increases induced by capsaicin and acids (pH 5.0) in HEK293 cells that expressed TRPV1 (Fig. 3c and e). The TRPV1 responses were inhibited by capsazepine. Since linalool elicited [Ca²⁺]_i increases in half of the TRPA1-expressing HEK293 cells, we analyzed its effects of on TRPA1 in non-responders to linalool. [Ca²⁺]_i responses to AITC were greatly suppressed in the presence of linalool (Fig. 3d). After washout of linalool, AITC responses appeared and were suppressed by A967079 (Fig. 3d). Linalool dose-dependently suppressed [Ca²⁺]_i increases induced by AITC and carvacrol with IC₅₀ values of 0.30 ± 0.02 mM and 0.29 ± 0.02 mM, respectively (Fig. 3d and f).

3.4. The effects of linalool on the [Ca²⁺]_i responses and nociceptive behaviors induced by PGJ₂, an endogenous TRPA1 agonist

As mentioned above, linalool inhibited exogenous agonist-induced TRPA1 activation in native and heterologously expressed channels. We next investigated the effect of linalool on the responses to PGJ₂, an endogenous TRPA1 agonist produced under inflammatory conditions [25]. In mouse sensory neurons, PGJ₂ (50 μM) evoked [Ca²⁺]_i increases that were inhibited by linalool. After washout of linalool, PGJ₂ responses appeared and were then suppressed by A967079 (Fig. 4a). The relative concentration-inhibition relations for linalool on PGJ₂ responses in sensory neurons and HEK293 cells expressing TRPA1 are illustrated in Fig. 4b. For comparison, the inhibitory effects of linalool on AITC and carvacrol are overwritten. Linalool suppressed the TRPA1 activation by PGJ₂, AITC and carvacrol to the same degree.

Fig. 4.

Effects of linalool on [Ca²⁺]_i responses and nociceptive behaviors induced by PGJ₂. (a) Representative averaged traces of [Ca²⁺]_i response to 50 μM PGJ₂ (left) and together with 1 mM linalool (right) in mouse sensory neurons. Vertical lines show mean ± SEM (left; n = 9, right; n = 7). After washout of linalool, PGJ₂ was applied alone and together with 10 μM A967079. (b) Relative amplitudes of [Ca²⁺]_i responses to PGJ₂ in sensory neurons (left) and TRPA1-HEK (right). For comparison, the concentration-inhibition relationships of linalool for AITC and carvacrol are overwritten (broken lines). **P < 0.01 vs. 0 mM linalool. Symbols with vertical lines show mean ± SEM (PGJ₂; n = 108–122, from 3 mice, n = 14–15 from 3 transfections). Some parts of the vertical lines are hidden by the symbols. (c) Time courses of nociceptive behaviors induced by PGJ₂ (10 nmol i. pl.) at every 2 min without (vehicle) and with linalool (200 nmol i. pl.). *P < 0.05, **P < 0.01 vs. vehicle. (d) Total times of nociceptive behaviors during 20 min after PGJ₂ injection into A1KO (hatched column) and wild-type mice (open columns) without and with linalool (130 and 200 nmol i. pl.). Lines with vertical lines show mean ± SEM for 4 mice per group. *P < 0.05, **P < 0.01 vs. 0 mM linalool in wild-type mice.

Based on the in vitro results, we examined the analgesic effect of linalool via TRPA1 inhibition in vivo. We measured the time of nociceptive behaviors (lifting and biting of the hind paw). Intraplantar injection of PGJ₂ (10 nmol) caused nociceptive behavior after its application and ceased within 10 min in wild-type mice (Fig. 4c). The total nociceptive behavior time was significantly decreased in A1KO mice (Fig. 4d), indicating that PGJ₂ elicits TRPA1-dependent nociceptive behaviors as reported previously [25]. Linalool (200 nmol) and vehicle evoked almost no nociceptive behaviors, but the former reduced PGJ₂-induced nociceptive behaviors (Fig. 4c). The total time of nociceptive behaviors for 20 min after the administration of PGJ₂ was significantly reduced by linalool (Fig. 4d). These results suggest that linalool is capable of suppressing TRPA1-mediated nociception in vivo.

4. Discussion

In the present study, we showed that linalool suppressed the [Ca²⁺]_i responses to TRPA1 agonists, but not TRPV1 ones, in native and heterologously expressed channels. Moreover, linalool decreased KCl-induced [Ca²⁺]_i increases and Cav currents in mouse sensory neurons. The present data focusing on nociceptors and their transduction mechanisms, our results indicated that linalool inhibited TRPA1 and Cav channels. Moreover, linalool decreased TRPA1-mediated nociceptive behaviors in vivo. There are a number of other mechanisms involved in the analgesic actions of linalool on TRPA1 and voltage-gated Ca²⁺ channels. For example, linalool reduces nitric oxide synthesis [26], activates adenosine receptors, opioid receptors, and the GABAergic system [[27], [28], [29]], and inhibits ionotropic glutamate receptors [30].

As shown in Fig. 1a, linalool at 1 mM did not induce [Ca²⁺]_i responses, but at 3 mM evoked [Ca²⁺]_i in some neurons and at 10 mM prominent [Ca²⁺]_i increases in almost all neurons. Since [Ca²⁺]_i overload is related to cell death [31], high concentrations of linalool may induce acute cytotoxicity. Indeed, linalool has been reported to induce anti-cancer activities at 2.5 mM in prostate cancer cells [32]. However, linalool (1 mM) failed to change [Ca²⁺]_i in untransfected naïve HEK293 cells, suggesting that at concentrations of linalool less than 1 mM linalool has little in the way of nonspecific effects.

It has been reported that linalool has a stimulatory action on TRPA1 [33,34]. In those reports, the authors used HEK293 cells expressing human TRPA1 for their experiments. On the other hand, we used mouse sensory neurons and HEK293 cells expressing mouse TRPA1. It has been reported that menthol, a monoterpene compound, activates human TRPA1, but inhibits mouse TRPA1 responses induced by AITC [35]. Thus, the opposite effect of linalool may be due to the difference of species.

The TRPA1 channel is desensitized by its agonists, [36]. In the present study, linalool activated TRPA1 in about half of the HEK293 cells expressing it with weaker agonistic action than the fully agonist AITC. However, the inhibitory effects of linalool on the TRPA1 were clearly observed in cells that did not respond to linalool. Thus, it is likely that linalool-induced TRPA1 inhibition is not related to channel desensitization. A recent paper reported that the odor-induced analgesic effect of linalool was independent of TRPA1 in mice [37]. The difference from our data obtained in vitro experiments may be derived from the different experimental settings. In trigeminal ganglion neurons, TRPM8 is expressed in addition to TRPA1 [38]. It has been reported that linalool induces Ca²⁺ responses through the activation of TRPM8 [39]. Linalool vaporized at room temperature might exert analgesic action via TRPM8 in the trigeminal nerve in the nasal epithelium.

There are two modes of action for TRPA1 activation dependent on agonists. Electrophilic agonists such as AITC activate TRPA1 through covalent modification of cysteine residues of the N terminal of the channel [40]. Non-electrophilic agonists such as carvacrol act on TRPA1 non-covalently [35]. Linalool inhibited TRPA1 activation induced by both AITC and carvacrol to almost the same extent in endogenously and heterogeneously expressed channels (Fig. 1, Fig. 3f). Moreover, linalool suppressed not only TRPA1 but also Cav channels (Fig. 2b). Since monoterpenes are highly hydrophobic and easily inserted into biological membranes, they produce numerous physicochemical interactions of lipid bilayers [41]. In the present study, however, linalool did not inhibit TRPV1 activation and the inhibitory action on Nav channels was slight (Fig. 2, Fig. 3e). Therefore, linalool may act on some specific sites or domains of TRPA1 and Cav channels. A recent paper indicated that specific amino acid residues were involved in the inhibitory action of monoterpenes on TRPV1 and TRPV3 channels [42]. Additional experiments may be needed to determine the molecular mechanisms responsible for the inhibitory actions of linalool on TRPA1 and Cav channels.

In mouse sensory neurons, linalool did not show agonistic effects on TRPA1, unlike heterologously expressed channels. Such differences between endogenously and heterologously expressed channels have been reported for the inhibitory effects on TRPA1 and this report suggests possible differences in expression levels [43]. Therefore, our inconsistent results may also be explained by the amount of TRPA1 expression and/or the difference of cell types. Nevertheless, the fact that linalool suppressed TRPA1 activation without an agonistic effect in sensory neurons could be beneficial for its an analgesic action.

We examined the effects of linalool on the responses to PGJ₂, which is an endogenous TRPA1 agonist produced under inflammatory conditions [25]. Similar to its inhibitory effects on exogenous agonists, linalool inhibited PGJ₂-induced TRPA1 activation in sensory neurons and heterologously expressed channels with similar potency (Fig. 4b). Moreover, it significantly decreased PGJ₂-induced nociceptive responses, which are mediated by TRPA1-activation because of the near disappearance of nociceptive behaviors in TRPA1 KO mice. These data suggest analgesic action of linalool via the suppression of nociceptive TRPA1 channels. It has been reported that intraplantar injection of linalool reduces acute pain induced by paclitaxel in mice [28]. Paclitaxel-induced pain is mediated by TRPA1 activation [44]. Therefore, it may be not necessary for linalool to interact with TRPA1 on olfactory neurons for the analgesic effect.

In sensory neurons, linalool suppressed the [Ca²⁺]_i responses to KCl and Cav currents with similar potency (Fig. 2b and f). Mouse DRG neurons express various subtypes of Cav channels such as T, N, P/Q, L and R [17]. Among these subtypes, inhibitory effects of linalool on T-type Cav channels have been reported in heterologous expressed Cav 3.2 [45]. In the present patch-clamp experiments, cells were voltage-clamped at −60 mV, at which voltage T-type Cav channels are almost inactivated [46]. Moreover, I–V relations show the properties of high voltage-activated Ca²⁺ channels [17] and these channels are also inhibited by linalool. Further experiments may be necessary to identify the subtypes of Cav susceptible to linalool. Unlike the effect on Cav currents, linalool showed only a slight inhibition of Nav currents up to 1 mM (Fig. 2e and f). A previous study showed that linalool at 6 mM blocked Nav currents in rat DRG neurons [47].

In this study, we demonstrated the analgesic mechanisms of linalool at the molecular and individual levels. The synthesis of linalool occurs in several ways [48]. In cosmetics, the toxicity of linalool is low because the amount used is about ten thousand times lower than the LD₅₀ and the mutagenesis of linalool is negative [49]. Therefore, linalool may provide a promising lead compound from herbal substances for analgesics targeting TRPA1 as well as Cav channels.

Author contributions

TO: designed the study; MH and TO: conducted experiments; MH, KT and TO: conducted data analyses and wrote the manuscript.

Declaration of competing interest

We have no conflict-of-interest to declare.