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A3812 - Nicotine Activates Epithelial TRPA1 Receptors to Cause Mucociliary Dysfunction
Author Block: S. Chung, R. Moore, J. S. Dennis, N. Baumlin, M. D. Kim, M. A. Salathe; University of Miami - Miller School of Medicine, Miami, FL, United States.
RATIONALE: Widespread e-cigarette use, touted as a ‘safer’ form of nicotine delivery, is confounded by high-dose nicotine deposition onto the airway epithelium. Historically, nicotine was studied for its properties on excitable systems (e.g., central nervous system), where nicotine-induced calcium (Ca2+) signaling is highly variable due to differential expression of nicotinic receptors. TRPA1, a Ca2+-selective ionophore receptor, was also reported to be nicotine-sensitive in chemosensory neurons. Here, we investigated whether TRPA1 is expressed and functional in the less excitable ciliated airway epithelia and whether TRPA1 contributes to effects of nicotine on mucociliary function.
METHODS: Primary normal human bronchial epithelial (NHBE) cells, isolated from appropriately consented donors whose lungs were found unsuitable for transplantation, were expanded and then re-differentiated at the air-liquid interface (ALI) on collagen-coated Transwell inserts. For Ca2+ measurements, GCaMP6s was transduced into NHBE cells. GCaMP6s emissions (Ex 495 nm) were recorded using MetaFluor software. Aerosolized treatments for assessing mucociliary parameters were deposited via a mesh nebulizer. Airway surface liquid (ASL) volume was measured by meniscus scanning. Ciliary beat frequency (CBF) was recorded and analyzed by SAVA software. Mucociliary transport (MCT) was assessed by recording movement of fluorescent micro-beads.
RESULTS: TRPA1 mRNA and protein were consistently expressed in low but detectable quantities in ALI-differentiated NHBE cells isolated from lungs of young non-smokers. A plasmid encoding a characterized human TRPA1 orthologue was used as a positive control for primer and antibody validation. TRPA1 function was probed by perfusing cultures with the TRPA1 agonist cinnamaldehyde, which caused robust Ca2+ influx in NHBE cells that was significantly reduced by the TRPA1 antagonist A967079. Nicotine induced a similar Ca2+ response that was also reduced by A967079 and HC030031, both TRPA1 antagonists. To assess Ca2+ signaling contributions from ER stores, experiments were repeated in the presence of thapsigargin: similar but smaller Ca2+ responses were again observed with nicotine but reduced with TRPA1 antagonists. Lastly, nicotine exposure via nebulization caused ASL volume loss, reduced CBF, and impaired MCT as early as 1 hr and up to 24 hrs post-exposure. Again, these parameters of mucociliary function were partially protected by pre-treatment with A967079.
CONCLUSIONS: Low but functional TRPA1 expression occurs in NHBE cells of non-smokers. Its expression is relevant since it contributes to nicotine signaling, which triggers a series of events that causes mucociliary dysfunction. Future studies will investigate how Ca2+ influx through TRPA1 impairs various molecular mechanisms related to airway hydration.