Figure 6.

CS extract induces PDE3 and PDE4 changes in 16HBE 14o⁻ cells. 16HBE 14o⁻ cells were incubated with control medium or medium containing CS extract (2.5% v/v; final concentration), for 24 h. FRET data analysis of 16HBE 14o⁻ (A–D) under basal conditions (A, B) and fenoterol stimulated conditions (C, D). Under the basal condition, clear increases of the PDE4, but not PDE3, on basal cAMP levels were detected after CS extract treatment (A, B). FRET experiments stimulated with fenoterol and different PDE inhibitors showed that after β₂‐adrenoceptor stimulation, none of the different treatments with cilostamide or rolipram was affected by CS extract (C, D). Number of independent experiments is indicated above the bars. (E) Gene expression of PDE3 (PDE3A and PDE3B) and PDE4 subfamilies (PDE4A, PDE4B and PDE4D) was analysed by real‐time quantitative PCR in lung slice lysates. CS increased the gene expression PDE4D. (F, G) Protein expression of PDE3A and PDE4 subfamilies (PDE4A, PDE4B and PDE4D) was determined by Western blot analysis in lung homogenates. Quantification and representative blots are shown. Data shown are from six independent experiments. Data are expressed as mean ± SEM, *P < 0.05; significantly different from control. One‐way ANOVA was used for simple two groups comparison; Rolipram, 10 μM; cilostamide, 10 μM; IBMX, 100 μM; fenoterol, 1 nM for 16HBE 14o⁻ cells.