Real-time monitoring of cAMP levels in living endothelial cells: thrombin transiently inhibits adenylyl cyclase 6

The crosstalk between Ca2+ and cAMP signals plays a significant role for the regulation of the endothelial barrier function. The Ca2+-elevating agent thrombin was demonstrated to increase endothelial permeability and to decrease cAMP levels. Since Ca2+ and cAMP signals are highly dynamic, we aimed to study the temporal resolution between thrombin-evoked Ca2+ signals and subsequent changes of cAMP levels. Here we conduct the first real-time monitoring of thrombin-mediated regulation of cAMP signals in intact human umbilical vein endothelial cells (HUVECs) by utilising the Ca2+-sensitive dye Fluo-4 and the fluorescence resonance energy transfer (FRET)-based cAMP sensor Epac1-camps. We calibrated in vitro FRET responses of Epac1-camps to [cAMP] in order to estimate changes in intracellular [cAMP] evoked by thrombin treatment of HUVECs. After increasing [cAMP] to 1.2 +/- 0.2 mu m by stimulation of HUVECs with isoproterenol (isoprenaline), we observed a transient decrease of cAMP levels by 0.4 +/- 0.1 mu m which reached a minimum value 30 s after thrombin application and 15 s after the thrombin-evoked Ca2+ peak. This transient decrease in [cAMP] was Ca2+-dependent and independent of a G(i)-mediated inhibition of adenylyl cyclases (ACs). Instead the knock down of the predominant subtype AC6 in HUVECs provided the first direct evidence that the Ca2+-mediated inhibition of AC6 accounts for the thrombin-induced decrease in cAMP levels.