title: Cigarette smoke extract activates human bronchial epithelial cells affecting non-neuronal cholinergic system signalling in vitro.
abstract: OBJECTIVE
  Acetylcholine (ACh) is synthesized by Choline Acetyl-Transferase (ChAT) that exerts its physiological effects in airway epithelial cells via muscarinic receptor (MR) activation. We evaluate the effect of ACh stimulation on human bronchial epithelial cells (16-HBE) and test whether cigarette smoke extract (CSE) can modify the basal cellular response to ACh affecting the non-neuronal cholinergic system signalling.
  
  
  METHODS
  ACh stimulated 16-HBE were tested for ACh-binding, Leukotriene B(4) (LTB(4)) release and ERK1/2 and NFkB pathway activation. Additionally, we investigated all the aforementioned parameters as well as ChAT and MR proteins and mRNA expression and endogenous ACh production in CSE-treated 16-HBE.
  
  
  RESULTS
  We showed that ACh induced in 16-HBE, in a concentration-dependent manner, LTB(4) release via the activation of ERK1/2 and NFkB pathways. The addition of Tiotropium (Spiriva®), Gallamine, Telenzepine and 4-DAMP (muscarinic receptor antagonists), as well as of PD 098059 (MAPKK inhibitor) and BAY117082 (inhibitor of IkBα phosphorilation), down-regulated the ACh-induced effects. Additionally, CSE treatment of 16-HBE increased the binding of ACh, and shifted the LTB4 release from the concentration ACh 1μM to 10nM. Finally, we observed that the treatment of 16-HBE with CSE increased the expression of ChAT, M(2) and M(3) and of endogenous ACh production in 16-HBE. Tiotropium regulated the LTB4 release and ACh production in CSE treated 16-HBE.
  
  
  CONCLUSIONS
  CSE increases the pro-inflammatory activity of human bronchial epithelial cells, and promotes the cellular response to lower concentrations of ACh, by affecting the expression of ChAT and MRs. Tiotropium might prevent pro-inflammatory events generated by ACh together with CSE.
authors.affiliation: Institute of Biomedicine and Molecular Immunology, Italian National Research Council, Via Ugo La Malfa 153, Palermo, Italy.
authors.forename: Mirella
authors.initials: M
authors.lastname: Profita
authors.forename: Anna
authors.initials: A
authors.lastname: Bonanno
authors.forename: Angela Marina
authors.initials: AM
authors.lastname: Montalbano
authors.forename: Maria
authors.initials: M
authors.lastname: Ferraro
authors.forename: Liboria
authors.initials: L
authors.lastname: Siena
authors.forename: Andreina
authors.initials: A
authors.lastname: Bruno
authors.forename: Stefania
authors.initials: S
authors.lastname: Girbino
authors.forename: Giusy Daniela
authors.initials: GD
authors.lastname: Albano
authors.forename: Paola
authors.initials: P
authors.lastname: Casarosa
authors.forename: Michael Paul
authors.initials: MP
authors.lastname: Pieper
authors.forename: Mark
authors.initials: M
authors.lastname: Gjomarkaj
mesh_terms: D000109:Acetylcholine / Q000378:metabolism*; D000704:Analysis of Variance; D015153:Blotting, Western; D001980:Bronchi / Q000166:cytology* / Q000187:drug effects; D002460:Cell Line; D002795:Choline O-Acetyltransferase / Q000378:metabolism; D045424:Complex Mixtures / Q000737:chemistry / Q000633:toxicity*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D007975:Leukotriene B4 / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D012906:Smoke / Q000032:analysis*; D014026:Nicotiana / Q000737:chemistry
chemical_list: D045424:Complex Mixtures; D016328:NF-kappa B; D011976:Receptors, Muscarinic; D012906:Smoke; D007975:Leukotriene B4; D002795:Choline O-Acetyltransferase; D048049:Extracellular Signal-Regulated MAP Kinases; D000109:Acetylcholine