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

J Physiol. 2009 Aug 15;587(Pt 16):4091-104. doi: 10.1113/jphysiol.2009.172957. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenylyl Cyclase Inhibitors*
  • Adenylyl Cyclases / metabolism*
  • Calcium / metabolism*
  • Cells, Cultured
  • Computer Systems
  • Cyclic AMP / metabolism*
  • Endothelial Cells / drug effects
  • Endothelial Cells / metabolism*
  • Humans
  • Signal Transduction / drug effects
  • Signal Transduction / physiology*
  • Thrombin / pharmacology*

Substances

  • Adenylyl Cyclase Inhibitors
  • Cyclic AMP
  • Thrombin
  • Adenylyl Cyclases
  • adenylyl cyclase 6
  • Calcium