title: 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.
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA. drgarcia@welch.jhu.edu
authors.forename: J G
authors.initials: JG
authors.lastname: Garcia
authors.forename: K L
authors.initials: KL
authors.lastname: Schaphorst
authors.forename: A D
authors.initials: AD
authors.lastname: Verin
authors.forename: S
authors.initials: S
authors.lastname: Vepa
authors.forename: C E
authors.initials: CE
authors.lastname: Patterson
authors.forename: V
authors.initials: V
authors.lastname: Natarajan
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD; D015820:Cadherins; D002199:Capillary Permeability / Q000187:drug effects*; D002417:Cattle; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002450:Cell Communication / Q000187:drug effects*; D002460:Cell Line; D017097:Electric Impedance; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D010545:Peroxides / Q000494:pharmacology*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D014443:Tyrosine / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology*
chemical_list: D000199:Actins; D015703:Antigens, CD; C050361:CDH2 protein, human; D015820:Cadherins; D015815:Cell Adhesion Molecules; D004791:Enzyme Inhibitors; D010545:Peroxides; D010750:Phosphoproteins; C091767:diperoxovanadate; C108082:focal adhesion protein, 52-kDa; D019000:Phosphotyrosine; D014638:Vanadates; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D020928:Mitogen-Activated Protein Kinases