Natarajan V, Jayaram H N, Scribner W M, Garcia J G
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.
Am J Respir Cell Mol Biol. 1994 Aug;11(2):221-9. doi: 10.1165/ajrcmb.11.2.8049083.
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.
我们研究了鞘氨醇及其衍生物对预先用[32P]正磷酸盐或[32P]溶血磷脂标记的牛肺动脉内皮细胞（BPAEC）中磷脂酶D（PLD）的激活作用。鞘氨醇以剂量和时间依赖性方式刺激[32P]磷脂酰胆碱（PC）的水解，导致[32P]磷脂酸（PA）的产生，提示PLD被激活。在存在乙醇（150 mM）的情况下，还观察到了[32P]磷脂酰乙醇的积累。鞘氨醇诱导的PLD活性刺激不受蛋白激酶C（PKC）抑制剂星形孢菌素处理的影响，也不受TPA对PKC的下调作用的影响，并且不依赖于细胞外Ca2+，这表明PLD的激活不依赖于PKC和Ca2+。用BAPTA螯合细胞内Ca2+实际上增强了鞘氨醇刺激的[32P]PC水解。此外，用霍乱毒素或百日咳毒素处理BPAEC并没有消除鞘氨醇对PLD的激活作用，这表明毒素敏感的G蛋白未参与其中。除了[32P]PC的水解外，鞘氨醇还刺激PLD介导的[32P]磷脂酰乙醇胺和[32P]磷脂酰肌醇的水解。在各种鞘氨醇类化合物中，除了鞘氨醇外，只有鞘氨醇-1-磷酸（Sph-1-P）激活内皮细胞PLD。使用[3H]甘油标记的PA测试了鞘氨醇和Sph-1-P对PA磷酸酶（PA Pase）活性的影响。不依赖Mg(2+)且与膜相关的PA Pase活性被鞘氨醇（IC50 = 200 microM）抑制，但不受Sph-1-P抑制。这意味着鞘氨醇和Sph-1-P具有相似的PLD刺激特性，但在PA Pase抑制活性方面有所不同。
