Figure 5.

Modulation of TRPA1 channel activity does not affect the anti-fibrotic properties of compound 145 in MRC-5 cells. (A) Lung fibroblasts were cultured for 24 h and then serum-deprived for 2 h. The cAMP concentration (fmol/mL/mg of protein; n = 4) was measured after 30 min of pre-incubation with HC-030031 (10 µM) or ASP7663 (10 µM) followed by 30 min of incubation with 145. (B) Lung fibroblasts were cultured for 24 h and then serum-deprived for 72 h. MRC-5 proliferation in the growing concentrations of the studied compounds (1–50 µM) was determined using alamarBlue^® assay after 48 h of incubation in the presence of FBS; n = 6. (C, D) MRC-5 contraction was determined after 30 min of pre-incubation with HC-030031 (10 µM) or ASP7663 (10 µM) followed by 30 min of incubation with 145 and 6 h exposure to TGF-β₁. (C) Representative pictures of collagen gel lattices taken at the beginning of the experiment (0 h) and after 6 h incubation. (D) Quantification of collagen gel area after a 6 h incubation in the presence of study compounds and TGF-β₁. Each bar represents the mean (± S.E.M.) (E) MRC-5 cells were treated for 200 s with 145 (10 µM) and then again with 145 (10 µM) in the presence of HC-030031 (10 µM); representative trace (left); bar charts showing average Ca²⁺ increase above the baseline recorded for 200 s during the first and the second application of 145 (n = 30, right). The results were considered statistically significant at the p level of 0.05 (*) or 0.01 (**).