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Journal of Lipid Research, Vol. 47, 614-621, March 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Sphingosine 1-phosphate is released from the cytosol of rat platelets in a carrier-mediated manner

Nobuyoshi Kobayashi^*,,, Tsuyoshi Nishi^*,,**, Takahiro Hirata^*,,, Akio Kihara, Takamitsu Sano, Yasuyuki Igarashi and Akihito Yamaguchi^1,*,,

^* Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki-shi, Osaka 567-0047, Japan
Graduate School of Pharmaceutical Sciences, Osaka University, Suita-shi, Osaka 565-0871, Japan
Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Saitama, Japan
^** Precursory Research for Embryonic Science and Technology(PRESTO), Japan Science and Technology Corporation, Saitama, Japan
Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12-jo, Nishi 6-choume, Kita-ku, Sapporo, Hokkaido 060-0812, Japan

Published, JLR Papers in Press, December 21, 2005.

¹ To whom correspondence should be addressed. e-mail: akihito{at}sanken.osaka-u.ac.jp

Sphingosine 1-phosphate (S1P) is accumulated in platelets and released on stimulation by thrombin or Ca²⁺. Thrombin-stimulated S1P release was inhibited by staurosporin, whereas Ca²⁺-stimulated release was not. When the platelet plasma membrane was permeabilized with streptolysin O (SLO), S1P leaked out with cytosol markers, whereas granular markers remained in the platelets. The SLO-induced S1P leakage required BSA, probably for solubilization of S1P in the medium. These results indicate that S1P is localized in the inner leaflet of the plasma membrane and that its release is a carrier-mediated process. We also used alpha-toxin (ATX), which makes smaller pores in the plasma membrane than SLO and depletes cytosolic ATP without BSA-dependent S1P leakage. The addition of ATP drove S1P release from ATX platelets. The ATP-driven S1P release from ATX platelets was greatly enhanced by thrombin. An ATP binding cassette transporter inhibitor, glyburide, prevents ATP- and thrombin-induced S1P release from platelets. Ca²⁺ also stimulated S1P release from ATX platelets without ATP, whereas the Ca²⁺-induced release was not inhibited by glyburide. Our results indicate that two independent S1P release systems might exist in the platelet plasma membrane, an ATP-dependent system stimulated by thrombin and an ATP-independent system stimulated by Ca²⁺.

Supplementary key words ATP binding cassette transporter • flip-flop • lipid mediator • streptolysin O • -toxin

Abbreviations: ABC, ATP binding cassette; ACD, acid citrate-dextrose solution; ATX, -toxin; LDH, lactate dehydrogenase; LTC4, leukotriene C4; PF4, platelet factor 4; PKC, protein kinase C; PLSCR, platelet phospholipid scramblase; PRP, platelet-rich plasma; SLO, streptolysin O; S1P, sphingosine 1-phosphate; TPA, 12-O-tetradecanoylphorbol-13-acetate

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