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1 Department of Medicine, Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
2 Department of Obstetrics and Gynecology, Indiana University, Indianapolis, IN, USA
* To whom correspondence should be addressed. E-mail: vnataraj{at}jhmi.edu.
Diperoxovanadate (DPV), a potent inhibitor of protein tyrosine phosphatases and activator of tyrosine kinases, alters endothelial barrier function via signaling pathways that are incompletely understood. One potential pathway is Src kinase mediated tyrosine phosphorylation of proteins such as cortactin that regulate endothelial cells (ECs) cytoskeleton assembly. As DPV modulates endothelial cell signaling via protein tyrosine phosphorylation, we determined the role of DPV-induced [Ca2+]i in activation of Src kinase, cytoskeletal remodeling and barrier function in bovine pulmonary artery endothelial cells (BPAECs). DPV in a dose- and time-dependent fashion increased [Ca2+]i, which was partially blocked by the calcium channel blockers nifedipine and Gd3+. Treatment of cells with thapsigargin released Ca2+ from endoplasmic reticulum and subsequent addition of DPV caused no further change in [Ca2+]i. These data suggest, that DPV-induced [Ca2+]i includes Ca-release from the endoplasmic reticulum, and Ca-influx through store-operated calcium entry. Furthermore, DPV induced an increase in protein tyrosine phosphorylation, phosphorylation of Src and cortactin, actin remodeling and altered transendothelial electrical resistance in BPAECs. These DPV-mediated effects were significantly attenuated by BAPTA (25 µM), a chelator of intracellular free Ca2+. Immunofluorescence studies revealed that the DPV-mediated co-localization of cortactin with peripheral actin was also prevented by BAPTA. Chelation of extracellular Ca2+ by EGTA had marginal effects on DPV-induced phosphorylation of Src and cortactin, actin stress fibers formation, however, affected ECs barrier function. These data suggest that DPV-induced changes in [Ca2+]i regulate endothelial barrier function using signaling pathways that involve Src and cytoskeleton remodeling.
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