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Real‐time monitoring of cAMP levels in living endothelial cells: thrombin transiently inhibits adenylyl cyclase 6
Author(s) -
Werthmann R.C.,
Von Hayn K.,
Nikolaev V.O.,
Lohse M.J.,
Bünemann M.
Publication year - 2009
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2009.172957
Subject(s) - thrombin , adenylyl cyclase , umbilical vein , chemistry , forskolin , microbiology and biotechnology , intracellular , biophysics , in vitro , medicine , signal transduction , biology , biochemistry , platelet
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 μ m by stimulation of HUVECs with isoproterenol (isoprenaline), we observed a transient decrease of cAMP levels by 0.4 ± 0.1 μ m 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.