J. Biochem, 2004, Vol. 136, No. 4 495-502
© 2004 The Japanese Biochemical Society
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Sphingosine 1-Phosphate Breakdown in Platelets
1 Department of Laboratory Medicine, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655; 2 Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, NY 10021, USA; 3 Department of Laboratory Medicine, University of Yamanashi Faculty of Medicine, Yamanashi 409-3898; and 4 Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Hokkaido 060-0812
We examined the formation of sphingolipid mediators in platelets, which abundantly store, and release extracellularly, sphingosine 1-phosphate (Sph-1-P). Challenging [3H]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 [3H]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 [3H]Sph-1-P (formed in platelets incubated with [3H]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 [3H]Sph-1-P radioactivity decreased, suggesting that the Sph-1-P released may undergo dephosphorylation extracellularly. To support this, [32P]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.
* To whom correspondence should be addressed. Tel: +81-3-5800- 8730; Fax: +81-3-5689-0495; E-mail: yatomiy-lab{at}h.u-tokyo.ac.jp
Present address: Japanese Red Cross Medical Center, Tokyo, Japan.
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