Hyperoxia alters phorbol ester-induced phospholipase D activation in bovine lung microvascular endothelial cells.
We investigated the effect of hyperoxia on phospholipase D (PLD) activation in bovine lung microvascular endothelial cells (BLMVECs). Generation of intracellular reactive oxygen species in BLMVECs exposed to hyperoxia for 2 or 24 h was three-fold higher compared with normoxic cells as measured by dichlorodihydrofluorescein di(acetoxymethyl ester) fluorescence. Exposure of BLMVECs to hyperoxia for 2 or 24 h attenuated 12-O-tetradecanoylphorbol 13-acetate (TPA)-mediated PLD activation compared with normoxic cells, however, hyperoxia did not alter basal PLD activity. Antioxidants, such as propyl gallate and pyrrolidine dithiocarbamate, reversed the effect of hyperoxia on TPA-induced PLD activity. Furthermore, the TPA-induced PLD activation was inhibited not only by the protein kinase C inhibitor, Go6976, but also by the tyrosine kinase inhibitor, genistein, and by the Src kinase specific inhibitor, PP-2, suggesting the involvement of protein kinase C and also tyrosine kinases in TPA-induced PLD activation. Western blot analysis of cell lysates from the hyperoxic (2 or 24 h) BLMVECs stimulated with TPA with anti-phosphotyrosine antibody showed an attenuation in overall tyrosine phosphorylation of proteins. In conclusion, we have demonstrated that hyperoxia enhanced the generation of reactive oxygen species in lung microvascular endothelial cells and attenuated TPA-induced protein tyrosine phosphorylation and PLD activation. As protein tyrosine phosphorylation and PLD play important roles in inflammatory responses, this could provide a mechanism for the regulation of endothelial barrier function during hyperoxic lung injury.
Roy
Shukla
S
Department of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University, Baltimore, MD 21224, USA.
Parinandi
Narasimham
N
Zeigelstein
Roy
R
Hu
Qinghua
Q
Pei
Yong
Y
Travers
Jeffrey B
JB
Natarajan
Viswanathan
V
D000818:Animals; D000975:Antioxidants / Q000378:metabolism / Q000494:pharmacology; D015153:Blotting, Western; D002227:Carbazoles / Q000494:pharmacology; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005452:Fluoresceins / Q000378:metabolism; D019833:Genistein / Q000494:pharmacology; D000860:Hypoxia; D007211:Indoles / Q000494:pharmacology; D015227:Lipid Peroxidation; D008168:Lung / Q000098:blood supply*; D055370:Lung Injury; D008833:Microcirculation / Q000378:metabolism*; D008954:Models, Biological; D010100:Oxygen / Q000378:metabolism; D010703:Phorbol Esters / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D017382:Reactive Oxygen Species; D013997:Time Factors
D000975:Antioxidants; D002227:Carbazoles; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D007211:Indoles; D010703:Phorbol Esters; D017382:Reactive Oxygen Species; C081021:Go 6976; C029569:diacetyldichlorofluorescein; D019833:Genistein; D011493:Protein Kinase C; D010739:Phospholipase D; D010100:Oxygen