Sphingosine 1-phosphate breakdown in platelets.
We examined the formation of sphingolipid mediators in platelets, which abundantly store, and release extracellularly, sphingosine 1-phosphate (Sph-1-P). Challenging [(3)H]Sph-labeled platelet suspensions with thrombin or 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in a decrease in Sph-1-P formation and an increase in sphingosine (Sph), ceramide (Cer), and sphingomyelin formation. Sph conversion into Cer, and Cer conversion into sphingomyelin were not affected upon activation, suggesting that Sph-1-P dephosphorylation may initiate the formation of sphingolipid signaling molecules. In fact, Sph-1-P phosphatase (but not lyase) activity was detected in platelets, but this activity was not enhanced by thrombin or TPA. When quantified with [(3)H]acetic anhydride acetylation, followed by HPLC separation, the amounts of Sph-1-P and Sph decreased and increased, respectively, upon stimulation with thrombin or TPA, and these changes were attenuated by staurosporine. Under these TPA treatment conditions, over half of the [(3)H]Sph-1-P (formed in platelets incubated with [(3)H]Sph) was detected extracellularly, possibly due to its release from platelets, which was completely inhibited by staurosporine pretreatment. Furthermore, when TPA-induced Sph-1-P release was blocked by staurosporine after the stimulation, the extracellular [(3)H]Sph-1-P radioactivity decreased, suggesting that the Sph-1-P released may undergo dephosphorylation extracellularly. To support this, [(32)P]Sph-1-P, when added extracellularly to platelet suspensions, was rapidly degraded, possibly due to the ecto-phosphatase activity. Our results suggest the presence in anucleate platelets of a transmembrane cycling pathway starting with Sph-1-P dephosphorylation and leading to the formation of other sphingolipid mediators.
Yatomi
Yutaka
Y
Department of Laboratory Medicine, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655, Japan. yatomiy-lab@h.u-tokyo.ac.jp
Yamamura
Soichiro
S
Hisano
Nobuo
N
Nakahara
Kazuhiko
K
Igarashi
Yasuyuki
Y
Ozaki
Yukio
Y
D001792:Blood Platelets / Q000201:enzymology* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002518:Ceramides / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008246:Lysophospholipids / Q000378:metabolism*; D008954:Models, Biological; D010711:Phosphatidate Phosphatase / Q000378:metabolism; D010766:Phosphorylation; D015539:Platelet Activation; D010974:Platelet Aggregation; D015398:Signal Transduction; D013107:Sphingolipids / Q000378:metabolism; D013109:Sphingomyelins / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
D002518:Ceramides; D008246:Lysophospholipids; D013107:Sphingolipids; D013109:Sphingomyelins; C060506:sphingosine 1-phosphate; C112948:lipid phosphate phosphatase; D010711:Phosphatidate Phosphatase; D013917:Thrombin; D019311:Staurosporine; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate