Real-time monitoring of cAMP levels in living endothelial cells:
thrombin transiently inhibits adenylyl cyclase 6
R. Werthmann, K. von Hayn, V. Nikolaev, M. Lohse, and M. Bunemann. J Physiol, 587 (Pt 16):
4091-104(August 2009)Werthmann, R C von Hayn, K Nikolaev, V O Lohse, M J Bunemann, M Research
Support, Non-U.S. Gov't England The Journal of physiology J Physiol.
2009 Aug 15;587(Pt 16):4091-104. Epub 2009 Jun 22..
Abstract
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 von Hayn, K Nikolaev, V O Lohse, M J Bunemann, M Research
Support, Non-U.S. Gov't England The Journal of physiology J Physiol.
2009 Aug 15;587(Pt 16):4091-104. Epub 2009 Jun 22.
%0 Journal Article
%1 Werthmann2009
%A Werthmann, R. C.
%A von Hayn, K.
%A Nikolaev, V. O.
%A Lohse, M. J.
%A Bunemann, M.
%D 2009
%J J Physiol
%K & AMP/*metabolism Adenylate Calcium/*metabolism Cells/drug Computer Cultured Cyclase/*antagonists Cyclic Endothelial Humans Signal Systems Thrombin/*pharmacology Transduction/drug effects/*metabolism effects/*physiology inhibitors/*metabolism Cell
%N Pt 16
%P 4091-104
%T Real-time monitoring of cAMP levels in living endothelial cells:
thrombin transiently inhibits adenylyl cyclase 6
%U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19546162
%V 587
%X 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.
@article{Werthmann2009,
abstract = {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.},
added-at = {2010-12-14T18:12:02.000+0100},
author = {Werthmann, R. C. and von Hayn, K. and Nikolaev, V. O. and Lohse, M. J. and Bunemann, M.},
biburl = {https://www.bibsonomy.org/bibtex/22687343ee738764ba74369e1a8067704/pharmawuerz},
endnotereftype = {Journal Article},
interhash = {2436f475f1e89846af0f7e5caadbb822},
intrahash = {2687343ee738764ba74369e1a8067704},
issn = {1469-7793 (Electronic) 1469-7793 (Linking)},
journal = {J Physiol},
keywords = {& AMP/*metabolism Adenylate Calcium/*metabolism Cells/drug Computer Cultured Cyclase/*antagonists Cyclic Endothelial Humans Signal Systems Thrombin/*pharmacology Transduction/drug effects/*metabolism effects/*physiology inhibitors/*metabolism Cell},
month = {Aug 15},
note = {Werthmann, R C von Hayn, K Nikolaev, V O Lohse, M J Bunemann, M Research
Support, Non-U.S. Gov't England The Journal of physiology J Physiol.
2009 Aug 15;587(Pt 16):4091-104. Epub 2009 Jun 22.},
number = {Pt 16},
pages = {4091-104},
shorttitle = {Real-time monitoring of cAMP levels in living endothelial cells: thrombin
transiently inhibits adenylyl cyclase 6},
timestamp = {2010-12-14T18:20:51.000+0100},
title = {Real-time monitoring of cAMP levels in living endothelial cells:
thrombin transiently inhibits adenylyl cyclase 6},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19546162},
volume = 587,
year = 2009
}