Title

 

Authors

S. Materazzi¹,  R. Nassini¹, P. Pedretti¹, N. Moretto², C. Fusi¹, G. Volpi², R. Patacchini² and P. Geppetti^1,3.

¹Dept. of Preclinical and Clinical Pharmacology, University of Florence, Italy, ²Pharmacology Dept., Chiesi Farmaceutici SpA, Italy, ³Headache Center, University of Florence, Italy.

 

Abstract

TRPA1 is an unselective cation channel co-expressed with TRPV1 on capsaicin-sensitive sensory neurons. TRPA1 activation, on primary sensory neurons, mediates pain, and also contributes to airway/pulmonary inflammation caused by oxidative chemical species (ROS, HClO^-, RNS, NO) and reactive carbonyl species like 4-hydroxynonenal and acrolein (Bessac & Jordt, 2008). Recent papers show that almost all major neuronal inflammatory signaling pathways converge on TRPA1 channel to increase C-fiber excitability during airway inflammation suggesting a role in the pathogenesis of diseases of the respiratory system, including asthma and chronic obstructive pulmonary disease (COPD). TRPA1 has been demonstrated to be a target for cough reflex generation (Andre et al., 2009), to mediate allergic inflammatory responses elicited by ovalbumin (Caceres et al., 2009) and acute airway inflammation caused by acetaminophen in mice (Nassini et al., 2010). In this study we have investigated both expression and function of TRPA1 in human primary small airways epithelial cells (SAEC), human alveolar type-II epithelial cell line (A549), in normal human pulmonary fibroblasts (NHLF) and in primary bronchial smooth muscle cells (HBSMCs). TRPA1 mRNA expression was detected by RT-PCR. Aqueous cigarette smoke extract (CSE) was obtained by bubbling cigarette smoke in cell culture medium. Intracellular calcium ([Ca²⁺]_i) fluorescence measurement was made in Fura-2 preloaded cells.  Release of cytokine IL-8 was measured by ELISA assay in the cell culture supernatants. We found constitutive TRPA1 mRNA levels in SAEC, A549, NHLF and HBSMCs cells. Functional experiments demonstrated that TRPA1 stimulation by the selective agonist acrolein, elevated [Ca²⁺]_i in SAEC, A549, NHLF and HBSMCs. Elevation of [Ca²⁺]_i was blocked by the TRPA1 selective antagonsits HC-030031  and AP-18. Overnight exposure to acrolein or CSE elicited a concentration-dependent release of IL-8 from SAEC, HNLF and HBSMCs, an effect that was significantly reduced by selective TRPA1 antagonists (HC-030031 or AP-18) and fully prevented by the α,β-unsaturated aldehyde scavenger N-acetylcysteine (NAC). We report here for the first time that TRPA1 mRNA is constitutively functional expressed in non-neuronal pulmonary cells. We also uncover a role of TRPA1 in mediating the release of IL-8, a major neutrophil chemoattractant known to be elevated in the airways of cigarette smokers and in patients affected with COPD. This novel mechanism, mediated by TRPA1 channel, might contribute to the pro-inflammatory effects caused by cigarette smoke, allergens and environmental irritants and take part in development of pulmonary diseases like asthma and COPD, in addition to the reported inflammatory responses elicited by activation of sensory fibres, the so-called “neurogenic inflammation” (Geppetti & Holzer, 1996).
 
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Andre (2009). Br J Pharmacol. 158, 1621-1628.
Caceres (2009). PNAS.106, 9099-104.
Nassini (2010). FASEB J. 24, 4904-16.
Geppetti & Holzer (1996). Press, Boca Raton CRC.