Fig. 5

Determination of acetylcholine release by TGF-β1-stimulated lung fibroblasts by using LC-MS/MS. HFL1 and A549 cells were grown to sub-confluence in monolayer culture and serum deprived for 24 h. Cells were stimulated with TGF-β1 and harvested for the detection of ChAT by immunoblotting. Vertical axis, relative intensity of ChAT/β-actin ratio; Horizontal axis, conditions. Representative immunoblots are shown at the top of the panel. All values represent the mean ± SEM of the three different strains (a). HFL1 cells (b) and primary lung fibroblast from non-smokers (c) and patients with COPD (d) were grown to sub-confluence and cultured in SF-DMEM with or without TGF-β1 for 24 h. Supernatants were then harvested into a 1.5 ml tube and acetyl- and butyrylcholinesterase inhibitor (1 μM rivastigmine) was added to prevent acetylcholine degradation before storing at − 80 °C until assay. For the acetylcholine assay, the aliquots were thawed immediately before analysis and 5 μl of the samples was injected directly into the LC-MS/MS system. Data shown are representative chromatograms (m/z 86.910–87.910), performed in each group (n = 3). To obtain a standard report, acetylcholine was added to SF-DMEM. The arrow indicates the peak of acetylcholine (e). Vertical axis, signal intensity; Horizontal axis, retention time. *P < 0.05, **P < 0.01, compared with control