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

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 K_m 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),² a bioactive lipid molecule present in the blood, plays an important role in diverse cellular responses, such as migration, proliferation, and differentiation (1, 2). These processes are triggered by the binding of S1P to its specific receptors (3), of which five subtypes (S1P₁-S1P₅) have been identified in endothelial and immune cells (4). Studies using S1P₁ receptor-deficient mice showed abnormalities in lymphocyte egress from lymph nodes, spleen, and thymus (5, 6). Whereas blood plasma contains a basal level of S1P from the nanomolar to the micromolar range (7–12), lymphoid tissues maintain a low S1P environment through the activity of S1P lyase (13). 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 (2, 5, 6, 13–15).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 (8, 16–18). These cells contain sphingosine kinase, which synthesizes S1P through the phosphorylation of sphingosine (16, 18, 19). Whereas platelets and mast cells release S1P in a stimulus-dependent manner (17, 20), erythrocytes, neutrophils, and mononuclear cells release S1P in a stimulus-independent manner (16). The roles of S1P derived from erythrocytes, the most abundant of these blood cells, have not been elucidated. However, recent reports suggest that S1P released from erythrocytes is a major source of plasma S1P (7, 9) and promotes lymphocyte egress to blood (9).Previously, we showed that S1P is released from rat platelets upon stimulation by thrombin or Ca²⁺ (21). We proposed that an ATP-dependent transporter plays a key role in S1P release from platelets (21). However, the detailed mechanism of S1P release is unclear because there is no way to assay the transport of S1P across the membrane. In this study we compared the properties of S1P release from erythrocytes with that of platelets and showed that S1P release from erythrocytes does not require any stimuli. We then established an assay to measure the ATP-dependent S1P uptake into inside-out membrane vesicles (IOVs) prepared from rat erythrocytes and characterized S1P transport in erythrocytes.