Abstract
Acetylcholine (ACh), synthesized by choline acetyltransferase (ChAT), and muscarinic M1, M2, and M3 receptors (MRs) are involved in fibroblast proliferation. We evaluated ChAT, MRs, and extracellular signal-regulated kinase (ERK) 1/2 and nuclear factor (NF) κB activation in lung fibroblasts from patients with chronic obstructive pulmonary disease (COPD), control smokers, and controls. Human fetal lung fibroblasts (HFL-1) stimulated with interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and cigarette smoke extracts (CSEs) were evaluated for ChAT and MR expression. We tested the effects of ACh on fibroblast proliferation and its ability to bind fibroblasts from patients with COPD, control smokers, controls, and HFL-1 stimulated with IL-1β, TNF-α, and CSE. ChAT, M1, and M3 expression and ERK1/2 and NFκB activation were increased, whereas M2 was reduced, in COPD and smoker subjects compared with controls. IL-1β increased the ChAT and M3, TNF-α down-regulated M2, and CSE increased ChAT and M3 expression while down-regulating the expression of M2 in HFL-1 cells. ACh stimulation increased fibroblast proliferation in patients with COPD, control smokers, and controls, with higher effect in control smokers and patients with COPD and increased HFL-1 proliferation only in CSE-treated cells. The binding of ACh was higher in patients with COPD and in control smokers than in controls and in CSE-treated than in IL-1β- and TNF-α-stimulated HFL-1 cells. Tiotropium (Spiriva; [1α,2β,4β,5α,7β-7-hydroxydi-2-thienylacetyl)oxy]-9,9-dimethyl-3-oxa-9-azoniatrcyclo[3.3.1.024], C19H22 NO4S2Br·H2O), gallamine triethiodide (C19H22N4O2S·2HCl·H2O), telenzepine [4,9-d-dihydro-3-methyl-4-[(4-methyl-1piperazinyl) acetyl]-10H-thieno [3,4-b][1,5]benzodiazepine-10-one dihydrobromide, C30H60I3N3O3], 4-diphenylacetoxy-N-methylpiperidine, PD098059 [2-(2-amino-3methoxyphenyl)-4H-1benzopyran-4-one, C16H13NO3], and BAY 11-7082 [(E)-3-(4-methylphenylsulfonyl)-2-propenetrile, C10H9NO2C], down-regulated the ACh-induced fibroblast proliferation, promoting the MRs and ERK1/2 and NFκB pathways involvement in this phenomenon. These results suggest that cigarette smoke might alter the expression of ChAT and MRs, promoting airway remodeling in COPD and that anticholinergic drugs, including tiotropium, might prevent these events.
Footnotes
-
This work was supported by a research grant from Boehringer Ingelheim Pharma GmbH and Co. KG (Biberach, Germany).
-
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
-
doi:10.1124/jpet.108.145888.
-
ABBREVIATIONS: M1, muscarinic M1 receptor; M2, muscarinic M2 receptor; M3, muscarinic M3 receptor; MR, muscarinic M1, M2, and M3 receptor; MAPK, mitogen-activated protein kinase; ACh, acetylcholine; ChAT, choline acetyltransferase; COPD, chronic obstructive pulmonary disease; IL, interleukin; TNF, tumor necrosis factor; CS, cigarette smoke; ERK, extracellular signal-regulated kinase; NF, nuclear factor; CSE, cigarette smoke extract; C, asymptomatic nonsmoking subject(s) with normal lung function; FBS, fetal bovine serum; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate; ELISA, enzyme-linked immunosorbent assay; pNFκB, phosphorylated NFκB; tNFκB, total NFκB; tiotropium, Spiriva, [1α,2β,4β,5α,7β-7-hydroxydi-2-thienylacetyl)oxy]-9,9-dimethyl-3-oxa-9-azoniatrcyclo[3.3.1.024], C19H22NO4S2Br·H2O; telenzepine, 4,9-d-dihydro-3-methyl-4-[(4-methyl-1piperazinyl) acetyl]-10H-thieno [3,4-b][1,5]benzodiazepine-10-one dihydrobromide, C30H60I3N3O3; gallamine triethiodide, C19H22N4O2S·2HCl·H2O; 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine; PD098059, 2-(2-amino-3methoxyphenyl)-4h-1benzopyran-4-one, C16H13NO3; BAY 11-7082, (E)-3-(4-methylphenylsulfonyl)-2-propenetrile, C10H9NO2C; hemicholinium-3, 2,2-(4,4′-biphenylene)bis(2-hydroxy-4,4-dimethylmorpholinium bromide, C24H34Br2N2O4; PBS, phosphate-buffered saline; ANOVA, analysis of variance; FEV1, forced expiratory volume in one second; FVC, forced vital capacity; pERK, phospho-ERK1/2; tERK, total ERK1/2.
- Received September 18, 2008.
- Accepted February 2, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|