title: Fluorescence-based rapid measurement of sphingosine-1-phosphate transport activity in erythrocytes.
abstract: Sphingosine-1-phosphate (S1P) is present in the blood plasma and acts as a pivotal intercellular signal transmitter in the immune system by recruiting lymphocytes from the thymus and secondary lymphoid tissues. The plasma S1P concentration is maintained by the supply of S1P from erythrocytes. Previously, we showed that S1P release from erythrocytes is mediated by an ATP-dependent transporter. In this study, we attempted to establish a rapid and reliable method for measuring the S1P transport activity in erythrocytes by using a fluorescent S1P analog, 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled S1P. NBD-S1P was released from erythrocytes in a time-dependent manner. The NBD-S1P release was reduced after exposure to glyburide, which is an inhibitor of the S1P transporter in erythrocytes. Moreover, the release of NBD-S1P and S1P from erythrocytes was competitively inhibited by intracellular S1P and NBD-S1P, respectively. These results showed that the erythrocyte S1P transporter exports NBD-S1P. We optimized the sample-preparation conditions and lipid extraction to increase the sensitivity of the assay. Furthermore, we successfully measured NBD-S1P release without lipid extraction by decreasing the concentration of BSA in the assay buffer to 0.1%. This method will be useful for the high-throughput screening of S1P transporter inhibitors using conventional fluorometers.
authors.affiliation: Department of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka 573-0101, Japan.|Faculty of Pharmaceutical Science, Teikyo Heisei University, Nakano-ku, Tokyo 164-8530, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Kobayashi
authors.affiliation: Department of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka 573-0101, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Otsuka
authors.affiliation: Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan.
authors.forename: Akihito
authors.initials: A
authors.lastname: Yamaguchi
authors.affiliation: Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan tnishi@sanken.osaka-u.ac.jp.|Faculty of Pharmaceutical Science, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Nishi
mesh_terms: D001792:Blood Platelets / Q000737:chemistry*; D004912:Erythrocytes / Q000737:chemistry*; D005453:Fluorescence; D006801:Humans; D008246:Lysophospholipids / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D008246:Lysophospholipids; C060506:sphingosine 1-phosphate; D013110:Sphingosine
keywords: 7-nitro-2-1,3-benzoxadiazol-4-yl; immunology; lipid transfer proteins; lipids/efflux; lysophospholipid; sphingolipids; transporter