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.     

A rapid radioassay for sphingosine kinase

A solvent-extraction-based radioassay for measuring sphingosine kinase (SKase) activity has been developed. The assay utilizes [3H]sphingosine substrate and differentially extracts the [3H]sphingosine-1-phosphate product. The extracted radioactivity is demonstrated to be primarily [3H]sphingosine-1-phosphate with less than 1% contamination by [3H]sphingosine. When assaying SKase activity in the soluble cell fraction, the extraction efficiency of the labeled sphingosine-1-phosphate product is a reproducible 78%, which allows for a simple back calculation to correct for the 22% extraction loss. With minor modification, the assay is also a reproducible procedure for determining SKase activity in subcellular membrane fractions. The assay is far more rapid than thin-layer chromatography and high-performance liquid chromatography methods, which makes it possible to do a large number of assays in a short period of time. The utility of the assay is demonstrated by using it to conduct a complete bisubstrate kinetic analysis of rat heart SKase.     

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.     

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。     

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.     

Role of sphingosine kinase localization in sphingolipid signaling

The sphingosine kinases, SK1 and SK2, produce the potent signaling lipid sphingosine-1-phosphate (S1P). These enzymes have garnered increasing interest for their roles in tumorigenesis, inflammation, vascular diseases, and immunity, as well as other functions. The sphingosine kinases are considered signaling enzymes by producing S1P, and their activity is acutely regulated by a variety of agonists. However, these enzymes are also key players in the control of sphingolipid metabolism. A variety of sphingolipids, such as sphingosine and the ceramides, are potent signaling molecules in their own right. The role of sphingosine kinases in regulating sphingolipid metabolism is potentially a critical aspect of their signaling function. A central aspect of signaling lipids is that their hydrophobic nature constrains them to membranes. Most enzymes of sphingolipid metabolism, including the enzymes that degrade S1P, are membrane enzymes. Therefore the localization of the sphingosine kinases and S1P is likely to be important in S1P signaling. Sphingosine kinase localization affects sphingolipid signaling in several ways. Translocation of SK1 to the plasma membrane promotes extracellular secretion of S1P. SK1 and SK2 localization to specific sites appears to direct S1P to intracellular protein effectors. SK localization also determines the access of these enzymes to their substrates. This may be an important mechanism for the regulation of ceramide biosynthesis by diverting dihydrosphingosine, a precursor in the ceramide biosynthetic pathway, from the de novo production of ceramide.     

A lipid binding domain in sphingosine kinase 2

The lipid second messenger sphingosine 1-phosphate (S1P) is a critical mediator of cellular proliferation and survival signals, and is essential for vasculogenesis and neurogenesis. S1P formation is catalysed by sphingosine kinases 1 and 2 (Sphk1 and Sphk2). We have found that the endogenous glycolipid sulfatide (3-O-sulfogalactosylceramide) binds to and inhibits the activity of Sphk2 and the closely related ceramide kinase (Cerk), but not Sphk1. Using sulfatide as a probe, we mapped the lipid binding domain to the N-terminus of Sphk2 (residues 1-175), a region of sequence that is absent in Sphk1, but aligns with a pleckstrin homology domain in Cerk. Accordingly, Sphk2 bound to phosphatidylinositol monophosphates but not to abundant cellular phospholipids. Deleting the N-terminal domain reduced Sphk2 membrane localisation in cells. We have therefore identified a lipid binding domain in Sphk2 that is important for the enzyme’s sub-cellular localisation.     

Roles of sphingosine 1-phosphate on tumorigenesis

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid with a variety of biological activities.It is generated from the conversion of ceramide to sphingosine by ceramidase and the subsequent conversion of sphingosine to S1P,which is catalyzed by sphingosine kinases.Through increasing its intracellular levels by sphingolipid metabolism and binding to its cell surface receptors,S1P regulates several physiological and pathological processes,including cell proliferation,migration,angiogenesis and autophagy.These processes are responsible for tumor growth,metastasis and invasion and promote tumor survival.Since ceramide and S1P have distinct functions in regulating in cell fate decision,the balance between the ceramide/sphingosine/S1P rheostat becomes a potent therapeutic target for cancer cells.Herein,we summarize our current understanding of S1P signaling on tumorigenesis and its potential as a target for cancer therapy.     

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.     

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.     

Sphingosine, W-7, and Trifluoperazine Inhibit the Elevation in Cytosolic Calcium Induced by High K+Depolarization in Synaptosomes

Abstract: A possible role for protein kinases in the regulation of free cytosolic Ca²⁺ levels in nerve endings was investigated by testing the effect of several kinase inhibitors on the increase in cytosolic Ca²⁺ (monitored with the Ca²⁺-sensitive dye fura-2) induced by depolarization with 15 or 30 mM K⁺. The ability of various drugs to inhibit the cytosolic Ca²⁺ response appeared to correlate with their reported mechanism of action in inhibiting protein kinases. W-7 and trifluoperazine, drugs reported to inhibit calmodulin-dependent events, were effective inhibitors of the increase in cytosolic Ca²⁺ induced by high K⁺ depolarization, as was sphingosine, a drug that inhibits protein kinase C by binding to the regulatory site, but which also inhibits calcium/calmodulin kinase. On the other hand, drugs that inhibit protein kinases by binding to the catalytic site, such as H-7 (1 m/W ), staurosporine (1μ M ), and K252_a(1μ M ), were ineffective. Activation of protein kinase C, which is blocked by each of these drugs, does not appear to be essential to the maintenance of elevated cytosolic Ca²⁺ in depolarized synaptosomes. All of the drugs, including sphingosine, that functionally inhibit the depolarization-induced elevation in cytosolic Ca²⁺ have in common the ability to bind to calmodulin. Because the drugs that inhibit protein kinases by competing with ATP binding at the active catalytic site did not block the response in this system, we suggest that a calmodulin or a calmodulin-like binding site participates in the regulation of Ca²⁺ increases after depolarization.     

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.     

Development of SKI-349, a dual-targeted inhibitor of sphingosine kinase and microtubule polymerization

Our sphingosine kinase inhibitor (SKI) optimization studies originated with the optimization of the SKI-I chemotype by replacement of the substituted benzyl rings with substituted phenyl rings giving rise to the discovery of SKI-178. We have recently reported that SKI-178 is a dual-targeted inhibitor of both sphingosine kinase isoforms (SphK1/2) and a microtubule disrupting agent (MDA). In mechanism-of-action studies, we have shown that these two separate actions synergize to induce cancer cell death in acute myeloid leukemia (AML) cell and animal models. Owning to the effectiveness of SKI-178, we sought to further refine the chemotype while maintaining “on-target” SKI and MDA activities. Herein, we modified the “linker region” between the substituted phenyl rings of SKI-178 through a structure guided approach. These studies have yielded the discovery of an SKI-178 congener, SKI-349, with log-fold enhancements in both SphK inhibition and cytotoxic potency. Importantly, SKI-349 also demonstrates log-fold improvements in therapeutic efficacy in a retro-viral transduction model of MLL-AF9 AML as compared to previous studies with SKI-178. Together, our results strengthen the hypothesis that simultaneous targeting of the sphingosine kinases (SphK1/2) and the induction of mitotic spindle assembly checkpoint arrest, via microtubule disruption, might be an effective therapeutic strategy for hematological malignancies including AML.     

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.     

Sphingosine kinases and their metabolites modulate endolysosomal trafficking in photoreceptors

Internalized membrane proteins are either transported to late endosomes and lysosomes for degradation or recycled to the plasma membrane. Although proteins involved in trafficking and sorting have been well studied, far less is known about the lipid molecules that regulate the intracellular trafficking of membrane proteins. We studied the function of sphingosine kinases and their metabolites in endosomal trafficking using Drosophila melanogaster photoreceptors as a model system. Gain- and loss-of-function analyses show that sphingosine kinases affect trafficking of the G protein-coupled receptor Rhodopsin and the light-sensitive transient receptor potential (TRP) channel by modulating the levels of dihydrosphingosine 1 phosphate (DHS1P) and sphingosine 1 phosphate (S1P). An increase in DHS1P levels relative to S1P leads to the enhanced lysosomal degradation of Rhodopsin and TRP and retinal degeneration in wild-type photoreceptors. Our results suggest that sphingosine kinases and their metabolites modulate photoreceptor homeostasis by influencing endolysosomal trafficking of Rhodopsin and TRP.     

Sphingosine can pre- and post-condition heart and utilizes a different mechanism from sphingosine 1-phosphate

Consistent with previous reports, sphingosine at a high concentration (5 microM) was cardiotoxic as evidenced by increased infarct size in response to ischemia/reperfusion in an ex vivo rat heart. Sphingosine 1-phosphate (S1P) at 5 microM was cardioprotective. However, at a physiologic concentration (0.4 microM) sphingosine as well as S1P was effective in protecting the heart from ischemia/reperfusion injury both when perfused prior to 40 min of ischemia (preconditioning) or when added to reperfusion media following ischemia (postconditioning). Protection by sphingosine and S1P was evidenced with both pre- and post-conditioning by a >75% recovery of left ventricular developed pressure during reperfusion and a decrease in infarct size from 45% of the risk area to less than 8%. When VPC23019, an S1P(1and3)G-protein coupled receptor antagonist, was added to the preconditioning or postconditioning medium along with S1P, it completely blocked S1P-induced protection. However, VPC 23019 did not affect the ability of 0.4 microM sphingosine to either precondition or postcondition hearts. Studies of preconditioning revealed that inhibition of protein kinase C with GF109203X blocked preconditioning by S1P. However, GF109203X did not affect preconditioning by 0.4 microM sphingosine. Likewise, cotreatment with the PI3 kinase inhibitor wortmanin blocked preconditioning by S1P but not by sphingosine. By contrast, inhibition of protein kinase G with KT5823 had no effect on S1P preconditioning but completely eliminated preconditioning by sphingosine. Also, the protein kinase A inhibitory peptide 14-22 amide blocked preconditioning by sphingosine but not S1P. These data reveal for the first time that sphingosine is not toxic at physiologic concentrations but rather is a potent cardioprotectant that utilizes a completely different mechanism than S1P; one that is independent of G-protein coupled receptors and utilizes cyclic nucleotide-dependent pathways.     

A high-performance liquid chromatographic method to measure sphingosine 1-phosphate and related compounds from sphingosine kinase assays and other biological samples

Sphingosine 1-phosphate is an intermediate of sphingosine catabolism as well as a potent signaling compound. Conditions were established for the extraction and analysis of sphingosine 1-phosphate and other sphingoid base 1-phosphates from in vitro sphingosine kinase assays and other biological samples. The sphingoid base 1-phosphates were extracted in high yield (85%) using small C-18 reverse-phase columns (LiChroprep RP-18). After the extracts were treated with 0.1 N KOH to remove glycerolipids, the sphingoid base 1-phosphates were converted to fluorescent o-phthalaldehyde derivatives that were separated by HPLC using C-18 columns with a mobile phase of methanol:10 mM potassium phosphate (pH 7.2):1 M tetrabutylammonium dihydrogen phosphate (in water) (83:16:1, v/v/v). The o-phthalaldehyde derivative of sphingosine 1-phosphate was reasonably stable (t(1/2) > or = 18 h) when EDTA was present and could be detected in picomole amounts. The HPLC retention time of the sphingoid base 1-phosphates could be shifted by adjusting the mobile phase to pH 5.5, which is useful in separating overlapping compounds (such as sphingosine 1-phosphate and 4-D-hydroxysphinganine) and in confirming the identity of sphingoid base 1-phosphates in biological samples. The extraction procedure and HPLC method facilitated assays of sphingosine kinase with different sphingoid bases as substrates and/or inhibitors and enabled the quantitation of sphingoid base 1-phosphates in human plasma, serum, and platelets as well as in strains of Saccharomyces cerevisae with mutations in sphingolipid metabolism.     

Prosaposin: a new player in cell death prevention of U937 monocytic cells

We report that prosaposin binds to U937 and is active as a protective factor on tumor necrosis factor alpha (TNFalpha)-induced cell death. The prosaposin-derived saposin C binds to U937 cells in a concentration-dependent manner, suggesting that prosaposin behaves similarly. Prosaposin binding induces U937 cell death prevention, reducing both necrosis and apoptosis. This effect was inhibited by mitogen-activated protein ERK kinase (MEK) and sphingosine kinase (SK) inhibitors, indicating that prosaposin prevents cell apoptosis by activation of extracellular signal-regulated kinases (ERKs) and sphingosine kinase. Prosaposin led to rapid ERK phosphorylation in U937 cells as detected by anti-phospho-p44/42 mitogen-activated protein (MAP) kinase and anti-phosphotyrosine reactivity on ERK immunoprecipitates. It was partially prevented by apo B-100 and pertussis toxin (PT), suggesting that both lipoprotein receptor-related protein (LRP) receptor and Go-coupled receptor may play a role in the prosaposin-triggered pathway. Moreover, sphingosine kinase activity was increased by prosaposin treatment as demonstrated by the enhanced intracellular formation of sphingosine-1-phosphate (S-1-P). The observation that the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin prevented the prosaposin effect on cell apoptosis suggests that sphingosine kinase exerts its anti-apoptotic activity by the PI3K-Akt pathway. Thus, cell apoptosis prevention by prosaposin occurs through ERK phosphorylation and sphingosine kinase. The biological effect triggered by prosaposin might be extended to primary cells because it triggers Erk phosphorylation in peripheral blood mononuclear cells (PBMCs). This is the first evidence of a biological effect consequent to a signal transduction pathway triggered by prosaposin in cells of non-neurological origin.     

Quantitative analysis of free sphingoid bases in the brain and spinal cord tissues by high-performance liquid chromatography with a fluorescence detection

The o-phthaldialdehyde precolumn derivatives of psychosine, sphinganine and sphingosine extracted from brain and spinal cord tissues were determined by high-performance liquid chromatography–fluorescence detection. This method was developed with the purpose of detecting an endogenous amount of psychosine, sphingosine and sphinganine using small aliquots of brain tissues and spinal cord in rats. These sphingolipid bases were extracted in various ratios of chloroform–methanol and several pH values. Recovery of the method is about 81% in 12 ng/tube (final volume, 320 μl), 90–95% in 45 ng/tube of sphingosine and sphinganine within 2–12% relative standard deviation. Detection limits of these sphingoid bases were about 0.05 pmol/mg brain tissue. In the forebrain, brainstem and spinal cord of rats at three different ages of postnatal days (PND) 1, PND 13 and 6 months old, the endogenous concentrations of psychosine, sphingosine and sphinganine were determined. From these results, this method is suitable for the determination of sphingoid bases in small aliquot of brain and spinal cord tissues.     

Inhibition of chemotactic motility and trans-endothelial migration of human neutrophils by sphingosine 1-phosphate

In previous studies, we reported that sphingosine 1-phosphate (Sph-1-P) inhibits the chemotactic motility of some cancer cell lines such as mouse melanoma cells, as well as human smooth muscle cells, at a very low concentration, as demonstrated by a transwell migration assay method (Proc. Natl. Acad. Sci. USA 89, 9698, 1992; J. Cell Biol. 130, 193, 1995). In this study, we investigated the effect of Sph-1-P on the chemotactic motility and invasiveness of human neutrophils, utilizing three different assay systems: (a) a transwell migration assay where IL-8 or fLMP was added as a chemotactic factor, (b) a phagokinetic assay with gold colloids, and (c) a trans-endothelial migration assay with human umbilical vein endothelial cells (HUVECs) plated on collagen layers. We found that among various sphingosine derivatives, Sph-1-P specifically inhibited the IL-8- or fLMP-induced chemotactic migration of neutrophils at concentrations below 1 μM. Phagokinetic activity of neutrophils was also suppressed by Sph-1-P, but more moderately than by the PKC inhibitory sphingosine analog, trimethylsphingosine. Finally, Sph-1-P inhibited trans-endothelial migration and invasiveness of neutrophils into HUVEC-covered collagen layers, whereas no effect on their adhesion to HUVECs was observed. These observations strongly suggest that Sph-1-P can act as a specific and effective motility regulator of human neutrophils, raising the possibility of future applications of Sph-1-P, or its analogs, as anti-inflammatory agents regulating invasive migration of neutrophils through endothelial layers at injured vascular sites.