Activation of endothelial cell phospholipase D by hydrogen peroxide and fatty acid hydroperoxide

Viswanathan Natarajan, Mohiuddin M. Taher, Bruce Roehm, Narasimham L. Parinandi, Harald H O Schmid, Zoltan Kiss, Joe GN Garcia

Research output: Contribution to journalArticle

206 Citations (Scopus)

Abstract

We have investigated oxidant-mediated stimulation of phospholipase D (PLD) activity in bovine pulmonary artery endothelial cells (BPAEC), prelabeled with [32P] orthophosphate or [32P]lysophospholipids. Treatment of cells incubated in Hanks' balanced salt solution (HBSS) containing 0.5% ethanol with hydrogen peroxide (H2O2) or linoleic acid hydroperoxide (18:2-OOH) enhanced the formation of 32P-labeled phosphatidylethanol (PEt) and phosphatidic acid (PA) in a dose-and time-dependent manner, indicating the activation of PLD. The H2O2- and 18:2-OOH-mediated PLD activation was not associated with cytotoxicity as determined by [3H]deoxyglucose release. The addition of ferrous chloride (50 μM) augmented H2O2-induced formation of [32P]PEt and [32P]PA about 2-fold, whereas the addition of the iron chelator desferoxamine blocked the potentiating effect of ferrous chloride. Replacement of the HBSS medium with Medium 199 containing 20% calf serum also potentiated the effect of H2O2-induced PLD activation. In addition to phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) were readily hydrolyzed by PLD in response to H2O2 and 18:2-OOH treatment. The substrate specificity for oxidant-stimulated PLD activity differed from that observed in the presence of bradykinin or exhibited by agonist stimulation with 12-O-tetradecanoylphorbol 13-acetate (TPA) where PC was the major phospholipid hydrolyzed by PLD. The formation of PEt in the presence of H2O2 and 18:2-OOH was not abolished by chelation of either extracellular Ca2+ with EGTA (5 mM) or intracellular Ca2+ with 1,2-bis-(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) (25 μM, 30 min). Furthermore, pretreatment of BPAEC with the protein kinase C (PKC) inhibitor staurosporine and down-regulation of PKC by chronic TPA treatment (100 nM, 18 hr) had no effect on H2O2-induced PLD activation, suggesting that PLD activation by H2O2 is independent of PKC activity. It is possible that H2O2- and 18:2-OOH-induced activation of PLD represents an important mechanism to produce PA and diacylglycerol in endothelial cells.

Original languageEnglish (US)
Pages (from-to)930-937
Number of pages8
JournalJournal of Biological Chemistry
Volume268
Issue number2
StatePublished - Jan 15 1993
Externally publishedYes

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Phospholipase D
Lipid Peroxides
Endothelial cells
Hydrogen Peroxide
Endothelial Cells
Chemical activation
Phosphatidic Acids
Protein Kinase C
Tetradecanoylphorbol Acetate
Phosphatidylcholines
Oxidants
Pulmonary Artery
Acetates
Lysophospholipids
Ethane
Staurosporine
Protein C Inhibitor
Egtazic Acid
Diglycerides
Deoxyglucose

ASJC Scopus subject areas

  • Biochemistry

Cite this

Natarajan, V., Taher, M. M., Roehm, B., Parinandi, N. L., Schmid, H. H. O., Kiss, Z., & Garcia, J. GN. (1993). Activation of endothelial cell phospholipase D by hydrogen peroxide and fatty acid hydroperoxide. Journal of Biological Chemistry, 268(2), 930-937.

Activation of endothelial cell phospholipase D by hydrogen peroxide and fatty acid hydroperoxide. / Natarajan, Viswanathan; Taher, Mohiuddin M.; Roehm, Bruce; Parinandi, Narasimham L.; Schmid, Harald H O; Kiss, Zoltan; Garcia, Joe GN.

In: Journal of Biological Chemistry, Vol. 268, No. 2, 15.01.1993, p. 930-937.

Research output: Contribution to journalArticle

Natarajan, V, Taher, MM, Roehm, B, Parinandi, NL, Schmid, HHO, Kiss, Z & Garcia, JGN 1993, 'Activation of endothelial cell phospholipase D by hydrogen peroxide and fatty acid hydroperoxide', Journal of Biological Chemistry, vol. 268, no. 2, pp. 930-937.
Natarajan V, Taher MM, Roehm B, Parinandi NL, Schmid HHO, Kiss Z et al. Activation of endothelial cell phospholipase D by hydrogen peroxide and fatty acid hydroperoxide. Journal of Biological Chemistry. 1993 Jan 15;268(2):930-937.
Natarajan, Viswanathan ; Taher, Mohiuddin M. ; Roehm, Bruce ; Parinandi, Narasimham L. ; Schmid, Harald H O ; Kiss, Zoltan ; Garcia, Joe GN. / Activation of endothelial cell phospholipase D by hydrogen peroxide and fatty acid hydroperoxide. In: Journal of Biological Chemistry. 1993 ; Vol. 268, No. 2. pp. 930-937.
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N2 - We have investigated oxidant-mediated stimulation of phospholipase D (PLD) activity in bovine pulmonary artery endothelial cells (BPAEC), prelabeled with [32P] orthophosphate or [32P]lysophospholipids. Treatment of cells incubated in Hanks' balanced salt solution (HBSS) containing 0.5% ethanol with hydrogen peroxide (H2O2) or linoleic acid hydroperoxide (18:2-OOH) enhanced the formation of 32P-labeled phosphatidylethanol (PEt) and phosphatidic acid (PA) in a dose-and time-dependent manner, indicating the activation of PLD. The H2O2- and 18:2-OOH-mediated PLD activation was not associated with cytotoxicity as determined by [3H]deoxyglucose release. The addition of ferrous chloride (50 μM) augmented H2O2-induced formation of [32P]PEt and [32P]PA about 2-fold, whereas the addition of the iron chelator desferoxamine blocked the potentiating effect of ferrous chloride. Replacement of the HBSS medium with Medium 199 containing 20% calf serum also potentiated the effect of H2O2-induced PLD activation. In addition to phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) were readily hydrolyzed by PLD in response to H2O2 and 18:2-OOH treatment. The substrate specificity for oxidant-stimulated PLD activity differed from that observed in the presence of bradykinin or exhibited by agonist stimulation with 12-O-tetradecanoylphorbol 13-acetate (TPA) where PC was the major phospholipid hydrolyzed by PLD. The formation of PEt in the presence of H2O2 and 18:2-OOH was not abolished by chelation of either extracellular Ca2+ with EGTA (5 mM) or intracellular Ca2+ with 1,2-bis-(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) (25 μM, 30 min). Furthermore, pretreatment of BPAEC with the protein kinase C (PKC) inhibitor staurosporine and down-regulation of PKC by chronic TPA treatment (100 nM, 18 hr) had no effect on H2O2-induced PLD activation, suggesting that PLD activation by H2O2 is independent of PKC activity. It is possible that H2O2- and 18:2-OOH-induced activation of PLD represents an important mechanism to produce PA and diacylglycerol in endothelial cells.

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