A Rapid Fluorescence Assay for Measuring Sphingosine-1-Phosphate Transporter Activity in Erythrocytes

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1697)

Abstract

Sphingosine-1-phosphate (S1P) is an intercellular signaling molecule that is present in the plasma and plays an important role in recruiting lymphocytes from the thymus and secondary lymphoid organs. Erythrocytes are the most abundant cells in the blood and substantially contribute to the S1P supply in the plasma by releasing intracellularly synthesized S1P via an S1P transporter. Thus, the S1P transporter in erythrocytes is a potential target for immuno-suppressing drugs.

In this chapter, we describe a rapid method for measuring the activity of the erythrocyte S1P transporter by using the fluorescent S1P analog, 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled S1P. This method does not require chromatography performed with high-performance liquid chromatography, liquid chromatography-tandem mass spectrometry, or thin-layer chromatography methods. Furthermore, S1P transporter activity can be detected by measuring the increase in fluorescence intensity in the extracellular buffer without performing lipid extraction.

Keywords

Sphingosine-1-phosphate Transporter Erythrocyte NBD Fluorescence 96-Well plate reader 

Notes

Acknowledgement

This work was supported by JSPS KAKENHI (grant number 25870695 to N. K.) and by the Cooperative Research Program of the “Network Joint Research Center for Materials and Devices” and AMED-CREST (to N. K. and T. N.).

References

  1. 1.
    Kobayashi N, Kobayashi N, Yamaguchi A, Nishi T (2009) Characterization of the ATP-dependent sphingosine 1-phosphate transporter in rat erythrocytes. J Biol Chem 284(32):21192–21200. doi:10.1074/jbc.M109.006163 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Kobayashi N, Otsuka M, Yamaguchi A, Nishi T (2016) Fluorescence-based rapid measurement of sphingosine-1-phosphate transport activity in erythrocytes. J Lipid Res 57(11):2088–2094. doi:10.1194/jlr.D071068 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Hisano Y, Kobayashi N, Kawahara A, Yamaguchi A, Nishi T (2011) The sphingosine 1-phosphate transporter, SPNS2, functions as a transporter of the phosphorylated form of the immunomodulating agent FTY720. J Biol Chem 286(3):1758–1766. doi:10.1074/jbc.M110.171116 CrossRefPubMedGoogle Scholar
  4. 4.
    Hisano Y, Kobayashi N, Yamaguchi A, Nishi T (2012) Mouse SPNS2 functions as a Sphingosine-1-phosphate transporter in vascular endothelial cells. PLoS One 7(6):e38941. doi:10.1371/journal.pone.0038941 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of Biochemistry, Faculty of Pharmaceutical SciencesSetsunan UniversityHirakataJapan
  2. 2.Department of Biomolecular Science and Regulation, Institute of Scientific and Industrial ResearchOsaka UniversityIbarakiJapan
  3. 3.Faculty of Pharmaceutical ScienceOsaka UniversitySuitaJapan

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