Zusammenfassung
The crosstalk between Ca(2+) and cAMP signals plays a significant
role for the regulation of the endothelial barrier function. The
Ca(2+)-elevating agent thrombin was demonstrated to increase endothelial
permeability and to decrease cAMP levels. Since Ca(2+) and cAMP signals
are highly dynamic, we aimed to study the temporal resolution between
thrombin-evoked Ca(2+) 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 Ca(2+)-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 microm
by stimulation of HUVECs with isoproterenol (isoprenaline), we observed
a transient decrease of cAMP levels by 0.4 +/- 0.1 microm which reached
a minimum value 30 s after thrombin application and 15 s after the
thrombin-evoked Ca(2+) peak. This transient decrease in cAMP was
Ca(2+)-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
Ca(2+)-mediated inhibition of AC6 accounts for the thrombin-induced
decrease in cAMP levels.
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