sphingosine-1-phosphate and safingol

This study aims to explore the potential biomarkers in the development of diabetes mellitus (DM) into diabetic retinopathy (DR).. Systematic review of diabetic metabolomics was used to screen the differential metabolites and related pathways during the development of DM. Non-targeted lipidomics of rat plasma was performed to explore the differential metabolites in the development of DM into DR in vivo. To verify the effects of differential metabolites in inducing retinal microvascular endothelial cells (RMECs) injury by increasing oxidative stress, high glucose medium containing differential metabolites was used to induce rat RMECs injury and cell viability, malondialdehyde (MDA) contents, superoxide dismutase (SOD) activities, reactive oxygen species (ROS) levels and mitochondrial membrane potential (MMP) were evaluated in vitro. Network pharmacology was performed to explore the potential mechanism of differential metabolites in inducing DR.. Through the systematic review, 148 differential metabolites were obtained and the sphingolipid metabolic pathway attracted our attention. Plasma non-targeted lipidomics found that sphingolipids were accompanied by the development of DM into DR. In vitro experiments showed sphinganine and sphingosine-1-phosphate aggravated rat RMECs injury induced by high glucose, further increased MDA and ROS levels, and further decreased SOD activities and MMP. Network pharmacology revealed sphinganine and sphingosine-1-phosphate may induce DR by regulating the AGE-RAGE and HIF-1 signaling pathways.. Integrated systematic review, lipidomics and experiment verification reveal that abnormal sphingolipid metabolism facilitates DR by inducing oxidative stress on RMECs. Our study could provide the experimental basis for finding potential biomarkers for the diagnosis and treatment of DR.

Topics: Animals; Biomarkers; Diabetes Mellitus; Diabetic Retinopathy; Endothelial Cells; Glucose; Lipidomics; Oxidative Stress; Rats; Reactive Oxygen Species; Sphingolipids; Superoxide Dismutase

Sphingolipids are a diverse set of structurally and metabolically related lipids that have numerous functions in cell structure and signaling. The regulation of these lipids is critical for normal cell function and disregulation has been implicated in pathophysiological conditions such as cancer and inflammation. Here we examine control of the initiating, and rate limiting, enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT). We find that de novo synthesis of sphingolipid is stimulated by a number of cancer chemotherapeutics, suggesting that this may be an important aspect of their cytotoxic effects. The three ORMDL proteins are membrane proteins of the endoplasmic reticulum related to the yeast Orm proteins, which have been shown to be homeostatic regulators of SPT. We find that the ORMDL proteins are also negative regulators of SPT that transmit cellular levels of sphingolipids to SPT. The three isoforms have redundant functions in this system. The sphingosine kinases (sphingosine kinase-1 and -2) phosphorylate both sphingosine, which is released from ceramide, but also dihydrosphingosine, which is in the de novo biosynthetic pathway. We therefore examined the role of the sphingosine kinases in controlling de novo ceramide biosynthesis and find that sphingosine kinase-1 does indeed act as a negative regulator of this pathway. This establishes that sphingosine kinase, in addition to producing sphingosine-1-phosphate as a signaling molecule, also consumes dihydrosphingosine to regulate ceramide synthesis. Our studies demonstrate that there are multiple mechanisms of regulation of SPT and suggest that these regulators are important mediators of cell stress responses.

Topics: Animals; Ceramides; Humans; Lysophospholipids; Membrane Proteins; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; Stress, Physiological

Aspirin is an antiplatelet drug which is commonly used in secondary prevention in ischemic heart disease and cerebrovascular events, and in newly diagnosed myocardial infarction. The aim of the present study was to examine effect of aspirin on the level of selected sphingolipid intermediates in plasma, erythrocytes and platelets.. Forty two healthy volunteers participated in the study. They were divided into two groups. In one group aspirin was given orally, daily, for one week in a dose of 75 mg (n=25). The subjects from the second group received one 300 mg dose of the drug (n=17). In both groups the blood was taken 4h after the last dose of aspirin. The following sphingolipid intermediates were quantified using high-pressure liquid chromatography: sphinganine, sphingosine, sphingosine-1-phosphate (S1P), sphinganine-1-phosphate (SA1P) and ceramide.. It was found that lower dose of aspirin increased the level of S1P and ceramide in erythrocytes (by 23 and 37%, respectively) having no effect on plasma and platelet sphingolipid levels. Higher dose of the drug reduced S1P and SA1P concentration in the plasma (by 16 and 10%, respectively).. We conclude that aspirin interferes with sphingolipid metabolism in blood and that this effect depends on a dose of the drug. Since S1P is a potent cardioprotectant, the reduction in its plasma concentration after the loading dose of aspirin could be undesired side effect of the drug.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Aspirin; Blood Platelets; Ceramides; Dose-Response Relationship, Drug; Erythrocytes; Female; Healthy Volunteers; Hemoglobins; Humans; Lysophospholipids; Male; Platelet Aggregation Inhibitors; Platelet Count; Sphingolipids; Sphingosine; Young Adult

Sphingosine 1-phosphate (S1P) is an important mediator of cancer cell growth and proliferation. Production of S1P is catalyzed by sphingosine kinase 1 (SphK). Safingol, (l-threo-dihydrosphingosine) is a putative inhibitor of SphK. We conducted a phase I trial of safingol (S) alone and in combination with cisplatin (C).. A 3 + 3 dose escalation was used. For safety, S was given alone 1 week before the combination. S + C were then administered every 3 weeks. S was given over 60 to 120 minutes, depending on dose. Sixty minutes later, C was given over 60 minutes. The C dose of 75 mg/m(2) was reduced in cohort 4 to 60 mg/m(2) due to excessive fatigue.. Forty-three patients were treated, 41 were evaluable for toxicity, and 37 for response. The maximum tolerated dose (MTD) was S 840 mg/m(2) over 120 minutes C 60 mg/m(2), every 3 weeks. Dose-limiting toxicity (DLT) attributed to cisplatin included fatigue and hyponatremia. DLT from S was hepatic enzyme elevation. S pharmacokinetic parameters were linear throughout the dose range with no significant interaction with C. Patients treated at or near the MTD achieved S levels of more than 20 μmol/L and maintained levels greater than and equal to 5 μmol/L for 4 hours. The best response was stable disease in 6 patients for on average 3.3 months (range 1.8-7.2 m). One patient with adrenal cortical cancer had significant regression of liver and lung metastases and another had prolonged stable disease. S was associated with a dose-dependent reduction in S1P in plasma.. Safingol, the first putative SphK inhibitor to enter clinical trials, can be safely administered in combination with cisplatin. Reversible dose-dependent hepatic toxicity was seen, as expected from preclinical data. Target inhibition was achieved with downregulation of S1P. The recommended phase II dose is S 840 mg/m(2) and C 60 mg/m(2), every 3 weeks.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Area Under Curve; Cisplatin; Dose-Response Relationship, Drug; Drug Administration Schedule; Fatigue; Female; Humans; Lymphopenia; Lysophospholipids; Male; Metabolic Clearance Rate; Middle Aged; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Treatment Outcome

Elimination strategies of chronic hepatitis C virus (HCV) infection aim to optimize the high antiviral potency of direct-acting antivirals (DAAs). Sphingolipids (SLs) constitute bioactive lipid compounds with a remarkable second messenger potential. SL levels associate with responsiveness to interferon treatment in HCV-patients, thus prompting the question whether failure to DAAs can be predicted by the serologic sphingolipidomic profile. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used to retrospectively quantify various sphingolipid metabolites in baseline serum samples of 97 chronic HCV patients with DAA failure compared with an age-matched cohort of 95 HCV-patients with sustained virological response (SVR). Sphingosine, sphinganine, sphingosine-1-phosphate (S1P) and sphinganine-1-phosphate (SA1P) serum concentrations were significantly upregulated at baseline in patients with DAA failure compared to patients with SVR. Similarly, GluC24:1Cer baseline levels were significantly upregulated in patients with DAA failure compared to the patients with SVR. However, GluC18Cer serum levels showed decreased baseline levels for patients with DAA failure compared to patients with SVR. In multivariate analysis sphinganine (OR 0.08494, CI 0.07393-0.9759, p = .021223), SA1P (OR 0.9818, CI 0.9653-0.9987, p = .034801), GluCerC18 (OR 1.0683, CI 1.0297-1.1104, p = .000786) and GluCer24:1 (OR 0.9961, CI 0.994-0.998, p = .000294) constituted independent predictors of treatment response. In conclusion, serum sphingolipid concentrations, in particular sphingosine, sphinganine and their derivatives S1P and SA1P as well as glucosylceramides may identify at baseline the minority of HCV patients with DAA failure. Serum sphingolipids could constitute additional biomarkers for national treatment strategies aiming to eliminate HCV infection.

Topics: Antiviral Agents; Biomarkers; Chromatography, Liquid; Hepacivirus; Hepatitis C; Hepatitis C, Chronic; Humans; Retrospective Studies; Sphingolipids; Sphingosine; Sustained Virologic Response; Tandem Mass Spectrometry

Psoriasis is a complex chronic immunologically mediated disease that may involve skin, nails, and joints. It is characterized by hyperproliferation, deregulated differentiation, and impaired apoptosis of keratinocytes. Sphingolipids, namely ceramide, sphingosine-1-phosphate, sphingosine, sphingomyelin, and sphinganine-1-phosphate, are signal molecules that may regulate cell growth, immune reactions, and apoptosis. Fifteen patients with psoriasis and seventeen healthy persons were enrolled in the study. Skin samples were taken from psoriatic lesions and non-lesional areas. Tissue concentration of ceramides, sphingosine-1-phosphate, sphingosine, sphingomyelin, and sphinganine-1-phosphate was measured by liquid chromatography. We assessed that all levels of ceramides, sphingosine-1-phosphate, sphingosine, sphingomyelin, and sphinganine-1-phosphate were higher in lesioned psoriatic skin than in non-affected skin. The profile of bioactive lipids in the lesional skin of patients with psoriasis differed significantly from non-involved psoriatic skin and skin in healthy subjects.

Topics: Ceramides; Humans; Phosphates; Psoriasis; Sphingolipids; Sphingomyelins; Sphingosine

Psoriasis is a chronic, complex, immunological disorder, which may lead to many different systemic complications. Sphingolipids, including ceramide, are bioactive lipids, which take part in the regulation of immune reactions, cell growth, and apoptosis. Twenty psoriatic patients and twenty-eight control subjects were included in the study. Skin (both lesional and non-lesional) and serum samples were collected from both the control group and the psoriatic patients. The levels of sphingosine (SFO), sphingosine-1-phosphate (S1P), sphingomyelin, sphinganine (SFA), sphinganine-1-phosphate (SFA1P), and ceramide (CER) were assessed in both tissue (t) and serum (s) samples using high-performance liquid chromatography (HPLC). We identified elevated serum levels of SFO, S1P, SFA, and SFA1P in psoriatic patients when compared to healthy individuals. As far as the lesional skin and serum of psoriatic patients are concerned, we demonstrated positive associations between CER_t and CER_s, SFA_t and CER_s, and SFO_t and CER_s. Additionally, we found negative correlations in the non-lesional skin and serum of psoriatic patients, including SFO_t vs. SFO_s, CER_t vs. SFA_s, CER_t vs. SFO_s, and SFO_t vs. SFA_s. Finally, we observed a positive correlation between S1P and SFA1P in both the serum samples of psoriatic patients and the serum samples of the control group. In this study, we did not observe any correlations between psoriasis area and severity index (PASI) scores and sphingolipid levels. In conclusion, our findings indicate an interplay between skin and serum lipids in psoriatic patients, which is not observed in healthy individuals.

Topics: Ceramides; Humans; Psoriasis; Skin; Sphingolipids; Sphingosine

Bioactive sphingolipids are emerging as key regulators of vascular function and homeostasis. While most of the clinical studies have been devoted to profile circulating sphingolipids in maternal plasma, little is known about the role of the sphingolipid at the feto-placental vasculature, which is in direct contact with the offspring circulation. Our study aims to compare the sphingolipid profile of normal with preeclamptic (PE) placental chorionic arteries and isolated endothelial cells, with the goal of unveiling potential underlying pathomechanisms in the vasculature. Dihydrosphingosine and sphingomyelin (SM) concentrations (C16:0-, C18:0-, and C24:0- sphingomyelin) were significantly increased in chorionic arteries of preeclamptic placentas, whereas total ceramide, although showing a downward trend, were not statistically different. Moreover, RNA and immunofluorescence analysis showed impaired sphingosine-1-phosphate (S1P) synthesis and signaling in PE vessels. Our data reveal that the exposure to a deranged maternal intrauterine environment during PE alters the sphingolipid signature and gene expression on the fetal side of the placental vasculature. This pathological remodeling consists in increased serine palmitoyltransferase (SPT) activity and SM accrual in PE chorionic arteries, with concomitance impairment endothelial S1P signaling in the endothelium of these vessels. The increase of endothelial S1P phosphatase, lyase and S1PR2, and blunted S1PR1 expression support the onset of the pathological phenotype in chorionic arteries.

Topics: Arteries; Ceramides; Chorion; Endothelial Cells; Endothelium, Vascular; Female; Fetus; Gene Expression; Humans; Lysophospholipids; Maternal-Fetal Exchange; Placenta; Placental Circulation; Pre-Eclampsia; Pregnancy; Signal Transduction; Sphingolipids; Sphingomyelins; Sphingosine

Sphingolipidoses are monogenic lipid storage diseases caused by variants in enzymes of lipid synthesis and metabolism. We describe an autosomal recessive complex neurological disorder affecting consanguineous kindred. All four affected individuals, born at term following normal pregnancies, had mild to severe intellectual disability, spastic quadriplegia, scoliosis and epilepsy in most, with no dysmorphic features. Brain MRI findings were suggestive of leukodystrophy, with abnormal hyperintense signal in the periventricular perioccipital region and thinning of the body of corpus callosum. Notably, all affected individuals were asymptomatic at early infancy and developed normally until the age of 8-18 months, when deterioration ensued. Homozygosity mapping identified a single 8.7 Mb disease-associated locus on chromosome 1q41-1q42.13 between rs1511695 and rs537250 (two-point LOD score 2.1). Whole exome sequencing, validated through Sanger sequencing, identified within this locus a single disease-associated homozygous variant in DEGS1, encoding C4-dihydroceramide desaturase, an enzyme of the ceramide synthesis pathway. The missense variant, segregating within the family as expected for recessive heredity, affects an evolutionary-conserved amino acid of all isoforms of DEGS1 (c.656A>G, c.764A>G; p.(N219S), p.(N255S)) and was not found in a homozygous state in ExAC and gnomAD databases or in 300 ethnically matched individuals. Lipidomcs analysis of whole blood of affected individuals demonstrated augmented levels of dihydroceramides, dihydrosphingosine, dihydrosphingosine-1-phosphate and dihydrosphingomyelins with reduced levels of ceramide, sphingosine, sphingosine-1-phosphate and monohexosylceramides, as expected in malfunction of C4-dihydroceramide desaturase. Thus, we describe a sphingolipidosis causing a severe regressive neurological disease.

Topics: Adolescent; Adult; Brain; Ceramides; Cerebrosides; Child; Child, Preschool; Exome Sequencing; Fatty Acid Desaturases; Female; Genetic Predisposition to Disease; Genetic Variation; Homozygote; Humans; Infant; Intellectual Disability; Lysophospholipids; Male; Mutation, Missense; Nervous System Diseases; Pedigree; Phenotype; Sequence Analysis, DNA; Sphingosine; Young Adult

Sphingosine-1-phosphate (S1P) is a bioactive lysosphingolipid that is found in high concentration in plasma. The majority of plasma S1P is transported bound to HDL and albumin. Although the major sources of circulating S1P have been identified, it remains obscure what is the contribution of different organs/tissues to S1P homeostasis in plasma. Answering this question was the major aim of the present study.. The experiment was performed on male Wistar rats from whom blood samples were taken from either: 1) femoral vein, right ventricle of the heart, and abdominal aorta (n=15) or 2) hepatic vein, portal vein, and abdominal aorta (n=11). Plasma was fractionated by sequential flotation ultracentrifugation and sphingolipids were quantified by a HPLC method.. Compared to the mixed venous blood sampled from the right ventricle, total plasma and lipoprotein-depleted plasma (LPDP) concentration of S1P in the arterial blood was lower. On the other hand, the level of S1P increased across the leg both in plasma and LPDP. The concentration of S1P, sphingosine, and sphinganine in the plasma, HDL, and LPDP isolated from the blood taken from the hepatic vein was markedly higher compared to both arterial and portal blood.. We conclude that, in contrast to HDL-bound S1P, albumin-associated S1P is very labile in the circulation. It is degraded in the pulmonary, and to a lesser extent, gastrointestinal circulation, and released across the liver and skeletal muscle. We also conclude that liver is an important source of HDL-bound S1P and circulating free sphingoid bases.

Topics: Animals; Aorta, Abdominal; Chromatography, High Pressure Liquid; Femoral Vein; Heart Ventricles; Hepatic Veins; Lipoproteins, HDL; Lipoproteins, LDL; Lysophospholipids; Male; Portal Vein; Protein Binding; Rats; Rats, Wistar; Sphingolipids; Sphingosine

Impaired apoptotic pathways in leukemic cells enable them to grow in an uncontrolled way. Moreover, aberrations in the apoptotic pathways are the main factor of leukemic cells drug resistance.. To assess the presence of potential abnormalities that might promote dysfunction of leukemic cells growth, HPLC system was used to determine sphingosine (SFO), sphinganine (SFA), sphingosine-1-phosphate (S1P) and ceramide (CER) concentration in the blood collected from patients diagnose with acute myeloblastic leukemia (AML; n = 49) and compare to values of control (healthily) group (n = 51). Additionally, in AML group concentration of SFO, SFA, S1P and CER was determined in bone marrow plasma and compared to respective values in blood plasma. The concentration of S1P and CER binding protein - plasma gelsolin (GSN) was also assessed in collected samples using immunoblotting assay.. We observed that in AML patients the average SFO, SFA and CER concentration in blood plasma was significantly higher (p < 0.001) compare to control group, when blood plasma S1P concentration was significantly lower (p < 0.001). At the same time the CER/S1P ratio in AML patient (44.5 ± 19.4) was about 54% higher compare to control group (20.9 ± 13.1). Interestingly the average concentration of S1P in blood plasma (196 ± 13 pmol/ml) was higher compare to its concentration in plasma collected from bone marrow (154 ± 21 pmol/ml).. We hypothesize that changes in profile of sphingolipids concentration and some of their binding protein partners such as GSN in extracellular environment of blood and bone marrow cells in leukemic patients can be targeted to develop new AML treatment method(s).

Topics: Adult; Aged; Aged, 80 and over; Bone Marrow; Case-Control Studies; Ceramides; Female; Gelsolin; Humans; Leukemia, Myeloid, Acute; Lysophospholipids; Male; Middle Aged; Sphingosine

Ozonated blood therapy is used in the treatment of several diseases, including superficial infections, burns, dental and intestinal conditions. Except that, the possibility of using ozone to sterilize blood supplies is under promising investigation. However, still little is known regarding the impact of blood ozonation, especially on biologically active serum sphinoglipids. In the present work we sought to investigate the contents of sphingolipids, such as sphingosine, sphingosine-1-phosphate (S-1-P), sphinganine, and ceramide (CER) in the plasma, after immediate and prolonged (1 h) ozonation of human whole blood. For the measurements liquid chromatography hyphenated with the mass spectrometry was applied. We demonstrated that only the content of sphingosine-1-phosphate in the plasma was increased significantly, possibly exerting its beneficial effect for various physiological and clinical events.

Topics: Adult; Ceramides; Humans; Lysophospholipids; Male; Ozone; Plasma; Sphingosine

Nowadays wrong nutritional habits and lack of physical activity give a rich soil for the development of insulin resistance and obesity. Many researches indicate lipids, especially the one from the sphingolipids class, as the group of molecules heavily implicated in the progress of insulin resistance in skeletal muscle. Recently, scientists have focused their scrutiny on myriocin, a potent chemical compound that inhibits ceramide (i.e., central hub of sphingolipids signaling pathway) de novo synthesis. In the present research we evaluated the effects of myriocin application on type 2 diabetes mellitus in three different types of skeletal muscles: (1) slow-oxidative (red gastrocnemius), (2) oxidative-glycolytic (soleus), and (3) glycolytic (white gastrocnemius). For these reasons the animals were randomly divided into four groups: "control" (C), "myriocin" (M), "high fat diet" (HFD), "high fat diet" (HFD), and "high fat diet + myriocin" (HFD + M). Our in vivo study demonstrated that ceramide synthesis inhibition reduces intramuscular ceramide, its precursor sphinganine, and its derivatives sphingosine and sphingosine-1-phosphate concentrations. Moreover, FFA and TG contents were also decreased after myriocin treatment. Thus, myriocin presents potential therapeutic perspectives with respect to the treatment of insulin resistance and its serious consequences in obese patients.

Topics: Animals; Ceramides; Diabetes Mellitus, Type 2; Diet, High-Fat; Disease Models, Animal; Fatty Acids, Monounsaturated; Glycolysis; Insulin Resistance; Lysophospholipids; Male; Muscle, Skeletal; Oxygen; Rats; Rats, Wistar; Signal Transduction; Sphingolipids; Sphingosine

Three bioactive sphingolipids, namely sphingosine-1-phosphate (S1P), ceramide (CER) and sphingosine (SPH) were shown to be involved in ischemia/reperfusion injury of the heart. S1P is a powerful cardioprotectant, CER activates apoptosis and SPH in a low dose is cardioprotective whereas in a high dose is cardiotoxic. The aim of the present study was to examine effects of experimental myocardial infarction on the level of selected sphingolipids in plasma, erythrocytes and platelets in the rat. Myocardial infarction was produced in male Wistar rats by ligation of the left coronary artery. Blood was taken from the abdominal aorta at 1, 6 and 24 h after the ligation. Plasma, erythrocytes and platelets were isolated and S1P, dihydrosphingosine-1-phosphate (DHS1P), SPH, dihydrosphingosine (DHS) and CER were quantified by means of an Agilent 6460 triple quadrupole mass spectrometer using positive ion electrospray ionization source with multiple reaction monitoring. The infarction reduced the plasma level of S1P, DHS1P, SPH and DHS but increased the level of total CER. In erythrocytes, there was a sharp elevation in the level of SPH and DHS early after the infarction and a reduction after 24 h whereas the level of S1P, DHS1P and total CER gradually increased. In platelets, the level of each of the examined compounds profoundly decreased 1 and 6 h after the infarction and partially normalized in 24 h. The results obtained clearly show that experimental heart infarction in rats produces deep changes in metabolism of sphingolipids in the plasma, platelets and erythrocytes.

Topics: Anesthesia; Animals; Ceramides; Coronary Vessels; Erythrocyte Count; Femoral Artery; Ligation; Lysophospholipids; Male; Myocardial Infarction; Phosphotransferases (Alcohol Group Acceptor); Platelet Count; Rats; Rats, Wistar; Sphingolipids; Sphingosine; Troponin T

A method for determination of two relevant sphingoid precursors such as sphingosine and sphinganine and the corresponding conjugates sphingosine 1-phosphate and sphinganine 1-phosphate in human urine and serum is here presented. The method is characterized by a solid- phase extraction step with in situ derivatization of the sphingolipids in the eluate (o-phthaldialdehyde derivatives) to obtain fluorescent compounds. In this way, sample preparation was completely performed in a single automated step by means of a lab-on-valve system. Derivatized analytes were injected into a liquid chromatography system operating at micro regime and detected by laser-induced fluorescence. For determination of sphingoid phosphates, they were enzymatically converted to free sphingoids to obtain stable fluorescent derivatives. The detection limits were in the range 4.2-10.2 ng mL(-1) for serum and 0.56-1.36 ng mL(-1) for urine, with repeatability ranging from 3.9% to 6.2% expressed as relative standard deviation. The method was validated by direct infusion tandem mass spectrometry in multiple reaction monitoring to compare results provided by analysis of biofluids and to confirm the identity of the target compounds. Sensitivity and precision were better than or similar to those provided by the confirmatory method. The automation of sample preparation enables to scale-down this step and improves precision by minimization of human intervention, being thus suitable for clinical analysis.

Topics: Chromatography, Liquid; Equipment Design; Humans; Lasers; Limit of Detection; Lysophospholipids; Solid Phase Extraction; Sphingosine; Tandem Mass Spectrometry

The sphingosine kinase (SPK)/sphingosine-1-phosphate (S1P) pathway recently has been associated with a variety of inflammatory-based diseases. The majority of these studies have been performed in vitro. Here, we have addressed the relevance of the SPK/S1P pathway in the acute inflammatory response in vivo by using different well known preclinical animal models. The study has been performed by operating a pharmacological modulation using 1) L-cycloserine and DL-threo-dihydrosphingosine (DTD), S1P synthesis inhibitors or 2) 2-undecyl-thiazolidine-4-carboxylic acid (BML-241) and N-(2,6-dichloro-4-pyridinyl)-2-[1,3-dimethyl-4-(1-methylethyl)-1H-pyrazolo[3,4-b]pyridin-6-yl]-hydrazinecarboxamide (JTE-013), specific S1P(2) and S1P(3) receptor antagonists. After local injection of carrageenan in mouse paw S1P release significantly increases locally and decreases during the resolution phase. Expression of SPKs and S1P(2) and S1P(3) receptors is increased in inflamed tissues. Administration of L-cycloserine or DTD caused a significant anti-inflammatory effect. By using different animal models we have also demonstrated that the SPK/S1P pathway contributes to changes in vascular permeability and promotes cell recruitment. The S1P effect on cell recruitment results is receptor-mediated because both JTE-013 and BML-241 inhibited zymosan-induced cell chemotaxis without effect on vascular leakage. Conversely, changes in vascular permeability involve mainly SPK activity, because compound 48/80-induced vascular leakage was significantly inhibited by DTD. In conclusion, the SPK/S1P pathway is involved in acute inflammation and could represent a valuable therapeutic target for developing a new class of anti-inflammatory drugs.

Topics: Animals; Capillary Permeability; Chemotaxis, Leukocyte; Cycloserine; Edema; Inflammation; Lysophospholipids; Male; Mice; Molecular Targeted Therapy; Phosphotransferases (Alcohol Group Acceptor); Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Thiazolidines

Consumption of high fat diet leads to muscle lipid accumulation which is an important factor involved in induction of insulin resistance. Ceramide is likely to partially inhibit insulin signaling cascade. The aim of this study was to examine the effect of different high fat diets on ceramide metabolism in rat skeletal muscles. The experiments were carried out on rats fed for 5 weeks: (1) a standard chow and (2) high fat diet rich in polyunsaturated fatty acids (PUFA) and (3) diet enriched with saturated fatty acids (SAT). Assays were performed on three types of muscles: slow-twitch oxidative (soleus), fast-twitch oxidative-glycolytic, and fast-twitch glycolytic (red and white section of the gastrocnemius, respectively). The activity of serine palmitoyltransferase (SPT), neutral and acid sphingomyelinase (n- and aSMase), and neutral and alkaline ceramidase (n- and alCDase) was examined. The content of ceramide, sphinganine, sphingosine, and sphingosine-1-phosphate was also measured. The ceramide content did not change in any muscle from PUFA diet group but increased in the SAT diet group by 46% and 52% in the soleus and red section of the gastrocnemius, respectively. Elevated ceramide content in the SAT diet group could be a result of increased SPT activity and simultaneously decreased activity of nCDase. Unchanged ceramide content in the PUFA diet group might be a result of increased activity of SPT and alCDase and simultaneously decreased activity of SMases. We conclude that regulation of muscle ceramide level depends on the diet and type of skeletal muscle.

Topics: Alkaline Ceramidase; Animals; Ceramides; Dietary Fats, Unsaturated; Fatty Acids; Fatty Acids, Nonesterified; Fatty Acids, Unsaturated; Glycolysis; Lysophospholipids; Male; Muscle Fibers, Fast-Twitch; Muscle Fibers, Slow-Twitch; Muscle, Skeletal; Neutral Ceramidase; Oxidation-Reduction; Rats; Rats, Wistar; Serine C-Palmitoyltransferase; Sphingolipids; Sphingomyelin Phosphodiesterase; Sphingosine

Liver regeneration after partial hepatectomy (PH) is achieved by intense cells proliferation. Sphingosine-1-phosphate stimulates proliferation but ceramide and sphingosine induce apoptosis. The aim of the study was to investigate the influence of high-fat diet (HFD) on the sphingolipid metabolism during the first 24h of liver regeneration in rats. Rats were fed HFD or standard diet for 7 days prior to the PH. The content of sphingolipids and the activity of sphingomyelinases (n and aSMase), ceramidases (n and aCDase) and sphingosine kinase (SPHK) were measured. It has been found that HFD increased the activity of aSMase and nCDase at 4th hour after PH. The content of ceramide and sphingosine decreased in HFD group at each time point. This was accompanied by elevated content of sphingosine-1-phosphate and sphinganine-1-phosphate. Decrease in SPHK activity in cytosol after partial hepatectomy was inversely correlated (r=-0.7538) with increase in S1P, which suggest translocation of SPHK to plasma membrane. Shingosine-1-phosphate to ceramide ratio was higher in rats fed HFD. It is concluded that HFD stimulates the pro-mitotic action of the sphingolipid signaling in regenerating rat liver.

Topics: Animals; Ceramidases; Ceramides; Diet; Dietary Fats; Hepatectomy; Hydrogen-Ion Concentration; Linoleic Acid; Liver; Liver Regeneration; Lysophospholipids; Male; Oleic Acid; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Wistar; Second Messenger Systems; Sphingolipids; Sphingomyelin Phosphodiesterase; Sphingosine

Identifying the cues required for the survival and development of photoreceptors is essential for treating retinal neurodegeneration. The authors previously established that glial-derived neurotrophic factor (GDNF) stimulates proliferation and that docosahexaenoic acid (DHA) promotes photoreceptor survival and differentiation. Later findings that ceramide triggers photoreceptor apoptosis suggested sphingolipids might also control photoreceptor development. The present study investigated whether sphingosine-1-phophate (S1P), which promotes survival and differentiation in several cell types, regulates photoreceptor proliferation and differentiation and whether it is a mediator in GDNF and DHA effects.. Rat retina neuronal cultures were supplemented at day 0 or 1 with S1P, GDNF, or DHA and were treated with DL-threo-dihydrosphingosine to inhibit S1P synthesis or with brefeldin A (BFA) to block intracellular trafficking. Proliferation was quantified to determine bromodeoxyuridine uptake and number of mitotic figures. Opsin, peripherin, and sphingosine kinase (SphK), the enzyme required for S1P synthesis, were quantified by immunocytochemistry and Western blot analysis.. S1P increased the proliferation of photoreceptor progenitors. It also stimulated the formation of apical processes, enhanced opsin and peripherin expression, and promoted their localization in these processes; DHA had similar effects. BFA prevented S1P and DHA enhancement of apical process formation without affecting opsin expression. GDNF and DHA enhanced SphK expression in photoreceptors, while inhibiting S1P synthesis blocked GDNF mitogenic effects and DHA effects on differentiation.. The authors propose S1P as a key regulator in photoreceptor development. GDNF and DHA might upregulate SphK levels to promote S1P synthesis, which would initially promote proliferation and then advance photoreceptor differentiation.

Topics: Animals; Blotting, Western; Brefeldin A; Cell Differentiation; Cell Proliferation; Cell Survival; Docosahexaenoic Acids; Enzyme Inhibitors; Glial Cell Line-Derived Neurotrophic Factor; Immunohistochemistry; Intermediate Filament Proteins; Lysophospholipids; Membrane Glycoproteins; Nerve Tissue Proteins; Opsins; Peripherins; Phosphotransferases (Alcohol Group Acceptor); Photoreceptor Cells, Vertebrate; Rats; Rats, Wistar; Sphingosine

Studies in skeletal muscle demonstrate that elevation of plasma FFAs increases the sphingolipid ceramide. We aimed to determine the impact of FFA oversupply on total sphingolipid profiles in a skeletal muscle model. C2C12 myotubes were treated with palmitate (PAL). Lipidomics analysis revealed pleiotropic effects of PAL on cell sphingolipids not limited to ceramides. (13)C labeling demonstrated that PAL activated several branches of sphingolipid synthesis by distinct mechanisms. Intriguingly, PAL increased sphingosine-1-phosphate independently of de novo synthesis. Quantitative real-time PCR demonstrated that PAL increased sphingosine kinase 1 (SK1) mRNA by approximately 4-fold. This was accompanied by a 2.3-fold increase in sphingosine kinase enzyme activity. This upregulation did not occur upon treatment with oleate, suggesting some level of specificity for PAL. These findings were recapitulated in the diet-induced obesity mouse model, in which high-fat feeding increased SK1 message in skeletal muscle over 2.3-fold. These data suggest that the impact of elevated FFA on sphingolipids reaches beyond ceramides and de novo sphingolipid synthesis. Moreover, these findings identify PAL as a novel regulatory stimulus for SK1.

Topics: Animals; Cell Line; Ceramides; Diet; Enzyme Activation; Humans; Isotope Labeling; Lysophospholipids; Mice; Muscle Fibers, Skeletal; Obesity; Oleic Acid; Palmitates; Phosphotransferases (Alcohol Group Acceptor); Rats; Serine C-Palmitoyltransferase; Signal Transduction; Sphingosine; Substrate Specificity; Up-Regulation

The sphingolipid sphingosine-1-phosphate (S1P) plays an important role in protecting the heart against ischemia-reperfusion injury. S1P is normally present in human plasma. However, there are no data available on the effect of myocardial infarction on the plasma concentrations of S1P and related sphingolipids. The aim of this study was to examine the concentrations of S1P, sphinganine-1-phosphate, free sphingosine, free sphinganine, and ceramide in the plasma of patients after myocardial infarction.. The study was performed on two groups of male subjects: controls with no specific complaints (n=21) and patients who had had acute myocardial infarction (n=22). In the latter group, blood was taken immediately after admission to the hospital and five days later. The concentrations of the above compounds were measured by high-pressure liquid chromatography.. The concentrations of S1P and sphinganine-1-phosphate were reduced by ca. 50% both early after infarction and five days later. The concentrations of the other compounds were not affected by myocardial infarction.. The reduction in plasma concentration of S1P after infarction could lessen its protective action on cardiomyocyte viability. The observed reduction in S1P level might be associated with the standard antiplatelet treatment given to patients since thrombocytes are one of the major sources of plasma S1P.

Topics: Aged; Animals; Ceramides; Humans; Lysophospholipids; Male; Mice; Middle Aged; Myocardial Infarction; Myocytes, Cardiac; Receptors, Lysosphingolipid; Reperfusion Injury; Sphingosine; Thrombolytic Therapy

Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates myriad important cellular processes, including growth, survival, cytoskeleton rearrangements, motility, and immunity. Here we report that treatment of Jurkat and U937 leukemia cells with the pan-sphingosine kinase (SphK) inhibitor N,N-dimethylsphingosine to block S1P formation surprisingly caused a large increase in expression of SphK1 concomitant with induction of apoptosis. Another SphK inhibitor, D,L-threo-dihydrosphingosine, also induced apoptosis and produced dramatic increases in SphK1 expression. However, up-regulation of SphK1 was not a specific effect of its inhibition but rather was a consequence of apoptotic stress. The chemotherapeutic drug doxorubicin, a potent inducer of apoptosis in these cells, also stimulated SphK1 expression and activity and promoted S1P secretion. The caspase inhibitor ZVAD reduced not only doxorubicin-induced lethality but also the increased expression of SphK1 and secretion of S1P. Apoptotic cells secrete chemotactic factors to attract phagocytic cells, and we found that S1P potently stimulated chemotaxis of monocytic THP-1 and U937 cells and primary monocytes and macrophages. Collectively, our data suggest that apoptotic cells may up-regulate SphK1 to produce and secrete S1P that serves as a "come-and-get-me" signal for scavenger cells to engulf them in order to prevent necrosis.

Topics: Antibiotics, Antineoplastic; Apoptosis; Chemotaxis, Leukocyte; Doxorubicin; Enzyme Inhibitors; Humans; In Vitro Techniques; Jurkat Cells; Lysophospholipids; Monocytes; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; U937 Cells; Up-Regulation

Fumonisin B(1), a mycotoxin, is an inhibitor of ceramide synthase causing marked dysregulation of sphingolipid metabolism in cells. This mycotoxin causes accumulation of free sphingoid bases (sphingosine and dihydrosphingosine or sphinganine) and their metabolites, important messengers involved in signal transduction leading to either cell survival or death. Free sphingoid bases are known apoptotic molecules whereas sphingosine 1-phosphate is protective. We previously reported that fumonisin B(1) caused sphingosine kinase (SPHK) induction along with the increase of serine palmitoyltransferase (SPT). Fumonisin B(1) also increased inducible nitric oxide synthase (iNOS) expression. In the current study we employed a mouse strain with the targeted deletion of iNOS gene (Nos-KO) to evaluate the role of nitric oxide (NO) on fumonisin B(1)-induced hepatotoxicity. The Nos-KO mice exhibited increased hepatotoxicity after subacute fumonisin B(1) exposure compared to their wild type counterparts, the liver regeneration was lower in Nos-KO compared to that in the WT mice. Increased hepatotoxicity in Nos-KO was not related to the extent of free sphingoid base accumulation after fumonisin B(1) treatment; however, it was accompanied by a lack of fumonisin B(1)-induced SPHK induction. The fumonisin B(1)-induced SPT was unaffected by lack of iNOS gene. Deletion of iNOS gene did not prevent fumonisin B(1)-dependent induction of inflammatory cytokines, namely tumor necrosis factor alpha, interferon gamma and interleukin-12. The lack of fumonisin B(1)-induced SPHK induction in Nos-KO was supported by a similar effect on phosphorylated metabolites of sphingoid bases; the equilibrium between sphingoid bases and their phosphates is maintained by SPHK. We therefore conclude that iNOS induction produced by fumonisin B(1) modulates SPHK activity; the lack of iNOS prevents generation of sphingosine 1-phosphate and deprives cells from its protective effects.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Carcinogens, Environmental; Cell Proliferation; Chemical and Drug Induced Liver Injury; Fumonisins; Hepatocytes; In Situ Nick-End Labeling; Interferon-gamma; Interleukin-12; Liver Diseases; Liver Regeneration; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide; Nitric Oxide Synthase Type II; Phosphotransferases (Alcohol Group Acceptor); RNA, Messenger; Sphingosine; Tumor Necrosis Factor-alpha; Weight Loss

Ceramide (CER) is an important mediator of lipotoxicity in the heart. It was found that Zucker diabetic fatty rats develop an age-dependent accumulation of myocardial CER leading to cardiomyocyte apoptosis. However, administration of peroxisome proliferator-activated receptor (PPAR) gamma agonist decreased the content of CER and prevented cardiomyocyte apoptosis [Zhou et al. Proc Natl Acad Sci USA 2000;97:1784-9]. These data suggest that PPARgamma activators affect myocardial CER metabolism. Therefore, the aim of our study was to examine the effects of pioglitazone, a selective PPARgamma agonist, on the content of CER and its metabolites and on the activity of key enzymes of CER metabolism in the heart. The experiments were conducted on rats fed either a standard chow (STD) or a high-fat diet (HFD) for 21 days. Each group was divided into two subgroups: control and treated with pioglitazone for 14 days. Surprisingly, administration of PPARgamma agonist significantly increased myocardial CER content in both STD and HFD rats. In the latter group an elevation in the amount of sphingomyelin was also observed. In STD rats pioglitazone treatment increased the activity of neutral sphingomyelinase and acid ceramidase. However, in HFD group the compound did not affect the activity of the aforementioned enzymes. Interestingly, the activity of serine palmitoyltransferase in both STD and HFD rats increased two-fold after pioglitazone treatment. We conclude that pioglitazone induced accumulation of CER in rat myocardium as a result of augmented CER synthesis de novo. However, in the STD group increased activity of neutral sphingomyelinase could also contributed to this effect.

Topics: Acid Ceramidase; Amidohydrolases; Animals; Catalytic Domain; Ceramidases; Ceramides; Dietary Fats; Heart; Hypoglycemic Agents; Lysophospholipids; Male; Myocardium; Palmitates; Pioglitazone; Rats; Rats, Wistar; Serine C-Palmitoyltransferase; Sphingomyelin Phosphodiesterase; Sphingomyelins; Sphingosine; Thiazolidinediones

It was shown that high-fat feeding of mice with cardiac-specific overexpression of peroxisome proliferator-activated receptor (PPAR) alpha but not wild type animals leads to the accumulation of ceramide (an important mediator of lipotoxicity) in the heart [Finck et al. 2003 Proc Natl Acad Sci USA]. To investigate the mechanism of this phenomenon we examined the effects of PPARalpha activation on ceramide metabolism in the myocardium. Male Wistar rats were fed either a standard chow or a high-fat diet. Each group was divided into two subgroups: control and treated with selective PPARalpha activator - WY-14643. In the rats fed on the standard diet WY-14643 did not affect the myocardial content of sphingomyelin and ceramide but reduced the content of sphinganine and sphingosine. It also inhibited the activity of neutral sphingomyelinase and increased the activity of acid sphingomyelinase, whereas the activity of ceramidases and serine palmitoyltransferase (SPT) remained stable. High-fat diet itself did not affect the content of the examined sphingolipids. However, it reduced the activity of sphingomyelinases and ceramidases having no effect on the activity of SPT. Administration of WY-14643 to this group significantly increased the content of myocardial free palmitate, ceramide, sphingomyelin and the activity of SPT. Our results demonstrated that PPARalpha activation modulates myocardial ceramide metabolism and leads to the accumulation of ceramide in the heart of the high-fat fed rats due to its increased synthesis de novo.

Topics: Amidohydrolases; Animals; Ceramidases; Ceramides; Dietary Fats; Enzyme Activation; Enzyme Inhibitors; Galactosylgalactosylglucosylceramidase; Heart; Lysophospholipids; Male; Myocardium; Palmitates; PPAR alpha; Pyrimidines; Rats; Rats, Wistar; Serine C-Palmitoyltransferase; Sphingomyelin Phosphodiesterase; Sphingomyelins; Sphingosine

Fumonisins (FNs) are ubiquitous contaminants of cereal grains. Fumonisin B(1) (FB(1)) was linked to several animal and human diseases. To validate FB(1) biomarkers for studying human disease risks, F344 rats were administered by gavage with either a single dose of 0, 10 or 25 mg FB(1)/kg body weight (BW) or repeated doses of 0, 1.0, or 2.5 mg FB(1)/kg BW/day for 5 weeks. FB(1) excretion and FB(1)-induced metabolic alterations of sphingolipids in rat urine, feces and serum were assessed. Dose-dependent urinary and fecal excretion of free FB(1) were found in both single-dose- and repeat-dose-treated rats. In the single-dose study, urinary sphinganine (Sa) to sphingosine (So) ratio (Sa/So) reached a maximum at day 7 for the high-dose group and at day 5 for the low-dose group, whereas serum Sa/So showed only marginal changes. In the repeat-dose study, urinary Sa/So was persistently elevated at 2 weeks, while serum Sa/So was unchanged. Time course changes of sphinganine 1-phosphate (SaP) and sphingosine 1-phosphate (SoP) were also examined. Although serum Sa/So and SaP/SoP ratios showed no signs of time- or dose-dependent changes, a 10-fold increase in urinary SaP/SoP was observed, suggesting that urinary SaP/SoP is a more sensitive biomarker for FB(1) exposure. The accumulation of SaP and SoP was evident in the time course of SaP/Sa and SoP/So, which may reflect activity changes of enzymes closely related to the metabolism and catabolism of SaP and SoP. These results provide concrete evidence towards the practical use of excreted FB(1), Sa/So and SaP/SoP as biomarkers of exposure to FNs.

Topics: Administration, Oral; Animals; Biomarkers; Carcinogens, Environmental; Dose-Response Relationship, Drug; Drug Administration Schedule; Food Contamination; Fumonisins; Humans; Lysophospholipids; Male; Mycotoxins; Random Allocation; Rats; Rats, Inbred F344; Sphingosine; Time Factors

Sa and the Sa/So ratio are very sensitive biomarkers of exposure to fumonisins in several species. We previously demonstrated that increases in Sa and in the Sa/So ratio in serum were less pronounced when ducks ingested fumonisins for more than 7 weeks than when animals were exposed for only 1-2 weeks [S.T. Tran, D. Tardieu, A. Auvergne, J.D. Bailly, R. Babilé, S. Durand, G. Benard, P. Guerre, Serum sphinganine and the sphinganine to sphingosine ratio as biomarker of dietary fumonisins during chronic exposure in ducks, Chem. Biol. Interact., in press]. The aim of this study was to investigate the kinetics of Sa and of the Sa/So in both liver and kidney of ducks that have been previously tested for Sa and the Sa/So ratio in serum. Analysis were performed on treatment days 0, 7, 14, 28 and 77 in five groups of ducks fed fumonisins obtained from an extract of Fusarium verticillioides culture material by daily gavage to obtain an exposure equal to 0, 2, 8, 32 and 128 mg FB1/kg feed. Sa and the Sa/So ratio in tissues were then correlated with Sa and the Sa/So ratio previously obtained in serum. The amounts on sphinganine 1-phosphate (Sa1P) and sphingosine1-phosphate (So1P) in the liver were also investigated. On day 7 of treatment, 2mg/kg FB1 in the feed were sufficient to increase Sa and the Sa/So ratio in liver (by 165 and 148%, respectively) and kidney (by 193 and 104%, respectively). At a rate of 128 mg/kg FB1 in the feed, a very high increase in Sa concentration was observed in both liver and kidney without mortality and/or signs of necrosis (respective increase of 2034 and 3768%). Although the precise mechanism of the resistance of ducks to fumonisin-induced hepatotoxicity is still uncertain, it might be linked to the rate at which the sphingoid bases sphinganine and sphingosine are converted to their 1-phosphate or other metabolite and eliminated from target tissues.

Topics: Administration, Oral; Animals; Biomarkers; Carcinogens, Environmental; Diet; Dose-Response Relationship, Drug; Ducks; Fumonisins; Kidney; Liver; Lysophospholipids; Mycotoxins; Organ Size; Sphingosine; Toxicity Tests

Dendritic cells (DC) are inducers of primary immune responses and represent an attractive vector for cancer immunotherapy. Sphingosine kinase (SphK) and its product sphingosine-1-phosphate (S1P) play an important role in the regulation of immune cells and cancer, affecting processes such as differentiation, growth or migration. We studied the role of SphK and S1P on migration of DC. RT-PCR showed mRNA expression of SphK in DC, declining from immature (iDC) to mature DC (mDC) to antigen-loaded mDC. Expression of S1P receptors was S1P(1) > S1P(2) = S1P(3), unrelated to maturation or antigen uptake. In transwell assays, iDC migrated towards SDF-1, MIP-1alpha, MCP and S1P, whereby S1P combined with a chemokine had a synergistic effect. mDC migrated towards 6Ckine and MIP-3beta, but not towards S1P. The SphK-inhibitor dihydro-sphingosine (DHS) reduced migration of iDC but not of mDC. In addition S1P(3)-inhibitor suramin inhibited DC migration in response to S1P. DHS had a reverse effect on endocytosis, enhancing the uptake of FITC dextran. We also observed an anti-apoptotic effect of S1P on mDC for the first time. This indicates that SphK/S1P may play a role in accumulation of peripheral iDC at the location of antigen and subsequent antigen-uptake. These findings may help to optimise DC-based cancer immunotherapy by modulation of SphK/S1P.

Topics: Apoptosis; Cell Movement; Cell Survival; Chemokines; Dendritic Cells; Endocytosis; Humans; Immunotherapy, Adoptive; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; RNA, Messenger; Sphingosine; Statistics, Nonparametric

Sphingosine-1-phosphate (S1P) and related compounds are important signaling molecules and are normal constituents of human plasma. So far, only a few methods exist for their determination specifically in plasma demanding radioactive agents, more or less time consuming extraction or derivatization procedures. Here, we describe a very simple, reliable, sensitive standard-addition method for the simultaneous determination of S1P, sphingosine (SPH), sphinganine (SAPH) and sphinganine-1-phosphate (SA1P) in human and rat plasma samples. After methanol precipitation of plasma samples the supernatants were directly assessed by liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS). HPLC analysis was done under gradient conditions using a C18 reversed phase column. The lower limit of quantification (LLOQ) was <10.2, <4.6, <1.9 and 0.57ng/ml for S1P, SPH, SAPH and SA1P, respectively. Variations in accuracy and intraday and interday precision were <15% over the range of calibration. All analytes were normal constituents both in human and rat plasma although the SA1P concentrations in a few rat plasma samples were below the lower limit of quantification. This validated method is suitable to generate new pharmacological findings by monitoring plasma concentrations of S1P and related compounds especially when low amounts of plasma samples are present (e.g. plasma samples from rodents).

Topics: Animals; Blood Chemical Analysis; Calibration; Chromatography, High Pressure Liquid; Humans; Lysophospholipids; Methanol; Rats; Reference Values; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization; Sphingosine; Time Factors

Fumonisin mycotoxicosis in pigs causes a decrease in mean aortic pressure, an increase in mean pulmonary arterial pressure, and increases in serum concentrations of sphinganine (3.2 microM) and sphingosine (1.4 microM). To determine a causal relationship between the hemodynamic changes and sphingolipid alterations, we examined the in vitro effects of sphinganine, sphingosine, and sphingosine-1-phosphate on porcine aortic and pulmonary arterial rings. Both sphinganine and sphingosine relaxed un-contracted and phenylephrine-contracted aortic rings at > or = 10 microM and > or = 1 microM, respectively. Sphingosine (> or = 10 microM) relaxed un-contracted and phenylephrine-contracted pulmonary arterial rings, whereas sphingosine-1-phosphate (10 microM) contracted pulmonary arterial rings. Sphingosine (3 microM) also impaired the contractile response of pulmonary artery rings to 60 mM KCl. The results suggested that the systemic hypotension caused by fumonisin is mediated, in part, by increases in serum sphinganine and sphingosine concentrations, and the pulmonary hypertension is mediated, in part, by increased sphingosine-1-phosphate concentrations.

Topics: Animals; Aorta, Thoracic; Dose-Response Relationship, Drug; In Vitro Techniques; Lysophospholipids; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Phenylephrine; Potassium Chloride; Pulmonary Artery; Sphingosine; Swine

The bioactive molecule sphingosine 1-phosphate (S1P) is abundantly stored in platelets and can be released extracellularly. However, although they have high sphingosine (Sph) kinase activity, platelets lack the de novo sphingolipid biosynthesis necessary to provide the substrates. Here, we reveal a generation pathway for Sph, the precursor of S1P, in human platelets. Platelets incorporated extracellular 3H-labeled Sph much faster than human megakaryoblastic cells and rapidly converted it to S1P. Furthermore, Sph formed from plasma sphingomyelin (SM) by bacterial sphingomyelinase (SMase) and neutral ceramidase (CDase) was rapidly incorporated into platelets and converted to S1P, suggesting that platelets use extracellular Sph as a source of S1P. Platelets abundantly express SM, possibly supplied from plasma lipoproteins, at the cell surface. Treating platelets with bacterial SMase resulted in Sph generation at the cell surface, conceivably by the action of membrane-bound neutral CDase. Simultaneously, a time-dependent increase in S1P levels was observed. Finally, we demonstrated that secretory acid SMase also induces S1P increases in platelets. In conclusion, our results suggest that in platelets, Sph is supplied from at least two sources: generation in the plasma followed by incorporation, and generation at the outer leaflet of the plasma membrane, initiated by cell surface SM degradation.

Topics: Acyltransferases; Amidohydrolases; Blood Platelets; Cell Line; Cell Membrane; Ceramidases; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Culture Media, Conditioned; Fumonisins; Glucosyltransferases; Humans; Hydrogen-Ion Concentration; Hydrolysis; Lipids; Lipoproteins; Lysophospholipids; Megakaryocytes; Models, Biological; Neutral Ceramidase; Plasmids; Serine C-Palmitoyltransferase; Sphingolipids; Sphingosine; Time Factors; Transfection; Transferases (Other Substituted Phosphate Groups)

Clostridium perfringens alpha-toxin induces hemolysis of rabbit erythrocytes through the activation of glycerophospholipid metabolism. Sheep erythrocytes contain large amounts of sphingomyelin (SM) but not phosphatidylcholine. We investigated the relationship between the toxin-induced hemolysis and SM metabolic system in sheep erythrocytes. Alpha-toxin simultaneously induced hemolysis and a reduction in the levels of SM and formation of ceramide and sphingosine 1-phosphate (S1P). N-Oleoylethanolamine, a ceramidase inhibitor, inhibited the toxin-induced hemolysis and caused ceramide to accumulate in the toxin-treated cells. Furthermore, dl-threo-dihydrosphingosine and B-5354c, isolated from a novel marine bacterium, both sphingosine kinase inhibitors, blocked the toxin-induced hemolysis and production of S1P and caused sphingosine to accumulate. These observations suggest that the toxin-induced activation of the SM metabolic system is closely related to hemolysis. S1P potentiated the toxin-induced hemolysis of saponin-permeabilized erythrocytes but had no effect on that of intact cells. Preincubation of lysated sheep erythrocytes with pertussis toxin blocked the alpha-toxin-induced formation of ceramide from SM. In addition, incubation of C. botulinum C3 exoenzyme-treated lysates of sheep erythrocytes with alpha-toxin caused an accumulation of sphingosine and inhibition of the formation of S1P. These observations suggest that the alpha-toxin-induced hemolysis of sheep erythrocytes is dependent on the activation of the SM metabolic system through GTP-binding proteins, especially the formation of S1P.

Topics: 4-Aminobenzoic Acid; ADP Ribose Transferases; Amidohydrolases; Animals; Bacterial Toxins; Botulinum Toxins; Calcium-Binding Proteins; Ceramidases; Chromatography, Thin Layer; Diglycerides; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Erythrocytes; Ethanolamines; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Hemolysis; Inositol 1,4,5-Trisphosphate; Lysophospholipids; Oleic Acids; para-Aminobenzoates; Pertussis Toxin; Phosphatidylcholines; Phosphorylcholine; Phosphotransferases (Alcohol Group Acceptor); Rabbits; Sheep; Sphingomyelins; Sphingosine; Time Factors; Toxins, Biological; Type C Phospholipases

Sphingolipids participate in membrane structure and signaling in neuronal cells, and an emerging strategy for control of gliomas is to inhibit growth and/or induce apoptosis using ceramide and ceramide analogs. Nonetheless, some sphingolipids (ceramides and sphingosine) induce and others (sphingosine 1-phosphate) inhibit apoptosis; therefore, when testing putative anti-cancer agents, it is critical to obtain precise knowledge of the types and quantities of not only the test compounds, but also their effects on endogenous species. Combination of liquid chromatography and tandem mass spectrometry affords a "metabolomic" profile of all of the intermediates of ceramide biosynthesis (3-ketosphinganine, sphinganine and dihydroceramides) and the direct products of ceramide metabolism (sphingomyelins and monohexosylceramides as well as sphingosine and sphingosine 1-phosphate). This method has been applied to four human glioma cell lines (LN18, LN229, LN319 and T98G), and differences in the amounts and types of sphingolipids were found. For example, LN229 and LN319 have approximately twice the sphingosine 1-phosphate of LN18 and T98G; LN229 and LN319 have more monohexosylceramides than lactosylceramides, whereas the opposite is the case for LN18 and T98G; and the fatty acyl chain distributions of the sphingolipids differ among the cell lines. The ability to obtain this type of "metabolomic" profile allows studies of how anti-cancer agents (especially sphingolipids and sphingolipid analogs) affect the amounts of these bioactive species, and may lead to a better understanding of the abnormal phenotypes of gliomas.

Topics: Astrocytoma; Cell Line, Tumor; Ceramides; Chromatography, High Pressure Liquid; Fatty Acids; Galactosylceramides; Glioblastoma; Glioma; Glucosylceramides; Humans; Lactosylceramides; Lysophospholipids; Molecular Structure; Spectrometry, Mass, Electrospray Ionization; Sphingolipids; Sphingomyelins; Sphingosine

Sphingosine kinase phosphorylates sphingosine to generate sphingosine 1-phosphate, a phospholipid that has been implicated in signaling by a number of transmembrane receptors and was recently shown to act as a ligand for a specific class of G protein-coupled receptors. Here we show that the G protein-coupled bradykinin B2 receptor activates sphingosine kinase leading to a time- and dose-dependent elevation of cellular sphingosine 1-phosphate levels that was blocked by the sphingosine kinase inhibitor dihydrosphingosine. Furthermore, increasing doses of this inhibitor partially affected the bradykinin-mediated ERK/MAP kinase activation and fully blocked the protein kinase C-independent component of the signaling pathway from the B2 receptor to the ERK/MAP kinase cascade. Overexpression of sphingosine kinase did not additionally increase the bradykinin-induced ERK/MAP kinase activity, indicating a permissive rather than activating role of sphingosine 1-phosphate in B2 receptor-mediated mitogenic signaling.

Topics: Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Humans; Lysophospholipids; Mitogen-Activated Protein Kinases; Phosphotransferases (Alcohol Group Acceptor); Receptor, Bradykinin B2; Receptors, Bradykinin; Signal Transduction; Sphingosine

Human hepatocytes usually are resistant to TNF-alpha cytotoxicity. In mouse or rat hepatocytes, repression of NF-kappaB activation is sufficient to induce TNF-alpha-mediated apoptosis. However, in both Huh-7 human hepatoma cells and Hc human normal hepatocytes, when infected with an adenovirus expressing a mutated form of IkappaBalpha (Ad5IkappaB), which almost completely blocks NF-kappaB activation, >80% of the cells survived 24 h after TNF-alpha stimulation. Here, we report that TNF-alpha activates other antiapoptotic factors, such as sphingosine kinase (SphK), phosphatidylinositol 3-kinase (PI3K), and Akt kinase. Pretreatment of cells with N,N-dimethylsphingosine (DMS), an inhibitor of SphK, or LY 294002, an inhibitor of PI3K that acts upstream of Akt, increased the number of apoptotic cells induced by TNF-alpha in Ad5IkappaB-infected Huh-7 and Hc cells. TNF-alpha-induced activations of PI3K and Akt were inhibited by DMS. In contrast, exogenous sphingosine 1-phosphate, a product of SphK, was found to activate Akt and partially rescued the cells from TNF-alpha-induced apoptosis. Although Akt has been reported to activate NF-kappaB, DMS and LY 294002 failed to prevent TNF-alpha-induced NF-kappaB activation, suggesting that the antiapoptotic effects of SphK and Akt are independent of NF-kappaB. Furthermore, apoptosis mediated by Fas ligand (FasL) involving Akt activation also was potentiated by DMS pretreatment in Hc cells. Sphingosine 1-phosphate administration partially protected cells from FasL-mediated apoptosis. These results indicate that not only NF-kappaB but also SphK and PI3K/Akt are involved in the signaling pathway(s) for protection of human hepatocytes from the apoptotic action of TNF-alpha and probably FasL.

Topics: Adenoviridae; Adjuvants, Immunologic; Apoptosis; Caspases; Cell Line; DNA Fragmentation; Enzyme Activation; Fas Ligand Protein; fas Receptor; Hepatocytes; Humans; I-kappa B Proteins; Ligands; Lysophospholipids; Membrane Glycoproteins; NF-kappa B; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphotransferases (Alcohol Group Acceptor); Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Sphingosine; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Formation of sphingosine-1-phosphate (SPP) by sphingosine kinase serves as a signalling pathway for various membrane receptors. Here, we show that membrane depolarisation is another mechanism by which this pathway can be activated. Formation of [(3)H]SPP as well as levels of endogenous SPP were rapidly and transiently increased in PC12 pheochromocytoma cells depolarised with high KCl. Time course and maximum were similar to those induced by bradykinin. Depolarisation-induced SPP production was also observed in RINm5F insulinoma cells, dependent on extracellular Ca(2+) and fully suppressed by verapamil, thus apparently caused by Ca(2+) influx via voltage-gated Ca(2+) channels. Studies with sphingosine kinase inhibitors and overexpression of sphingosine kinase revealed a partial contribution of this pathway to depolarisation-induced noradrenaline release and Ca(2+) increase.

Topics: Animals; Bradykinin; Calcium Channels; Calcium Signaling; Cell Membrane; Lysophospholipids; Norepinephrine; PC12 Cells; Phosphotransferases (Alcohol Group Acceptor); Potassium Chloride; Rats; Recombinant Proteins; Sphingosine; Verapamil

Sphingosine inhibits the activity of the skeletal muscle Ca2+ release channel (ryanodine receptor) and is a noncompetitive inhibitor of [3H]ryanodine binding (Needleman et al., Am. J. Physiol. 272, C1465-1474, 1997). To determine the contribution of other sphingolipids to the regulation of ryanodine receptor activity, several sphingolipid bases were assessed for their ability to alter [3H]ryanodine binding to sarcoplasmic reticulum (SR) membranes and to modulate the activity of the Ca2+ release channel. Three lipids, N,N-dimethylsphingosine, dihydrosphingosine, and phytosphingosine, inhibited [3H]ryanodine binding to both skeletal and cardiac SR membranes. However, the potency of these three lipids and sphingosine was lower in rabbit cardiac membranes when compared to rabbit skeletal muscle membranes and when compared to sphingosine. Like sphingosine, the lipids inhibited [3H]ryanodine binding by greatly increasing the rate of dissociation of bound [3H]ryanodine from SR membranes, indicating that these three sphingolipid bases were noncompetitive inhibitors of [3H]ryanodine binding. These bases also decreased the activity of the Ca2+ release channel incorporated into planar lipid bilayers by stabilizing a long closed state. Sphingosine-1-PO4 and C6 to C18 ceramides of sphingosine had no significant effect on [3H]ryanodine binding to cardiac or skeletal muscle SR membranes. Saturation of the double bond at positions 4-5 decreased the ability of the sphingolipid bases to inhibit [3H]ryanodine binding 2-3 fold compared to sphingosine. In summary, our data indicate that other endogenous sphingolipid bases are capable of modulating the activity of the Ca2+ release channel and as a class possess a common mechanism of inhibition.

Topics: Animals; Calcium Channel Blockers; In Vitro Techniques; Kinetics; Lipid Bilayers; Lysophospholipids; Muscle, Skeletal; Myocardium; Rabbits; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sphingolipids; Sphingosine

Sphingosine-1-phosphate (SPP) produced from sphingosine by sphingosine kinase has recently been reported to act as intracellular second messenger for a number of plasma membrane receptors. In the present study, we investigated whether the sphingosine kinase/SPP pathway is involved in cellular signaling of the Gi protein-coupled formyl peptide receptor in myeloid differentiated human leukemia (HL-60) cells. Receptor activation resulted in rapid and transient production of SPP by sphingosine kinase, which was abolished after pertussis toxin treatment. Direct activation of heterotrimeric G proteins by AlF4- also rapidly increased SPP formation in intact HL-60 cells. In cytosolic preparations of HL-60 cells, sphingosine kinase activity was stimulated by the stable GTP analog, guanosine 5'-O-(3-thiotriphosphate). Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,N-dimethylsphingosine did not affect phospholipase C stimulation and superoxide production but markedly inhibited receptor-stimulated Ca2+ mobilization and enzyme release. We conclude that the formyl peptide receptor stimulates through Gi-type G proteins SPP production by sphingosine kinase, that the enzyme is also stimulated by direct G protein activation, and that the sphingosine kinase/SPP pathway apparently plays an important role in chemoattractant signaling in myeloid differentiated HL-60 cells.

Topics: Calcium; Enzyme Inhibitors; GTP-Binding Proteins; HL-60 Cells; Humans; Lysophospholipids; N-Formylmethionine Leucyl-Phenylalanine; Phosphotransferases (Alcohol Group Acceptor); Receptors, Formyl Peptide; Receptors, Immunologic; Receptors, Peptide; Second Messenger Systems; Signal Transduction; Sphingosine; Superoxides; Type C Phospholipases

Sphingosine 1-phosphate (Sph-1-P) is considered to play a dual role in cellular signaling, acting intercellularly as well as intracellularly. In this study, we examined the role of Sph-1-P as a signaling molecule in human platelets, using DL-threo-dihydrosphingosine (DHS) and N,N-dimethylsphingosine (DMS), inhibitors of Sph kinase and protein kinase C. Both DMS and DL-threo-DHS were confirmed to be competitive inhibitors of Sph kinase obtained from platelet cytoplasmic fractions. In intact platelets labeled with [3H]Sph, stimulation with 12-O-tetradecanoylphorbol 13-acetate or thrombin did not affect [3H]-Sph-1-P formation. While both DMS and DL-threo-DHS inhibited not only [3H]Sph-1-P formation but also protein kinase C-dependent platelet aggregation, staurosporine, a potent protein kinase inhibitor, only inhibited the protein kinase C-dependent reaction. Hence, it is unlikely that Sph kinase activation and the resultant Sph-1-P formation are mediated by protein kinase C in platelets. Furthermore, Ca2+ mobilization induced by platelet agonists that act on G protein-coupled receptor was not affected by DMS or DL-threo-DHS. Our results suggest that Sph-1-P does not mediate intracellular signaling, including Ca2+ mobilization, in platelets.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adult; Blood Platelets; Calcium Signaling; Enzyme Inhibitors; GTP-Binding Proteins; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Signal Transduction; Sphingosine; Thrombin

Recent studies have demonstrated that ceramide plays an important role as a second messenger in many kinds of cells. However, it is not known whether apoptosis of and bone resorption by mature osteoclasts are mediated via sphingomyelinase (SMase) and ceramide. Thus, we examined the possible involvement of SMase and ceramide in the induction of apoptosis in and bone resorption by rabbit mature osteoclasts. SMase and C2-ceramide inhibited strongly F-actin ring formation of and bone resorption by the osteoclasts. However, the osteoclast apoptosis was not induced by C2-ceramide. The ceramide inhibition of the bone resorption was suppressed by DL-threodihydrosphingosine, an inhibitor of sphingosine kinase. In addition, we observed that sphingosine-1-phosphate is able to inhibit bone resorption by the osteoclasts. These results suggest an important role of the sphingomyelin pathway in bone resorption by rabbit mature osteoclasts.

Topics: Actins; Animals; Animals, Newborn; Apoptosis; Bone Resorption; Cells, Cultured; Enzyme Inhibitors; Lysophospholipids; Osteoclasts; Rabbits; Sphingomyelin Phosphodiesterase; Sphingosine

Formation of inositol 1,4,5-trisphosphate (IP3) by phospholipase C (PLC) with subsequent release of Ca2+ from intracellular stores, is one of the major Ca2+ signalling pathways triggered by G-protein-coupled receptors (GPCRs). However, in a large number of cellular systems, Ca2+ mobilization by GPCRs apparently occurs independently of the PLC-IP3 pathway, mediated by an as yet unknown mechanism. The present study investigated whether sphingosine kinase activation, leading to production of sphingosine-1-phosphate (SPP), is involved in GPCR-mediated Ca2+ signalling as proposed for platelet-derived growth factor and FcepsilonRI antigen receptors. Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,N-dimethylsphingosine markedly inhibited [Ca2+]i increases elicited by m2 and m3 muscarinic acetylcholine receptors (mAChRs) expressed in HEK-293 cells without affecting mAChR-induced PLC stimulation. Activation of mAChRs rapidly and transiently stimulated production of SPP in HEK-293 cells. Finally, intracellular injection of SPP induced a rapid and transient Ca2+ mobilization in HEK-293 cells which was not antagonized by heparin. We conclude that mAChRs utilize the sphingosine kinase-SPP pathway in addition to PLC-IP3 to mediate Ca2+ mobilization. As Ca2+ signalling by various, but not all, GPCRs in different cell types was likewise attenuated by the sphingosine kinase inhibitors, we suggest a general role for sphingosine kinase, besides PLC, in mediation of GPCR-induced Ca2+ signalling.

Topics: Animals; Calcium; Cattle; Cell Line; Enzyme Activation; Enzyme Inhibitors; GTP-Binding Proteins; Humans; Lysophospholipids; Microinjections; Phosphotransferases (Alcohol Group Acceptor); Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Bradykinin; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophosphatidic Acid; Receptors, Muscarinic; Signal Transduction; Sphingosine; Tumor Cells, Cultured

Sphingosine kinase (SK) catalyses the phosphorylation of sphingosine to generate sphingosine 1-phosphate, which is a second messenger involved in the proliferative responses of mammalian cells. Although the yeast Saccharomyces cerevisiae has similar phosphorylated sphingoid bases which appear to be involved in growth regulation and the response to stress, SK activity had not been previously demonstrated in yeast. In this study, an in vitro system was set up to characterize yeast SK activity. Activity was detected in the cytosol at neutral pH and 37 degreesC. Yeast SK phosphorylated the sphingoid bases sphingosine, dihydrosphingosine and phytosphingosine. (d,l)-threo-dihydrosphingosine, an inhibitor of mammalian SK, did not inhibit the yeast enzyme. Unique properties of yeast SK were an optimal temperature of 43 degreesC, and in vivo activation during nutrient deprivation. Spontaneous mutants with diminished SK activity were isolated utilizing a screen for resistance to sphingosine in a sphingosine-phosphate-lyase deletion background. Abnormal growth and heat sensitivity were observed in these mutants. These findings suggest that SK may function as a stress-response protein in yeast.

Topics: Cell Division; Cell Survival; Enzyme Activation; Enzyme Inhibitors; Heat-Shock Proteins; Hydrogen-Ion Concentration; Kinetics; Lysophospholipids; Mutation; Phenotype; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Saccharomyces cerevisiae; Sphingosine; Spores; Temperature

The phosphorylated derivative of sphingosine, sphingosine-1-phosphate, is a short-living metabolite of ultimate ceramide degradation and was shown to act as an intracellular signaling molecule, stimulating cell proliferation in quiescent Swiss 3T3 fibroblasts and inducing the release of calcium from intracellular stores (Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell. Biol. 114, 155-167). In the present study, 24-h treatment of Swiss 3T3 fibroblasts with the synthetic sphingosine analogue cis-4-methylsphingosine resulted in proliferation of quiescent Swiss 3T3 fibroblasts that was 3-fold stronger than that of equimolar sphingosine-1-phosphate. The phosphorylated derivative of cis-4-methylsphingosine accumulated drastically in the cells. Simultaneous treatment with the sphingosine kinase inhibitor L-threo-sphinganine reduced both the amount of phosphorylated cis-4-methylsphingosine and cell proliferation induced by this compound by about 50%, indicating that the phosphorylated derivative mediated the proliferative stimulus. The mitogenic effect of cis-4-methylsphingosine was associated with a mobilization of intracellular calcium in Swiss 3T3 fibroblasts that was similar to that induced by sphingosine-1-phosphate. The results demonstrate that the phosphorylated derivative of cis-4-methylsphingosine mimics the previously reported mitogenic action of sphingosine-1-phosphate in Swiss 3T3 cells, and the stronger effect most likely corresponds to the unusual accumulation of this compound.

Topics: 3T3 Cells; Animals; Apoptosis; Calcium; Cell Division; Fibroblasts; Isomerism; Lysophospholipids; Mice; Mitogens; Molecular Mimicry; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

Platelet-derived growth factor (PDGF) and serum, but not epidermal growth factor (EGF), stimulated sphingosine kinase activity in Swiss 3T3 fibroblasts and increased intracellular concentrations of sphingosine 1-phosphate (SPP), a sphingolipid second messenger (Olivera, A., and Spiegel, S. (1993) Nature 365, 557-560). We report herein that DL-threo-dihydrosphingosine (DHS), a competitive inhibitor of sphingosine kinase that prevents PDGF-induced SPP formation, specifically inhibited the activation of two cyclin-dependent kinases (p34(cdc2) kinase and Cdk2 kinase) induced by PDGF, but not by EGF. SPP reversed the inhibitory effects of DHS on PDGF-stimulated cyclin-dependent kinases and DNA synthesis, demonstrating that the DHS effects were mediated via inhibition of sphingosine kinase. DHS also markedly reduced PDGF-stimulated but not EGF-stimulated mitogen-activated protein kinase activity and DNA binding activity of activator protein-1. Examination of the early signaling events of PDGF action revealed that DHS did not affect PDGF-induced autophosphorylation of the growth factor receptor or phosphorylation of the SH2/SH3 adaptor protein Shc and its association with Grb2. This sphingosine kinase inhibitor did not abrogate activation of phosphatidylinositol 3-kinase by PDGF. In agreement, treatment with SPP had no effect on these responses but did, however, potently stimulate phosphorylation of Crk, another SH2/SH3 adaptor protein. Moreover, DHS inhibited PDGF-stimulated, but not EGF-stimulated, Crk phosphorylation. Thus, regulation of sphingosine kinase activity defines divergence in signal transduction pathways of PDGF and EGF receptors leading to mitogen-activated protein kinase activation.

Topics: 3T3 Cells; Animals; Calcium-Calmodulin-Dependent Protein Kinases; CDC2 Protein Kinase; CDC2-CDC28 Kinases; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; DNA; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Kinetics; Lysophospholipids; Mice; Phosphatidylinositol 3-Kinases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Platelet-Derived Growth Factor; Protein Serine-Threonine Kinases; Receptors, Platelet-Derived Growth Factor; Second Messenger Systems; Signal Transduction; Sphingosine; Transcription Factor AP-1

Calcium mobilization through antigen receptors, including high-affinity IgE receptors (Fc epsilon RI), is thought to be mediated by inositol-1,4,5-trisphosphate production (InsP3). Here we show that antigen clustering of Fc epsilon RI on the rat mast-cell line (RBL-2H3) activates a sphingosine kinase (SK) and produces sphingosine-1-phosphate (S1P), and alternative second messenger for intracellular calcium mobilization. The sphingosine analogue, D-L-threo-dihydrosphingosine (DHS), inhibits the SK enzyme competitively with a dissociation constant, K1, of 5 to 18 microM. This inhibition substantially suppresses the Fc epsilon RI-mediated calcium signal, but leaves intact the syk tyrosine kinase activation and the small InsP3 production. The entire InsP3-dependent pathway activated by a transfected G-protein coupled receptor, used here as a positive control, also remained intact. Thus Fc epsilon RI principally utilizes a SK pathway to mobilize calcium.

Topics: 3T3 Cells; Animals; Calcium; Cell Line; Cytosol; Enzyme Activation; Enzyme Inhibitors; Genistein; Humans; Inositol 1,4,5-Trisphosphate; Isoflavones; Lysophospholipids; Mice; Phosphotransferases (Alcohol Group Acceptor); Protein-Tyrosine Kinases; Rats; Receptors, IgE; Receptors, Muscarinic; Signal Transduction; Sphingosine

The B subunit of cholera toxin, which binds specifically to ganglioside GM1, is mitogenic for quiescent Swiss 3T3 fibroblasts. Recently, sphingolipids metabolites, ceramide, sphingosine and sphingosine-1-phosphate, have been implicated as second messengers in cell growth regulation and differentiation. In this paper, we examined the possibility that interaction of the B subunit with membrane GM1 leads to alterations in metabolism of glycosphingolipids and that increased levels of sphingolipids metabolites may mediate the biological effects of the B subunit. While the B subunit did not induce a change in the level of ceramide or sphingosine, the level of sphingosine-1-phosphate was rapidly and transiently increased. The B subunit also transiently activated cytosolic sphingosine kinase activity, which catalyzes the phosphorylation of the primary hydroxyl group of sphingosine to produce sphingosine-1-phosphate. To determine whether the increase in sphingosine-1-phosphate level plays a role in B subunit-induced mitogenicity, we used a competitive inhibitor of sphingosine kinase, D,L-threo-dihydrosphingosine. D,L-thereo-Dihydrosphingosine not only inhibited B subunit-induced DNA synthesis by 26%, it also reduced its ability to stimulate DNA-binding activity of the transcription factor AP-1. This sphingosine kinase inhibitor also inhibited B subunit-induced increases in the activity of cell cycle-regulated, cyclin-dependent serine/threonine kinases, cdk2 and p34cdc2. These findings suggest that sphingosine-1-phosphate may play a role in the signal transduction pathways activated by binding of the B subunit to endogenous ganglioside GM1.

Topics: 3T3 Cells; Animals; Antibodies; Binding, Competitive; CDC2 Protein Kinase; CDC2-CDC28 Kinases; Cell Division; Ceramides; Cholera Toxin; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; DNA; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; G(M1) Ganglioside; Lysophospholipids; Mice; Mitogens; Peptide Fragments; Phosphotransferases (Alcohol Group Acceptor); Platelet-Derived Growth Factor; Protein Serine-Threonine Kinases; Sphingolipids; Sphingosine; Tetradecanoylphorbol Acetate; Transcription Factor AP-1

The syntheses of D-erythro-sphingosine-1-phosphate and DL-erythro-sphinganine-1-phosphate are described starting from the commercially available D-erythro-sphingosine and DL-erythro-sphinganine. The phosphate group is introduced via phosphoramidite chemistry using bis(2-cyanoethyl)-N,N-diisopropylamino-phosphoramidite as the monophosphorylating reagent. The procedure generates the phosphorylated sphingoid bases in three steps and 32-39% overall yields.

Topics: Amides; Lysophospholipids; Molecular Structure; Phosphoric Acids; Phosphorylation; Sphingosine

Growth signalling networks that use glycerophospholipid metabolites as second messengers have been well characterized, but less is known of the second messengers derived from sphingolipids, another major class of membrane lipids. A tantalizing link between sphingolipids and cellular proliferation has emerged from the discovery that the sphingolipid metabolites sphingosine and sphingosine-1-phosphate stimulate growth of quiescent Swiss 3T3 fibroblasts by a pathway that is independent of protein kinase C. Sphingosine-1-phosphate is rapidly produced from sphingosine and may mediate its biological effects. Furthermore, sphingosine-1-phosphate triggers the dual signal transduction pathways of calcium mobilization and activation of phospholipase D, prominent events in the control of cellular proliferation. Here we report that activation of sphingosine kinase and the formation of sphingosine-1-phosphate are important in the signal transduction pathways activated by the potent mitogens platelet-derived growth factor (PDGF) and fetal calf serum (FCS).

Topics: 3T3 Cells; Animals; Cattle; Cell Division; DNA; Enzyme Activation; Epidermal Growth Factor; Fetal Blood; Humans; Lysophospholipids; Mice; Phosphotransferases (Alcohol Group Acceptor); Platelet-Derived Growth Factor; Second Messenger Systems; Sphingosine