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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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
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
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
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
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.).
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Kobayashi, N., Nishi, T. (2017). A Rapid Fluorescence Assay for Measuring Sphingosine-1-Phosphate Transporter Activity in Erythrocytes. In: Pébay, A., Turksen, K. (eds) Sphingosine-1-Phosphate. Methods in Molecular Biology, vol 1697. Humana Press, New York, NY. https://doi.org/10.1007/7651_2017_25
Download citation
DOI: https://doi.org/10.1007/7651_2017_25
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7412-2
Online ISBN: 978-1-4939-7413-9
eBook Packages: Springer Protocols