Assay to measure the secretion of sphingosine-1-phosphate from cells induced by S1P lyase inhibitors

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.