Endothelial cells form the innermost layer of blood vessels and build a barrier between blood and tissue. The permeability of this barrier is antagonistically controlled by the intracellular signalling molecules Ca2+ and cAMP. A rise in Ca2+ concentration increases permeability, whereas increased cAMP levels strengthen the endothelial cell barrier. In this study we investigated the impact of the coagulation factor thrombin that is known to increase Ca2+ concentrations and endothelial permeability, on cAMP levels. Surprisingly we detected that thrombin also led to a delayed and slow increase of cAMP concentrations. We discovered that this increase is due to the production of prostacyclin and a subsequent stimulation of endothelial prostacyclin receptors that finally induce cAMP production. This thrombin-mediated increase of cAMP levels might constitute a negative feedback control to protect endothelial barrier function despite a rise of Ca2+ concentrations.
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].