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

Sandoval, Raudel, Asrar B. Malik, Tabassum Naqvi, Dolly Mehta, and Chinnaswamy Tiruppathi. Requirement for Ca signaling in the mechanism of thrombininduced increase in endothelial permeability. Am J Physiol Lung Cell Mol Physiol 280: L239–L247, 2001.—We compared the thrombin-activated responses in human umbilical vein endothelial cells (HUVECs) and a HUVEC-derived cell line, ECV304. Thrombin induced a 40–50% decrease in transendothelial monolayer electrical resistance and a twofold increase in I-albumin permeability in HUVECs, whereas it failed to alter the endothelial barrier function in ECV304 cells. Thrombin produced a brisk intracellular Ca concentration transient and phosphorylation of 20-kDa myosin light chain in HUVECs but not in ECV304 cells. Thrombin-induced phosphoinositide hydrolysis was comparable in ECV304 cells and HUVECs, indicating the activation of thrombin receptors in both cell types. La reduced both the thrombin-induced decrease in endothelial monolayer electrical resistance and the increase in I-albumin permeability in HUVECs. Because the absence of Ca signaling could explain the impairment in the permeability response in ECV304 cells, we studied the effect of increasing intracellular Ca concentration in ECV304 cells with thapsigargin. Exposure of ECV304 cells to thapsigargin caused decreased endothelial monolayer electrical resistance and increased I-albumin permeability. These results indicate that Ca influx and activation of Ca-dependent signaling pathways are important determinants of the thrombin-induced increase in endothelial permeability.