Figure 2
Cyclic AMP activation induced by a range of doses of isoproterenol in hASM cells using (A) CFP-Epac(dDEP,CD)-VENUS activation or (B) 3H-cyclic AMP formation as a readout. (A) For Epac-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 maximal ratio was recorded and plotted. Data are expressed as fold over basal. Each data point represents the mean (± SEM) of 3-9 separate experiments. (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 isoproterenol 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 3-6 experiments.