15-hydroxy-11-alpha-9-alpha-(epoxymethano)prosta-5-13-dienoic-acid and sphingosine-kinase

We analyzed the intracellular action of sphingosine 1-phosphate (Sph-1-P), formed from sphingosine (Sph) by sphingosine kinase (SPHK), in platelets. When sphingosine kinase activity was inhibited by N,N-dimethylsphingosine (DMS), Ca2+ mobilization induced by convulxin, an agonist of the collagen receptor glycoprotein VI (GPVI), was moderately but specifically abolished; that induced via G protein-coupled receptors was not affected. Under the same conditions, however, tyrosine phosphorylation of Syk and phospholipase Cgamma2, which is essential for the GPVI-mediated signaling, was not inhibited. Sphingosine kinase activity of the platelet membrane fraction increased specifically upon stimulation with convulxin or collagen. Our results suggest that intracellular sphingosine 1-phosphate is related to Ca2+ mobilization in GPVI-mediated signaling pathways.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Blood Platelets; Calcium; Calcium Signaling; Collagen; Crotalid Venoms; Humans; Lectins, C-Type; Lysophospholipids; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Platelet Membrane Glycoproteins; Receptors, Thrombin; Sphingosine; Tyrosine

Sphingosine 1-phosphate (Sph-1-P) is considered to play a dual role in cellular signaling, acting intercellularly as well as intracellularly. In this study, we examined the role of Sph-1-P as a signaling molecule in human platelets, using DL-threo-dihydrosphingosine (DHS) and N,N-dimethylsphingosine (DMS), inhibitors of Sph kinase and protein kinase C. Both DMS and DL-threo-DHS were confirmed to be competitive inhibitors of Sph kinase obtained from platelet cytoplasmic fractions. In intact platelets labeled with [3H]Sph, stimulation with 12-O-tetradecanoylphorbol 13-acetate or thrombin did not affect [3H]-Sph-1-P formation. While both DMS and DL-threo-DHS inhibited not only [3H]Sph-1-P formation but also protein kinase C-dependent platelet aggregation, staurosporine, a potent protein kinase inhibitor, only inhibited the protein kinase C-dependent reaction. Hence, it is unlikely that Sph kinase activation and the resultant Sph-1-P formation are mediated by protein kinase C in platelets. Furthermore, Ca2+ mobilization induced by platelet agonists that act on G protein-coupled receptor was not affected by DMS or DL-threo-DHS. Our results suggest that Sph-1-P does not mediate intracellular signaling, including Ca2+ mobilization, in platelets.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adult; Blood Platelets; Calcium Signaling; Enzyme Inhibitors; GTP-Binding Proteins; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Signal Transduction; Sphingosine; Thrombin