Wunen, a Drosophila lipid phosphate phosphatase, is required for septate junction-mediated barrier function

Lipid phosphate phosphatases (LPPs) are integral membrane enzymes that regulate the levels of bioactive lipids such as sphingosine 1-phosphate and lysophosphatidic acid. The Drosophila LPPs Wunen (Wun) and Wunen-2 (Wun2) have a well-established role in regulating the survival and migration of germ cells. We now show that wun has an essential tissue-autonomous role in development of the trachea: the catalytic activity of Wun is required to maintain septate junction (SJ) paracellular barrier function, loss of which causes failure to accumulate crucial luminal components, suggesting a role for phospholipids in SJ function. We find that the integrity of the blood-brain barrier is also lost in wun mutants, indicating that loss of SJ function is not restricted to the tracheal system. Furthermore, by comparing the rescue ability of different LPP homologs we show that wun function in the trachea is distinct from its role in germ cell migration.     

Compartmentalizing the embryo

Lipid phosphate phosphatases (LPPs) are a class of enzymes that can dephosphorylate a number of lysophopholipids in vitro. Analysis of knockouts of LPP family members has demonstrated striking but diverse developmental roles for these enzymes. LPP3 is required for mouse vascular development while the Drosophila LPPs Wunen (Wun) and Wunen2 (Wun2) are required during embryogenesis for germ cell migration and survival. In a recent publication we examined if these fly LPPs have further developmental roles and found that Wun is required for proper tracheal formation. In particular we highlight a role for Wun in septate junction mediated barrier function in the tracheal system. In this paper we discuss further the possible mechanisms by which LPPs may influence barrier activity.     

FTY720-phosphate is dephosphorylated by lipid phosphate phosphatase 3

FTY720 is a novel immunomodulatory drug efficacious in the treatment of multiple sclerosis. The drug is converted in vivo to the monophosphate, FTY720-P, by sphingosine kinase 2. This conversion is incomplete, suggesting opposing actions of kinase and phosphatase activities. To address which of the known lipid phosphatases might dephosphorylate FTY720-P, we overexpressed the broad specificity lipid phosphatases LPP1-3, and the specific S1P phosphatases (SPP1 and 2) in HEK293 cells, and performed in vitro assays using lysates of transfected cells. Among LPPs, only LPP3 was able to dephosphorylate FTY720-P; among SPPs, only SPP1 showed activity against FTY720-P. On intact cells, LPP3 acted as an ecto-phosphatase or FTY720-P, thus representing the major phosphatase involved in the equilibrium between FTY720 and FTY720-P observed in vivo.     

Germ cell-autonomous Wunen2 is required for germline development in Drosophila embryos

In many animals, primordial germ cells (PGCs) migrate through the embryo towards the future gonad, a process guided by attractive and repulsive cues provided from surrounding somatic cells. In Drosophila, the two related lipid phosphate phosphatases (LPPs), Wunen (Wun) and Wun2, are thought to degrade extracellular substrates and to act redundantly in somatic cells to provide a repulsive environment to steer the migration of PGCs, or pole cells. Wun and Wun2 also affect the viability of pole cells, because overexpression of either one in somatic cells causes pole cell death. However, the means by which they regulate pole cell migration and survival remains elusive. We report that Wun2 has a maternal function required for the survival of pole cells during their migration to the gonad. Maternal wun2 RNA was found to be concentrated in pole cells and pole cell-specific expression of wun2 rescued the pole cell death phenotype of the maternal wun2 mutant, suggesting that wun2 activity in pole cells is required for their survival. Furthermore, we obtained genetic evidence that pole cell survival requires a proper balance of LPP activity in pole cells and somatic cells. We propose that Wun2 in pole cells competes with somatic Wun and Wun2 for a common lipid phosphate substrate, which is required by pole cells to produce their survival signal. In somatic cells, Wun and Wun2 may provide a repulsive environment for pole cell migration by depleting this extracellular substrate.     

Structural organization of mammalian lipid phosphate phosphatases: implications for signal transduction.

This article describes the regulation of cell signaling by lipid phosphate phosphatases (LPPs) that control the conversion of bioactive lipid phosphates to their dephosphorylated counterparts. A structural model of the LPPs, that were previously called Type 2 phosphatidate phosphatases, is described. LPPs are characterized by having no Mg(2+) requirement and their insensitivity to inhibition by N-ethylmaleimide. The LPPs have six putative transmembrane domains and three highly conserved domains that define a phosphatase superfamily. The conserved domains are juxtaposed to the proposed membrane spanning domains such that they probably form the active sites of the phosphatases. It is predicted that the active sites of the LPPs are exposed at the cell surface or on the luminal surface of intracellular organelles, such as Golgi or the endoplasmic reticulum, depending where various LPPs are expressed. LPPs could attenuate cell activation by dephosphorylating bioactive lipid phosphate esters such as phosphatidate, lysophosphatidate, sphingosine 1-phosphate and ceramide 1-phosphate. In so doing, the LPPs could generate alternative signals from diacylglycerol, sphingosine and ceramide. The LPPs might help to modulate cell signaling by the phospholipase D pathway. For example, phosphatidate generated within the cell by phospholipase D could be converted by an LPP to diacylglycerol. This should change the relative balance of signaling by these two lipids. Another possible function of the LPPs relates to the secretion of lysophosphatidate and sphingosine 1-phosphate by activated platelets and other cells. These exogenous lipids activate phospholipid growth factor receptors on the surface of cells. LPP activities could attenuate cell activation by lysophosphatidate and sphingosine 1-phosphate through their respective receptors.     

Structural organization of mammalian lipid phosphate phosphatases: implications for signal transduction

This article describes the regulation of cell signaling by lipid phosphate phosphatases (LPPs) that control the conversion of bioactive lipid phosphates to their dephosphorylated counterparts. A structural model of the LPPs, that were previously called Type 2 phosphatidate phosphatases, is described. LPPs are characterized by having no Mg²⁺ requirement and their insensitivity to inhibition by N-ethylmaleimide. The LPPs have six putative transmembrane domains and three highly conserved domains that define a phosphatase superfamily. The conserved domains are juxtaposed to the proposed membrane spanning domains such that they probably form the active sites of the phosphatases. It is predicted that the active sites of the LPPs are exposed at the cell surface or on the luminal surface of intracellular organelles, such as Golgi or the endoplasmic reticulum, depending where various LPPs are expressed. LPPs could attenuate cell activation by dephosphorylating bioactive lipid phosphate esters such as phosphatidate, lysophosphatidate, sphingosine 1-phosphate and ceramide 1-phosphate. In so doing, the LPPs could generate alternative signals from diacylglycerol, sphingosine and ceramide. The LPPs might help to modulate cell signaling by the phospholipase D pathway. For example, phosphatidate generated within the cell by phospholipase D could be converted by an LPP to diacylglycerol. This should change the relative balance of signaling by these two lipids. Another possible function of the LPPs relates to the secretion of lysophosphatidate and sphingosine 1-phosphate by activated platelets and other cells. These exogenous lipids activate phospholipid growth factor receptors on the surface of cells. LPP activities could attenuate cell activation by lysophosphatidate and sphingosine 1-phosphate through their respective receptors.     

Fly and mammalian lipid phosphate phosphatase isoforms differ in activity both in vitro and in vivo

Wunen (Wun), a homologue of a lipid phosphate phosphatase (LPP), has a crucial function in the migration and survival of primordial germ cells (PGCs) during Drosophila embryogenesis. Past work has indicated that the LPP isoforms may show functional redundancy in certain systems, and that they have broad-range lipid phosphatase activities in vitro, with little apparent specificity between them. We show here that there are marked differences in biochemical activity between fly Wun and mammalian LPPs, with Wun having a narrower activity range than has been reported for the mammalian LPPs. Furthermore, although it is active on a range of substrates in vitro, mouse Lpp1 has no activity on an endogenous Drosophila germ-cell-specific factor in vivo. Conversely, human LPP3 is active, resulting in aberrant migration and PGC death. These results show an absolute difference in bioactivity among LPP isoforms for the first time in a model organism and may point towards an underlying signalling system that is conserved between flies and humans.     

Roles for lipid phosphate phosphatases in regulation of cellular signaling

Lipid phosphate phosphatases (LPPs) are a family of integral membrane glycoproteins that catalyze the dephosphorylation of a number of bioactive lipid mediators including lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P) and phosphatidic acid (PA). These mediators exert complex effects on cell function through both actions at cell surface receptors and on intracellular targets. The LPP-catalyzed dephosphorylation of these substrates can both terminate their signaling actions and itself generate further molecules with biological activity. Recent advances have revealed that a family of structurally related genes is responsible for LPP activities in species from yeast to mammals. These genes exhibit distinct but overlapping expression patterns and their products appear to be heterogeneous with respect to their posttranslational modification and subcellular localizations. Here we review the structure and catalytic properties of the LPPs and consider recent developments in understanding their cellular biology and functions.     

Lipid phosphate phosphatase (LPP3) and vascular development

Lipid phosphate phosphatases (LPP) are integral membrane proteins with broad substrate specificity that dephosphorylate lipid substrates including phosphatidic acid, lysophosphatidic acid, ceramide 1-phosphate, sphingosine 1-phosphate, and diacylglycerol pyrophosphate. Although the three mammalian enzymes (LPP1-3) demonstrate overlapping catalytic activities and substrate preferences in vitro, the phenotypes of mice with targeted inactivation of the Ppap2 genes encoding the LPP enzymes reveal nonredundant functions. A specific role for LPP3 in vascular development has emerged from studies of mice lacking Ppap2b. A meta-analysis of multiple, large genome-wide association studies identified a single nucleotide polymorphism in PPAP2B as a novel predictor of coronary artery disease. In this review, we will discuss the evidence that links LPP3 to vascular development and disease and evaluate potential molecular mechanisms. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Identification and characterization of a novel human sphingosine-1-phosphate phosphohydrolase,hSPP2

Sphingosine 1-phosphate (S1P) is a bioactive lipid molecule that acts as both an extracellular signaling mediator and an intracellular second messenger. S1P is synthesized from sphingosine by sphingosine kinase and is degraded either by S1P lyase or by S1P phosphohydrolase. Recently, mammalian S1P phosphohydrolase (SPP1) was identified and shown to constitute a novel lipid phosphohydrolase family, the SPP family. In this study we have identified a second human S1P phosphohydrolase, SPP2, based on sequence homology to human SPP1. SPP2 exhibited high phosphohydrolase activity against S1P and dihydrosphingosine 1-phosphate. The dihydrosphingosine-1-phosphate phosphohydrolase activity was efficiently inhibited by excess S1P but not by lysophosphatidic acid, phosphatidic acid, or glycerol 3-phosphate, indicating that SPP2 is highly specific to sphingoid base 1-phosphates. Immunofluorescence microscopic analysis demonstrated that SPP2 is localized to the endoplasmic reticulum. Although the enzymatic properties and localization of SPP2 were similar to those of SPP1, the tissue-specific expression pattern of SPP2 was different from that of SPP1. Thus, SPP2 is another member of the SPP family that may play a role in attenuating intracellular S1P signaling.     

Sphingosine 1-phosphate phosphatase 2 is induced during inflammatory responses

Sphingosine 1-phosphate (S1P) levels in cells and, consequently, its bioactivity as a signalling molecule are controlled by the action of enzymes responsible for its synthesis and degradation. In the present report, we examined alterations in expression patterns of enzymes involved in S1P-metabolism (sphingosine kinases including their splice variants, sphingosine 1-phosphate phosphatases, and sphingosine 1-phosphate lyase) under certain inflammatory conditions. We found that sphingosine kinase type 1 (SPHK1) mRNA could be triggered in a cell type-specific manner; individual SPHK1 splice variants were induced with similar kinetics. Remarkably, expression and activity of S1P phosphatase 2 (SPP2) was found to be highly upregulated by inflammatory stimuli in a variety of cells (e.g., neutrophils, endothelial cells). Bandshift analysis using oligonucleotides spanning predicted NFkappaB sites within the SPP2 promoter and silencing of NFkappaB/RelA via RelA-directed siRNA demonstrated that SPP2 is an NFkappaB-dependent gene. Silencing of SPP2 expression in endothelial cells, in turn, led to a marked reduction of TNF-alpha-induced IL-1beta mRNA and protein and to a partial reduction of induced IL-8, suggesting a pro-inflammatory role of SPP2. Notably, up-regulation of SPP2 was detected in samples of lesional skin of patients with psoriasis, an inflammatory skin disease. This study provides detailed insights into the regulation of SPP2 gene expression and suggests that SPP2 might be a novel player in pro-inflammatory signalling.     

Lipid phosphate phosphatases and their roles in mammalian physiology and pathology

Lipid phosphate phosphatases (LPPs) are a group of enzymes that belong to a phosphatase/phosphotransferase family. Mammalian LPPs consist of three isoforms: LPP1, LPP2, and LPP3. They share highly conserved catalytic domains and catalyze the dephosphorylation of a variety of lipid phosphates, including phosphatidate, lysophosphatidate (LPA), sphingosine 1-phosphate (S1P), ceramide 1-phosphate, and diacylglycerol pyrophosphate. LPPs are integral membrane proteins, which are localized on plasma membranes with the active site on the outer leaflet. This enables the LPPs to degrade extracellular LPA and S1P, thereby attenuating their effects on the activation of surface receptors. LPP3 also exhibits noncatalytic effects at the cell surface. LPP expression on internal membranes, such as endoplasmic reticulum and Golgi, facilitates the metabolism of internal lipid phosphates, presumably on the luminal surface of these organelles. This action probably explains the signaling effects of the LPPs, which occur downstream of receptor activation. The three isoforms of LPPs show distinct and nonredundant effects in several physiological and pathological processes including embryo development, vascular function, and tumor progression. This review is intended to present an up-to-date understanding of the physiological and pathological consequences of changing the activities of the different LPPs, especially in relation to cell signaling by LPA and S1P.     

Sphingosine-1-phosphate phosphatases

Sphingosine-1-phosphate is a potent proliferative, survival, and morphogenetic factor, acting as an extracellular ligand for the EDG family of G-protein-coupled receptors and possibly intracellularly through as yet, unidentified targets. It is produced within most, if not all cells by phosphorylation of sphingosine, and is an abundant serum lipid that is released from activated platelets. Sphingosine and sphingosine-1-phosphate are in dynamic equilibrium with each other due to the activities of sphingosine kinase and sphingosine-1-phosphate phosphatase (SPPase). Several SPPase genes have now been cloned, first from yeast and more recently from mammalian cells. By sequence homology, these enzymes can be classified as a subset of membrane bound, Type 2 lipid phosphohydrolases that contain conserved residues within three domains predicted to be at the active site of the enzyme. Outside of the consensus motif, there is very little homology between SPPases and the other type 2 lipid phosphohydrolases in the LPP/PAP family. Type 2 phosphatase activity is Mg+-independent and insensitive to N-ethylmaleimide, and substrate specificity is broad for LPP enzymes, whereas SPPases are highly selective for sphingolipid substrates. SPPase activity in yeast and mammalian cells regulates intracellular sphingosine-1-phosphate levels, and also alters the levels of sphingosine and ceramide, two other signaling molecules that often oppose the actions of sphingosine-1-phosphate. Thus, loss of SPPase in yeast results in high sphingosine-1-phosphate levels and cells are more resistant to stress, and in mammalian cells, overexpression of SPPase elevates ceramide levels and provokes apoptosis.     

Sphingosine-1-Phosphate Phosphatases

Sphingosine-1-phosphate is a potent proliferative, survival, and morphogenetic factor, acting as an extracellular ligand for the EDG family of G-protein-coupled receptors and possibly intracellularly through as yet, unidentified targets. It is produced within most, if not all cells by phosphorylation of sphingosine, and is an abundant serum lipid that is released from activated platelets. Sphingosine and sphingosine-1-phosphate are in dynamic equilibrium with each other due to the activities of sphingosine kinase and sphingosine-1-phosphate phosphatase (SPPase). Several SPPase genes have now been cloned, first from yeast and more recently from mammalian cells. By sequence homology, these enzymes can be classified as a subset of membrane bound, Type 2 lipid phosphohydrolases that contain conserved residues within three domains predicted to be at the active site of the enzyme. Outside of the consensus motif, there is very little homology between SPPases and the other type 2 lipid phosphohydrolases in the LPP/PAP family. Type 2 phosphatase activity is Mg(+)-independent and insensitive to N-ethylmaleimide, and substrate specificity is broad for LPP enzymes, whereas SPPases are highly selective for sphingolipid substrates. SPPase activity in yeast and mammalian cells regulates intracellular sphingosine-1-phosphate levels, and also alters the levels of sphingosine and ceramide, two other signaling molecules that often oppose the actions of sphingosine-1-phosphate. Thus, loss of SPPase in yeast results in high sphingosine-1-phosphate levels and cells are more resistant to stress, and in mammalian cells, overexpression of SPPase elevates ceramide levels and provokes apoptosis.     

Lipid phosphate phosphatases and related proteins: signaling functions in development, cell division, and cancer

Lipid phosphates initiate key signaling cascades in cell activation. Lysophosphatidate (LPA) and sphingosine 1-phosphate (S1P) are produced by activated platelets. LPA is also formed from circulating lysophosphatidylcholine by autotaxin, a protein involved tumor progression and metastasis. Extracellular LPA and S1P stimulate families of G-protein coupled receptors that elicit diverse responses. LPA is involved in wound repair and tumor growth. Exogenous S1P is a potent stimulator of angiogenesis, a process vital in development, tissue repair and the growth of aggressive tumors. Inside the cell, phosphatidate (PA), ceramide 1-phosphate (C1P), LPA, and S1P act as signaling molecules with distinct functions including the stimulation of cell division, cytoskeletal rearrangement, Ca(2+) transients, and membrane movement. These observations imply that phosphatases that degrade lipid phosphates on the cell surface, or inside the cell, regulate cell signaling under physiological and pathological conditions. This occurs through attenuation of signaling by the lipid phosphates and by the production of bioactive products (diacylglycerol, ceramide, and sphingosine). Three lipid phosphate phosphatases (LPPs) and a splice variant dephosphorylate LPA, PA, CIP, and S1P. Two S1P phosphatases (SPPs) act specifically on S1P. In addition, there is family of four LPP-related proteins (LPRs, or plasticity-related genes, PRGs). PRG-1 expression in neurons has been reported to increase extracellular LPA breakdown and attenuate LPA-induced axonal retraction. It is unclear whether the LRPs dephosphorylate LPA directly, stimulate LPP activity, or bind LPA and S1P. Also, the importance of extra- versus intra-cellular actions of the LPPs and SPPs, and the individual roles of different isoforms is not firmly established. Understanding the functions and regulation of the LPPs, SPPs and related proteins will hopefully contribute to interventions to correct dysfunctions in conditions such as wound repair, inflammation, angiogenesis, tumor growth, and metastasis.     

Role of the autotaxin–lysophosphatidate axis in cancer resistance to chemotherapy and radiotherapy

High expression of autotaxin in cancers is often associated with increased tumor progression, angiogenesis and metastasis. This is explained mainly since autotaxin produces the lipid growth factor, lysophosphatidate (LPA), which stimulates cell division, survival and migration. It has recently become evident that these signaling effects of LPA also produce resistance to chemotherapy and radiation-induced cell death. This results especially from the stimulation of LPA₂ receptors, which depletes the cell of Siva-1, a pro-apoptotic signaling protein and stimulates prosurvival kinase pathways through a mechanism mediated via TRIP-6. LPA signaling also increases the formation of sphingosine 1-phosphate, a pro-survival lipid. At the same time, LPA decreases the accumulation of ceramides, which are used in radiation therapy and by many chemotherapeutic agents to stimulate apoptosis. The signaling actions of extracellular LPA are terminated by its dephosphorylation by a family of lipid phosphate phosphatases (LPP) that act as ecto-enzymes. In addition, lipid phosphate phoshatase-1 attenuates signaling downstream of the activation of both LPA receptors and receptor tyrosine kinases. This makes many cancer cells hypersensitive to the action of various growth factors since they often express low LPP1/3 activity. Increasing our understanding of the complicated signaling pathways that are used by LPA to stimulate cell survival should identify new therapeutic targets that can be exploited to increase the efficacy of chemo- and radio-therapy. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Enzymatic analysis of lipid phosphate phosphatases

Lipid phosphate monoesters including phosphatidic acid, lysophosphatidic acid, sphingosine 1-phosphate and ceramide 1-phosphate are intermediates in phosho- and sphingo-lipid biosynthesis and also play important roles in intra- and extra-cellular signaling. Dephosphorylation of these lipids terminates their signaling actions and, in some cases, generates products with additional biological activities or metabolic fates. The key enzymes responsible for dephosphorylation of these lipid phosphate substrates are collectively termed lipid phosphate phosphatases (LPPs). They are integral membrane enzymes with a core domain of six transmembrane spanning alpha-helices linked by extramembrane loops. LPPs are oriented in the membrane with their N- and C-termini facing the cytoplasm. LPPs exhibit isoform and cell specific localization patterns being variably distributed between endomembrane compartments (primarily the endoplasmic reticulum and Golgi apparatus) and the plasma membrane. The active site of these enzymes is formed from residues within two of the extramembrane loops and faces the lumen of endomembrane compartments or, when localized to the plasma membrane, towards, the extracellular space. Biochemical, pharmacological, cell biological and genetic studies identify roles for LPPs in both intracellular lipid metabolism and the regulation of both intra- and extra-cellular signaling pathways that control cell growth, survival and migration. This article describes procedures for the expression of LPPs in insect and mammalian cells and their analysis by SDS-PAGE and Western blotting. The most straightforward way to determine LPP activity is to measure release of the substrate phosphate group. We described methods for the synthesis and purification of [(32)P]-labeled LPP substrates. We describe the use of both radiolabeled and fluorescent lipid substrates for the detection, quantitation and analysis of the enzymatic activities of the LPPs measured using intact or broken cell preparations as the source of enzyme.     

G-protein-coupled receptor stimulation of the p42/p44 mitogen-activated protein kinase pathway is attenuated by lipid phosphate phosphatases 1, 1a, and 2 in human embryonic kidney 293 cells

Sphingosine 1-phosphate, lysophosphatidic acid, and phosphatidic acid bind to G-protein-coupled receptors to stimulate intracellular signaling in mammalian cells. Lipid phosphate phosphatases (1, 1a, 2, and 3) are a group of enzymes that catalyze de-phosphorylation of these lipid agonists. It has been proposed that the lipid phosphate phosphatases exhibit ecto activity that may function to limit bioavailability of these lipid agonists at their receptors. In this study, we show that the stimulation of the p42/p44 mitogen-activated protein kinase pathway by sphingosine 1-phosphate, lysophosphatidic acid, and phosphatidic acid, all of which bind to G(i/o)-coupled receptors, is substantially reduced in human embyronic kidney 293 cells transfected with lipid phosphate phosphatases 1, 1a, and 2 but not 3. This was correlated with reduced basal intracellular phosphatidic acid and not ecto lipid phosphate phosphatase activity. These findings were supported by results showing that lipid phosphate phosphatases 1, 1a, and 2 also abrogate the stimulation of p42/p44 mitogen-activated protein kinase by thrombin, a peptide G(i/o)-coupled receptor agonist whose bioavailability at its receptor is not subject to regulation by the phosphatases. Furthermore, the lipid phosphate phosphatases have no effect on the stimulation of p42/p44 mitogen-activated protein kinase by other agents that do not use G-proteins to signal, such as serum factors and phorbol ester. Therefore, these findings show that the lipid phosphate phosphatases 1, 1a, and 2 may function to perturb G-protein-coupled receptor signaling per se rather than limiting bioavailability of lipid agonists at their respective receptors.     

Enzymatic measurement of sphingosine 1-phosphate.

Sphingosine 1-phosphate (SPP) is a sphingolipid metabolite which has novel dual actions acting as both an intracellular second messenger and a ligand for a family of G protein-coupled receptors. This paper describes a rapid enzymatic method to quantify mass levels of SPP in serum, mammalian tissues, and cultured cells. The assay utilizes an alkaline lipid extraction to selectively separate SPP from other phospholipids and sphingolipids, including sphingosine. Extracted SPP is efficiently converted to sphingosine by alkaline phosphatase treatment. Sphingosine thus formed is then quantitatively phosphorylated to [(32)P]SPP using recombinant sphingosine kinase and [gamma-(32)P]ATP. With this procedure we were able to obtain reproducible measurements of SPP over a broad range from 0.25 pmol to 2.5 nmol. In various rat tissues, levels of SPP varied between 0. 5 and 6 pmol/mg wet wt. The lowest levels were found in heart and testes, while brain contained the highest levels. The method was adapted easily to measure minute amounts of SPP present in various cultured cell types. The amount of SPP in cell extracts was proportional to the cell number and varied between 0.04 and 2 pmol/10(6) cells. Concurrent measurements of sphingosine levels revealed that its concentration was significantly higher than SPP in most cells and tissues. Furthermore, with this assay we were able to measure increases in intracellular SPP levels in rat pheochromocytoma PC12 cells after treatment with exogenous sphingosine or with nerve growth factor which stimulates sphingosine kinase activity.     

Involvement of Lysophosphatidic Acid,Sphingosine 1-Phosphate and Ceramide 1-Phosphate in the Metabolization of Phosphatidic Acid by Lipid Phosphate Phosphatases in Bovine Rod Outer Segments

The aim of the present research was to evaluate the generation of [2-3H]diacylglycerol ([2-3H]DAG) from [2-3H]-Phosphatidic acid ([2-3H]PA) by lipid phosphate phosphatases (LPPs) at different concentrations of lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P), and ceramide 1-phosphate (C1P) in purified 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. Western blot analysis revealed the presence of LPP3 exclusively in all membrane preparations. Immunoblots of entire ROS and depleted ROS did not show dark-light differences in LPP3 levels. LPPs activities were diminished by 53% in BLROS with respect to DROS. The major competitive effect on PA hydrolysis was exerted by LPA and S1P in DROS and by C1P in BLROS. LPPs activities in depleted ROS were similar to the activity observed in entire DROS and BLROS, respectively. LPA, S1P and C1P competed at different extent in depleted DROS and BLROS. Sphingosine and ceramide inhibited LPPs activities in entire and depleted DROS. Ceramide also inhibited LPPs activities in entire and in depleted BLROS. Our findings are indicative of a different degree of competition between PA and LPA, S1P and C1P by LPPs depending on the illumination state of the retina.