Functional expression of the transient receptor potential channel TRPA1, a sensor for toxic lung inhalants, in pulmonary epithelial cells Thomas Robert Heinrich Büch a,1 , Eva Anna Maria Schäfer b,1 , Maria-Theresia Demmel b , Ingrid Boekhoff b , Horst Thiermann c , Thomas Gudermann b,d,e , Dirk Steinritz b,c,⇑ , Annette Schmidt c,f a Rudolf-Boehm Institute of Pharmacology and Toxicology, Clinical Pharmacology, University Leipzig, Härtelstraße 16-18, 04107 Leipzig, Germany b Walther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilian University Munich, Goethestraße 33, 80336 Munich, Germany c Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstraße 11, 80937 Munich, Germany d Comprehensive Pneumology Center Munich (CPC-M), German Center for Lung Research, Munich, Germany e DZHK (German Centre for Cardiovascular Research), Munich Heart Alliance, Munich, Germany f Department for Molecular and Cellular Sport Medicine, German Sport University Cologne, Am Sportplatz Müngersdorf, 50933 Cologne, Germany article info Article history: Available online 30 August 2013 Keywords: Non-neuronal TRPA1 Calcium channel signaling Chemosensation Lung Toxic inhalation hazards ERK1/2 abstract The cation channel TRPA1 functions as a chemosensory protein and is directly activated by a number of noxious inhalants. A pulmonary expression of TRPA1 has been described in sensory nerve endings and its stimulation leads to the acceleration of inflammatory responses in the lung. Whereas the function of TRPA1 in neuronal cells is well defined, only few reports exist suggesting a role in epithelial cells. The aim of the present study was therefore (1) to evaluate the expression of TRPA1 in pulmonary epithelial cell lines, (2) to characterize TRPA1-promoted signaling in these cells, and (3) to study the extra-neuronal expression of this channel in lung tissue sections. Our results revealed that the widely used alveolar type II cell line A549 expresses TRPA1 at the mRNA and protein level. Furthermore, stimulating A549 cells with known TRPA1 activators (i.e., allyl isothiocyanate) led to an increase in intracellular calcium levels, which was sensitive to the TRPA1 blocker ruthenium red. Investigating TRPA1 coupled downstream sig- naling cascades it was found that TRPA1 activation elicited a stimulation of ERK1/2 whereas other MAP kinases were not affected. Finally, using epithelial as well as neuronal markers in immunohistochemical approaches, a non-neuronal TRPA1 protein expression was detected in distal parts of the porcine lung epithelium, which was also found examining human lung sections. TRPA1-positive staining co-localized with both epithelial and neuronal markers underlining the observed epithelial expression pattern. Our findings of a functional expression of TRPA1 in pulmonary epithelial cells provide causal evidence for a non-neuronal TRPA1-mediated control of inflammatory responses elicited upon TRPA1-mediated regis- tration of toxic inhalants in vivo. Ó 2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Acute lung injury by toxic inhalational hazards (TIH) is a major problem in toxicology since exposure towards these substances is a relatively common phenomenon and therapeutic options are still very limited. In most cases, acute treatment is restricted to symp- tomatic measures, e.g., inhalational application of steroids and supply of oxygen [3,17]. After toxic smoke inhalation mortality was found to be 12%; if smoke inhalation was associated with ther- mal burns even 61% of the cases were fatal [10]. Thus, with regard to the missing therapeutic options toxic lung injury is associated with significant health risks. Although TIH represent a divergent group of substances in terms of their chemical structure, electrophilic or oxidizing com- pounds like methylisocyanate, phosgene, chlorine, or ozone repre- sent one of the most important classes of TIH [3]. Toxic lung injury evoked by these substances has long been regarded as a result of unspecific cellular damage due to structural alterations of the cell membrane, DNA modifications, or undirected effects on cellular energy metabolism, respectively. However, in recent years, polymodal chemosensory receptors have been identified in the air- ways, which are activated by toxic chemical cues and subsequently transduce these stimuli to defined downstream signaling path- ways. These chemoreceptor-promoted signaling cascades are involved in adaptive or defensive mechanisms as well as in the 0009-2797/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbi.2013.08.012 Abbreviations: AITC, allyl isothiocyanate; EGF, epithelial growth factor; ERK1/2, extracellular signal-regulated protein kinase ½; FCS, fetal calf serum HBS, HEPES buffered saline; MAPK, mitogen activated protein kinase; RR, ruthenium red; SDS, sodium dodecyl sulfate; TRP, transient receptor potential channel; ICC, immuno- cytochemistry; IHC, immunohistochemistry; WB, Western blot. ⇑ Corresponding author. Address: Neuherbergstr 11, 80937 Munich, Germany. Tel.: +49 89 3168 2304; fax: +49 89 3168 2333. E-mail addresses: dirk.steinritz@lrz.uni-muenchen.de, dirksteinritz@bundes- wehr.org (D. Steinritz). 1 These authors contributed equally to this work. Chemico-Biological Interactions 206 (2013) 462–471 Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint