Cigarette smoke–induced neurogenic inflammation is mediated by α,β-unsaturated aldehydes and the TRPA1 receptor in rodents

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 α,β-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 α,β-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.

Inhaled cigarette smoke (CS) causes an early inflammatory response in the airways that is partly neurogenic (1). In rodents, CS-evoked neurogenic plasma protein leakage (1) is mediated by release of the neuropeptides substance P (SP) and neurokinin A (NKA) from capsaicin-sensitive sensory nerves that activate tachykinin NK1 receptors in postcapillary venules to cause edema (2). The neurogenic mechanism of CS-evoked plasma extravasation in the airways was discovered because it was absent in capsaicin-pretreated rats, in which SP-containing C-fiber nociceptors in the respiratory tract had degenerated (1). Although desensitization of sensory nerves by capsaicin is due to its unique ability to stimulate the transient receptor potential cation channel, subfamily V, member 1 (TRPV1) (3, 4), TRPV1 does not mediate the neuronal action of CS, because the selective TRPV1 receptor antagonist capsazepine (5) failed to prevent CS-evoked plasma protein extravasation (6). Hence, the molecular entity expressed by sensory neurons, which mediates CS-evoked neurogenic inflammation, is unknown. In addition, the chemical components among the approximately 5,000 constituents of CS that cause airway neurogenic inflammation are unknown. Thus, some 25 years after the original observation made by Lundberg and Saria (1), both the chemical components of CS and their molecular target on sensory neurons remain to be identified. Ruthenium red (RR), an inorganic dye initially reported to prevent capsaicin-evoked responses (7) and now recognized as a nonspecific blocker of TRP channels, reportedly reduces CS-induced tracheal plasma extravasation (6). From this apparently contradictory observation, made at a time (1993) when none of the presently recognized TRP channels had been described, we now hypothesize that one or more additional TRPs expressed by sensory neurons could be the molecular target(s) of CS in producing airway neurogenic inflammation.

In addition to TRPV1 (3), other TRP channels are expressed by C-fiber nociceptors. These include TRPV2, TRPV3, and TRPV4 (gated by warm, non-noxious, and noxious temperatures and small reductions in tonicity) and TRPM8 (activated by menthol and moderate low temperature) (8–13). ANKTM1, renamed TRPA1 (TRP cation channel, subfamily A, member 1), is a recently identified channel that is almost entirely coexpressed with TRPV1 on sensory neurons, including vagal neurons innervating the airways (14–16). TRPA1 is activated by isothiocyanates, thiosulfinate, or cinnamaldehyde compounds, which are the pungent ingredients found in mustard, garlic, and cinnamon, respectively (17–20). A recent study showed that the irritant α,β-unsaturated aldehyde, acrolein, is a TRPA1 stimulant (18). Since acrolein and another α,β-unsaturated aldehyde, crotonaldehyde, are abundantly contained in CS (21, 22), we hypothesized that stimulation of TRPA1 on capsaicin-sensitive sensory neurons by α,β-unsaturated aldehydes is the underlying mechanism of CS-evoked neurogenic inflammation in the airways. Herein we present direct pharmacological and genetic evidence that supports our hypothesis. This evidence clarifies the molecular mechanisms underlying the neurogenic inflammatory response activated by the exposure to CS and might be relevant for understanding the pathogenesis of smoke-induced diseases, including chronic obstructive pulmonary disease (COPD).

Ca²⁺ mobilization in guinea pig jugular ganglia neurons. Exposure to CS aqueous extract (CSE) (0.001–0.1 OD) caused a rapidly developing and concentration-dependent mobilization of intracellular Ca²⁺ ([Ca²⁺]_i) in cultured guinea pig jugular ganglia (JG) neurons (Figure 1, A and C). CSE vehicle had no effect (data not shown). All cells that responded to CSE invariably responded to capsaicin (1 μM). Crotonaldehyde and acrolein have been previously found to be the most abundant α,β-unsaturated aldehydes present in CSE prepared according to the previously described method (21, 22) Therefore, we decided to challenge guinea pig JG neurons with these aldehydes to determine whether they mimic the CSE-evoked effect. In cultured neurons, acrolein and crotonaldehyde caused a concentration-dependent, prompt, and sustained mobilization of [Ca²⁺]_i. EC₅₀ were 5 μM (CI, 4–8) and 16 μM (CI, 7–36), respectively (Figure 1B). Ca²⁺ responses to CSE, acrolein, or crotonaldehyde in guinea pig JG neurons were inhibited by the nonselective TRPA1 antagonists, RR (1 μM; >80% reduction; Figure 1, C–F and H) or camphor (23) (1 mM; >80% reduction; data not shown), whereas the selective TRPV1 antagonist, capsazepine (10 μM), was ineffective (Figure 1, C–F and H). More importantly, the selective TRPA1 receptor antagonist, HC-030031 (30 μM) (24), abated by more than 80% the responses evoked by CSE, acrolein, or crotonaldehyde (Figure 1, C–F and H). The unsaturated aldehyde and ROS scavenger, glutathione monoethylester (GSH, 10 mM), also reduced these evoked responses by about 90% (Figure 1, D, F, and H), whereas a combination of scavengers, reportedly effective in scavenging a variety of ROS, including mannitol (20 mM), ascorbic acid (100 μM), lycopene (10 μM), and β-carotene (10 μM), did not affect the Ca²⁺ responses to CSE, acrolein, or crotonaldehyde (Figure 1, C–F and H).

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).

A recent paper (25) showed that H₂O₂ produces a delayed Ca²⁺ response in mouse dorsal root ganglia (DRG) neurons. This response was ascribed to the TRPA1 channel stimulation, because it was absent in neurons from TRPA1-deficient mice. In guinea pig JG neurons, we found that H₂O₂ causes a concentration-related (0.1–5 mM) response. EC₅₀ was 0.65 mM (CI, 0.32–1.8). The response to H₂O₂, as shown previously (25), was delayed in onset (responses initiated 20–40 seconds and reached a plateau 60–200 seconds after the addition of the stimulus). The Ca²⁺ response to 1 mM H₂O₂ (change in F₃₄₀/F₃₈₀, 0.503 ± 0.08; n = 28) was markedly reduced by the combination of the 4 ROS scavengers: mannitol (20 mM), ascorbic acid (100 μM), lycopene (10 μM), and β-carotene (10 μM) (change in F₃₄₀/F₃₈₀, 0.176 ± 0.02; n = 32; P < 0.01).

The selective agonist of the TRPA1 channel, cinnamaldehyde (17), evoked a robust Ca²⁺ response that was abolished by RR, HC-030031, and GSH and was not affected by capsazepine or by ROS scavengers (Figure 1G). Acetaldehyde, the most abundant saturated aldehyde present in CSE (21, 26, 27), has recently been reported to stimulate, at mM concentrations, recombinant and wild-type (mouse DRG neurons) TRPA1 (28, 29). Acetaldehyde in concentrations up to 1 mM did not cause any Ca²⁺ response in guinea pig JG neurons but caused a moderate response at 5-mM concentration (change in F₃₄₀/F₃₈₀, 0.4 ± 0.07; n = 40). However, that moderate response was resistant to HC-030031 (100 μM; change in F₃₄₀/F₃₈₀, 0.36 ± 0.08; n = 32). Nicotine has been recently shown to mobilize [Ca²⁺]_i in cultured primary sensory neurons in a hexamethonium-sensitive manner (30). In the present study, nicotine (10 μM) increased [Ca²⁺]_i mobilization (change in F₃₄₀/F₃₈₀, 0.89 ± 0.12; n = 43) in JG neurons, a response that was inhibited by 100-μM hexamethonium (change in F₃₄₀/F₃₈₀, 0.12 ± 0.02; n = 37; P < 0.05) and by 2-μM mecamylamine (change in F₃₄₀/F₃₈₀, 0.18 ± 0.03; n = 19; P < 0.05) but not by 30-μM HC-030031 (change in F₃₄₀/F₃₈₀, 0.78 ± 0.17; n = 18; NS). In contrast, CSE-evoked (0.1 OD) increase in [Ca²⁺]_i (change in F₃₄₀/F₃₈₀, 1.32 ± 0.23; n = 28) was not affected by either hexamethonium (100 μM; change in F₃₄₀/F₃₈₀, 1.44 ± 0.19; n = 33) or mecamylamine (change in F₃₄₀/F₃₈₀, 1.28 ± 0.23; n = 16). Thus, the role of ROS, saturated aldehydes, and nicotine in CSE-evoked Ca²⁺ mobilization seems to be absent or minor.

Ca²⁺ mobilization in HEK293 cells and mouse DRG neurons. In HEK293 cells transfected with rat TRPA1 cDNA (rTRPA1-HEK293) and treated with tetracycline to induce TRPA1, exposure to CSE, acrolein, or crotonaldehyde caused a remarkable increase in [Ca²⁺]_i mobilization. rTRPA1-HEK293 cells also responded to cinnamaldehyde (Figure 2, A and C). EC₅₀ for acrolein and crotonaldehyde were 0.8 μM (CI, 0.7–0.9) and 23 μM (CI, 16–33), respectively (Figure 1B). Ca²⁺ responses to CSE (0.1 OD) (change in F₃₄₀/F₃₈₀, 1.42 ± 0.21; n = 38), acrolein (1 μM; change in F₃₄₀/F₃₈₀, 1.08 ± 0.16; n = 24), or crotonaldehyde (25 μM; change in F₃₄₀/F₃₈₀, 1.41 ± 0.28; n = 25) were not affected by a combination of ROS scavengers (mannitol, 20 mM; ascorbic acid, 100 μM; lycopene, 10 μM; and β-carotene, 10 μM) (change in F₃₄₀/F₃₈₀, 1.38 ± 0.2; n = 35; 0.97 ± 0.12, n = 23; and 1.32 ± 0.22, n = 27; respectively). rTRPA1-HEK293 cells that were not treated with tetracycline (and therefore did not express TRPA1) (Figure 2A, bottom panel) and HEK293 cells transfected with empty vector (data not shown) failed to respond to CSE, crotonaldehyde, acrolein, or cinnamaldehyde. Thus, expression of TRPA1 was the necessary condition to confer onto HEK293 cells the ability to respond to unsaturated aldehydes and CSE.

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.

In cultured DRG neurons from Trpa1^+/+ mice, crotonaldehyde induced a prompt and sustained Ca²⁺ response in some neurons that responded to capsaicin (Figure 2, D, bottom panel, and E). In contrast, crotonaldehyde was completely inactive in neurons from Trpa1^–/– mice (Figure 2, D, bottom panel, and E). CSE (0.05 OD) induced a rapidly developing and sustained increase in baseline fluorescence in some neurons from Trpa1^+/+ mice (Figure 2, D, top panel, and E). All neurons from Trpa1^+/+ mice that responded to CSE were sensitive to capsaicin. However, CSE was completely inactive in neurons from Trpa1^–/– mice, neurons that had responded normally to capsaicin (Figure 2, D, top panel, and E). Overall, these results show that both crotonaldehyde and CSE effects in mouse DRG neurons are mediated by TRPA1.

Calcitonin gene–related peptide and SP release from guinea pig airways. CSE strongly stimulated the outflow of SP-like immunoreactivity (SP-LI) and of another major sensory neuropeptide (2), the calcitonin gene–related peptide-LI (CGRP-LI), from slices of guinea pig airways (Figure 3). This effect was significantly reduced when tissues were incubated in Ca²⁺-free medium (93% and 87% reduction, respectively) or in preparations desensitized to capsaicin (20 μM for 20 minutes) (77% and 80% reduction, respectively) (Figure 3). Exposure to acrolein or crotonaldehyde for 10 minutes also caused a significant increase in the outflow of SP-LI and CGRP-LI. These responses were significantly reduced (>80% reduction) when experiments were performed in a Ca²⁺-free medium or after capsaicin desensitization (Figure 3). Thus, CSE and unsaturated aldehydes cause a Ca²⁺-dependent release of neuropeptides from capsaicin-sensitive airway sensory nerve terminals.

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).

Organ bath studies. Exposure of isolated guinea pig bronchial rings to CSE (0.02–0.1 OD) in the presence of indomethacin (5 μM) and atropine (1 μM) caused a concentration-dependent (data not shown), rapidly developing, and sustained contraction that at 0.1 OD was 68 ± 5% of that produced by carbachol (1 μM; n = 12; added before atropine) (Figure 4A). The response to CSE was inhibited by capsaicin desensitization (20 μM for 20 minutes) of sensory nerve terminals, RR (30 μM), the combination of SR 140333 and SR 48968 (NK1 and NK2 tachykinin receptor antagonists, respectively; both 1 μM), HC-030031 (50 μM), camphor (1 mM; data not shown), and GSH (10 mM) (Figure 4, A and B). In contrast, the response to CSE was unaffected by either capsazepine (10 μM) or the combination of 4 ROS scavengers: mannitol (20 mM), ascorbic acid (100 μM), lycopene (10 μM), and β-carotene (10 μM) (Figure 4B). As with CSE, contractions evoked by acrolein and crotonaldehyde were inhibited by capsaicin pretreatment, RR, the combination of NK1 and NK2 tachykinin receptor antagonists, HC-030031, camphor (1 mM; data not shown), and GSH but were unaffected by capsazepine or a combination of 4 ROS scavengers (Figure 4, C, D, and F). Cinnamaldehyde caused a capsaicin-sensitive contraction of isolated guinea pig bronchi, which was inhibited by RR, NK1 and NK2 tachykinin receptor blockade, HC-030031, camphor (1 mM, not shown), and GSH but was not affected by capsazepine, mannitol, or a combination of 4 ROS scavengers (Figure 4E). Thus, CSE and unsaturated aldehydes contract the guinea pig bronchus by a neurogenic mechanism mediated by TRPA1 stimulation.

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).

Plasma extravasation. Evans blue dye extravasation was significantly greater in the trachea (26.1 ± 5.3 ng/g of tissue; n = 11) of anesthetized guinea pigs that inhaled CS than in those that inhaled room air (8 ± 1.6 ng/g of tissue; n = 9; P < 0.05) (Figure 5A). This response was significantly reduced by pretreatment with RR via intratracheal instillation (4.5 nmol/150 μl; 74% reduction) or HC-030031 (45 nmol/150 μl; 91% reduction). In contrast, pretreatment with intratracheal instillation of capsazepine (45 nmol/150 μl) failed to prevent CS-induced dye extravasation (Figure 5A). Selectivity of HC-030031 and capsazepine was tested in guinea pigs treated with capsaicin. The increase in plasma extravasation evoked by capsaicin (1 μmol/kg, i.v.) was abolished by capsazepine and was unaffected by HC-030031 (Figure 5B). Intratracheal instillation of RR, HC-030031, capsazepine, or their respective vehicles did not affect baseline plasma protein extravasation. Instillation of CSE (1 OD; 25 μl) into the trachea of Trpa1^+/+ mice strongly stimulated extravasation of the Evans blue dye, a response that was completely absent in Trpa1^–/– mice (Figure 5C). These results provide both pharmacological and genetic evidence that the TRPA1 ion channel is required to evoke airway inflammation by CS.

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).

Our study resulted in 2 important findings that we believe to be novel that are supported by pharmacological and genetic data. First, CS has the unique ability to excite capsaicin-sensitive primary sensory neurons by activating TRPA1 to induce neurogenic inflammation in the airways. Second, among approximately 5,000 constituents, acrolein and crotonaldehyde, which are the most abundant α,β-unsaturated aldehydes in CS (21, 22), are the major mediators of CS-induced activation of TRPA1-expressing neurons. The 3 stimuli used in our experiments, CSE, acrolein, and crotonaldehyde, caused a robust and pharmacologically identical Ca²⁺ response in guinea pig JG neurons. The observations that the selective TRPV1 antagonist, capsazepine, was ineffective and that nonselective (camphor and RR) or selective (HC-030031) (24) TRPA1 antagonists blocked the Ca²⁺ response to CSE, crotonaldehyde, or acrolein, indicate that TRPA1 is the mediator of the neuronal excitation by the 3 stimuli. Ca²⁺ entry in nociceptors sensitive to capsaicin is associated with the release of the sensory neuropeptides SP, NKA, and CGRP. Our findings confirm previous reports that CS releases sensory neuropeptides (31) and show that in the guinea pig airways, a Ca²⁺-dependent neurosecretion from capsaicin-sensitive sensory nerve endings is evoked by the unsaturated aldehydes acrolein and crotonaldehyde.

The tachykinins SP and NKA mediate neurogenic inflammatory responses in the guinea pig airways, which consist mainly of bronchoconstriction (mediated by tachykinin NK1 and NK2 receptors) and plasma protein extravasation (mediated by NK1 receptors) (2). Inhaled CS reportedly produces an early phase of bronchoconstriction, mediated by both a cholinergic reflex and tachykinin release from sensory neurons, and a late phase caused by cyclooxygenase metabolites of arachidonic acid (32). The component of bronchoconstriction, mediated by SP/NKA release from sensory nerve terminals, seems to be the most prominent contributing mechanism (31). Our results show that the rapidly developing, indomethacin- and atropine-resistant in vitro bronchoconstrictions evoked by CSE, acrolein, and crotonaldehyde were abated by capsaicin desensitization and by a combination of tachykinin NK1 and NK2 receptor antagonists. Capsaicin has the unique property to excite and desensitize sensory neurons by stimulating TRPV1, a nonselective cation channel that is also activated by noxious heat (3), low extracellular pH (33, 34), and some lipid molecules (35–37). Capsaicin desensitization, which results from prolonged contact of isolated tissues with high capsaicin concentrations, is a widely used procedure that renders sensory nerve terminals unresponsive to capsaicin and to any other irritant stimulus (4, 38). Therefore, our findings indicate that bronchoconstriction evoked by CSE or unsaturated aldehydes is a neurogenic inflammatory response. The observation that responses to CSE and unsaturated aldehydes in isolated bronchi, like the Ca²⁺ response in cultured neurons, were insensitive to capsazepine but were completely abated by HC-030031 suggests a major role for TRPA1 in these responses.

CS contains a large array of chemical species that potentially may stimulate sensory neurons and TRPA1. Nicotine has been recently shown to excite rat sensory neurons in a hexamethonium-sensitive manner (30). However, both hexamethonium and mecamylamine, 2 nicotinic receptor antagonists, at concentrations that blocked nicotine-evoked effects, failed to affect the Ca²⁺ response elicited by CSE in guinea pig JG neurons. Moreover, because the Ca²⁺ response to nicotine was insensitive to HC-030031, the contribution of nicotine to CS-evoked neurogenic inflammation can be considered absent or minor. Glutathione, a natural scavenger of unsaturated aldehydes (21), abated either CSE-evoked Ca²⁺ response in neurons or bronchoconstriction, thus corroborating the hypothesis that acrolein and crotonaldehyde mediate the effects of CSE. Nevertheless, it should be considered that glutathione can also scavenge ROS and that CS contains ROS (39). However, 4 diverse ROS scavengers (mannitol, ascorbic acid, lycopene, and β-carotene), which target different oxygen species, including superoxide, hydroxyl radical, peroxyl radicals, sulphur radicals, nitrogen-oxygen radicals, and singlet oxygen (40–43), failed to affect the Ca²⁺ response or the bronchoconstrictor effect of CSE. H₂O₂, which produces ROS, has been recently reported to stimulate TRPA1 (25). We confirmed these findings in guinea pig JG neurons. However, important differences were observed between responses evoked by H₂O₂ and CSE. As reported previously (25), the Ca²⁺ response to H₂O₂ was delayed in onset, because it became visible after 20–40 seconds and reached a maximum effect within 60–200 seconds. In contrast, the response to CSE was immediate, reaching a maximum effect within 5–10 seconds. The response to H₂O₂ was inhibited by ROS scavengers, whereas that to CSE was not affected by this pharmacological intervention. Finally, the levels of H₂O₂ in CSE were reported to be in the low μM range (44). These low concentrations in our and previous (25) experiments failed to stimulate TRPA1.

Acetaldehyde, the most abundant saturated aldehyde contained in CSE (21, 26, 27), has been recently reported to activate TRPA1 expressed in recombinant systems (28, 29). In mouse DRG neurons, acetaldehyde reportedly caused both TRPA1-dependent and -independent responses only at very high concentrations (≥1 mM) (28). Our present observation in guinea pig JG neurons confirms that acetaldehyde (5 mM concentration) can evoke a weak Ca²⁺ response. Because this response was insensitive to HC-030031, the hypothesis that acetaldehyde targets TRPA1 in guinea pig neurons is not supported by present results. One possible explanation for these conflicting findings is that in guinea pig neurons, the non-TRPA1–dependent response to acetaldehyde prevails. Regardless of the conflicting findings, the acetaldehyde concentration (~0.1 mM; ref. 21) contained in the most concentrated (0.1 OD) CSE preparation used throughout our study was unable to produce any measurable response in guinea pig (present data) or mouse (28) neurons. Finally, unlike unsaturated aldehydes, acetaldehyde in CSE is resistant to the scavenging action of GSH (21), which did abate CSE-evoked responses. Overall, present evidence indicates that nicotine, ROS, or acetaldehyde do not play a major role in CSE-evoked neurogenic inflammation, although a minor contribution by these agents contained in the inhaled CS gas phase can not be excluded.

CS stimulates extravasation of plasma proteins from postcapillary venules by releasing SP, which activates NK1 receptors (1). We confirmed our previous findings that TRPV1 is not the neuronal target of CS in this in vivo assay (6), because capsazepine was ineffective. We also observed that the nonselective TRP channel antagonist, RR, greatly prevented CS-induced plasma protein extravasation. The ability of the TRPA1 blocker, HC-030031, to selectively prevent CS-evoked plasma extravasation, without affecting the response to capsaicin, adds strong circumstantial evidence to the proposal that TRPA1 mediates this response. We believe that conclusive proof of the unique role of TRPA1 and α,β-unsaturated aldehydes in CS-evoked neuronal excitation was obtained from genetically manipulated bioassays. First, transfection of the TRPA1 into HEK293 cells conferred to these cells the ability to respond to unsaturated aldehydes and CSE. Second, DRG neurons from TRPA1-deficient mice failed to respond to acrolein (18), crotonaldehyde, and CSE (present study). Finally, and more importantly, in contrast with what occurred in wild-type mice, CSE failed to produce any plasma protein extravasation in the trachea of TRPA1-deficient mice. Thus, our genetic results demonstrate that TRPA1 is necessary and sufficient to mediate the sensory neuronal excitation by CS to produce neurogenic inflammation.

Acrolein and crotonaldehyde recapitulated all the effects evoked by CSE in all the bioassays. However, the important question arises as to whether the effects produced by CSE are entirely mediated by the unsaturated aldehydes, acrolein, and crotonaldehyde contained therein. Concentrations of unsaturated aldehydes in CSE (0.1 OD) used in the present experiments (21) were approximately 15 μM. Concentration-response curves to acrolein and crotonaldehyde in neurons and in TRPA1-HEK293 cells indicated EC₅₀ values ranging from 1 to 23 μM, thus making plausible the hypothesis that levels of unsaturated aldehydes in CSE are sufficient to activate TRPA1 and evoke neurogenic inflammation. Nevertheless, a minor contribution of components of CS, sensitive to the scavenging action of GSH and different from unsaturated aldehydes acting via TRPA1 receptor stimulation, can not be excluded completely.

The general hypothesis that protracted and uncontrolled neurogenic inflammation may contribute to various diseases, including asthma or COPD, is supported by a large body of evidence (2, 45). However, neurogenic inflammation in various tissues, including the airways, has also been proposed to be part of a protective mechanism, referred to as the nocifensor system (46–48). Thus, plasma protein extravasation and other neurogenic inflammatory responses evoked by TRPA1 stimulation by unsaturated aldehydes contained in CS may be an early protective response against the detrimental action of CS to the airways and lung tissue (49). Loss of sensitivity to the irritant effect of unsaturated aldehydes at the TRPA1 channel might reduce or abolish the protective action of the nocifensor system. Currently, the prevalent pathophysiological role of capsaicin-sensitive sensory nerves in chronic models of COPD is unknown. However, our present study that identifies the molecular mechanism by which CS stimulates airway sensory nerves is the first step toward a better understanding of the role of neurogenic inflammation in the pathophysiology of CS-evoked diseases.

Supported by grants from MiUR, Rome (FIRB, RBIP06YM29 to P. Geppetti and R. Patacchini), PRIN (2003054380), Ente Cassa di Risparmio di Firenze (to P. Geppetti), and NIH (DK39957, DK43207, DK57840 to N.W. Bunnett) We are grateful to David Julius (UCSF Departments of Physiology and Cellular and Molecular Pharmacology) for the generous gift of TRPA1-deficient mice, Fabrizio Facchinetti (Chiesi Pharmaceuticals, Parma, Italy) for useful suggestions on CSE preparation, and Pamela Derish (Publications Manager, UCSF Department of Surgery) for the revision of the English text.

Address correspondence to: Pierangelo Geppetti, Department of Preclinical and Clinical Pharmacology, Viale Pieraccini 6, I-50139 Florence, Italy. Phone: 39-055-427-1329; Fax: 39-055-427-1280; E-mail: pierangelo.geppetti@unifi.it.

Nonstandard abbreviations used: [Ca²⁺]_i, intracellular Ca²⁺; CGRP, calcitonin gene–related peptide; CS, cigarette smoke; CSE, CS aqueous extract; DRG, dorsal root ganglia; GSH, glutathione monoethylester; JG, jugular ganglia; -LI, -like immunoreactivity; NKA, neurokinin A; RR, ruthenium red; SP, substance P; TRP, transient receptor potential; TRPA1, TRP cation channel, subfamily A, member 1; TRPV1, TRP cation channel, subfamily V, member 1.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J. Clin. Invest.118:2574–2582 (2008). doi:10.1172/JCI34886

Serena Materazzi and Pierangelo Geppetti’s present address is: Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy.

Eunice Andrè and Barbara Campi contributed equally to this work.

See the related articles at TRPA1 is a major oxidant sensor in murine airway sensory neurons, How irritating: the role of TRPA1 in sensing cigarette smoke and aerogenic oxidants in the airways, and TRPA1 is a major oxidant sensor in murine airway sensory neurons.

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