Sphingosine 1-phosphate formation and intracellular Ca2+ mobilization in human platelets: evaluation with sphingosine kinase inhibitors.
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.
Department of Laboratory Medicine, Yamanashi Medical University, Nakakoma, Yamanashi, 409-3898, Japan.
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Yang
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Yatomi
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Satoh
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Igarashi
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Ozaki
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D000328:Adult; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D008246:Lysophospholipids; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology; D000099157:Sphingosine Kinase
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D004791:Enzyme Inhibitors; D019204:GTP-Binding Proteins; D008246:Lysophospholipids; D017853:Phosphotransferases (Alcohol Group Acceptor); D011493:Protein Kinase C; D013110:Sphingosine; D013917:Thrombin; D000099157:Sphingosine Kinase; C060506:sphingosine 1-phosphate; C061800:N,N-dimethylsphingosine; C005682:safingol