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Tyrosine kinases and calcium dependent activation of endothelial cell phospholipase D by diperoxovanadate

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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 α and ε, 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.

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Authors and Affiliations

  1. Department of Medicine, Indiana University School of Medicine, Indianapolis, USA

    V. Natarajan, S. Vepa, R. Shamlal, M. Al-Hassani & W.M. Scribner

  2. Department of Biochemistry, Indian Institute of Science, Bangalore, India

    T. Ramasarma & H.N. Ravishankar

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  1. V. Natarajan
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  2. S. Vepa
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  3. R. Shamlal
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  4. M. Al-Hassani
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  5. T. Ramasarma
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  6. H.N. Ravishankar
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  7. W.M. Scribner
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Natarajan, V., Vepa, S., Shamlal, R. et al. Tyrosine kinases and calcium dependent activation of endothelial cell phospholipase D by diperoxovanadate. Mol Cell Biochem 183, 113–124 (1998). https://doi.org/10.1023/A:1006872230910

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