Characterization of the ATP-dependent Sphingosine 1-Phosphate Transporter in Rat Erythrocytes

Abstract

Sphingosine 1-phosphate (S1P) is a bioactive lipid signal transmitter present in blood. Blood plasma S1P is supplied from erythrocytes and plays an important role in lymphocyte egress from lymphoid organs. However, the S1P export mechanism from erythrocytes to blood plasma is not well defined. To elucidate the mechanism of S1P export from erythrocytes, we performed the enzymatic characterization of S1P transporter in rat erythrocytes. Rat erythrocytes constitutively released S1P without any stimulus. The S1P release was reduced by an ABCA1 transporter inhibitor, glyburide, but not by a multidrug resistance-associated protein inhibitor, MK571, or a multidrug resistance protein inhibitor, cyclosporine A. Furthermore, we measured S1P transport activity using rat erythrocyte inside-out membrane vesicles (IOVs). Although the effective S1P transport into IOVs was observed in the presence of ATP, this activity was also supported by dATP and adenosine 5′-(Β,γ-imido)triphosphate. The rate of S1P transport increased depending on S1P concentration, with an apparent Km value of 21 μm. Two phosphorylated sphingolipids, dihydrosphingosine 1-phosphate and ceramide 1-phosphate, did not inhibit S1P transport. Similar to the intact erythrocytes, the uptake of S1P into IOVs was inhibited by glyburide and vanadate but not by the other ABC transporter inhibitors. These results suggest that S1P is exported from the erythrocytes by a novel ATP-dependent transporter. Sphingosine 1-phosphate (S1P) is a bioactive lipid signal transmitter present in blood. Blood plasma S1P is supplied from erythrocytes and plays an important role in lymphocyte egress from lymphoid organs. However, the S1P export mechanism from erythrocytes to blood plasma is not well defined. To elucidate the mechanism of S1P export from erythrocytes, we performed the enzymatic characterization of S1P transporter in rat erythrocytes. Rat erythrocytes constitutively released S1P without any stimulus. The S1P release was reduced by an ABCA1 transporter inhibitor, glyburide, but not by a multidrug resistance-associated protein inhibitor, MK571, or a multidrug resistance protein inhibitor, cyclosporine A. Furthermore, we measured S1P transport activity using rat erythrocyte inside-out membrane vesicles (IOVs). Although the effective S1P transport into IOVs was observed in the presence of ATP, this activity was also supported by dATP and adenosine 5′-(Β,γ-imido)triphosphate. The rate of S1P transport increased depending on S1P concentration, with an apparent Km value of 21 μm. Two phosphorylated sphingolipids, dihydrosphingosine 1-phosphate and ceramide 1-phosphate, did not inhibit S1P transport. Similar to the intact erythrocytes, the uptake of S1P into IOVs was inhibited by glyburide and vanadate but not by the other ABC transporter inhibitors. These results suggest that S1P is exported from the erythrocytes by a novel ATP-dependent transporter. Sphingosine 1-phosphate (S1P), 2The abbreviations used are: S1Psphingosine 1-phosphateIOVinside-out membrane vesicleABCATP binding cassetteAMP-PNPadenosine 5′-(Β,γ-imido)triphosphateMRPmultidrug resistance-associated proteinTPA12-O-tetradecanoylphorbol-13-acetateDHS1Pdihydrosphingosine 1-phosphateATPγSadenosine 5′-O-(thiotriphosphate)BSAbovine serum albuminmAbmonoclonal antibody. a bioactive lipid molecule present in the blood, plays an important role in diverse cellular responses, such as migration, proliferation, and differentiation (1Kihara A. Mitsutake S. Mizutani Y. Igarashi Y. Prog. Lipid Res. 2007; 46: 126-144Crossref PubMed Scopus (141) Google Scholar, 2Rosen H. Goetzl E.J. Nat. Rev. Immunol. 2005; 5: 560-570Crossref PubMed Scopus (623) Google Scholar). These processes are triggered by the binding of S1P to its specific receptors (3Hla T. Semin. Cell Dev. Biol. 2004; 15: 513-520Crossref PubMed Scopus (350) Google Scholar), of which five subtypes (S1P1-S1P5) have been identified in endothelial and immune cells (4Spiegel S. Milstien S. Nat. Rev. Mol. Cell Biol. 2003; 4: 397-407Crossref PubMed Scopus (1768) Google Scholar). Studies using S1P1 receptor-deficient mice showed abnormalities in lymphocyte egress from lymph nodes, spleen, and thymus (5Matloubian M. Lo C.G. Cinamon G. Lesneski M.J. Xu Y. Brinkmann V. Allende M.L. Proia R.L. Cyster J.G. Nature. 2004; 427: 355-360Crossref PubMed Scopus (2091) Google Scholar, 6Allende M.L. Dreier J.L. Mandala S. Proia R.L. J. Biol. Chem. 2004; 279: 15396-15401Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). Whereas blood plasma contains a basal level of S1P from the nanomolar to the micromolar range (7Ito K. Anada Y. Tani M. Ikeda M. Sano T. Kihara A. Igarashi Y. Biochem. Biophys. Res. Commun. 2007; 357: 212-217Crossref PubMed Scopus (159) Google Scholar, 8Lee Y.M. Venkataraman K. Hwang S.I. Han D.K. Hla T. Prostaglandins Other Lipid Mediat. 2007; 84: 154-162Crossref PubMed Scopus (99) Google Scholar, 9Pappu R. Schwab S.R. Cornelissen I. Pereira J.P. Regard J.B. Xu Y. Camerer E. Zheng Y.W. Huang Y. Cyster J.G. Coughlin S.R. Science. 2007; 316: 295-298Crossref PubMed Scopus (735) Google Scholar, 10Butter J.J. Koopmans R.P. Michel M.C. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2005; 824: 65-70Crossref PubMed Scopus (29) Google Scholar, 11Allende M.L. Sasaki T. Kawai H. Olivera A. Mi Y. van Echten-Deckert G. Hajdu R. Rosenbach M. Keohane C.A. Mandala S. Spiegel S. Proia R.L. J. Biol. Chem. 2004; 279: 52487-52492Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 12Schmidt H. Schmidt R. Geisslinger G. Prostaglandins Other Lipid Mediat. 2006; 81: 162-170Crossref PubMed Scopus (53) Google Scholar), lymphoid tissues maintain a low S1P environment through the activity of S1P lyase (13Schwab S.R. Pereira J.P. Matloubian M. Xu Y. Huang Y. Cyster J.G. Science. 2005; 309: 1735-1739Crossref PubMed Scopus (654) Google Scholar). It has been proposed that a higher concentration of S1P in the blood plasma than in the lymphoid organs establishes an essential gradient along which lymphocytes expressing the S1P1 receptor on cell surfaces migrate (2Rosen H. Goetzl E.J. Nat. Rev. Immunol. 2005; 5: 560-570Crossref PubMed Scopus (623) Google Scholar, 5Matloubian M. Lo C.G. Cinamon G. Lesneski M.J. Xu Y. Brinkmann V. Allende M.L. Proia R.L. Cyster J.G. Nature. 2004; 427: 355-360Crossref PubMed Scopus (2091) Google Scholar, 6Allende M.L. Dreier J.L. Mandala S. Proia R.L. J. Biol. Chem. 2004; 279: 15396-15401Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 13Schwab S.R. Pereira J.P. Matloubian M. Xu Y. Huang Y. Cyster J.G. Science. 2005; 309: 1735-1739Crossref PubMed Scopus (654) Google Scholar, 14Lo C.G. Xu Y. Proia R.L. Cyster J.G. J. Exp. Med. 2005; 201: 291-301Crossref PubMed Scopus (258) Google Scholar, 15Cyster J.G. Annu. Rev. Immunol. 2005; 23: 127-159Crossref PubMed Scopus (722) Google Scholar). sphingosine 1-phosphate inside-out membrane vesicle ATP binding cassette adenosine 5′-(Β,γ-imido)triphosphate multidrug resistance-associated protein 12-O-tetradecanoylphorbol-13-acetate dihydrosphingosine 1-phosphate adenosine 5′-O-(thiotriphosphate) bovine serum albumin monoclonal antibody. The source of plasma S1P remains unclear despite its importance in the cellular responses of endothelial cells and lymphocytes. Unlike most cells, blood cells, astrocytes, and vascular endothelial cells are reported to release S1P (8Lee Y.M. Venkataraman K. Hwang S.I. Han D.K. Hla T. Prostaglandins Other Lipid Mediat. 2007; 84: 154-162Crossref PubMed Scopus (99) Google Scholar, 16Yang L. Yatomi Y. Miura Y. Satoh K. Ozaki Y. Br. J. Haematol. 1999; 107: 282-293Crossref PubMed Scopus (112) Google Scholar, 17Yatomi Y. Ruan F. Hakomori S. Igarashi Y. Blood. 1995; 86: 193-202Crossref PubMed Google Scholar, 18Anelli V. Bassi R. Tettamanti G. Viani P. Riboni L. J. Neurochem. 2005; 92: 1204-1215Crossref PubMed Scopus (97) Google Scholar). These cells contain sphingosine kinase, which synthesizes S1P through the phosphorylation of sphingosine (16Yang L. Yatomi Y. Miura Y. Satoh K. Ozaki Y. Br. J. Haematol. 1999; 107: 282-293Crossref PubMed Scopus (112) Google Scholar, 18Anelli V. Bassi R. Tettamanti G. Viani P. Riboni L. J. Neurochem. 2005; 92: 1204-1215Crossref PubMed Scopus (97) Google Scholar, 19Ancellin N. Colmont C. Su J. Li Q. Mittereder N. Chae S.S. Stefansson S. Liau G. Hla T. J. Biol. Chem. 2002; 277: 6667-6675Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Whereas and cells release S1P in a Y. Ruan F. Hakomori S. Igarashi Y. Blood. 1995; 86: 193-202Crossref PubMed Google Scholar, R. V. T. J. Exp. Med. 1999; PubMed Scopus Google Scholar), erythrocytes, and cells release S1P in a (16Yang L. Yatomi Y. Miura Y. Satoh K. Ozaki Y. Br. J. Haematol. 1999; 107: 282-293Crossref PubMed Scopus (112) Google Scholar). The of S1P from erythrocytes, the most of blood cells, have not been However, suggest that S1P released from erythrocytes is a source of plasma S1P (7Ito K. Anada Y. Tani M. Ikeda M. Sano T. Kihara A. Igarashi Y. Biochem. Biophys. Res. Commun. 2007; 357: 212-217Crossref PubMed Scopus (159) Google Scholar, 9Pappu R. Schwab S.R. Cornelissen I. Pereira J.P. Regard J.B. Xu Y. Camerer E. Zheng Y.W. Huang Y. Cyster J.G. Coughlin S.R. Science. 2007; 316: 295-298Crossref PubMed Scopus (735) Google and lymphocyte egress to blood R. Schwab S.R. Cornelissen I. Pereira J.P. Regard J.B. Xu Y. Camerer E. Zheng Y.W. Huang Y. Cyster J.G. Coughlin S.R. Science. 2007; 316: 295-298Crossref PubMed Scopus (735) Google Scholar). we showed that S1P is released from rat by or N. T. T. Kihara A. Sano T. Igarashi Y. A. J. Lipid Res. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). proposed that an ATP-dependent transporter plays a role in S1P release from N. T. T. Kihara A. Sano T. Igarashi Y. A. J. Lipid Res. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). However, the mechanism of S1P release is unclear is to the transport of S1P the this we the of S1P release from erythrocytes with that of and showed that S1P release from erythrocytes not any an to the ATP-dependent S1P uptake into inside-out membrane vesicles from rat erythrocytes and S1P transport in erythrocytes. ATP, ceramide 1-phosphate, glyburide, and cyclosporine from and from was from S1P was from and dihydrosphingosine 1-phosphate was from and from was from Life was from and and from Other of and from or and blood was from using an as an by the S1P release erythrocytes with a of and by in the in by a or an in and was to concentration of and an erythrocytes and the by from the and erythrocytes and by in with a IOVs to a of and PubMed Scopus Google Scholar). Rat erythrocytes by of blood as with of and of and with of and of erythrocytes in of and The was and the in was the the was in of and on we the IOVs without in the the was performed using the without the the was and the was in of The was through a The of vesicles and was a in and The vesicles the with of and The was and the vesicles with a The vesicles the S1P uptake membrane vesicles of with or in of ATP in the and The by the of and The was The of in the vesicles was by of the S1P from the IOVs was measured as membrane vesicles with or without ATP in the as the to the S1P in the and was and the was in the with or without ATP and the was and the was The of in the vesicles was as with with a and Rat was and The was and to the membrane Rat in the plasma by of blood The plasma was and the in the a by and the membrane was by on an and with the Rat monoclonal by of the to with S1P export from erythrocytes was that plays an important role in lymphocyte egress into blood R. Schwab S.R. Cornelissen I. Pereira J.P. Regard J.B. Xu Y. Camerer E. Zheng Y.W. Huang Y. Cyster J.G. Coughlin S.R. Science. 2007; 316: 295-298Crossref PubMed Scopus (735) Google Scholar). To this we measured the S1P release from rat erythrocytes was to the was into rat erythrocytes and to 1-phosphate The of S1P in the erythrocyte its the of release was observed and increased in a The of and which the S1P release in N. T. T. Kihara A. Sano T. Igarashi Y. A. J. Lipid Res. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar), did not the of released from erythrocytes, that cell is not of the S1P from cells we showed that and release S1P from rat N. T. T. Kihara A. Sano T. Igarashi Y. A. J. Lipid Res. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). erythrocytes, release was not by the of an inhibitor, MK571, or a multidrug resistance protein inhibitor, cyclosporine but was inhibited by an ABCA1 inhibitor, glyburide These results suggest that an but not a a role in S1P from of and on the S1P release from of in a Although characterization of the S1P transporter in intact cells is of with the from the of the IOVs this to the that erythrocytes S1P without any in to and IOVs from erythrocytes to S1P transport was into IOVs in the presence of ATP in a but not in the presence of or in the of ATP The of was from the in the IOVs measured by the To that the into the IOVs and using of from the and the and that most The ATP-dependent transport of showed with to the S1P and ATP on the the apparent Km to 21 and S1P and ATP, However, observed of S1P to a in the and a of S1P a concentration of its or of the S1P from the vesicles was not observed in the presence or of ATP, that this ATP-dependent S1P transport was To S1P is by or by that are with of which are in erythrocyte we the of and on ATP-dependent transport. and and did not S1P did the and or or to inhibit to a inhibitor, inhibited S1P transport activity in a However, and which also inhibit did not inhibit S1P a of the S1P transporter by These results that ATP-dependent S1P transport was by of and on the ATP-dependent uptake of in a ABC and reported as the S1P transporter N. T. T. Kihara A. Sano T. Igarashi Y. A. J. Lipid Res. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, K. E. Y. C. H. M. Y. A. F. J. Neurochem. 2007; Scholar, P. C.A. Milstien S. Spiegel S. Sci. 2006; PubMed Scopus (350) Google Scholar). S1P was in an ATP-dependent we the of ABC in rat erythrocytes. IOVs from rat erythrocytes by using monoclonal and the ABCA1 protein in and the protein in ABCA1 is from by its ABCA1 was not in rat or The and the and and and and in and These results suggest that the and in S1P transport in erythrocytes. which is S1P release from N. T. T. Kihara A. Sano T. Igarashi Y. A. J. Lipid Res. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar), was to the as an S1P in to the of S1P in the of the we the of on ATP-dependent S1P uptake into the was the uptake was increased than However, the ATP-dependent uptake of a the erythrocyte into IOVs was by These results that is the uptake of such as into the the of the uptake activity was a uptake of the presence of in the of the These results suggest that plays an important role in the release of from the erythrocyte membrane and in the effective transport of the the is a phosphorylated with a to It is released from rat the uptake and phosphorylation of and T. was also by rat erythrocytes and phosphorylated to was released to the in a the that released from erythrocytes through the S1P transporter. To the of the S1P transporter in erythrocytes, the uptake of into IOVs with phosphorylated sphingolipids, which have a to that of S1P performed using and ceramide Although the uptake was reduced to by and ceramide 1-phosphate on the transport of that the erythrocyte ATP-dependent S1P transporter the S1P as its Furthermore, we used and ATP to the and the of ATP in S1P transport uptake of S1P was observed in the presence of ATP, and or low S1P uptake was observed in the presence of the other These results that S1P transport is by and that is not essential transport. To the S1P transport mechanism in we the of on the uptake of inhibited S1P release from intact erythrocytes and also inhibited the transport of into the the other ABC transporter or cyclosporine multidrug resistance protein in IOVs the or of inhibitor, the uptake of which is by was inhibited Furthermore, a which a of also reduced S1P uptake and vanadate reduced the transport activity in a the of the transport activity by vanadate of the activity with glyburide to S1P transport activity was inhibited by vanadate These results that glyburide and vanadate of the S1P transport mechanism and that an ATP-dependent and transporter is the S1P transporter in of of glyburide and vanadate on the ATP-dependent transport of S1P into of of glyburide ATP-dependent uptake of the in IOVs was performed with ATP as of of vanadate S1P in plasma is to an important role in the of cellular responses in vascular endothelial cells and lymphocytes (5Matloubian M. Lo C.G. Cinamon G. Lesneski M.J. Xu Y. Brinkmann V. Allende M.L. Proia R.L. Cyster J.G. Nature. 2004; 427: 355-360Crossref PubMed Scopus (2091) Google Scholar, 6Allende M.L. Dreier J.L. Mandala S. Proia R.L. J. Biol. Chem. 2004; 279: 15396-15401Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, Y. T. G. F. H. Sano T. Satoh K. S. G. Igarashi Y. Ozaki Y. Blood. PubMed Google Scholar), and is a concentration Y. Igarashi Y. L. N. R. N. Satoh K. Ozaki Y. S. J. Biochem. PubMed Scopus Google Scholar). have that plasma S1P is supplied from erythrocytes (7Ito K. Anada Y. Tani M. Ikeda M. Sano T. Kihara A. Igarashi Y. Biochem. Biophys. Res. Commun. 2007; 357: 212-217Crossref PubMed Scopus (159) Google Scholar, 9Pappu R. Schwab S.R. Cornelissen I. Pereira J.P. Regard J.B. Xu Y. Camerer E. Zheng Y.W. Huang Y. Cyster J.G. Coughlin S.R. Science. 2007; 316: 295-298Crossref PubMed Scopus (735) Google Scholar, P. P. J. 2007; PubMed Scopus Google Scholar). However, a mechanism of the S1P release from the cells is To the the S1P transport the erythrocyte we measured the ATP-dependent S1P transport into IOVs from rat erythrocytes was the effective transport of S1P into the IOVs and the of S1P from the membrane N. T. T. Kihara A. Sano T. Igarashi Y. A. J. Lipid Res. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). plasma most of the S1P to the and S. Yatomi Y. M. M. F. Satoh K. K. Ozaki Y. J. Biochem. 2005; PubMed Scopus Google Scholar). most an important role in the serum S1P level by S1P from and by S1P from the membrane in of S1P was in IOVs a without ATP, than of the S1P in presence of It was that in the of ATP, was also into IOVs by Although ATP-dependent uptake was with the the of the S1P in IOVs without ATP was not with the of S1P transport is and S1P from IOVs was not These results suggest that S1P is to the vesicle and not into the IOVs without an ABC transporter inhibitor, and a inhibitor, inhibited the ATP-dependent S1P transport activity ABC export lipid and are inhibited by glyburide or ABCA1 is in from cells and is inhibited by glyburide N. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). an ABC transporter is a the S1P transporter in erythrocytes. However, the S1P transporter did not ATP as was not with the ATP but also with dATP and to the ATP did not the S1P transport the ATP, and dATP the S1P transport low or transport activity was observed with and to the presence of a in the of this a inhibited the ATP-dependent S1P transport activity These results suggest that ATP is not essential but that a the of ATP, and is important S1P transport most ABC ATP the effective transport of However, of the ABC are by ATP the by is by ATP and I. M. Li C. Y. J. 2003; PubMed Scopus Google Scholar). It is that an ABC transporter in S1P transport in erythrocytes without ATP to a (7Ito K. Anada Y. Tani M. Ikeda M. Sano T. Kihara A. Igarashi Y. Biochem. Biophys. Res. Commun. 2007; 357: 212-217Crossref PubMed Scopus (159) Google Scholar), have S1P activity not which to of erythrocyte The used erythrocytes, we erythrocyte IOVs using the PubMed Scopus Google Scholar), which in vesicles with activity from other cells such as (7Ito K. Anada Y. Tani M. Ikeda M. Sano T. Kihara A. Igarashi Y. Biochem. Biophys. Res. Commun. 2007; 357: 212-217Crossref PubMed Scopus (159) Google Scholar). is that this activity was inhibited with ATP and the ATP-dependent S1P transport activity was by and vanadate but not by the of of the ATP-dependent transport activity was observed with the of the of the S1P activity These results that S1P not the S1P transport S1P release from the cells was of S1P concentration the erythrocyte cells that S1P transport the S1P concentration gradient and that ATP as a to the S1P transport of the transporter. on reported we and as the S1P transporter N. T. T. Kihara A. Sano T. Igarashi Y. A. J. Lipid Res. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, K. E. Y. C. H. M. Y. A. F. J. Neurochem. 2007; Scholar, P. C.A. Milstien S. Spiegel S. Sci. 2006; PubMed Scopus (350) Google Scholar). S1P release from erythrocytes and the ATP-dependent S1P transport in IOVs inhibited by an ABCA1 inhibitor, glyburide, was that S1P is by ABCA1 in erythrocytes. However, ABCA1 was not in the K. E. Y. C. H. M. Y. A. F. J. Neurochem. 2007; reported that ABCA1 plays an important role in S1P release from The apparent Km value ATP is also with the reported Km value of ABCA1 K. Y. N. M. K. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). However, the from cells is on but not N. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Y. T. Y. K. N. H. K. M. J. Lipid Res. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar), S1P release from the and erythrocytes was with but not with N. T. T. Kihara A. Sano T. Igarashi Y. A. J. Lipid Res. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, and activity of ABCA1 inhibited by glyburide but not by vanadate N. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. Y. N. M. K. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). These results suggest that ABCA1 is not the S1P transporter of the blood P. C.A. Milstien S. Spiegel S. Sci. 2006; PubMed Scopus (350) Google reported that plays an important role in S1P release from showed that S1P release from cells was inhibited by an inhibitor, MK571, and the of Although was in erythrocytes S1P release from erythrocytes and S1P uptake into IOVs not by that S1P is not to exported by in erythrocytes. we showed that which has a to is in M. A. Y. S. A. Biochem. Biophys. Res. Commun. 2003; PubMed Scopus Google Scholar), that in S1P release in this we showed that is in rat and erythrocytes has been reported to in lipid release from cells N. M. P. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. S. Y. R. S. A. K. T. N. M. S. K. Biochem. Biophys. Res. Commun. 2003; PubMed Scopus Google Scholar, S. 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The erythrocyte S1P transporter ATP but not ATP its It to the ATP binding its The of the protein that as the S1P transporter in blood cells is to of cells have cell S1P The of ABC transporter on S1P release from erythrocytes and S1P uptake into erythrocyte membrane vesicles to that of N. T. T. Kihara A. Sano T. Igarashi Y. A. J. Lipid Res. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). is that the to ATP-dependent S1P transport in erythrocytes and are However, is a in the of the on S1P release erythrocytes and N. T. T. Kihara A. Sano T. Igarashi Y. A. J. Lipid Res. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). the S1P the cells and depending on Y. Ruan F. Hakomori S. Igarashi Y. Blood. 1995; 86: 193-202Crossref PubMed Google Scholar). However, erythrocytes the S1P its (7Ito K. Anada Y. Tani M. Ikeda M. Sano T. Kihara A. Igarashi Y. Biochem. Biophys. Res. Commun. 2007; 357: 212-217Crossref PubMed Scopus (159) Google Scholar, 16Yang L. Yatomi Y. Miura Y. Satoh K. Ozaki Y. Br. J. Haematol. 1999; 107: 282-293Crossref PubMed Scopus (112) Google Scholar). by the source of the S1P and the in the of S1P in erythrocytes and The S1P transporter in erythrocytes constitutively by a of the the transporter in is depending on the and such as that ATP-dependent S1P release from the was by which the protein through the receptor N. T. T. Kihara A. Sano T. Igarashi Y. A. J. Lipid Res. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). ABC such as ABCA1 and are depending on phosphorylation by protein and protein S. A. J. R. V. M. G. S.R. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, K. T. T. Life Sci. 2005; PubMed Scopus Google Scholar). Similar present in the S1P transporter in blood that an ATP-dependent transport in S1P release from erythrocytes To elucidate the of the ATP-dependent S1P we the of the ATP-dependent transport to the S1P release from intact erythrocytes. The rate of the S1P release from the erythrocytes was from in erythrocytes reported by (7Ito K. Anada Y. Tani M. Ikeda M. Sano T. Kihara A. Igarashi Y. Biochem. Biophys. Res. Commun. 2007; 357: 212-217Crossref PubMed Scopus (159) Google Scholar). the of S1P the erythrocytes was the rate of S1P release was The erythrocyte and the of IOVs from erythrocyte cell to and the S1P concentration the erythrocyte was (7Ito K. Anada Y. Tani M. Ikeda M. Sano T. Kihara A. Igarashi Y. Biochem. Biophys. Res. Commun. 2007; 357: 212-217Crossref PubMed Scopus (159) Google Scholar), and the rate of S1P release was to the rate of S1P uptake is to S1P the rate of the S1P release from intact erythrocytes S1P was to the the S1P uptake rate by the rate of membrane vesicle was to the rate of the S1P uptake into IOVs was with that of the released S1P from erythrocytes, that S1P was released from erythrocytes through the ATP-dependent transporter. results that S1P is the transport is but did not ATP the S1P transport of the of the erythrocyte transporter that S1P is by an ATP-dependent and transporter. is the characterization of the S1P with