Because activation of the coagulation cascade and the generation of thrombin coexist with sepsis and the release of tumor necrosis factor (TNF)-α, we determined the effects of TNF-α on the mechanism of thrombin-induced increase in endothelial permeability. We assessed Ca2+signaling in human umbilical vein endothelial cells. In human umbilical vein endothelial cells exposed to TNF-α for 2 h, thrombin produced a rise in the intracellular Ca2+concentration ([Ca2+]i) lasting up to 10 min. In contrast, thrombin alone produced a rise in [Ca2+]ilasting for 3 min, whereas TNF-α alone had no effect on [Ca2+]i.Thrombin-induced inositol 1,4,5-trisphosphate generation was not different between control and TNF-α-exposed cells. In the absence of extracellular Ca2+, thrombin produced similar increases in [Ca2+]iin both control and TNF-α-exposed cells. In TNF-α-exposed cells, the thrombin-induced Ca2+influx after intracellular Ca2+store depletion was significantly greater and prolonged compared with control cells. Increased Ca2+entry was associated with an approximately fourfold increase in Src activity and was sensitive to the Src kinase inhibitor PP1. After TNF-α exposure, thrombin caused increased tyrosine phosphorylation of junctional proteins and actin stress fiber formation as well as augmented endothelial permeability. These results suggest that TNF-α stimulation of endothelial cells results in amplification of the thrombin-induced Ca2+influx by an Src-dependent mechanism, thereby promoting loss of endothelial barrier function.
Protocatechuic aldehyde inhibits TNF-α-induced fibronectin expression in human umbilical vein endothelial cells via a c-Jun N-terminal kinase dependent pathway
