Menthol attenuates α7-nACh receptor activity

At the highest concentration used in this study, 1 mM acetylcholine (ACh) did not cause detectable currents in un-injected oocytes (n = 7) or in oocytes injected with distilled water (n = 6) (data not shown). Application of 100 µM ACh for 3 to 4 sec activated fast inward currents that desensitized rapidly in oocytes injected with cRNA transcribed from cDNA encoding the α 7 -subunit of human nACh receptor. Moreover, ACh-induced inward currents were abolished completely with 100 nM α-bungarotoxin (n = 7, data not shown), indicating that the α-bungarotoxin-sensitive α7-nACh receptor-ion channel mediates these responses.

The effects of 10 min incubation with menthol (30 µM) on α 7 -nACh receptor mediated currents are shown in Fig. 1A. A time-course plot showing the effect of menthol application on the amplitudes of ACh-induced currents is presented in Fig. 1B. Whereas the vehicle solution did not alter ACh-induced currents, application of menthol (30 µM) caused a significant inhibition of the current. This inhibition by menthol was partially reversed during a washout period of 10 to 15 min. In the absence of these drugs, maximal amplitudes of currents elicited by the application of 100 µM ACh every 5 min remained unchanged during the course of the experiments (Fig. 1B, controls).

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larger image TIFF original image Download: Figure 1. Effect of menthol on α 7 -nicotinic acetylcholine receptor-mediated ion currents. (A) Records of currents activated by acetylcholine (ACh, 100 µM) in control conditions (left), during co-application of 30 µM menthol and acetylcholine after 10 min pretreatment with 30 µM menthol (middle), and 15 min following menthol washout (right). (B) Time-course of the effect of menthol (100 µM) on the peaks of the acetylcholine-induced currents. Each data point represents the normalized mean ± S.E.M. of 4 to 5 oocytes. Duration of drug application is indicated by the horizontal bar in the figure. (C) Comparison of the extent of inhibition caused by 100 µM of (+), (−), and racemic forms of menthol application for 15 min. Bars represent the means ± S.E.M. from 6 to 7 cells. (D) Comparison of the effect of 30 µM of racemic menthol application for 15 min on the currents activated by 100 µM acetylcholine or 10 µM nicotine. Bars represent the means ± S.E.M. from 5 to 6 cells. https://doi.org/10.1371/journal.pone.0067674.g001

Some of the biological actions of menthol have been shown to be stereo-specific (Eccles, 1994). For this reason, we compared the effects of 100 µM of (−) and (+) stereoisomers, and racemic (±) menthol on human α7-nACh receptors. Results show that the 2 stereoisomers and the racemic menthol (100 µM) inhibit nACh receptor currents to a similar extent with no statistical significant detected between the compounds (Fig. 1C; n = 6–7, F (2, 16) = 0.322; ANOVA, P>0.05). In all subsequent experiments, unless stated, racemic (±) menthol was employed.

Menthol is often delivered with tobacco products that contain nicotine. Therefore we tested the effect of menthol on nicotine-activated currents in oocytes. As shown in Fig. 1D, we did not find a statistically significant difference in menthol-mediated inhibition of α7-nACh receptor currents between cells treated with ACh or nicotine (n = 5–6, F (1, 9) = 0.052; ANOVA, P>0.05). It is noteworthy that the inhibitory effect of menthol was dependent on the application mode. Without menthol pre-incubation, a co-application of menthol (30 µM) and ACh (100 µM) did not alter the amplitudes of maximal currents (Fig. 2A). However after pre-incubation, menthol inhibited the maximal responses in a time-dependent manner. As incubation time was prolonged, the extent of menthol inhibition was enhanced and reached a maximum level at 10 to 15 min (Fig. 1B). Close examination of the time course of menthol actions indicated that the inhibition occurs at fast and slow phases with the respective time constants of τ 1/2fast = 23 sec. and τ 1/2slow = 5.2 min (Fig. 2A). Since the magnitude of the effect was time-dependent, menthol was applied for 10 to 15 min to ensure equilibrium conditions. Menthol inhibited the function of α7-nACh receptor in a concentration-dependent manner with respective IC 50 and slope values of 32.6±2.3 µM and 1.7, respectively (Fig. 2B).

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larger image TIFF original image Download: Figure 2. Time and concentration-dependence of menthol inhibition of α 7 -nicotinic acetylcholine receptor-mediated ion currents. (A) Inhibition of the α 7 -nicotinic acetylcholine receptor increases with the prolongation of menthol pre-application time. Each data point represents the mean ± S.E.M. of 5 to 6 oocytes. (B) Menthol inhibits α 7 -nicotinic acetylcholine receptor function in a concentration-dependent manner. Each data point represents the mean ± S.E.M. of 7 to 9 oocytes. The curve is the best fit of the data to the logistic equation described in the methods section. https://doi.org/10.1371/journal.pone.0067674.g002

G-protein coupled receptors [32] have been shown to be involved in cellular and behavioral effects of menthol. Thus, we tested the effect of menthol in control (distilled-water injected) and pertussis toxin (PTX) - injected oocytes expressing nACh receptors. There was no significant difference in menthol inhibition of ACh responses between controls and PTX-injected cells (Figure 3A, n = 7–8; F (1, 14) = 0.692, ANOVA, P>0.05 for the significance of menthol inhibition between controls and PTX group).

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larger image TIFF original image Download: Figure 3. Inhibition of acetylcholine-induced currents by menthol is independent of the activation of pertussis toxin sensitive receptors, membrane potential and Ca2+-activated Cl− channels. (A) Bar presentation of the effects of 30 µM menthol application (15 min) on the maximal amplitudes of ACh-induced currents in oocytes injected with 50 nl distilled-water, controls (n = 5) or 50 nl of PTX (50 µg/ml, n = 6). Bars represent the means ± S.E.M. (B) α 7 -nicotinic acetylcholine receptor expressing oocytes injected with 50 nl distilled water and recorded in 2 mM Ca2+ containing MBS solution (control) or injected with 50 nl of BAPTA (100 mM) and recorded in 2 mM Ba2+ containing MBS solution (BAPTA). Bars represent the means ± S.E.M. of 6 to 8 oocytes. The numbers of oocytes are presented on top of each bar. There was no statistically significant difference between menthol (30 µM) inhibition in the presence and in the absence of BAPTA injections (P>0.05, n = 5–8, ANOVA). (C) Current-voltage relationships of acetylcholine-activated currents in the absence and presence of menthol (30 µM). Normalized currents activated by 100 µM acetylcholine before (control,•) and after 15 min treatment with menthol (○). Each data point presents the normalized means and S.E.M. of five to six oocytes. (D) Quantitative evaluation of the effect of menthol as percent inhibition at different voltages. https://doi.org/10.1371/journal.pone.0067674.g003

Since activation of α 7 -nACh receptors allows sufficient Ca2+ entry to activate endogenous Ca2+-dependent Cl− channels in Xenopus oocytes (for a recent review: [33]), it was important to determine whether the effect of menthol was exerted on nACh receptor-mediated currents or on Cl− currents induced by Ca2+ entry in the cell. Thus, extracellular Ca2+ was replaced with Ba2+ since Ba2+ can pass through α 7 -nicotinic acetylcholine receptors but causes a negligible activation of Ca2+-dependent Cl− channels [34]. In addition to Ba2+ replacement, a small contribution of remaining Ca2+-dependent Cl− channel activity has been shown to be abolished by the injection of the Ca2+ chelator BAPTA [34]. We tested the effect of menthol in a solution containing 2 mM Ba2+ in BAPTA-injected oocytes. Menthol (30 µM) produced the same level of inhibition (67±5 in controls versus 65±5 in BAPTA-injected oocytes; n = 7; F (1, 12) = 0.863; ANOVA, P<0.05) on ACh-induced currents when BAPTA-injected oocytes were recorded in Ca2+ free solutions containing 2 mM Ba2+ (Fig. 3B). Menthol has also been reported to alter intracellular Ca2+ homeostasis in various preparations [2]. In the oocyte expression system, an increased level of intracellular Ca2+ can be detected by Ca2+-activated Cl− channels and concomitant alteration in the holding current [35], [36]. However, in control experiments, the menthol used in this study (30 µM for 15 min) did not alter the magnitudes of holding-currents in oocytes voltage-clamped at −70 mV (n = 12–14) suggesting that Ca2+-dependent Cl− channels are not involved in the effect of menthol in our system.

Recent electrophysiological studies report that menthol inhibits the functions of Na+ [37], [38] and Ca2+ channels [38] in a voltage-dependent manner. We examined if menthol-inhibition of α7-nACh receptors was dependent on the membrane potential. As indicated in Fig. 3C, menthol (30 µM) was able to inhibit ACh (100 µM)-induced currents at all of the tested potentials and thus seemingly can act independent of voltage changes. Indeed, an evaluation of the current-voltage relationship (Fig. 3D) shows that α7-nACh receptor inhibition by menthol does not change significantly at varying holding potentials (n = 6–7, inhibition at −20 mV versus −120 mV; F (1, 11) = 0.058; ANOVA, P>0.05).

It is possible that menthol decreases the binding of ACh to the nACh receptor by acting as a competitive antagonist at the same binding site. Concentration-response curves for ACh in the absence and presence of 30 µM menthol are presented in Fig. 4A. Menthol did not cause a significant change in the affinity of ACh for the receptor (EC 50 values of 63±12 µM versus controls 76±11 µM; n = 6–8; F (1, 12) = 1.126, ANOVA, P>0.05), but inhibited the maximal ACh response by about 47±4% of controls (n = 6), suggesting that menthol inhibits the α7-nACh receptor response in a non-competitive manner.

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larger image TIFF original image Download: Figure 4. Concentration-response curves for acetylcholine-induced currents and binding of [125I] α-bungarotoxin in control and in the presence of menthol. (A) Effect of menthol on the acetylcholine concentration-response relationship. Oocytes were voltage-clamped at −70 mV and currents were activated by applying acetylcholine (1 µM to 3 mM). Oocytes were exposed to 100 µM menthol for 15 min and acetylcholine was reapplied. Paired concentration-response curves were constructed and responses normalized to maximal response under control conditions. EC 50 and slope values were determined by fitting the curves from 6 to 8 oocytes to the standard logistic equation as described in the methods section. Data points obtained before (control) and after 15 min treatment with menthol (100 µM) were indicated by filled circles, open circles, and open triangles, respectively. Each data point presents the normalized means and S.E.M. of five to six experiments. (B) The effects of menthol on the specific binding of [125I] α-bungarotoxin to oocyte membrane preparations. In the presence and absence of menthol, specific binding as a function of the concentration of [125I] α-bungarotoxin is presented. Data points for controls and menthol (100 µM) are indicated by filled circles, and open circles, respectively. Data points are the means of three independent experiments carried out in triplicate. https://doi.org/10.1371/journal.pone.0067674.g004

We determined the effects of 30 µM menthol in radioligand binding studies using [125I] α-bungarotoxin. Equilibrium curves for the binding of [125I] α-bungarotoxin, in the presence and absence (controls) of menthol are presented in Fig. 4B. At a concentration of 30 µM, menthol did not cause a significant inhibition of the specific binding of [125I] α-bungarotoxin. Maximum binding activities (B max ) of [125I] α-bungarotoxin were 1.9±0.3 and 1.7±0.2 pM/mg (means ± S.E.M.) for controls and menthol-treated preparations, respectively (Fig. 4B). The apparent affinity (K D ) of the receptor for [125I] α-bungarotoxin was 854±236 and 716±213 pM for controls and menthol, respectively. There was no statistically significant difference between controls and menthol-treated groups with respect to K D (n = 5–6, F (1, 9) = 1.023; ANOVA, P<0.05) and B max K D (n = 5–6, F (1, 9) = 1.066; ANOVA, P<0.05) values.

Because radioligand-binding in oocyte membrane homogenates is known to disrupt cellular integrity, the subcellular fractions used in the binding assay are likely to contain both intracellular as well as plasma membranes. To determine menthol binding and actions at the cell surface, we also performed radioligand-binding assays in intact oocytes. In these experiments, menthol (30 µM) did not cause a significant inhibition of the specific binding of [125I] α-bungarotoxin (20 nM) in oocytes injected with the α7-nicotinic acetylcholine receptor cRNA. Specific binding of [125I] α-bungarotoxin was 1576±201 cpm, 1438±189 cpm (means ± S.E.M.) for controls and menthol (30 µM)-treated oocytes, respectively. In the presence of menthol (30 µM), we did not detect a significant alteration in the specific binding of [125I] α-bungarotoxin in intact oocytes (n = 12–14; F (1, 24) = 0.026, ANOVA; P>0.05). Since α-bungarotoxin competes with ACh at the same binding site on the α7-nACh receptor, the current data suggests that menthol does not interact with the ACh binding site; i.e. acts as a noncompetitive antagonist.