Real-time monitoring of cAMP levels in living endothelial cells: thrombin transiently inhibits adenylyl cyclase 6.
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.
Werthmann
R C
RC
Department of Pharmacology and Toxicology, University of Würzburg, Versbacherstrasse 9, 97078 Würzburg, Germany.
von Hayn
K
K
Nikolaev
V O
VO
Lohse
M J
MJ
Bünemann
M
M
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003199:Computer Systems; D000242:Cyclic AMP / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013917:Thrombin / Q000494:pharmacology*
D000067956:Adenylyl Cyclase Inhibitors; D000242:Cyclic AMP; D013917:Thrombin; D000262:Adenylyl Cyclases; C121100:adenylyl cyclase 6; D002118:Calcium