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PubMed1995

Activation of a retinoblastoma-protein-dependent pathway by sphingosine.

The retinoblastoma protein (Rb) is a tumour suppressor that is activated by dephosphorylation the function of which appears to be mediated, at least partly, through the inhibition of several transcription factors, such as E2F. We have recently described sphingosine, a sphingolipid-breakdown product, as a potent and specific inducer of Rb dephosphorylation resulting in inhibition of cell growth and a specific arrest in the G0/G1 phase of the cell cycle. Here we examine the role of Rb and its interaction with E2F in mediating the effects of sphingosine on cell growth. Sphingosine potently inhibited growth of lymphoblastic leukaemic cells, Molt-4, at submicromolar concentrations but showed a 10-fold reduced potency in inhibiting growth of retinoblastoma cells, WERI-Rb-1, which lack functional Rb. In addition, sphingosine's ability to inhibit growth of mink lung epithelial cells was significantly attenuated in cells overexpressing simian virus 40 large T antigen which binds Rb and related proteins. Sphingosine treatment of Molt-4 cells, but not WERI-Rb-1 cells, resulted in the loss of the specific E2F bands produced by the interaction of E2F and its specific DNA sequence element on gel-shift assays. The concentration (submicromolar) and kinetics (4 h) of sphingosine treatment were identical with those required to induce Rb dephosphorylation. In addition, at similar concentrations, sphingosine caused c-myc down-regulation in Molt-4 cells starting at 6 h after treatment. These results demonstrate that activation of Rb by sphingosine leads to sequestration of E2F by the active (hypophosphorylated) form of Rb with the resultant loss of its DNA-binding and genetranscribing abilities. A functional Rb is required to mediate the specific effects of sphingosine on growth arrest.

Amines↗
PubMed1996

Biosynthesis of N-acetylsphingosine by platelet-activating factor: sphingosine CoA-independent transacetylase in HL-60 cels.

We have previously identified a novel CoA-independent transacetylase in the membrane fraction of HL-60 cells that transfers the acetate group from platelet activating factor (PAF) to a variety of lysophospholipid acceptors (Lee, T.-c., Uemura, Y., and Snyder, F. (1992) J. Biol. Chem. 267, 19992-20001). In the present study, we demonstrate that a similar transacetylase can transfer the acetate group from PAF to sphingosine forming N-acetylsphingosine (C2-ceramide). The chemical structure of the reaction product, C3-ceramide, was established by its identical Rf value with authentic C2-ceramide standard on thin-layer plate, sensitivity to acid treatment, resistance to alkaline hydrolysis, and ability to form the C2-ceramide dibenzoate derivative. Nonspecific transfer of the acetate from PAF to sphingosine in the absence of enzyme and nonlinearity of the reaction rates were rectified by complexing sphingosine to bovine serum albumin in a 1:1 molar ratio. Under these conditions, the apparent Km for PAF is 5.4 microM, which is in the same range as the Km (12.0 microM) when lysoplasmalogen is the acetate acceptor. PAF:sphingosine transacetylase has a narrow substrate specificity and strict stereochemical configuration requirements. Ceramide, sphingosylphosphocholine, stearylamine, sphingosine 1-phosphate, or sphingomyelin are not substrates, whereas sphinganine has a limited capacity to accept the acetate from PAF. Also, only the naturally synthesized D-erythroisomer but not the synthetic L-erythro-, D-threo-, or L-threosiomers of sphingosine can serve as a substrate. PAF transacetylase activity is widely distributed among several tissues and may involve histidine and cysteine for its catalytic activity due to inhibitory effects to the enzyme by diethyl pyrocarbonate and N-ethylmaleimide, respectively. C2-ceramide is produced via PAF:sphingosine transacetylase, and physiological levels of C2-ceramide are detected in both undifferentiated and differentiated intact HL-60 cells. Collectively, because C2-ceramide has many biological activities that differ from that of PAF and sphingosine, the CoA-independent, PAF-dependent transacetylase serves as a modifier of PAF, and sphingosine functions by generating a variant lipid mediator, C2-ceramide.

Acetyltransferases↗
PubMed1997

Sphingosine mediates the immediate negative inotropic effects of tumor necrosis factor-alpha in the adult mammalian cardiac myocyte.

To determine whether activation of the neutral sphingomyelinase pathway was responsible for the immediate (<30 min) negative inotropic effects of tumor necrosis factor-alpha (TNF-alpha), we examined sphingosine levels in diluent and TNF-alpha-stimulated cardiac myocytes. TNF-alpha stimulation of adult feline cardiac myocytes provoked a rapid (<15 min) increase in the hydrolysis of [14C]sphingomyelin in cell-free extracts, as well as an increase in ceramide mass, consistent with cytokine-induced activation of the neutral sphingomyelinase pathway. High performance liquid chromatographic analysis of lipid extracts from TNF-alpha-stimulated cardiac myocytes showed that TNF-alpha stimulation produced a rapid (<30 min) increase in free sphingosine levels. Moreover, exogenous D-sphingosine mimicked the effects of TNF-alpha on intracellular calcium homeostasis, as well as the negative inotropic effects of TNF-alpha in isolated contracting myocytes; time course studies showed that exogenous D-sphingosine produced abnormalities in cell shortening that were maximal at 5 min. Finally, blocking sphingosine production using an inhibitor of ceramidase, n-oleoylethanolamine, completely abrogated the negative inotropic effects of TNF-alpha in isolated contracting cardiac myocytes. Additional studies employing biologically active ceramide analogs and sphingosine 1-phosphate suggested that neither the immediate precursor of sphingosine nor the immediate metabolite of sphingosine, respectively, were likely to be responsible for the immediate negative inotropic effects of TNF-alpha. Thus, these studies suggest that sphingosine mediates the immediate negative inotropic effects of TNF-alpha in isolated cardiac myocytes.

Animals↗
PubMed2005

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

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

Aldehyde-Lyases↗
PubMed2007

Sphingosine kinase-1--a potential therapeutic target in cancer.

Sphingolipid metabolites play critical functions in the regulation of a number of fundamental biological processes including cancer. Whereas ceramide and sphingosine mediate and trigger apoptosis or cell growth arrest, sphingosine 1-phosphate promotes proliferation and cell survival. The delicate equilibrium between the intracellular levels of each of these sphingolipids is controlled by the enzymes that either produce or degrade these metabolites. Sphingosine kinase-1 is a crucial regulator of this two-pan balance, because it produces the prosurvival sphingosine 1-phosphate, and reduces the content of both ceramide and sphingosine, the proapoptotic sphingolipids. Sphingosine kinase-1 controls the levels of sphingolipids having opposite effects on cell survival/death, its gene was found to be of oncogenic nature, its mRNA is overexpressed in many solid tumors, its overexpression protects cells from apoptosis and its activity is decreased during anticancer treatments. Therefore, sphingosine kinase-1 appears to be a target of interest for therapeutic manipulation via its pharmacological inhibition. Strategies to kill tumor cells by increasing their ceramide and/or sphingosine content while blocking sphingosine 1-phosphate generation should have a favorable therapeutic index.

Animals↗
PubMed1997

Modulation of skeletal muscle Ca2(+)-release channel activity by sphingosine.

The effect of D-erythro-C18-sphingosine (sphingosine) and related compounds on the Ca(2+)-release channel (ryanodine binding protein) was examined on rabbit skeletal muscle membranes, on the purified ryanodine binding protein, and on the channel reconstituted into planar lipid bilayers. Sphingosine inhibited [3H]ryanodine binding to sarcoplasmic reticulum (SR) membranes in a dose-dependent manner similar to published results (R. A. Sabbadini, R. Betto, A. Teresi, G. Fachechi-Cassano, and G. Salviati. J. Biol. Chem. 267: 15475-15484, 1992). The sphingolipid also inhibited [3H]ryanodine binding to the purified ryanodine binding protein. Our results demonstrate that the inhibition of [3H]ryanodine binding by sphingosine is due to an increased rate of dissociation of bound [3H]ryanodine from SR membranes and a decreased rate of association of [3H]ryanodine to the high-affinity site. Unlike other modulators of the Ca(2+)-release channel, sphingosine can remove bound [3H]ryanodine from the high-affinity site within minutes. Sphingosine increased the rate of dissociation of [3H]ryanodine bound to a solubilized proteolytic fragment derived from the carboxy terminus of the ryanodine binding protein (cleavage at Arg4475). Sphingosine also inhibited the activity of the Ca(2+)-release channel incorporated into planar lipid bilayers. Taken together, the data provide evidence for a direct effect of sphingosine on the Ca(2+)-release channel. Sphingosine is a noncompetitive inhibitor at the high-affinity ryanodine binding site, and it interacts with a site between Arg4475 and the carboxy terminus of the Ca(2+)-release channel.

Animals↗
PubMed2004

Modulation by sphingosine of phosphorylation of substrate proteins by protein kinase C in nuclei from cow mammary gland.

Protein kinase C (PKC) is an enzyme activated by diacylglycerols such as 1-oleoyl-2-acetyl-sn-glycerol (OAG), phospholipids (in particular phosphatidylserine; PS) and Ca2+, which regulate a wide variety of intracellular functions by phosphorylating multiple substrate proteins and enzymes. The effect of sphingosine, the backbone moiety of sphingolipids, on PKC activity and phosphorylation of endogenous proteins catalyzed by PKC was investigated in nuclei of cow mammary gland. Sphingosine inhibited nuclear PKC activity when lysine-rich histone was used as the substrate. The sphingosine inhibition of the PKC activity was reversed by the excess addition of PS, but not by OAG or Ca2+. Several nuclear proteins, including 56-kDa, 43-kDa, 38-kDa and 36-kDa proteins, were shown to be substrates for PKC. Of the substrate proteins, the 38-kDa and 36-kDa proteins were identified as annexin I, the Ca2+/phospholipid-binding protein; the 56-kDa and 43-kDa proteins have not yet been identified. Sphingosine inhibited phosphorylation of the 56-kDa protein and the 36-kDa annexin I, whereas it enhanced that of the 43-kDa protein. The 38-kDa annexin I species was unaffected by sphingosine. As with the PKC activity, inhibition by sphingosine of phosphorylation of the 56-kDa protein and 36-kDa annexin I was reversed by the excess addition of PS, but not by OAG or Ca2+. In addition, by the excess addition of PS and not by OAG or Ca2+, the sphingosine-enhanced phosphorylation of the 43-kDa protein was reversed and returned to near the level in the absence of sphingosine. It is suggested that sphingosine is involved in the regulation of PKC-dependent phosphorylation in the nucleus by modulating the association of PKC or its substrates, particularly annexin I, with membrane phospholipids in cow mammary gland.

Animals↗
PubMed1990

Sphingosine stimulates cellular proliferation via a protein kinase C-independent pathway.

Sphingosine, a metabolite of membrane sphingolipids, is generally considered to be cytotoxic for a variety of cell types. However, we have found that sphingosine at low concentrations stimulates DNA synthesis and acts synergistically with known growth factors to induce proliferation of quiescent Swiss 3T3 fibroblasts. Structurally related analogs of sphingosine, such as N-stearoylsphingosine and other long chain aliphatic amines, had no mitogenic effects, suggesting that sphingosine did not induce nonspecific membrane perturbations. Sphingosine, which has been proposed to be a physiological inhibitor of protein kinase C, also markedly potentiates the mitogenic effect of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Sphingosine still stimulates DNA synthesis in cells made protein kinase C deficient by prolonged treatment with phorbol ester. At mitogenic concentrations, sphingosine does not bind to protein kinase C as shown by its lack of effect on phorbol dibutyrate binding. Only at higher concentrations, in the cytotoxic range, was there a displacement of phorbol dibutyrate from its cellular-binding sites. In contrast to sphingosine, H-7, a known inhibitor of protein kinase C, inhibited the mitogenic response to TPA and the TPA-induced phosphorylation of the 80 kDa cellular substrate of protein kinase C. Our results suggest that sphingosine may play an important role as a positive regulator of cell growth acting in a fundamentally different, protein kinase C-independent pathway.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Sphingosine-1-phosphate, a putative second messenger, mobilizes calcium from internal stores via an inositol trisphosphate-independent pathway.

Sphingosine-1-phosphate, a metabolite of sphingolipids which has previously been shown to stimulate DNA synthesis and cell division in quiescent cultures of Swiss 3T3 fibroblasts (Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell Biol. 114, 155-167), induced a transient increase in intracellular free calcium independent of extracellular calcium. The increase in calcium was completely abolished when intracellular calcium pools were depleted with thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase. The dose-response for calcium release induced by sphingosine-1-phosphate correlated closely with the concentration required for stimulation of DNA synthesis. The magnitude of the calcium response decreased with successive challenges, although sphingosine-1-phosphate did not attenuate the responses to either bradykinin or ionomycin. Conversely, prior stimulation of the cells with bradykinin had no effect on the sphingosine-1-phosphate-induced calcium signal. Although sphingosine-1-phosphate increased inositol (1,4,5)-trisphosphate levels, complete inhibition of inositol phosphate formation by pretreatment with 12-O-tetradecanoylphorbol-13-acetate did not block sphingosine-1-phosphate-mediated calcium responses. Moreover, in permeabilized cells, heparin, an inositol (1,4,5)-trisphosphate antagonist, blocked Ca2+ release induced by inositol (1,4,5)-trisphosphate, but did not significantly alter the Ca2+ release induced by sphingosine-1-phosphate. Sphingosine-1-phosphate did not stimulate the release of arachidonic acid, another signaling molecule known to elevate [Ca2+]i without inositol lipid turnover or calcium influx. Our data suggest that sphingosine-1-phosphate mobilizes Ca2+ from internal stores primarily through a mechanism independent of inositol lipid hydrolysis and arachidonic acid release and that sphingolipid metabolism may be important in calcium homeostasis.

3T3 Cells↗
PubMed1993

Sphingosine metabolism induces Ca2+ oscillations in rat pancreatic acinar cells.

The possibility that sphingosine or a related metabolite could mediate inositol-1,4,5-triphosphate (1,4,5-IP3)-independent Ca2+ release in pancreatic acinar cells was investigated. In intact rat pancreatic acini superfused at 37 degrees C, sphingosine (10-100 microM) evoked, after a delay of several minutes, an increase in the cytosolic [Ca2+]i which was oscillatory below concentrations of 50 microM. At room temperature no increase in [Ca2+]i was observed. The increase in [Ca2+]i evoked by sphingosine was not inhibited by U73122, and thus was unlikely to be due to phosphatidylinositol (PI) hydrolysis. Furthermore, no PI hydrolysis or 1,4,5-IP3 production was detected on incubation with sphingosine. As metabolism of sphingosine could explain the temperature-dependent action, the effects of sphingosine and related sphingoid bases were tested in permeabilized cells. After a delay of 30 s at 37 degrees C, sphingosine evoked a slow increase in [Ca2+] into the medium. In contrast, addition of sphingosylphosphorylcholine (SPC) resulted in a rapid increase in [Ca2+]. This response was mimicked by 1,4,5-IP3 but not by addition of sphingosine phosphate (1-10 microM), ceramide (30 microM), or sphingomyelin (50 micrograms/ml). SPC appeared to induce release of Ca2+ from the same store as 1,4,5-IP3, as SPC failed to evoke any further increase in Ca2+ from cells previously depleted by addition of a maximal concentration of 2,4,5-IP3 or thapsigargin. 1,4,5-IP3 but not SPC-induced Ca2+ release was blocked by heparin, indicating that SPC-induced Ca2+ release was not mediated through the IP3 receptor-channel complex. This study demonstrates that SPC, a sphingosine metabolite, could be responsible for IP3-independent Ca2+ oscillations previously seen in pancreatic acinar cells.

Animals↗
PubMed1999

Predominance of the acylation route in the metabolic processing of exogenous sphingosine in neural and extraneural cells in culture.

The metabolic fate of exogenous [3H]sphingosine was investigated in five types of cultured cells: primary cultures of neurons and astrocytes, murine and human neuroblastoma cells and human skin fibroblasts. After administration of 40 nM [3-3H]sphingosine into a cell-conditioned medium containing fetal calf serum, all cell types rapidly and efficiently incorporated the long-chain base in a time-dependent fashion. In all cases, after a 120 min pulse, the amount of radioactivity taken up was in the range of the endogenous sphingosine content. However, unchanged [3H]sphingosine represented only a very minor portion of the label incorporated into cells throughout the pulse period (10-120 min), indicating rapid and efficient sphingosine metabolism in these cells. Most of the [3H]sphingosine taken up was metabolically processed, either by degradation (assessed as 3H2O release into the culture medium) or by N-acylation (mainly to radioactive ceramide, sphingomyelin, neutral glycolipids and gangliosides). [3H]Sphingosine 1-phosphate accounted for less than 2% of the total radioactivity incorporated in all cases. Throughout the pulse period and in all cell types, 3H-labelled organic metabolites largely prevailed over 3H2O, indicating that N-acylation is the major metabolic fate of sphingosine in these cells under apparently physiological conditions. These results are consistent with the notion that sphingosine has a rapid turnover in the cells studied, and indicate that regulation of the basal level of this bioactive molecule occurs mainly through N-acylation.

Acylation↗
PubMed2005

Regulation of sphingosine-1-phosphate lyase gene expression by members of the GATA family of transcription factors.

Sphingosine-1-phosphate is a bioactive sphingolipid that regulates proliferation, differentiation, migration, and apoptosis. Sphingosine-1-phosphate is irreversibly degraded by the highly conserved enzyme sphingosine-1-phosphate lyase. Recent studies have suggested that sphingosine-1-phosphate lyase expression affects animal development and cell fate decisions. Despite its crucial role, mechanisms affecting expression of sphingosine-1-phosphate lyase remain poorly understood. In this study, regulation of sphingosine-1-phosphate lyase gene expression was investigated in Caenorhabditis elegans, where lyase expression is spatially restricted to cells of the developing and adult gut and is essential for normal development. Deletion analysis and generation of transgenic worms combined with fluorescence microscopy identified a 350-nucleotide sequence upstream of the ATG start site necessary for maximal lyase expression in adult worms. Site-specific mutagenesis of a GATA transcription factor-binding motif in the promoter led to loss of reporter expression. Knockdown of the gut-specific GATA transcription factor ELT-2 by RNA interference similarly led to loss of reporter expression. ELT-2 interacted with the GATA factor-binding motif in vitro and was also capable of driving expression of a Caenorhabditis elegans lyase promoter-beta-galactosidase reporter in a heterologous yeast system. These studies demonstrate that ELT-2 regulates sphingosine-1-phosphate lyase expression in vivo. Additionally, we demonstrate that the human sphingosine-1-phosphate lyase gene is regulated by a GATA transcription factor. Overexpression of GATA-4 led to both an increase in activity of a reporter gene as well as an increase in endogenous sphingosine-1-phosphate lyase protein.

Aldehyde-Lyases↗
PubMed1999

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.

Adenosine Triphosphate↗
PubMed2005

Sphingosine kinase regulates the sensitivity of Dictyostelium discoideum cells to the anticancer drug cisplatin.

The drug cisplatin is widely used to treat a number of tumor types. However, resistance to the drug, which remains poorly understood, limits its usefulness. Previous work using Dictyostelium discoideum as a model for studying drug resistance showed that mutants lacking sphingosine-1-phosphate (S-1-P) lyase, the enzyme that degrades S-1-P, had increased resistance to cisplatin, whereas mutants overexpressing the enzyme were more sensitive to the drug. S-1-P is synthesized from sphingosine and ATP by the enzyme sphingosine kinase. We have identified two sphingosine kinase genes in D. discoideum--sgkA and sgkB--that are homologous to those of other species. The biochemical properties of the SgkA and SgkB enzymes suggest that they are the equivalent of the human Sphk1 and Sphk2 enzymes, respectively. Disruption of the kinases by homologous recombination (both single and double mutants) or overexpression of the sgkA gene resulted in altered growth rates and altered response to cisplatin. The null mutants showed increased sensitivity to cisplatin, whereas mutants overexpressing the sphingosine kinase resulted in increased resistance compared to the parental cells. The results indicate that both the SgkA and the SgkB enzymes function in regulating cisplatin sensitivity. The increase in sensitivity of the sphingosine kinase-null mutants was reversed by the addition of S-1-P, and the increased resistance of the sphingosine kinase overexpressor mutant was reversed by the inhibitor N,N-dimethylsphingosine. Parallel changes in sensitivity of the null mutants are seen with the platinum-based drug carboplatin but not with doxorubicin, 5-fluorouracil, and etoposide. This pattern of specificity is similar to that observed with the S-1-P lyase mutants and should be useful in designing therapeutic schemes involving more than one drug. This study identifies the sphingosine kinases as new drug targets for modulating the sensitivity to platinum-based drugs.

Adenosine Triphosphate↗
PubMed2001

Activation of sphingosine kinase by the bradykinin B2 receptor and its implication in regulation of the ERK/MAP kinase pathway.

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.

Enzyme Activation↗
PubMed1993

Sphingosine is endogenous to cardiac and skeletal muscle.

Endogenous sphingosine levels have been determined for rabbit skeletal and cardiac muscle. The sphingosine content of whole muscle tissue was compared to cytosol fractions and to particulate fractions enriched in transverse tubules or sarcoplasmic reticulum membranes. Cardiac and skeletal muscle tissue exhibited high levels of sphingosine (17 pmol/g). Muscle cytosol fractions contained potentially significant levels of sphingosine when expressed as a concentration (0.6-1.3 microM). Isolated T-tubule membranes displayed high sphingosine contents (1400 pmol/mg), making the T-tubules a major source of sphingosine in the muscle cell. The presence of sphingosine in muscle cells suggests that sphingosine may be a physiologically important second messenger in cardiac and striated muscle.

Animals↗
PubMed1999

Sphingosine 1-phosphate regulates heat shock protein 27 induction by a p38 MAP kinase-dependent mechanism in aortic smooth muscle cells.

In an aortic smooth muscle cell line, A10 cells, we investigated the effect of sphingosine 1-phosphate on the induction of heat shock protein 27 (HSP27), a low-molecular-weight heat shock protein. Sphingosine 1-phosphate significantly induced the accumulation of HSP27 in a pertussis toxin-sensitive manner. The effect was dose-dependent in the range between 0.1 and 30 microM. Sphingosine 1-phosphate stimulated an increase in the levels of mRNA for HSP27. Sphingosine 1-phosphate stimulated both p42/p44 mitogen-activated protein (MAP) kinase and p38 MAP kinase activation. PD98059, an inhibitor of the upstream kinase that activates p42/p44 MAP kinase, did not affect sphingosine 1-phosphate-stimulated HSP27 induction. In contrast, SB203580, an inhibitor of p38 MAP kinase, reduced sphingosine 1-phosphate-induced HSP27 induction. SB203580 reduced the levels of mRNA for HSP27 induced by sphingosine 1-phosphate. These results indicate that sphingosine 1-phosphate stimulates the induction of HSP27 via p38 MAP kinase activation in aortic smooth muscle cells.

Animals↗
PubMed1993

Modulation by sphingosine of substrate phosphorylation by protein kinase C in bovine mammary gland.

The effect of sphingosine on the phosphorylation of endogenous proteins by protein kinase C (PKC) was investigated in bovine mammary gland. Several proteins were shown to be substrates for PKC in both cytosolic and total particulate fractions by phosphorylation in the absence or presence of 1-oleoyl-2-acetyl-sn-glycerol, phosphatidylserine (PS) and Ca2+. At concentrations of 83 microM or less, sphingosine inhibited phosphorylation of several substrates for PKC in both fractions. Phosphorylation of cytosolic 36 kDa, 21 kDa and particulate 36 kDa proteins was particularly sensitive to sphingosine. Cytosolic 97 kDa phosphorylation (which was enhanced by Ca2+ alone) was also sensitive to sphingosine. The inhibition was reversed by excess addition of lipid cofactors, particularly PS, but not by Ca2+. At higher concentrations (167 and 417 microM), in addition to the inhibition seen at lower concentrations, sphingosine stimulated phosphorylation of several proteins, including cytosolic 19 kDa and particulate 53 kDa, which were not detected in the absence of sphingosine. The sphingosine-induced phosphorylation disappeared with excess addition of PS, but not with addition of Ca2+. The results point toward the importance of the interaction of sphingosine with membrane phospholipids in the signal transduction pathway mediated by PKC-dependent phosphorylation in bovine mammary gland.

Animals↗