Activation of transient receptor potential ankyrin-1 by wood smoke particulate material

honors thesisCollege of ScienceBiologyChristopher A. ReillyExposure to wood smoke particulate matter (WSPM) has been linked to exacerbation of pre-existing respiratory conditions such as asthma, development of chronic obstructive pulmonary disease (COPD), and premature deaths. While it is clear that WSPM exposure is hazardous to human health, the molecular and cellular mechanisms through which it causes these adverse respiratory effects are not well understood. Transient receptor potential ankyrin-1 (TRPA1) is a cation channel that is expressed in sensory neurons, small airway epithelial cells, smooth muscle cells, and fibroblasts. TRPA1 has been implicated as a mediator of toxicity for several combustion-derived particulate materials (cdPM), including diesel exhaust (DEP) and cigarette smoke (CS). The hypothesis of this project was that WSPM would selectively activate TRPA1 through direct binding to ligand binding sites, including sites of covalent binding by electrophiles, and/or mechanical contact, which would then initiate cellular processes that culminate in pulmonary inflammation, lung injury, and respiratory dysfunction. Pine and mesquite PM were generated in the laboratory. These PM activated TRPA1 in a manner similar to DEP and CS in all cell lines tested: TRPA1 over-expressing HEK-293, primary mouse trigeminal (TG) neurons, and human alveolar adenocarcinoma (A549) cells. TRPA1 activation by WSPM was attenuated by a TRPA1 antagonist, HC-030031, in both A459 cells and TG neurons. Differential activation of TRPA1, as a function of particle size, demonstrated that respirable PM≤2.5 μm were most potent. Additionally, several known chemical components of WSPM were TRPA1 agonists. Both WSPM and agathic acid activated TRPA1 primarily though the electrophile/oxidant sensing site, while 3,5-ditert-butylphenol activated TRPA1 through the menthol-binding site. This study establishes WSPM as a potent and selective activator of TRPA1 and outlines a specific biochemical mechanism for how WSPM and associated chemical components activate TRPA1. These results provide key insights into how one could potentially develop therapeutics to reduce WSPM toxicity in the respiratory tract

Activation of Transient Receptor Potential Ankyrin‑1 (TRPA1) in Lung Cells by Wood Smoke Particulate Material

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-<i>ditert</i>-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-<i>ditert</i>-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

Activation of TRPV3 by Wood Smoke Particles and Roles in Pneumotoxicity

Wood/biomass smoke particulate materials (WBSPM) are pneumo­toxic, but the mechanisms by which these materials affect lung cells are not fully understood. We previously identified transient receptor potential (TRP) ankyrin-1 as a sensor for electrophiles in WBSPM and hypothesized that other TRP channels expressed by lung cells might also be activated by WBSPM, contributing to pneumo­toxicity. Screening TRP channel activation by WBSPM using calcium flux assays revealed TRPV3 activation by materials obtained from burning multiple types of wood under fixed conditions. TRPV3 activation by WBSPM was dependent on the chemical composition, and the pattern of activation and chemical components of PM agonists was different from that of TRPA1. Chemical analysis of particle constituents by gas chromatography–mass spectrometry and principal component analysis indicated enrichment of cresol, ethyl­phenol, and xylenol analogues, plus several other chemicals among the most potent samples. 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-xylenol, 2-, 3-, and 4-ethyl­phenol, 2-methoxy-4-methyl­phenol, and 5,8-dihydro­naphthol were TRPV3 agonists exhibiting preferential activation versus TRPA1, M8, V1, and V4. The concentration of 2,3- and 3,4-xylenol in the most potent samples of pine and mesquite smoke PM (<3 μm) was 0.1–0.3% by weight, while that of 5,8-dihydro­naphthol was 0.03%. TRPV3 was expressed by several human lung epithelial cell lines, and both pine PM and pure chemical TRPV3 agonists found in WBSPM were more toxic to TRPV3-over-expressing cells via TRPV3 activation. Finally, mice treated sub-acutely with pine particles exhibited an increase in sensitivity to inhaled methacholine involving TRPV3. In summary, TRPV3 is activated by specific chemicals in WBSPM, potentially contributing to the pneumo­toxic properties of certain WBSPM