Figure 8.

CS extract induces local PDE changes in the presence of fenoterol in the ex vivo model. (A) Experimental design. Lung slices were treated with CS extract ex vivo exposure for 24 h, and PDE3 and PDE4 FRET activities were monitored after β₂‐adrenoceptor stimulation with fenoterol. β₂‐AR, β₂‐adrenoceptor; AC, adenylyl cyclase. (B, D) Representative FRET traces from slices after 24 h incubation with control medium or (C, E) medium containing CS extract (2.5% v/v; final concentration) . After treatment with fenoterol, PDE3 and PDE4 were inhibited with cilostamide (B, C) and rolipram (D, E) respectively. Finally, the non‐selective PDE inhibitor IBMX was used to inhibit multiple PDEs. (F, G) Quantification of FRET experiments is shown in B–E. Maximal FRET response was considered as the response of fenoterol, cilostamide/rolipram and IBMX. IBMX FRET response was calculated as the response of cilostamide/rolipram and IBMX. It indicated a significant increase of PDE3 contribution to cAMP hydrolysis after β₂‐adrenoceptor stimulation. No differences could be obtained on the PDE4 level in either maximal FRET response or IBMX‐induced FRET change. Number of slices/mice is indicated above the bars. On average n = 9–36 independent slices from three to seven mice have been used. Data are expressed as mean ± SEM, *P < 0.05; significantly different from control; One‐way ANOVA was used for simple two groups comparison. Fenoterol, 1 μM; cilostamide, 10 μM; rolipram, 10 μM; IBMX, 100 μM.