Fluorescence-based rapid measurement of sphingosine-1-phosphate transport activity in erythrocytes
Autor: | Naoki Kobayashi, Akihito Yamaguchi, Tsuyoshi Nishi, Masato Otsuka |
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Rok vydání: | 2016 |
Předmět: |
Blood Platelets
0301 basic medicine Erythrocytes QD415-436 lipid transfer proteins Biochemistry Fluorescence immunology 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Endocrinology Sphingosine Blood plasma Methods Humans Platelet Sphingosine-1-phosphate sphingolipids organic chemicals Transporter Cell Biology Sphingolipid 030104 developmental biology lysophospholipid chemistry transporter 030220 oncology & carcinogenesis lipids/efflux lipids (amino acids peptides and proteins) Lysophospholipids Plant lipid transfer proteins Intracellular |
Zdroj: | Journal of Lipid Research, Vol 57, Iss 11, Pp 2088-2094 (2016) |
ISSN: | 0022-2275 |
Popis: | 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. |
Databáze: | OpenAIRE |
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