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Assay to measure the secretion of sphingosine-1-phosphate from cells induced by S1P lyase inhibitors.
Biochem Biophys Res Commun 2013; 433(3):345-8BB

Abstract

Inhibitors of the sphingosine-1-phosphate (S1P) degrading enzyme S1P lyase (SPL) may be useful in the therapy of inflammatory diseases by preventing lymphocyte recruitment to diseased tissues. Here we describe a cellular assay for such inhibitors, which takes advantage of the observation that a fraction of the intracellular S1P accumulated in the presence of SPL inhibitors is secreted into the medium of cultured cells. The secreted S1P is then quantified using an S1P-sensitive reporter cell line. In the routine assay protocol, human HEK293T cells are treated with SPL inhibitors in the presence of phosphatase inhibitors and sphingosine; while the phosphatase inhibitors are included to prevent the degradation of S1P secreted from the cells, sphingosine is added as source for intracellular S1P that is prone to SPL degradation. The secreted S1P in the supernatant of the cell cultures is then quantified by measuring calcium flux induced in CHO-K1 cells expressing the human S1P3 receptor. Using this method SPL inhibitors were shown to induce a concentration-dependent increase of extracellular S1P under the conditions used; thus, the assay allows for the ranking of SPL inhibitors according to their potency on living cells.

Authors+Show Affiliations

Novartis Institutes for BioMedical Research, Basel, Switzerland.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

23499842

Citation

Loetscher, Erika, et al. "Assay to Measure the Secretion of Sphingosine-1-phosphate From Cells Induced By S1P Lyase Inhibitors." Biochemical and Biophysical Research Communications, vol. 433, no. 3, 2013, pp. 345-8.
Loetscher E, Schneider K, Beerli C, et al. Assay to measure the secretion of sphingosine-1-phosphate from cells induced by S1P lyase inhibitors. Biochem Biophys Res Commun. 2013;433(3):345-8.
Loetscher, E., Schneider, K., Beerli, C., & Billich, A. (2013). Assay to measure the secretion of sphingosine-1-phosphate from cells induced by S1P lyase inhibitors. Biochemical and Biophysical Research Communications, 433(3), pp. 345-8. doi:10.1016/j.bbrc.2013.03.004.
Loetscher E, et al. Assay to Measure the Secretion of Sphingosine-1-phosphate From Cells Induced By S1P Lyase Inhibitors. Biochem Biophys Res Commun. 2013 Apr 12;433(3):345-8. PubMed PMID: 23499842.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Assay to measure the secretion of sphingosine-1-phosphate from cells induced by S1P lyase inhibitors. AU - Loetscher,Erika, AU - Schneider,Karolina, AU - Beerli,Christian, AU - Billich,Andreas, Y1 - 2013/03/14/ PY - 2013/02/25/received PY - 2013/03/05/accepted PY - 2013/3/19/entrez PY - 2013/3/19/pubmed PY - 2013/6/5/medline SP - 345 EP - 8 JF - Biochemical and biophysical research communications JO - Biochem. Biophys. Res. Commun. VL - 433 IS - 3 N2 - Inhibitors of the sphingosine-1-phosphate (S1P) degrading enzyme S1P lyase (SPL) may be useful in the therapy of inflammatory diseases by preventing lymphocyte recruitment to diseased tissues. Here we describe a cellular assay for such inhibitors, which takes advantage of the observation that a fraction of the intracellular S1P accumulated in the presence of SPL inhibitors is secreted into the medium of cultured cells. The secreted S1P is then quantified using an S1P-sensitive reporter cell line. In the routine assay protocol, human HEK293T cells are treated with SPL inhibitors in the presence of phosphatase inhibitors and sphingosine; while the phosphatase inhibitors are included to prevent the degradation of S1P secreted from the cells, sphingosine is added as source for intracellular S1P that is prone to SPL degradation. The secreted S1P in the supernatant of the cell cultures is then quantified by measuring calcium flux induced in CHO-K1 cells expressing the human S1P3 receptor. Using this method SPL inhibitors were shown to induce a concentration-dependent increase of extracellular S1P under the conditions used; thus, the assay allows for the ranking of SPL inhibitors according to their potency on living cells. SN - 1090-2104 UR - https://www.unboundmedicine.com/medline/citation/23499842/Assay_to_measure_the_secretion_of_sphingosine_1_phosphate_from_cells_induced_by_S1P_lyase_inhibitors_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0006-291X(13)00397-5 DB - PRIME DP - Unbound Medicine ER -