V Natarajan, HN Jayaram, WM Scribner and JG Garcia
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.

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.

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