Scintillation Proximity Assay to Detect the Changes in Cellular Dihydrosphingosine 1‐Phosphate Levels

Abstract

Compounds that modulate the activity of sphingosine 1-phosphate (S1P)-metabolizing enzymes are expected to be potential therapeutic agents for various diseases. Investigation of their potencies requires not only cell-free but also cell-based assays in which intracellular accumulation/depletion of S1P could be monitored. However, conventional methods have limitations to their simplicity, mainly due to the necessity of a separation process that separates S1P from its related substances. Here, we describe a method utilizing a scintillation proximity assay (SPA) for semi-quantifying intracellular [(3)H]-labeled dihydroS1P ([(3)H]dhS1P), which is also a substrate for S1P-metabolizing enzymes. We found that uncoated yttrium silicate SPA beads could selectively bind to and detect [(3)H]dhS1P rather than [(3)H]dihydrosphingosine (the non-phosphorylated form of [(3)H]dhS1P). Based on this, we developed a novel cell-based assay system which does not require any organic solvent extraction or chromatographic separation, and confirmed its practicality by using siRNA targeting S1P lyase (S1PL) and known S1PL inhibitors as models. Our results demonstrated that this assay is useful for rapid and easy evaluation of S1PL inhibitors, and could be potentially applicable for all compounds that modulate the activity of S1P-metabolizing enzymes.

Component of Caramel Food Coloring, THI, Causes Lymphopenia Indirectly via a Key Metabolic Intermediate

“…Firstly, to confirm that A6770 can also be phosphorylated in the same way, we conducted a cell-free phosphorylation experiment using recombinant PDXK and high-performance liquid chromatography (HPLC) analysis. In this system, it was previously observed that DOP was converted to DOP-P (Ohtoyo et al, 2015). After incubation of A6770 with PDXK we detected the shifted peak, which was identified as [2-acetyl-1H-imidazole-4(5)-yl]methoxyphosphate (A6770-P) by the consistency of retention time with chemically synthesized A6770-P (Fig-Figure 2.…”

“…To enhance the dhSph-to-dhS1P conversion, we pre-treated cells with the ceramide synthase inhibitor fumonisin B1 (FB1). Under conditions of both normal and VB6 deficiency (Ohtoyo et al, 2015), A6770 increased [ 3 H]dhS1P/S1P and simultaneously decreased [ 3 H]glycerophospholipids in a concentration-dependent manner ( Figure 2C). The increase in dhS1P/S1P suggests that A6770 inhibits degradation of dhS1P/S1P while the amounts of dhSph/Sph and ceramide remain largely unaltered.…”

“…Also in a rat in vivo study, A6770-P was detected in the thymus homogenate after oral administration of A6770, showing that the conversion from A6770 to A6770-P can occur even in vivo ( Figures 3B and 3C). Secondly, we examined the inhibitory activity of A6770-P on S1PL by using a membrane fraction (MF) derived from cells cultured in a VB6-deficient medium (Ohtoyo et al, 2015). This MF includes a decreased amount of holoenzyme (with PLP, active form) and an increased amount of apoenzyme (without PLP, inactive form), and therefore showed slight dhS1P degradation activity, whereas the enzymatic activity was restored by the addition of PLP.…”

“…In Vitro Phosphorylation Analysis Purified recombinant human PDXK was mixed with A6770 in the presence of 1 mM ATP, and incubated at 37 C for 1 hr. HPLC analysis was performed as previously described (Ohtoyo et al, 2015). (A-C) THI was orally administered to rats at 50 mg/kg under a fed condition, with or without pre-administration of antibiotics.…”

“…However, it is still controversial whether THI (and its derivatives) can bind to S1PL directly or whether it first becomes modified in vivo or acts by an indirect mechanism, because THI did not show any in vitro inhibitory activity on S1PL in both cell-free (Schwab et al, 2005;Bagdanoff et al, 2009;Reina et al, 2012) and cell-based systems (Billich et al, 2013a;Loetscher et al, 2013). Most recently we reported that THI itself shows a weak inhibitory effect in a cell-based system under a vitamin B6 (VB6)-deficient condition, but this solely cannot explain all of the mechanism because of the discrepancy found between the effective concentrations of the in vitro assay (R100 mM) (Ohtoyo et al, 2015) and those found in the plasma of the in vivo study (approximately 0.1-10 mM up to 24 hr following oral administration) (Yu et al, 2010). It was also reported that THI inhibits pyridoxal kinase (PDXK), which provides pyridoxal 5 0 -phosphate (PLP), a co-factor of various PLP-dependent enzymes including S1PL (Elsinghorst et al, 2015).…”