Diperoxovanadate alters endothelial cell focal contacts and barrier function: role of tyrosine phosphorylation

Abstract

Diperoxovanadate (DPV), a potent tyrosine kinase activator and protein tyrosine phosphatase inhibitor, was utilized to explore bovine pulmonary artery endothelial cell barrier regulation. DPV produced dose-dependent decreases in transendothelial electrical resistance (TER) and increases in permeability to albumin, which were preceded by brief increases in TER (peak TER effect at 10-15 min). The significant and sustained DPV-mediated TER reductions were primarily the result of decreased intercellular resistance, rather than decreased resistance between the cell and the extracellular matrix, and were reduced by pretreatment with the tyrosine kinase inhibitor genistein but not by inhibition of p42/p44 mitogen-activating protein kinases. Immunofluorescent analysis after DPV challenge revealed dramatic F-actin polymerization and stress-fiber assembly and increased colocalization of tyrosine phosphoproteins with F-actin in a circumferential pattern at the cell periphery, changes that were abolished by genistein. The phosphorylation of focal adhesion and adherens junction proteins on tyrosine residues was confirmed in immunoprecipitates of focal adhesion kinase and cadherin-associated proteins in which dramatic dose-dependent tyrosine phosphorylation was observed after DPV stimulation. We speculate that DPV enhances endothelial cell monolayer integrity via focal adhesion plaque phosphorylation and produces subsequent monolayer destabilization of adherens junctions initiated by adherens junction protein tyrosine phosphorylation catalyzed by p60(src) or Src-related tyrosine kinases.

Pulmonary Endothelial Cell Barrier Enhancement by Sphingosine 1-Phosphate

“…The role of EC MLCK in vascular barrier regulation has previously been confined to its involvement as a central participant in several models of EC permeability (10). However, we previously noted the critical importance of the cortical actin ring in S1P-mediated barrier enhancement, which was accompanied at this site by strong increases in Ser 19 and Thr 18 phosphorylation of MLC that are highly suggestive of EC MLCK activation (2). CBP-mediated inhibition of MLCK interaction with cortactin appears to have important functional consequences on this MLC phosphorylation.…”

“…2C) (22) and shear stress (7). Diperoxovanadate (DPV) is a potent up-regulator of cellular tyrosine phosphorylation, which produces a brief period of EC barrier enhancement (0 -30 min) followed by prolonged increased permeability associated with p60 src activation and focal contact alteration (19,23). DPV induces substantial early translocation of cortactin to the periphery (Fig.…”

“…TER values from each microelectrode were pooled at discrete time points and plotted versus time as the mean Ϯ S.E. (19).…”

“…Fig. 8A depicts significant CBP-mediated attenuation of the S1P-induced (but not thrombin-induced) increased MLC phosphorylation on Ser 19 and Thr 18 as detected by Western blotting (quantified by densitometry and normalized to total MLC content in Fig. 8B).…”

Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependent cytoskeletal rearrangement

“…TER was monitored for 30 minutes to establish a baseline resistance (R 0 ) which, for bovine pulmonary endothelium, was typically between 8 to 12 × 10 3 Ω (wells with R 0 < 7 × 10 3 Ω were rejected). As cells adhere and spread out on the microelectrode, TER increases (maximal at confluence), whereas cell retraction, rounding, or loss of adhesion is reflected by a decrease in TER (32). These measurements provide a highly sensitive biophysical assay that indicates the state of cell shape and focal adhesion (29).…”