Activation of transient receptor potential ankyrin-1 (TRPA1) in lung cells by wood smoke particulate material

Abstract

Cigarette smoke, diesel exhaust, and other combustion-derived particles activate the calcium channel transient receptor potential ankyrin-1 (TRPA1), causing irritation and inflammation in the respiratory tract. It was hypothesized that wood smoke particulate and select chemical constituents thereof would also activate TRPA1 in lung cells, potentially explaining the adverse effects of wood and other forms of biomass smoke on the respiratory system. TRPA1 activation was assessed using calcium imaging assays in TRPA1-overexpressing HEK-293 cells, mouse primary trigeminal neurons, and human adenocarcinoma (A549) lung cells. Particles from pine and mesquite smoke were less potent agonists of TRPA1 than an equivalent mass concentration of an ethanol extract of diesel exhaust particles; pine particles were comparable in potency to cigarette smoke condensate, and mesquite particles were the least potent. The fine particulate (PM < 2.5 μm) of wood smoke were the most potent TRPA1 agonists and several chemical constituents of wood smoke particulate, 3,5-ditert-butylphenol, coniferaldehyde, formaldehyde, perinaphthenone, agathic acid, and isocupressic acid, were TRPA1 agonists. Pine particulate activated TRPA1 in mouse trigeminal neurons and A549 cells in a concentration-dependent manner, which was inhibited by the TRPA1 antagonist HC-030031. TRPA1 activation by wood smoke particles occurred through the electrophile/oxidant-sensing domain (i.e., C621/C641/C665/K710), based on the inhibition of cellular responses when the particles were pretreated with glutathione; a role for the menthol-binding site of TRPA1 (S873/T874) was demonstrated for 3,5-ditert-butylphenol. This study demonstrated that TRPA1 is a molecular sensor for wood smoke particulate and several chemical constituents thereof, in sensory neurons and A549 cells, suggesting that TRPA1 may mediate some of the adverse effects of wood smoke in humans.

Figure 1

Anderson cascade impactor stages (silver collection disks) showing the relative mass and size distribution of pine PM collected for this study. The residue on the stages indicate deposited WSPM with the black denoting highest concentrations of particle, brown (medium), and silver is the lowest.

Figure 2

(A) Quantitative comparison of TRPA1-mediated calcium flux in human TRPA1-overexpressing HEK-293 cells using pine PM, mesquite PM, cigarette smoke condensate (CSC), and diesel exhaust-ethanol extract (DEP-EtOH) at different concentrations. Data were collected using a NOVOstar plate reader. (*) Represents a statistical difference relative to DEP-EtOH, (∧) indicates a difference relative to pine PM, and (○) represents a difference relative to CSC using two-way ANOVA with Bonferroni post-test, p<0.05. (B) Kinetic comparison of TRPA1 activation by 1.15 mg/mL pine PM, mesquite PM, CSC, and DEP-EtOH.

Figure 3

Comparison of TRPA1 activation by different size fractions of pine PM, ranging from 0.43 μm (left) to 10 μm (right), at 0.73 mg/mL in TRPA1-overexpressing HEK-293 cells. LHC-9 represents the vehicle control. AITC (150 μM) was used as the positive control and to determine the maximum value for TRPA1-dependent calcium flux. Data were collected using a NOVOstar plate reader. *Indicates a significant response (p<0.01 using one-way ANOVA and Dunnett’s post-test) compared to vehicle control

Figure 4

(A) Concentration-response analysis for pine PM-induced calcium flux, using Fura-2, in isolated mouse TG neurons. Each data point represents the mean ± SEM value from TG neurons isolated from ≥ three animals. *Indicates a significant increase (p<0.01 using one-way ANOVA and Dunnett’s post-test) in response versus vehicle control. (B) Inhibition of AITC (50 μM)- and pine PM (0.073mg/mL)-induced calcium flux by the selective TRPA1 antagonist HC-030031 (50 μM). Data are expressed as percentage of maximum response in viable neurons determined using KCl (50 mM). Each data point represents the mean ± SEM response from ≥ three animals. *Indicates a significant inhibition of calcium flux by HC-030031 (p<0.01 using two-way ANOVA with Bonferroni post-test).

Figure 5

(A) Expression of TRPA1 mRNA in A549 cells. qPCR analysis of TRPA1 expression in TRPA1-overexpressing (TRPA1-OE) HEK-293, A549, and HEK-293 cells. N.D.=none detected. (B) Inhibition of AITC (200 μM)- and pine PM (0.73, 1.5, and 2.3 mg/mL)-induced calcium flux in A549 cells by HC-030031 (200 μM). Data were collected using a NOVOstar plate reader. *Indicates a significant inhibition of calcium flux by HC-030031 (p<0.05 using two-way ANOVA with Bonferroni post-test).

Figure 6

Comparison of coniferaldehyde and formaldehyde in pine and mesquite PM of various sizes. Data are the mean ± SEM representative of three replicates. Formaldehyde in pine PM is represented as red squares, and open red squares for mesquite PM. Coniferaldehyde in pine PM is represented as black circles, and open circles for mesquite PM.

Figure 7

Elucidation of the mechanism of activation of TRPA1 by pine PM (0.09 mg/mL), mesquite PM (0.19 mg/mL), agathic acid (75 μM), and 3,5-ditert-butylphenol (250 μM). Responses were compared using HEK-293 cells transiently transfected with either wild-type TRPA1 or TRPA1-ST mutant plasmids. Treatments were with and without pre-incubation of the PM/agonist for 10 min with 20 mM GSH. Changes in cellular fluorescence were determined microscopically and are expressed as the percentage of cellular fluorescence elicited by ionomycin (10 μM) and normalized to the positive control for TRPA1, AITC (150 μM). *Indicates significant reduction in calcium flux due to GSH pre-treatment (p<0.05 using one-way ANOVA with Bonferroni post-test). **Indicates a significant reduction in calcium flux between wild-type TRPA1 and the TRPA1-ST mutant (p<0.05 using one-way ANOVA and Bonferroni post-test).