Requirement for Ca2+ signaling in the mechanism of thrombin-induced increase in endothelial permeability

We compared the thrombin-activated responses in human umbilical vein endoth elial cells (HUVECs) and a HUVEC-derived cell line, ECV304. Thrombin induce d a 40-50% decrease in transendothelial monolayer electrical resistance and a twofold increase in I-125-albumin permeability in HUVECs, whereas it fai led to alter the endothelial barrier function in ECV304 cells. Thrombin pro duced a brisk intracellular Ca2+ concentration transient and phosphorylatio n of 20-kDa myosin light chain in HUVECs but not in ECV304 cells. Thrombin- induced phosphoinositide hydrolysis was comparable in ECV304 cells and HUVE Cs, indicating the activation of thrombin receptors in both cell types. La3 + reduced both the thrombin-induced decrease in endothelial monolayer elect rical resistance and the increase in I-125-albumin permeability in HUVECs. Because the absence of Ca2+ signaling could explain the impairment in the p ermeability response in ECV304 cells, we studied the effect of increasing i ntracellular Ca2+ concentration in ECV304 cells with thapsigargin. Exposure of ECV304 cells to thapsigargin caused decreased endothelial monolayer ele ctrical resistance and increased I-125-albumin permeability. These results indicate that Ca2+ influx and activation of Ca2+-dependent signaling pathwa ys are important determinants of the thrombin-induced increase in endotheli al permeability.