Cigarette smoke-induced neurogenic inflammation is mediated by alpha,beta-unsaturated aldehydes and the TRPA1 receptor in rodents

Abstract

Cigarette smoke (CS) inhalation causes an early inflammatory response in rodent airways by stimulating capsaicin-sensitive sensory neurons that express transient receptor potential cation channel, subfamily V, member 1 (TRPV1) through an unknown mechanism that does not involve TRPV1. We hypothesized that 2 alpha,beta-unsaturated aldehydes present in CS, crotonaldehyde and acrolein, induce neurogenic inflammation by stimulating TRPA1, an excitatory ion channel coexpressed with TRPV1 on capsaicin-sensitive nociceptors. We found that CS aqueous extract (CSE), crotonaldehyde, and acrolein mobilized Ca2+ in cultured guinea pig jugular ganglia neurons and promoted contraction of isolated guinea pig bronchi. These responses were abolished by a TRPA1-selective antagonist and by the aldehyde scavenger glutathione but not by the TRPV1 antagonist capsazepine or by ROS scavengers. Treatment with CSE or aldehydes increased Ca2+ influx in TRPA1-transfected cells, but not in control HEK293 cells, and promoted neuropeptide release from isolated guinea pig airway tissue. Furthermore, the effect of CSE and aldehydes on Ca2+ influx in dorsal root ganglion neurons was abolished in TRPA1-deficient mice. These data identify alpha,beta-unsaturated aldehydes as the main causative agents in CS that via TRPA1 stimulation mediate airway neurogenic inflammation and suggest a role for TRPA1 in the pathogenesis of CS-induced diseases.

Figure 1. CSE and unsaturated aldehydes increase intracellular Ca²⁺ in guinea pig JG neurons.

[Ca²⁺]_i mobilization in cultured guinea pig JG neurons by CSE and capsaicin (CPS) (A) or acrolein (ACR) and crotonaldehyde (CRA) (B). Typical traces (C and E) and pooled data (D and F–H), showing the effect of RR (1 μM), HC-030031 (HC; 30 μM), GSH (10 mM), capsazepine (CPZ; 10 μM), or a combination of ROS scavengers (RS; mannitol, 20 mM; ascorbic acid, 100 μM; lycopene, 10 μM; and β-carotene, 10 μM) on [Ca²⁺]_i mobilization in cultured JG neurons from the guinea pig, induced by CSE (0.1 OD), crotonaldehyde (100 μM; circles), cap­saicin (0.1 μM; triangles), acrolein (30 μM), and cinnamaldehyde (CNM; 30 μM). Each column represents the mean ± SEM value of at least 21 cells. *P < 0.05 versus vehicle (Veh) (1-way ANOVA, followed by Bonferroni’s test).

Figure 2. CSE and unsaturated aldehydes increase intracellular Ca²⁺ in TRPA1-transfected cells.

(A) Typical traces and (C) pooled data of [Ca²⁺]_i mobilization in HEK293 cells transfected with the cDNA of the rat TRPA1 (rTRPA1-HEK cells) in absence or presence of tetracycline and exposed to CSE (0.1 OD), crotonaldehyde (50 μM), acrolein (30 μM) or cinnamaldehyde (50 μM). (B) Concentration-response curves to acrolein or crotonaldehyde in rTRPA1-HEK cells. Pooled data are expressed as mean ± SEM of at least 25 cells. FIU, fluorescence intensity unit. Typical traces (D) and pooled data (E) of [Ca²⁺]_i mobilization in DRG neurons from Trpa1^+/+ or Trpa1^–/– mice exposed to CSE, crotonaldehyde, capsaicin, or KCl. Numbers in E indicate the number of responding cells/total cells examined.

Figure 3. CSE and unsaturated aldehydes promote neuropeptide release from guinea pig airways.

(A) CGRP-LI and (B) SP-LI release induced by CSE, acrolein, or crotonaldehyde from slices of guinea pig airways. Experiments were performed in a Ca²⁺-free medium plus 1 mM EDTA (Ca²⁺ free), after capsaicin desensitization (CPS des; 10 μM for 20 minutes), or in a normal medium with the vehicle of capsaicin. Each column represents the mean ± SEM of at least 5 experiments. *P < 0.05 versus Veh (1-way ANOVA, followed by Bonferroni’s test).

Figure 4. CSE and unsaturated aldehydes contract guinea pig isolated bronchus.

(A and C) Typical traces and (B and D–F) pooled data of contractile responses to carbachol (CCh; 1 μM; triangle), CSE (0.1 OD; filled circles), crotonaldehyde (100 μM; open circles), acrolein (20 μM), and cinnamaldehyde (30 μM) in guinea pig isolated bronchus after capsaicin desensitization (20 μM for 20 minutes) or in the presence of RR (30 μM), HC-030031 (50 μM), capsazepine (10 μM), SR 140333 plus SR 48968 (SR; both 1 μM), glutathione (10 mM), or a combination of ROS scavengers (mannitol, 20 mM; ascorbic acid, 100 μM; lycopene, 10 μM; and β-carotene, 10 μM) or their respective vehicles. Each column represents the mean ± SEM for at least 4 experiments. *P < 0.05 versus Veh (1-way ANOVA, followed by Bonferroni’s test).

Figure 5. CS and CSE increase tracheal plasma extravasation.

Effect of intratracheal RR (4.5 nmol/150 μl), HC-030031 (45 nmol/150 μl), capsazepine (45 nmol/150 μl), or their respective vehicles (150 μl) on the increase in plasma extravasation produced by (A) CS inhalation (2 puffs = 60 ml) or (B) capsaicin administration (1 μmol/kg, i.v.) in anesthetized guinea pigs. Columns represent the mean ± SEM of a least 4 experiments. Veh0, vehicles of RR, HC, and CPZ; Veh1, vehicle of CPS; Veh2, vehicles of HC and CPZ. *P < 0.05 versus Veh; ^#P < 0.05 versus Veh2. C shows plasma extravasation evoked by intratracheal instillation of CSE vehicle (WT Veh; 25 μl) or CSE in wild-type or CSE in TRPA1-deficient mice. ^ΧP < 0.05 versus WT (1-way ANOVA, followed by Bonferroni’s test).