title: Tyrosine kinases and calcium dependent activation of endothelial cell phospholipase D by diperoxovanadate.
abstract: Reactive oxygen species (ROS) mediated modulation of signal transduction pathways represent an important mechanism of cell injury and barrier dysfunction leading to the development of vascular disorders. Towards understanding the role of ROS in vascular dysfunction, we investigated the effect of diperoxovanadate (DPV), derived from mixing hydrogen peroxide and vanadate, on the activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAECs). Addition of DPV to BPAECs in the presence of .05% butanol resulted in an accumulation of [32P] phosphatidylbutanol (PBt) in a dose- and time-dependent manner. DPV also caused an increase in tyrosine phosphorylation of several protein bands (Mr 20-200 kD), as determined by Western blot analysis with antiphosphotyrosine antibodies. The DPV-induced [32P] PBt-accumulation was inhibited by putative tyrosine kinase inhibitors such as genistein, herbimycin, tyrphostin and by chelation of Ca2+ with either EGTA or BAPTA, however, pretreatment of BPAECs with the inhibitor PKC bisindolylmaleimide showed minimal inhibition. Also down-regulation of PKC alpha and epsilon, the major isotypes of PKC in BPAECs, by TPA (100 nM, 18 h) did not attenuate the DPV-induced PLD activation. The effects of putative tyrosine kinase and PKC inhibitors were specific as determined by comparing [32P] PBt formation between DPV and TPA. In addition to tyrosine kinase inhibitors, antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate also attenuated DPV-induced protein tyrosine phosphorylation and PLD stimulation. These results suggest that oxidation, prevented by reduction with thiol compounds, is involved in DPV-dependent protein tyrosine phosphorylation and PLD activation.
authors.affiliation: Department of Medicine, Indiana University School of Medicine, Indianapolis 46202-2879, USA.
authors.forename: V
authors.initials: V
authors.lastname: Natarajan
authors.forename: S
authors.initials: S
authors.lastname: Vepa
authors.forename: R
authors.initials: R
authors.lastname: Shamlal
authors.forename: M
authors.initials: M
authors.lastname: Al-Hassani
authors.forename: T
authors.initials: T
authors.lastname: Ramasarma
authors.forename: H N
authors.initials: HN
authors.lastname: Ravishankar
authors.forename: W M
authors.initials: WM
authors.lastname: Scribner
mesh_terms: D000818:Animals; D002118:Calcium / Q000502:physiology*; D002417:Cattle; D002460:Cell Line; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007424:Intracellular Fluid / Q000737:chemistry; D010084:Oxidation-Reduction / Q000187:drug effects; D010545:Peroxides / Q000494:pharmacology*; D010739:Phospholipase D / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011651:Pulmonary Artery / Q000166:cytology; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology*
chemical_list: D004791:Enzyme Inhibitors; D010545:Peroxides; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds; C091767:diperoxovanadate; D014638:Vanadates; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D010739:Phospholipase D; D002118:Calcium