Fluorescence-based assay of sphingosine kinases

Sphingosine kinase enzymatic activity is commonly measured using radiolabeled substrates, with thin-layer chromatography and/or solvent extraction needed to detect the reaction product sphingosine-1-phosphate. We developed a fluorescence-based assay, using a sphingosine derivative labeled with a 7-nitrobenz-2-oxa-1,3-diazole moiety (15-NBD-Sph). Separation of substrate (15-NBD-Sph) from product (the corresponding phosphate) is achieved by extraction with chloroform/methanol at pH 8.5. The phosphate derivative is recovered by >98% in the aqueous phase and is directly detected and quantified by its fluorescence. 15-NBD-Sph is readily phosphorylated by human and murine sphingosine kinases 1 and 2. The suitability of the assay for measuring the activity of the kinases, both in the purified state and when contained in lysates of mammalian cells, was demonstrated. The present method is a convenient alternative to the radiometric assays and is particularly suited to the search for inhibitors of sphingosine kinases.     

A rapid assay for assessment of sphingosine kinase inhibitors and substrates

Sphingosine kinases (SphKs) catalyze the transfer of phosphate from adenosine triphosphate (ATP) to sphingosine to generate sphingosine 1-phosphate (S1P), an important bioactive lipid molecule that mediates a diverse range of cell signaling processes. The conventional assay of SphK enzymatic activity uses [γ-³²P]ATP and sphingosine as substrates, with the radiolabeled S1P product recovered by organic extraction, displayed by thin layer chromatography, and quantified by liquid scintillation counting. Although this assay is sensitive and accurate, it is slow and labor-intensive; thus, it precludes the simultaneous screening of more than a few inhibitor compounds. Here we describe a 96-well assay for SphKs that is rapid and reproducible. Our method, which takes advantage of the limited solubility of S1P, detects radioactive S1P adhering to the plate by scintillation proximity counting. Our procedure obviates extraction into organic solvents, postreaction transfers, and chromatography. Furthermore, our assay enables assessment of both inhibitors and substrates, and it can detect endogenous SphK activity in cell and tissue extracts. The SphK kinetic parameter, K_m, and the K_i values of inhibitors determined with our assay and the conventional assay were indistinguishable. These results document that our assay is well-suited for the screening of chemical libraries of SphK inhibitors.     

Dimethylsphingosine and FTY720 inhibit the SK1 form but activate the SK2 form of sphingosine kinase from rat heart

Fractionation of cytosolic sphingosine kinase (SKase) activity by gel filtration chromatography gave rise to a 96-kDa peak that contained only the SK2 form of SKase (by Western analysis) and a broad ca. 46 kDa peak that contained only SK1 forms. SK2 appeared to have a bound accessory protein. When tested with the classic SKase inhibitor dimethylsphingosine (DMS), SK1 was extensively inhibited; however, SK2 was not inhibited but unexpectedly was activated. Activation of SK2 was the result of DMS enhancing the affinity of the enzyme for sphingosine, and, at low concentrations of ATP and sphingosine, activated by more than 100%. Activation of SK2 could be demonstrated in the cytosolic fraction indicating it was unrelated to the purification step. The immunomodulator FTY720 also activated SK2 (although to a lesser extent), but was a potent inhibitor of SK1. SK2 from rat liver and spleen was also not inhibited by DMS. L-Sphingosine and to a lesser extent dihydrosphingosine and phytosphingosine were effective inhibitors of both forms.     

An assay for sphingosine kinase activity using biotinylated sphingosine and streptavidin-coated membranes

Sphingosine kinase catalyses the phosphorylation of sphingosine to generate sphingosine 1-phosphate, a lipid signaling molecule implicated in roles in a diverse range of mammalian cell processes through its action as both a ligand for G-protein-coupled cell-surface receptors and an apparent intracellular second messenger. This paper describes a rapid, sensitive, and reproducible assay for sphingosine kinase activity using biotinylated sphingosine (biotinyl-Sph) as a substrate and capturing the phosphorylated product with streptavidin-coated membranes. We have shown that both human sphingosine kinase 1 and 2 (hSK1 and hSK2) can efficiently phosphorylate biotinyl-Sph, with K(m) values similar to those of sphingosine. The assay utilizing this substrate has high sensitivity for hSK1 and hSK2, with detection limits in the low-femtomole range for both purified recombinant enzymes. Importantly, we have also demonstrated the capacity of this assay to measure endogenous sphingosine kinase activity in crude cell extracts and to follow changes in this activity following sphingosine kinase activation. Together, these results demonstrate the potential utility of this assay in both cell-based analysis of sphingosine kinase signaling pathways and high-throughput screens for agents affecting sphingosine kinase activity in vitro.     

A sphingosine kinase activity assay using direct infusion electrospray ionization tandem mass spectrometry

D-erythro-Sphingosine is known to be phosphorylated by sphingosine kinase to yield sphingosine-1-phosphate. With the importance of sphingosine-1-phosphate in biological functions being made evident by recent research, a selective and convenient method of assay to measure sphingosine kinase activity is required. Here we developed a new sphingosine kinase assay using murine teratocarcinoma mutant F9-12 cells and electrospray ionization tandem mass spectrometry (ESI–MS/MS) with direct infusion. Sphingosine-1-phosphate in the crude extract of enzyme reaction mixture was selectively characterized and quantitated using precursor ion scanning for [PO₃]^- in the negative electrospray ionization mode. The method was successfully validated for an activator and an inhibitor of sphingosine kinase. Direct quantitation of S1P without the use of radioactive reagents, chemical derivatization, and extensive chromatographic separation enables simplified assay for sphingosine kinase activity at the cellular system level, and the use of a structural analog as an internal standard provides robustness to the assay.     

Simultaneous quantitative analysis of ceramide and sphingosine in mouse blood by naphthalene-2,3-dicarboxyaldehyde derivatization after hydrolysis with ceramidase

Ceramide and sphingosine are sphingolipids with important functional and structural roles in cells. In this paper we report a new enzyme-based method to simultaneously quantify the levels of ceramide and sphingosine in biological samples. This method utilizes purified human recombinant acid ceramidase to completely hydrolyze ceramide to sphingosine, followed by derivatization of the latter with naphthalene-2,3-dialdehyde (NDA) and quantification by reverse-phase high-performance liquid chromatography. The limits of detection for sphingosine-NDA and ceramidase-derived sphingosine-NDA were 9.6 and 12.3 fmol, respectively, and the limits of quantification were 34.2 and 45.7 fmol, respectively. The recovery of sphingosine and ceramide standards quantified by this assay were between 95.6 and 104.6%. The relative standard deviations for the intra- and interday sphingosine assay were 2.1 and 4.5%, respectively, and those for the ceramide assay were 3.3 and 4.1%, respectively. To validate this procedure, we quantified ceramide and sphingosine in mouse plasma, white blood cells, and hemoglobin, the first reported time that the amounts of these lipids have been documented in individual blood components. We also used this technique to evaluate the ability of a novel ceramide analog, AD2646, to inhibit the hydrolytic activity of acid ceramidase. The results demonstrate that this new procedure can provide sensitive, reproducible, and simultaneous ceramide and sphingosine quantification. The technique also may be used for determining the activity and inhibition of ceramidases and may be adapted for quantifying sphingomyelin and sphingosine-1-phosphate levels. In the future it could be an important tool for investigators studying the role of ceramide/sphingosine metabolism in signal transduction, cell growth and differentiation, and cancer pathogenesis and treatment.     

Cultured granule cells and astrocytes from cerebellum differ in metabolizing sphingosine

Sphingosine metabolism was studied in primary cultures of differentiated cerebellar granule cells and astrocytes. After a 2-h pulse with [C3-(3)H]sphingosine at different doses (0.1-200 nmol/mg of cell protein), both cell types efficiently incorporated the long chain base; the percentage of cellular [(3)H]sphingosine over total label incorporation was extremely low at sphingosine doses of <10 nmol/mg of cell protein and increased at higher doses. Most of the [(3)H]sphingosine taken up underwent metabolic processing by N-acylation, 1-phosphorylation, and degradation (assessed as (3)H(2)O released in the medium). The metabolic processing of exogenous sphingosine was extremely efficient in both cells, granule cells and astrocytes being able to metabolize, respectively, an amount of sphingosine up to 80- and 300-fold the cellular content of this long chain base in 2 h. At the different doses, the prevailing metabolic route of sphingosine was different. At lower doses and in a wide dose range, the major metabolic fate of sphingosine was N-acylation. With increasing doses, there was first increased sphingosine degradation and then increased levels of sphingosine-1-phosphate. The data demonstrate that, in neurons and astrocytes, the metabolic machinery devoted to sphingosine processing is different, astrocytes possessing an overall higher capacity to synthesize the bioactive compounds ceramide and sphingosine-1-phosphate.     

Globular adiponectin induces adhesion molecule expression through the sphingosine kinase pathway in vascular endothelial cells

The signaling pathways that couple adiponectin receptors to functional, particularly inflammatory, responses have remained elusive. We report here that globular adiponectin induces endothelial cell activation, as measured by the expression of adhesion proteins such as vascular adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), E-selectin and MCP-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with globular adiponectin resulted in NF-kappaB activation and increased mRNA levels of VCAM-1, ICAM-1, E-selectin and MCP-1. Sphingosine 1-phosphate (S1P), but not ceramide or sphingosine, was a potent stimulator of adhesion protein expression. As S1P is generated from sphingosine by SKase, we treated cells with siRNA for SKase to silence the effects of S1P in the endothelial cells. Treatment with SKase siRNA inhibited globular adiponectin-induced NF-kappaB activation and markedly decreased the globular adiponectin-induced mRNA levels of adhesion protein. Thus, we demonstrated that the SKase pathway, through the generation of S1P, is critically involved in mediating globular adiponectin-induced endothelial cell activation.     

An assay system for measuring the acute production of sphingosine 1-phosphate in intact monolayers

Sphingosine kinase (SK) is a signaling enzyme that phosphorylates sphingosine to produce sphingosine 1-phosphate. Sphingosine and sphingosine 1-phosphate (S1P) belong to a class of bioactive sphingolipid metabolites that are critical in a number of cellular processes, yet often have opposing biological functions. The intracellular localization of sphingosine kinase has been demonstrated in multiple studies to be a critical aspect of its signaling function. To date, assays of sphingosine kinase activity have been developed for measuring activity in lysates, where the effects of localization are lost. Here we outline a system in which the rate of production of S1P can be measured in intact cells using exogenously added radiolabeled ATP instead of tritiated sphingosine. The surprising ability of ATP to enter unpermeabilized monolayers is one aspect that makes this assay simple, efficient, and inexpensive, yet sensitive enough to measure endogenous enzyme activity. The assay is well behaved in terms of kinetics and substrate dependence. Overall, this assay is ideal for future studies to identify changes in S1P production in intact cells such as those that result from the differential intracellular targeting of sphingosine kinase.     

Sphingosine-1-phosphate, a metabolite of sphingosine, increases phosphatidic acid levels by phospholipase D activation.

Sphingosine and sphingosine-1-phosphate, metabolites of membrane sphingolipids, have recently been shown to stimulate release of calcium from internal sources and to increase proliferation of quiescent Swiss 3T3 fibroblasts (Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell Biol. 114, 155-167). The present study demonstrates that mitogenic concentrations of sphingosine induce early increases in sphingosine-1-phosphate levels which precede the increase in the potent mitogen, phosphatidic acid. Sphingosine-1-phosphate itself induces a more rapid increase in phosphatidic acid, thus suggesting that it may mediate the effects of sphingosine on phosphatidic acid accumulation. The concentration dependence for the formation of phosphatidic acid induced by sphingosine-1-phosphate correlates with its effect on DNA synthesis. Similar to sphingosine, sphingosine-1-phosphate also stimulates the activity of phospholipase D, although a significant effect is observed at a much lower concentration. However, in contrast to previous reports with sphingosine, sphingosine-1-phosphate does not inhibit the phosphatidic acid phosphohydrolase activity in cell homogenates. Thus, in addition to its effect on mobilization of calcium, sphingosine-1-phosphate can increase the level of phosphatidic acid, most likely via activation of phospholipase D. We suggest that sphingosine-1-phosphate mediates the effect of sphingosine on phosphatidic acid accumulation in Swiss 3T3 fibroblasts and may regulate cellular proliferation by affecting multiple transmembrane signaling pathways.     

Inhibition of sphingosine kinase in vitro and in platelets. Implications for signal transduction pathways.

Sphingosine kinase was partially purified and characterized from rat brain microsomes. A new assay, utilizing octyl-beta-D-glucopyranoside and sphingosine mixed micelles, was developed to quantitate formation of the sphingosine-1-phosphate product. The assay was proportional with respect to time and protein, displayed Michaelis-Menten kinetics, and was subject to surface dilution in regard to the sphingosine substrate. Investigations into substrate specificity showed that the enzyme is specific for the erythro-enantiomers of sphingosine and dihydrosphingosine. Neither of the threo-enantiomers were phosphorylated in this system, but both were found to be potent competitive inhibitors of sphingosine kinase activity. Human platelet sphingosine kinase activity displayed substrate and inhibitor specificities similar to the rat brain enzyme. A mixture of DL-threo-dihydrosphingosine competitively inhibited sphingosine kinase activity in a dose dependent manner in isolated platelets. DL-Threo-dihydrosphingosine caused a prolongation of the inhibition of thrombin-induced protein kinase C-dependent 40 (47)-kDa protein phosphorylation in platelets. D-, L-, or DL-Threo-dihydrosphingosine may be useful as a tool to investigate D-Erythrosphingosine metabolism and the function of sphingosine-1-phosphate in signal transduction processes.     

Sphingosine-1-phosphate, a novel lipid, involved in cellular proliferation

Sphingosine, a metabolite of membrane sphingolipids, regulates proliferation of quiescent Swiss 3T3 fibroblasts (Zhang, H., N. E. Buckley, K. Gibson. and S. Spiegel. 1990. J. Biol. Chem. 265:76-81). The present study provides new insights into the formation and function of a unique phospholipid, a metabolite of sphingosine, which was unequivocally identified as sphingosine-1-phosphate. The rapid increase in 32P-labeled sphingosine-1-phosphate levels induced by sphingosine was concentration dependent and correlated with its effect on DNA synthesis. Similar to the mitogenic effects of sphingosine, low concentrations of sphingosine-1-phosphate stimulated DNA synthesis and induced pronounced morphological alterations. Both sphingosine and sphingosine-1-phosphate stimulated DNA synthesis in cells made protein kinase C deficient by prolonged treatment with phorbol ester and sphingosine still elicited similar increases in sphingosine-1-phosphate levels in these cells. Although both sphingosine and sphingosine-1-phosphate acted synergistically with a wide variety of growth factors, there was no additive or synergistic effect in response to a combination of sphingosine and sphingosine-1-phosphate. Using a digital imaging system for measurement of calcium changes, we observed that both sphingosine and sphingosine-1-phosphate are potent calcium-mobilizing agonists in viable 3T3 fibroblasts. The rapid rise in cytosolic free calcium was independent of the presence of calcium in the external medium, indicating that the response is due to the mobilization of calcium from internal store. Our results suggest that sphingosine-1-phosphate may be a component of the intracellular second messenger system that is involved in calcium release and the regulation of cell growth induced by sphingosine.     

Sphingosine releases Ca2+ from intracellular stores via the ryanodine receptor in sea urchin egg homogenates

Various reports have demonstrated that the sphingolipids sphingosine and sphingosine-1-phosphate are able to induce Ca2+ release from intracellular stores in a similar way to second messengers. Here, we have used the sea urchin egg homogenate, a model system for the study of intracellular Ca2+ release mechanisms, to investigate the effect of these sphingolipids. While ceramide and sphingosine-1-phosphate did not display the ability to release Ca2+, sphingosine stimulated transient Ca2+ release from thapsigargin-sensitive intracellular stores. This release was inhibited by ryanodine receptor blockers (high concentrations of ryanodine, Mg2+, and procaine) but not by pre-treatment of homogenates with cADPR, 8-bromo-cADPR or blockers of other intracellular Ca2+ channels. However, sphingosine rendered the ryanodine receptor refractory to cADPR. We propose that, in the sea urchin egg, sphingosine is able to activate the ryanodine receptor via a mechanism distinct from that used by cADPR.     

Sphingosine 1-phosphate lyase enzyme assay using a BODIPY-labeled substrate

Sphingosine 1-phosphate lyase (SPL) is responsible for the irreversible catabolism of sphingosine 1-phosphate, which signals through five membrane receptors to mediate cell stress responses, angiogenesis, and lymphocyte trafficking. The standard assay for SPL activity utilizes a radioactive dihydrosphingosine 1-phosphate substrate and is expensive and cumbersome. In this study, we describe an SPL assay that employs an ω-labeled BODIPY-sphingosine 1-phosphate substrate, allowing fluorescent product detection by HPLC and incorporating advantages of the BODIPY fluorophore. The major aldehyde product is confirmed by reaction with 2,4-dinitrophenylhydrazine. The SPL-catalyzed reaction is linear over a 30 min time period and yields a K_m of 35 μM for BODIPY-sphingosine 1-phosphate.     

鞘磷脂代谢与脑缺血

鞘磷脂特别是鞘脂是髓鞘的主要成分,高度集中在中枢神经系统。在生理和病理生理条件下,具有生物活性的鞘磷脂及其代谢产物以及信号传导过程的重要性正在逐步被人们所认识。鞘脂代谢产物鞘氨醇及其前体物质神经酰胺与细胞生长停滞和凋亡有关,而1-磷酸鞘氨醇与增强细胞增殖、分化和细胞生存以及调节细胞的生理和病理过程有关,具有细胞外第一信使和细胞内第二信使的双重功能。这三者之间的相互转换、鞘脂代谢物的相对水平以及细胞的命运,受到鞘氨醇激酶的活性的强烈影响。鞘氨醇激酶可催化磷酸鞘氨醇产生1-磷酸鞘氨醇。1-磷酸鞘氨醇在中枢神经系统中与G蛋白偶联受体家族结合对中枢神经系统发挥作用。本文对鞘磷脂代谢过程中的鞘氨醇激酶、1-磷酸鞘氨醇及其受体与脑缺血之间的关系进行概述。     

The immune modulator FTY720 inhibits sphingosine-1-phosphate lyase activity

FTY720 is a novel immunomodulatory agent that inhibits lymphocyte trafficking and prevents allograft rejection. FTY720 is phosphorylated in vivo, and the phosphorylated drug acts as agonist for a family of G protein-coupled receptors that recognize sphingosine 1-phosphate. Evidence suggests that FTY720-phosphate-induced activation of S1P1 is responsible for its mechanism of action. FTY720 was rationally designed by modification of myriocin, a naturally occurring sphingoid base analog that causes immunosuppression by interrupting sphingolipid metabolism. In this study, we examined interactions between FTY720, FTY720-phosphate, and sphingosine-1-phosphate lyase, the enzyme responsible for irreversible sphingosine 1-phosphate degradation. FTY720-phosphate was stable in the presence of active sphingosine-1-phosphate lyase, demonstrating that the lyase does not contribute to FTY720 catabolism. Conversely, FTY720 inhibited sphingosine-1-phosphate lyase activity in vitro. Treatment of mice with FTY720 inhibited tissue sphingosine-1-phosphate lyase activity within 12 h, whereas lyase gene and protein expression were not significantly affected. Tissue sphingosine 1-phosphate levels remained stable or increased throughout treatment. These studies raise the possibility that disruption of sphingosine 1-phosphate metabolism may account for some effects of FTY720 on immune function and that sphingosine-1-phosphate lyase may be a potential target for immunomodulatory therapy.     

Diacylglyceride lipase activity in rod outer segments depends on the illumination state of the retina

We have demonstrated that the competition between phosphatidic acid (PA) and lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) for lipid phosphate phosphatases (LPP) generates different levels of diacylglycerol (DAG) depending on the illumination state of the retina. The aim of the present research was to determine the diacylglyceride lipase (DAGL) activity in purified rod outer segments (ROS) obtained from dark-adapted retinas (DROS) or light-adapted retinas (BLROS) as well as in ROS membrane preparations depleted of soluble and peripheral proteins. [2-(3)H]monoacylglycerol (MAG), the product of DAGL, was evaluated from [2-(3)H]DAG generated by LPP action on [2-(3)H]PA in the presence of either LPA, S1P or C1P. MAG production was inhibited by 55% in BLROS and by 25% when the enzymatic assay was carried out in ROS obtained from dark-adapted retinas and incubated under room light (LROS). The most important events occurred in DROS where co-incubation of [2-(3)H]PA with LPA, S1P or C1P diminished MAG production. A higher level of DAGL activity was observed in LROS than in BLROS, though this difference was not apparent in the presence of LPA, S1P or C1P. DAGL activity in depleted DROS was diminished with respect to that in entire DROS. LPA, S1P and C1P produced a similar decrease in MAG production in depleted DROS whereas only C1P significantly diminished MAG generation in depleted BLROS. Sphingosine and ceramide inhibited MAG production in entire DROS and stimulated its generation in BLROS. Sphingosine and ceramide stimulated MAG generation in both depleted DROS and BLROS. Under our experimental conditions the degree of MAG production depended on the illumination state of the retina. We therefore suggest that proteins related to phototransduction phenomena are involved in the effects observed in the presence of S1P/sphingosine or C1P/ceramide.     

Sphingosine kinase-dependent directional migration of leukocytes in response to phorbol ester

Syndecan-4 participates in focal adhesion by non-G protein-dependent activation of protein kinase C. Ligation of syndecan-4 with antithrombin elicits pertussis toxin-sensitive chemotaxis of leukocytes. As activation of protein kinase C stimulates release of sphingosine-1-phosphate, a chemoattracting G protein-coupled receptor agonist, we studied directional migration of leukocytes in response to phorbol myristate acetate (PMA), a direct activator of protein kinase C. Human peripheral blood neutrophils, monocytes, and lymphocytes were purified and tested for chemotactic migration in micropore filter assays in response to PMA. Dose-dependent stimulation of migration was seen only when leukocytes were exposed to concentration gradients of PMA; in the absence of such a gradient, inhibition of random migration was induced. Dimethylsphingosine inhibited PMA-induced leukocyte chemotaxis, indicating that activation of sphingosine kinase for enhanced production of sphingosine-1-phosphate mediates the chemotactic response to PMA. Pertussis toxin abrogated the chemotactic response to PMA, suggesting involvement of G protein-coupled sphingosine-1-phosphate receptor. Dimethylsphingosine also inhibited leukocyte chemotaxis toward antithrombin, indicating that similar mechanisms may be involved upon syndecan-4 ligation. Data show that protein kinase C-dependent activation of sphingosine kinase may play a central role in leukocyte chemotaxis toward non-G protein-coupled receptor agonists.     

Sphingosine-phosphate lyase enhances stress-induced ceramide generation and apoptosis

Sphingosine-1-phosphate lyase is a widely expressed enzyme that catalyzes the essentially irreversible cleavage of the signaling molecule sphingosine 1-phosphate. To investigate whether sphingosine-1-phosphate lyase influences mammalian cell fate decisions, a recombinant human sphingosine-1-phosphate lyase fused to green fluorescent protein was expressed in HEK293 cells. The recombinant enzyme was active, localized to the endoplasmic reticulum, and reduced baseline sphingosine and sphingosine 1-phosphate levels. Stable overexpression led to diminished viability under stress, which was attributed to an increase in apoptosis and was reversible in a dose-dependent manner by exogenous sphingosine 1-phosphate. In contrast to sphingosine 1-phosphate, the products of the lyase reaction had no effect on apoptosis. Lyase enzymatic activity was required to potentiate apoptosis, because cells expressing a catalytically inactive enzyme behaved like controls. Stress increased the amounts of long- and very long-chain ceramides in HEK293 cells, and this was enhanced in cells overexpressing wild type but not catalytically inactive lyase. The ceramide increases appeared to be required for apoptosis, because inhibition of ceramide synthase with fumonisin B1 decreased apoptosis in lyase-overexpressing cells. Thus, sphingosine-1-phosphate lyase overexpression in HEK293 cells decreases sphingosine and sphingosine 1-phosphate amounts but elevates stress-induced ceramide generation and apoptosis. This identifies sphingosine-1-phosphate lyase as a dual modulator of sphingosine 1-phosphate and ceramide metabolism as well as a regulator of cell fate decisions and, hence, a potential target for diseases with an imbalance in these biomodulators, such as cancer.