Cigarette smoke altered the expression of choline acetyl transferase and muscarinic receptors promoting the LTB4 release from human bronchial epithelial cells

Acetylcholine (ACh) may be involved in the pathogenesis of chronic inflammatory disorders through the activation of airway epithelial cells. It is synthesized by choline acetyl transferase (ChAT), and exerts its physiological effects via the activation of muscarinic receptors (MRs). We used an in vitro model to test the effect of cigarette smoke extracts (CSE) in the ChAT and MRs expression and in the activation of human bronchial epithelial cell line (16-HBE). We evaluated the expression of both protein and mRNA of ChAT and MRs and the binding of ACh in the 16HBE stimulated with CSE. Then we stimulated the 16HBE, in the presence or absence of CSE, with ACh and we tested the ERK1/2 pathway activation and the Leukotriene B4 (LTB4) release. Our results show that CSE increases the expression of ChAT, M2 and M3 in the 16HBE and ACh (10mM) induced the release of LTB4 via the activation of ERK1/2. In addition, the preincubation of 16HBE with CSE increases the binding of ACh and shifts the cellular response from the ACh dose 10mM to 10nM. Finally, the addition of PDO98059 (MAPK inhibitor) and Tiotropium (Spiriva®) in the experimental condition prevents the effect of ACh. In conclusion, the cigarette smoke might altered the expression of muscarinic receptors M2 and M3 in the airway epithelial cells which in turn become more responsiveness to the lower doses of ACh in the release of proinflammatory mediators.