Fig. 1

Effect of roflumilast on real-time [cAMP]_i, estimated by FRET with and without smoke. a Treatment with 100 nM roflumilast alone (10 min) does not increase [cAMP]_i, but roflumilast enhances forskolin- and albuterol-mediated [cAMP]_i increases (10 μM forskolin and 20 μM albuterol in control cultures without airflow exposure (white bars; forskolin three lungs, n = 21; albuterol one lung, n = 8). b Representative tracings of FRET ratios over time as a reflection of changes in [cAMP]_i upon stimulation with forskolin in the presence (red) and absence (black) of roflumilast. c Smoke exposed cells (black bar) show a decreased baseline FRET ratio compared to cultures exposed to airflow (grey bar), possibly indicating a decreased intracellular cAMP level (three different lungs, n = 30). However, FRET ratios under these conditions cannot be calibrated to [cAMP]_i. d Roflumilast leads to a significant increase in the forskolin-induced ΔFRET ratio in air control (gray) and smoke exposed (black) cultures using the Vitrocell VC-10 smoke exposure system (three different lungs, n = 21–43). e Roflumilast treated cultures show a prolonged elevation of the FRET ratio after washout of forskolin in air control and smoke exposed cells (three different lungs, n = 21–43). *p < 0.05