Menthol attenuates respiratory irritation responses to multiple cigarette smoke irritants

Abstract

Menthol, the cooling agent in peppermint, is added to almost all commercially available cigarettes. Menthol stimulates olfactory sensations, and interacts with transient receptor potential melastatin 8 (TRPM8) ion channels in cold-sensitive sensory neurons, and transient receptor potential ankyrin 1 (TRPA1), an irritant-sensing channel. It is highly controversial whether menthol in cigarette smoke exerts pharmacological actions affecting smoking behavior. Using plethysmography, we investigated the effects of menthol on the respiratory sensory irritation response in mice elicited by smoke irritants (acrolein, acetic acid, and cyclohexanone). Menthol, at a concentration (16 ppm) lower than in smoke of mentholated cigarettes, immediately abolished the irritation response to acrolein, an agonist of TRPA1, as did eucalyptol (460 ppm), another TRPM8 agonist. Menthol's effects were reversed by a TRPM8 antagonist, AMTB. Menthol's effects were not specific to acrolein, as menthol also attenuated irritation responses to acetic acid, and cyclohexanone, an agonist of the capsaicin receptor, TRPV1. Menthol was efficiently absorbed in the respiratory tract, reaching local concentrations sufficient for activation of sensory TRP channels. These experiments demonstrate that menthol and eucalyptol, through activation of TRPM8, act as potent counterirritants against a broad spectrum of smoke constituents. Through suppression of respiratory irritation, menthol may facilitate smoke inhalation and promote nicotine addiction and smoking-related morbidities.

Figure 1.

Breathing patterns of mice at baseline, during acrolein exposure, or during coexposure with acrolein and menthol. Representative recording of digitized respiratory flow signals obtained from mice during exposure to clean air (A), acrolein (B) or menthol-acrolein (C) after metyrapone pretreatment (see text). The 0 point on the flow axis separates inspiration (downward) from expiration (upward). Each tick mark represents 0.5 s. Breathing frequency was ∼240, 100, and 200 breaths/min in clean air, acrolein, and acrolein-menthol, respectively. The prolonged DB, indicated by black bars, at the onset of expiration during acrolein exposure is readily apparent.

Figure 2.

Effect of menthol vapor on the respiratory irritation response to acrolein. A) Average DB response during 15-min exposure to acrolein (1.9 ppm), menthol (4.1 or 16 ppm), or the combination. Response in the acrolein and acrolein-menthol groups did not differ (P=0.07) at the low menthol concentration; at the high menthol concentration, the response in the acrolein-menthol group was significantly attenuated and not different from the response to menthol alone. P values are indicated. B) Time course of the DB response in the high concentration menthol study. In all groups, DB was increased over baseline during exposure. As indicated by the asterisks, from min 3 to 15 of exposure the response in the combined group was less than in the acrolein group (P<0.05). Data are shown as means ± se; 4–6 mice/group.

Figure 3.

Effect of menthol vapor on respiratory irritation by acrolein in metyrapone-treated mice. A) Average DB during exposure to 2.1 ppm acrolein, 16 ppm menthol, and the combination. All mice were pretreated with metyrapone to inhibit nasal CYP450 metabolism. Coexposure with menthol significantly inhibited irritation to levels observed with menthol alone. P values are indicated. B) Time course of DB. DB was increased over baseline throughout acrolein exposure, but only during min 1–2 of exposure in the other groups. As indicated by the asterisks, from min 2 to 15 of exposure, response in the combined group was smaller than in the acrolein group (P<0.05). Data are shown as means ± se; 4–6 mice/group.

Figure 4.

Effect of eucalyptol vapor on respiratory irritation by acrolein. A) Average DB response during exposure to 2.1 ppm acrolein, 464 ppm eucalyptol, or combined vapors. Coexposure with eucalyptol significantly attenuated respiratory irritation to levels observed with eucalyptol alone. P values are indicated; 5 mice/group. B) Time course of DB in the same groups of mice shown in A. DB was increased over baseline throughout acrolein exposure. Coexposure with eucalyptol immediately and completely inhibited the respiratory irritation response. Eucalyptol alone did not cause any increase in DB. *P < 0.05 vs. acrolein + eucalyptol; ^#P < 0.05 vs. eucalyptol. Data are shown as means ± se.

Figure 5.

Cyclohexanone, a tobacco smoke irritant, activates TRPV1 channels in sensory neurons. A) Ca²⁺ imaging experiments showing averaged amplitudes of Fura-2 ratios in HEK 293 cells expressing mouse TRPV1 (mTRPV1, red) or pCDNA3.1 vector control (vector, black) in response to applications of cyclohexanone (cyclohex, 10 mM), capsaicin (cap, 1 μM), and ionomycin (iono, 1.5 μM). B) Steady-state Ca²⁺ responses in TRPV1-, TRPA1- or vector-transfected HEK 293 cells to cyclohexanone, measured 1 min after application at the indicated concentrations. Responses are indicated as percentage ratio of ionomycin response. C) Ca²⁺ responses in cyclohexanone-sensitive DRG neurons from wild-type and Trpv1^−/− mice superfused with cyclohexanone (10 mM), capsaicin (1 μM), and KCl (40 mM). Averages of 20 to 30 neurons are shown for each group. Trpv1^−/− neurons show strongly diminished Ca²⁺ transients. Only the neurons that showed a minimum 20% increase in Fura-2 ratio above baseline on agonist applications are included. D) Population analysis of cyclohexanone-sensitive cultured primary sensory neurons derived from wild-type and Trpv1^−/− mice. Experiments were performed as in C. Bar graph displays percentage of K⁺-sensitive cells responding to the given stimulus (cyclohexanone, 10 mM; or capsaicin 1 μM). Responsiveness was defined by a 20% increase in Fura-2 ratio above baseline following agonist application. *P < 0.05; **P < 0.01. E) Venn diagrams of cyclohexanone and capsaicin-responsive sensory neurons from wild-type (top panel) or Trpv1^−/− mice (bottom panel). A total of 265 neurons from wild-type mice, identified through K⁺ responsiveness, were analyzed from 8 fields. Of these neurons, 54 were responsive to cyclohexanone (10 mM) and 148 were responsive to capsaicin (1 μM). A large majority of cyclohexanone-sensitive neurons (n=50) were contained within the capsaicin-sensitive population.

Figure 6.

Effects of menthol or eucalyptol vapor on respiratory irritation responses elicited by acetic acid or cyclohexanone vapors. A) Average DB response during exposure to 149 ppm acetic acid or 1483 ppm cyclohexanone with and without 17 ppm menthol vapor. Menthol significantly attenuated responses to both exposures. All mice were pretreated with metyrapone to focus on the effect of parent menthol. B) Average DB response during exposure to 110 ppm acetic acid or 1472 ppm cyclohexanone with or without coexposure to 464 ppm eucalyptol. Eucalyptol significantly attenuated responses to both irritants. Mice were not pretreated with metyrapone. P values are indicated.

Figure 7.

Effects of l-menthol and the TRPM8 antagonist, AMTB, on respiratory irritation responses elicited by acrolein. Average DB response during exposure to 2.2 ppm acrolein with and without 7 ppm l-menthol. l-Menthol concentration averaged 6.4, 7.6, and 6.8 ppm in the l-menthol, combination and combination+AMTB groups, respectively (P>0.1). AMTB (3 mg/kg, s.c.) did not affect the response in the acrolein or l-menthol groups; therefore, data from nontreated and treated-animals were pooled for these groups. l-Menthol significantly attenuated the response to acrolein, while AMTB significantly diminished the menthol-induced attenuation of the irritant response to acrolein.