Receptor-dependent activation of store-operated calcium entry increases endothelial cell permeability

The present study evaluated the necessity of store-operated Ca2+ entry in mediating thrombin-induced 20-kDa myosin light chain (MLC20) phosphorylation and increased permeability in bovine pulmonary artery endothelial cells (BPAECs). Thrombin (7 U/ml) and thapsigargin (1 mu M) activated Ca2+ entry through a common pathway in confluent BPAECs. Similar increases in MLC20 phosphorylation were observed 5 min after thrombin and thapsigargin challenge, although thrombin produced a sustained increase in MLC20 phosphorylation that was not observed in response to thapsigargin. Neither agonist increased MLC20 phosphorylation when Ca2+ influx was inhibited. Thrombin and thapsigargin induced inter-endothelial cell gap formation and increased FITC-dextran (molecular radii 23 Angstrom) transfer across confluent BPAEC monolayers. Activation of store-operated Ca2+ entry was required for thapsigargin and thrombin receptor-activating peptide to increase permeability, demonstrating that activation of store-operated Ca2+ entry is coupled with MLC20 phosphorylation and is associated with intercellular gap formation and increased barrier transport of macromolecules. Unlike thrombin receptor-activating peptide, thrombin increased permeability without activation of store-operated Ca2+ entry, suggesting that it partly disrupts the endothelial barrier through a proteolytic mechanism independent of Ca2+ signaling.