Crosstalk between mAChRM3 and ?2AR, via acetylcholine PI3/PKC/PBEP1/Raf-1 MEK1/2/ERK1/2 pathway activation, in human bronchial epithelial cells after long-term cigarette smoke exposure

[Background: Cigarette smoke extract (CSE) affects the expression of non-neuronal components of cholinergic system in bronchial epithelial cells and, as PEBP1/Raf-mediated MAPK1/2 and ERK1/2 pathway, promotes inflammation and oxidative stress. Aims: We studied whether Acetylcholine (ACh) is involved in the mechanism of crosstalk between mAChRM3 and ?2Adrenergic receptors (?2AR) promoting, via PI3/PKC/PBEP1/Raf/MEK1/2/ERK1/2 activation, ?2AR desensitization, inflammation and, oxidative stress in a bronchial epithelial cell line (16HBE) after long-term exposure to cigarette smoke extract (LECSE). Methods: We evaluated mAChRM3 and Choline Acetyltransferase (ChAT) expression, ACh production, PEBP1, ERk1/2, and ?2AR phosphorylation, as well as NOX-4, ROS production and IL-8 release in 16HBE after LECSE. The inhibitory activity of Hemicholinium (HCh-3) (a potent choline uptake blocker), LY294002 (a highly selective inhibitor of PI3 kinase), Tiotropium (Spiriva®) (anticholinergic drug) and Olodaterol (?2AR agonist), were tested in 16HBE after LECSE. Results: mAChRM3, ChAT, ACh activity, pPEBP1, p?2AR, pERK1/2, ROS, NOX-4 and IL-8 increased after LECSE in 16HBE LECSE compared to untreated cells. HCh-3 and LY294002 (alone or in combination) as well as Tiotropium (Spiriva®) or Olodaterol (alone or in combination) all reduced the levels of pPEBP1, p?2AR, pERK1/ 2, ROS, NOX-4, and IL-8 in 16HBE LECSE compared to untreated cells. Conclusions: LECSE promotes ACh production which enhances PI3/PKC/PEBP1/Raf-ERK1/2 pathway activation, heterologous ?2AR desensitization, as well as release of inflammatory and oxidative mediators in bronchial epithelial cells. The use of anticholinergic drugs and long-acting ?2-agonists, alone or in combination may be dampen these inflammatory mechanisms when used in combination in some epithelial cell types.object Object]