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 [P-32]orthophosphate or [P-32]lyso phospholipids. Sphingosine, in a dose- and time-dependent manner, stimulated the hydrolysis of [P-32]phosphatidylcholine (PC) resulting in the production of [P-32]phosphatidic acid (PA), suggesting PLD activation. In the presence of ethanol (150 mM), the accumulation of [P-32]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 [P-32]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 [(32)p]PC, sphingosine also stimulated PLD-mediated hydrolysis of [P-32] phosphatidylethanolamine and [P-32]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 [H-3]glycerol-labeled PA. The Mg2+-independent and membrane-associated PA Pase activity was inhibited by sphingosine (IC50 = 200 mu M) 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.