Phospholipase D activation in endothelial cells is redox sensitive.
Reactive oxygen species (ROS) are implicated in the pathophysiology of a number of vascular disorders, including atherosclerosis. Recent studies indicate that ROS modulate signal transduction in mammalian cells. Previously, we have shown that ROS (hydrogen peroxide, fatty acid hydroperoxide, diperoxovanadate, and 4-hydroxynonenal) enhance protein tyrosine phosphorylation and activate phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAECs). In the present study, our aim was to investigate the role of exogenous thiol agents on ROS-induced PLD activation in conjunction with the role of cellular thiols--glutathione (GSH) and protein thiols--on PLD activation and protein tyrosine phosphorylation. Pretreatment of BPAECs with N-acetyl-L-cysteine (NAC) or 2-mercaptopropionylglycine (MPG) blocked ROS-induced changes in intracellular GSH and PLD activation. Also, pretreatment with NAC attenuated diperoxovanadate-induced protein tyrosine phosphorylation. Pretreatment of BPAECs with diamide or L-buthionine-(S,R)-sulfoximine (BSO), agents that lower intracellular GSH and thiols, enhanced PLD activity. Furthermore, NAC blocked diamide- or BSO-mediated changes in GSH levels, PLD activity, and protein tyrosine phosphorylation. NAC also attenuated diamide-induced tyrosine phosphorylation of proteins between 69 and 118 KDa. These results support the hypothesis that modulation of thiol-redox status (cellular nonprotein and protein thiols) may contribute to the regulation of ROS-induced protein tyrosine phosphorylation and PLD activation in vascular endothelium.
Department of Medicine, Division of Pulmonary & Critical Care, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA.
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Parinandi
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Scribner
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Vepa
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Shi
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Natarajan
D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000378:metabolism* / Q000494:pharmacology; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007424:Intracellular Fluid / Q000378:metabolism; D010084:Oxidation-Reduction; D010739:Phospholipase D / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D011651:Pulmonary Artery; D017382:Reactive Oxygen Species / Q000502:physiology; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology; D008625:Tiopronin / Q000494:pharmacology
D000975:Antioxidants; D004791:Enzyme Inhibitors; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D019000:Phosphotyrosine; D019328:Buthionine Sulfoximine; C038983:1,3-dimethylthiourea; D008625:Tiopronin; D010739:Phospholipase D; D013890:Thiourea; D000111:Acetylcysteine