Figure 3
Cyclic AMP activation induced by single doses of a range of β 2 -adrenoceptor agonists in hASM cells using (A) CFP-Epac(dDEP,CD)-VENUS activation or (B) 3H-cyclic AMP formation as a readout. β2-adrenoceptor agonists studied were isoproterenol (10 μM), salbutamol (1 μM), salmeterol (100 nM), indacaterol (1 μM) and formoterol (1 μM). (A) For FRET-based studies single hASM cells expressing CFP-Epac(dDEP,CD)-VENUS were excited at 440 nm and the emission ratio (470/535) changes collected in real time. The maximum ratio observed was recorded and plotted. Data are expressed as fold over basal. Each data point represents the mean (± SEM) of 6-16 separate experiments. **denotes P < 0.01. (B) For studies investigating 3H-cyclic AMP formation, monolayers of hASM cells in 24 well plates were labelled with 3H-adenine for 2 hours and then exposed to the appropriate concentrations of β2-adrenoceptor agonist for 5 minutes. The reaction was terminated by addition of hydrochloric acid and total 3H-labelled cyclic AMP was collected via column-based separation and quantified by scintillation counting [13]. Data are expressed as fold over basal. Each data point represents the mean (± SEM) of 3-9 experiments. * denotes P < 0.05, **denotes P < 0.01.