title: Vitamin C-induced activation of phospholipase D in lung microvascular endothelial cells: regulation by MAP kinases.
abstract: Our earlier studies have shown that vitamin C at pharmacological doses (mM) induces loss of redox-dependent viability in bovine lung microvascular endothelial cells (BLMVECs) that is mediated by oxidative stress. Therefore, here, we investigated the vitamin C-induced activation of the lipid signaling enzyme, phospholipase D (PLD) in BLMVECs. Monolayer cultures of BLMVECs were treated with vitamin C (0-10 mM) for different time periods (0-2 h) and the activity of PLD was determined. Vitamin C induced activation of PLD in BLMVECs in a time- and dose-dependent fashion that was significantly attenuated by antioxidants, p38 mitogen-activated protein kinase (p38 MAPK)-specific inhibitor (SB203580), extracellular signal-regulated protein kinase (ERK)-specific inhibitor (PD98059), and transient transfection of cells with dominant-negative (DN)-p38 MAPK and DN-ERK1/ERK2. Vitamin C also induced phosphorylation and enhanced the activities of p38 MAPK and ERK in BLMVECs in a time-dependent fashion. It was also evident that vitamin C induced translocation of PLD(1) and PLD(2), association of p38 MAPK and ERK with PLD(1) and PLD(2), threonine phosphorylation of PLD(1) and PLD(2) and SB203580- and PD98059-inhibitable threonine phosphorylation of PLD(1) in BLMVECs. Transient transfection of BLMVECs with DN-p38 MAPK and DN-ERK1/ERK2 resulted in marked attenuation of vitamin C-induced phosphorylation of threonine in PLD(1) and PLD(2). We, for the first time, showed that vitamin C at pharmacological doses, activated PLD in the lung microvascular ECs through oxidative stress and MAPK activation.
authors.affiliation: Lipid Signaling and Lipomics Laboratory, Division of Pulmonary, Critical Care and Sleep Medicine, College of Medicine and Public Health, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Saradhadevi
authors.initials: S
authors.lastname: Varadharaj
authors.forename: Emily
authors.initials: E
authors.lastname: Steinhour
authors.forename: Melissa G
authors.initials: MG
authors.lastname: Hunter
authors.forename: Tonya
authors.initials: T
authors.lastname: Watkins
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Baran
authors.forename: Ulysses
authors.initials: U
authors.lastname: Magalang
authors.forename: Periannan
authors.initials: P
authors.lastname: Kuppusamy
authors.forename: Jay L
authors.initials: JL
authors.lastname: Zweier
authors.forename: Clay B
authors.initials: CB
authors.lastname: Marsh
authors.forename: Viswanathan
authors.initials: V
authors.lastname: Natarajan
authors.forename: Narasimham L
authors.initials: NL
authors.lastname: Parinandi
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D001205:Ascorbic Acid / Q000494:pharmacology*; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D004789:Enzyme Activation; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
chemical_list: D000975:Antioxidants; D047428:Protein Kinase Inhibitors; D013912:Threonine; D020928:Mitogen-Activated Protein Kinases; D010739:Phospholipase D; D001205:Ascorbic Acid