Temporally resolved cAMP monitoring in endothelial cells uncovers a thrombin-induced [cAMP] elevation mediated via the Ca²+-dependent production of prostacyclin.
The barrier function of the endothelium is controlled by the second messengers Ca2+ and cAMP that differentially regulate the permeability of endothelial cells. The Ca2+-elevating agent thrombin has been demonstrated to increase endothelial permeability and to decrease cAMP levels as detected via enzyme immunoassays. To study the effects of thrombin on cAMP with high temporal resolution, we utilised the FRET-based cAMP sensor Epac1-camps in single intact human umbilical vein endothelial cells (HUVECs). In these cells, thrombin induced a delayed increase in [cAMP], initiating after about 40 s, with maximum cAMP levels after 130 s of thrombin application. This increase of cAMP levels was Ca2+-dependent, but did not require calmodulin (CaM). Pharmacological approaches revealed that phospholipase A2 (PLA2) activity and cyclooxygenase (COX)-mediated synthesis of prostaglandins was required for the thrombin-induced elevation of [cAMP]. Furthermore, preincubation of HUVECs with a prostacyclin-receptor antagonist significantly reduced the thrombin-induced increase in [cAMP]. We conclude that thrombin causes the synthesis of prostacyclin in endothelial cells and that the subsequent stimulation of Gs-coupled prostacyclin receptors then results in an increase in [cAMP].
Institute of Pharmacology and Toxicology, University of Würzburg, Versbacherstrasse 9, 97078 Würzburg, Germany.
R C
RC
Werthmann
M J
MJ
Lohse
M
M
Bünemann
D015374:Biosensing Techniques; D002118:Calcium / Q000378:metabolism*; D002199:Capillary Permeability; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D042783:Endothelial Cells / Q000378:metabolism*; D011464:Epoprostenol / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D054467:Phospholipases A2 / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin / Q000378:metabolism; D015398:Signal Transduction; D013917:Thrombin / Q000378:metabolism*; D013997:Time Factors; D015854:Up-Regulation
C113936:PTGIR protein, human; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin; D011464:Epoprostenol; D000242:Cyclic AMP; D011451:Prostaglandin-Endoperoxide Synthases; D054467:Phospholipases A2; D013917:Thrombin; D002118:Calcium