Phospholipase D Activation in Endothelial Cells Is Redox Sensitive

Abstract

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.

Hypoxia-Induced Generation of Methane in Mitochondria and Eukaryotic Cells - An Alternative Approach to Methanogenesis

“…PLD hydrolyzes PC to phosphatidic acid and choline, and it has been implicated as a signal-activated key enzyme in a wide range of physiological responses [15]. In this respect, the redox regulation of PLD is well defined, and it has additionally been proposed that oxidants per se have significant effects on PLD activation in a variety of tissues [16,17].…”

Redox Regulation of 4-Hydroxy-2-nonenal-mediated Endothelial Barrier Dysfunction by Focal Adhesion, Adherens, and Tight Junction Proteins

“…Substances containing -SH groups such as aminoethanethiol, dimethylthiourea, NAC, and MPG may confer protective antioxidants effects (51). NAC blocks 4-HNE-induced depletion in cellular glutathione (2,19,20), apoptosis (53), suppresses activation of MAPKs (20), and phospholipase D activation (14,57). MPG administration to the heart, immediately before ischemia, reduces 4-HNE protein adducts by 75% (7).…”

“…Moreover, activation of focal adhesion kinase (FAK) by oxidative stress is an important event in ROS-mediated vascular EC bar-rier function that is regulated by cell-cell and cell-matrix contacts (13). Because 4-HNE not only forms Schiff base adducts with -NH 2 residues of proteins (14) but also alters cellular redox status due to loss of cellular sulfhydryl compounds (2), in our present study, we investigated the mechanisms of redox regulation of 4-HNE-mediated EC barrier dysfunction. The present study shows that: (i) 4-HNE, in a dose-dependent fashion, modulates cell-cell adhesion contacts as measured by transendothelial electrical resistance (TER); (ii) thiol protectants, such as N-acetylcysteine (NAC) and mercaptopropionyl glycine (MPG), attenuate 4-HNEmediated ROS formation, activation of ERK, JNK, and p38 MAPK signaling pathways, Michael protein adduct formation, and cytoskeleton rearrangement; and (iii) 4-HNE affects focal adhesion, adherent and tight junction proteins in lung microvascular ECs involving integrin signal transduction.…”

Role of Mitogen-activated Protein Kinases in 4-Hydroxy-2-nonenal-induced Actin Remodeling and Barrier Function in Endothelial Cells

“…Although the current study has not characterized any of the Michael adducts formed in lung microvascular ECs, it has been demonstrated that HNE-protein adduct accumulation reflects cellular toxicity compromising tissue survival during heart ischemia, ischemia/reperfusion injury, or pulmonary diseases (18, 19, 38 -40). Exogenously added or endogenously generated in cells, 4-HNE modulates protein function; examples include Na-K-ATPase (57), glucose transporter (58), MAPKs (7)(8)(9)(10)(11)24), phospholipases (1,4,5,59,60), protein kinase C (6), IK␤ kinase (61), and gene expression of ␥-glutamylcysteine synthetase (62). Thus, 4-HNE generated during lipid peroxidation can serve as an extracellular and intracellular signaling molecule altering cellular responses to stress and toxicity.…”