Activation of endothelial cell phospholipase D by sphingosine and sphingosine-1-phosphate.
We have investigated the activation of phospholipase D (PLD) by sphingosine and its derivatives in bovine pulmonary artery endothelial cells (BPAEC) prelabeled with [32P]orthophosphate or [32P]lyso phospholipids. Sphingosine, in a dose- and time-dependent manner, stimulated the hydrolysis of [32P]phosphatidylcholine (PC) resulting in the production of [32P]phosphatidic acid (PA), suggesting PLD activation. In the presence of ethanol (150 mM), the accumulation of [32P]phosphatidylethanol was also observed. The sphingosine-induced stimulation of PLD activity was not affected by treatment with the protein kinase C (PKC) inhibitor staurosporine or by down-regulation of PKC with TPA and was independent of extracellular Ca2+, suggesting that the PLD activation was independent of PKC and Ca2+. Chelation of intracellular Ca2+ with BAPTA actually potentiated the sphingosine-stimulated [32P]PC hydrolysis. Furthermore, the activation of PLD by sphingosine was not abolished by treatment of BPAEC with either cholera or pertussis toxin, indicating noninvolvement of toxin-sensitive G-proteins. In addition to hydrolysis of [32P]PC, sphingosine also stimulated PLD-mediated hydrolysis of [32P]phosphatidylethanolamine and [32P]phosphatidylinositol. Among the various sphingoid compounds, in addition to sphingosine, only sphingosine-1-phosphate (Sph-1-P) activated the endothelial cell PLD. The effect of sphingosine and Sph-1-P on PA phosphatase (PA Pase) activity was tested using [3H]glycerol-labeled PA. The Mg(2+)-independent and membrane-associated PA Pase activity was inhibited by sphingosine (IC50 = 200 microM) but not by Sph-1-P. This implies that sphingosine and Sph-1-P share a similar PLD-stimulating property but differ in their PA Pase inhibitory activity.
Natarajan
V
V
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.
Jayaram
H N
HN
Scribner
W M
WM
Garcia
J G
JG
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003847:Deoxyglucose / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation; D007700:Kinetics; D008246:Lysophospholipids / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D010743:Phospholipids / Q000096:biosynthesis / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D011493:Protein Kinase C / Q000378:metabolism; D011651:Pulmonary Artery; D012265:Ribonucleotides / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D008246:Lysophospholipids; D010710:Phosphates; D010712:Phosphatidic Acids; D010743:Phospholipids; D010761:Phosphorus Radioisotopes; D012265:Ribonucleotides; C060506:sphingosine 1-phosphate; D004533:Egtazic Acid; D003847:Deoxyglucose; D011493:Protein Kinase C; D010739:Phospholipase D; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium