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PubMed1996

Sphingosine enhances phosphatidylinositol 4-kinase activity in rabbit platelets.

The modulating effect of sphingosine on the metabolism of inositol phospholipids was investigated using rabbit platelets. When [3H]arachidonic acid- or [3H]inositol-labeled platelets were incubated at 37 degrees C with sphingosine, the radioactivity of the phosphatidylinositol (PI) fraction obtained on TLC decreased time-dependently up to 5 min, and phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP2) increased concomitantly, though neither arachidonic acid nor 1,2-diacylglycerol was formed. The effect of sphingosine was dose-dependent, the maximum effect being observed at 20 microM. Treatment with a sphingosine derivative, sphingosine-1-phosphate (Sph-1-P) or N-hexanoyl-sphingosine (C6-ceramide), did not result in an increase in PIP. The increased radioactivity of PIP with sphingosine was attributable to an increase in phosphatidylinositol 4-phosphate, but not phosphatidylinositol 3-phosphate. Furthermore, wortmannin, an inhibitor of PI 3-kinase, did not affect the modulating effect of sphingosine at 100 nM, at which the enzyme is known to be completely inhibited. The activity of PI 4-kinase in the platelet lysate was increased by sphingosine but not by Sph-1-P. These results suggest that sphingosine enhances the activity of PI 4-kinase and thereby contributes to the regulation of inositol phospholipid metabolism.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2004

Activation of the melastatin-related cation channel TRPM3 by D-erythro-sphingosine [corrected].

TRPM3, a member of the melastatin-like transient receptor potential channel subfamily (TRPM), is predominantly expressed in human kidney and brain. TRPM3 mediates spontaneous Ca2+ entry and nonselective cation currents in transiently transfected human embryonic kidney 293 cells. Using measurements with the Ca2+-sensitive fluorescent dye fura-2 and the whole-cell patch-clamp technique, we found that D-erythro-sphingosine, a metabolite arising during the de novo synthesis of cellular sphingolipids, activated TRPM3. Other transient receptor potential (TRP) channels tested [classic or canonical TRP (TRPC3, TRPC4, TRPC5), vanilloid-like TRP (TRPV4, TRPV5, TRPV6), and melastatin-like TRP (TRPM2)] did not significantly respond to application of sphingosine. Sphingosine-induced TRPM3 activation was not mediated by inhibition of protein kinase C, depletion of intracellular Ca2+ stores, and intracellular conversion of sphingosine to sphingosine-1-phosphate. Although sphingosine-1-phosphate and ceramides had no effect, two structural analogs of sphingosine, dihydro-D-erythro-sphingosine and N,N-dimethyl-D-erythro-sphingosine, also activated TRPM3. Sphingolipids, including sphingosine, are known to have inhibitory effects on a variety of ion channels. Thus, TRPM3 is the first ion channel activated by sphingolipids.

Calcium↗
PubMed2000

The role of sphingosine kinase in the signaling initiated at the high-affinity receptor for IgE (FcepsilonRI) in mast cells.

Over the last few years, sphingolipids have emerged as an additional class of lipids participating in signaling events in various cell types. The best-investigated examples so far are ceramide and sphingosine-1-phosphate. Ceramide-activated protein kinase and sphingosine kinase are two enzymes which respond to and generate these mediators. In particular, sphingosine kinase, its substrate sphingosine and the product sphingosine-1-phosphate have recently been implicated in the signaling cascades initiated at the FcepsilonRI of mast cells. High intracellular levels of sphingosine seem to serve as an 'intracellular' inhibitor which is 'deactivated' by the action of sphingosine kinase, due to the conversion to sphingosine-1-phosphate. One mode of action of the inhibitory process in this cell type is prevention of the activation of the mitogen-activated protein (MAP) kinase pathway. Sphingosine-1-phosphate itself, the product of this enzymatic reaction, is believed to lead to Ca(2+) mobilization and to stimulate the MAP kinase pathway. The existence and function of this second messenger explains the 'IP3 gap' described in mast cells after FcepsilonRI activation. Therefore, a picture emerges whereby the balance of these two lipid molecules seems to be decisive for the activation of mast cells by IgE plus antigen, with sphingosine kinase acting as a permissive switch for stimulation.

Animals↗
PubMed1990

Sphingosine differentially inhibits activation of the Na+/H+ exchanger by phorbol esters and growth factors.

The role of protein kinase C in activation of the plasma membrane Na+/H+ exchanger was studied in cultured vascular smooth muscle cells. The basic lipid, sphingosine, was used to block enzymatic activity of protein kinase C. Na+/H+ exchange was activated by phorbol 12-myristate 13-acetate (PMA), diacylglycerols, platelet-derived growth factor (PDGF), thrombin, or by osmotically-induced cell shrinkage. Intracellular pH and Na+/H+ exchange activity were measured using the intracellular pH indicator, 2',7'-bis(carboxyethyl)-5(6) carboxyfluorescein. Acting alone, both crude sphingosine and pure, synthetic C18 D-(+)-erythro-sphingosine raised pHi in a dose-dependent manner (from 6.95 +/- 0.02 to 7.19 +/- 0.09 over 10 min for 10 microM sphingosine). This alkalinization was not due to Na+/H+ exchange as it was not altered by t-butylamiloride (50 microM) nor by replacement of the assay medium with a Na(+)-free solution. Sphingosine-induced alkalinization did not require protein kinase C activity, since it was fully intact in protein kinase C-depleted cells. It was also not due to a detergent action of sphingosine on the cell membrane, since both ionic and non-ionic detergents caused cell acidification. Rather, alkalinization induced by sphingosine appeared to be due to cellular uptake of NH3 groups since N-acetylsphingosine showed no alkalinization. After the initial cell alkalinization, cellular uptake of [3H]sphingosine continued slowly for up to 24 h. The ability of PMA or dioctanoylglycerol to activate Na+/H+ exchange fell to 20% of control after 24 h of sphingosine exposure. At all times, C11 and N-acetylsphingosine failed to block PMA-induced activation of the exchanger. Activation of the Na+/H+ exchanger by sucrose, which does not depend on protein kinase C activity, was unaffected by sphingosine. Activation of Na+/H+ exchange by thrombin and PDGF was partially inhibited by 30 and 20%, respectively. These data indicate that both thrombin and PDGF activate Na+/H+ exchange by pathway(s) that are primarily independent of protein kinase C.

Animals↗
PubMed1988

Suppression of nerve growth factor-directed neurite outgrowth in PC12 cells by sphingosine, an inhibitor of protein kinase C.

The formation of vertebrate neural circuitry is regulated in part by neurotrophic agents, such as nerve growth factor (NGF); however, the biochemical mechanisms involved in neurite outgrowth have yet to be completely resolved. Phorbol ester tumor promoters are known to influence the extension of neurites in a variety of neurodevelopmental systems, and protein kinase C, the major phorbol ester receptor, has been implicated in this process. In the present study, sphingosine, a specific pharmacological inhibitor of protein kinase C, was employed to investigate the role of this enzyme in the elaboration of neurites in PC12 pheochromocytoma cells. Normally, PC12 cells respond to NGF by morphologically differentiating into sympathetic neuron-like cells, exhibiting a marked hypertrophy, and extending slender neurites piloted by well defined growth cones. The elaboration of NGF-induced neurites was found to be reversibly inhibited by sphingosine in a dose-dependent manner (IC50 = 2.5-5 microM), while similar concentrations of several structural analogs were inactive. The suppression of neurite outgrowth by sphingosine was antagonized by the addition of 12-O-tetradecanoylphorbol 13-acetate (TPA), which binds to and directly activates protein kinase C. In the presence of NGF, TPA treatment increased the incidence of neurite outgrowth, and this increase, in turn, was antagonized by sphingosine. The binding of [3H]phorbol 12,13-dibutyrate to specific phorbol ester binding sites in PC12 cells was inhibited by sphingosine at concentrations similar to those which inhibited neurite outgrowth. The effects of sphingosine on TPA-directed protein phosphorylation were examined in situ, revealing inhibition of [32P]phosphate incorporation into cellular proteins. The specific TPA-directed phosphorylation of tyrosine hydroxylase was inhibited by sphingosine, as was the resulting increase in enzyme activity. The effects of sphingosine on the levels of alpha- and beta-tubulin mRNAs were also examined in an effort to delimit the locus of protein kinase C action. Concentrations of sphingosine which suppressed neurite outgrowth did not inhibit the NGF-directed elevation of tubulin transcript levels. Taken together, these results reveal the presence of a sphingosine-sensitive pathway in neurite outgrowth and indicate that protein kinase C plays a role in mediating the neuritogenic effects of NGF. Furthermore, the results suggest that protein kinase C acts at a distal segment of the neurite growth pathway.

Adrenal Gland Neoplasms↗
PubMed1988

Regulation of the epidermal growth factor receptor phosphorylation state by sphingosine in A431 human epidermoid carcinoma cells.

The regulation of protein phosphorylation by sphingosine in A431 human epidermoid carcinoma cells was examined. Sphingosine is a competitive inhibitor of phorbol ester binding to protein kinase C (Ca2+/phospholipid-dependent enzyme) and potently inhibits phosphotransferase activity in vitro. Addition of sphingosine to intact A431 cells caused an inhibition of the phorbol ester-stimulated phosphorylation of two protein kinase C substrates, epidermal growth factor (EGF) receptor threonine 654 and transferrin receptor serine 24. We conclude that sphingosine inhibits the activity of protein kinase C in intact A431 cells. However, further experiments demonstrated that sphingosine-treatment of A431 cells resulted in the regulation of the EGF receptor by a mechanism that was independent of protein kinase C. First, sphingosine caused an increase in the threonine phosphorylation of the EGF receptor on a unique tryptic peptide. Second, sphingosine caused an increase in the affinity of the EGF receptor in A431 and in Chinese hamster ovary cells expressing wild-type (Thr654) and mutated (Ala654) EGF receptors. Sphingosine was also observed to cause an increase in the number of EGF-binding sites expressed at the surface of A431 cells. Examination of the time course of sphingosine action demonstrated that the effects on EGF binding were rapid (maximal at 2 mins) and were observed prior to the stimulation of receptor phosphorylation (maximal at 20 mins). We conclude that sphingosine is a potently bioactive molecule that modulates cellular functions by: 1) inhibiting protein kinase C; 2) stimulating a protein kinase C-independent pathway of protein phosphorylation; and 3) increasing the affinity and number of cell surface EGF receptors.

Carcinoma, Squamous Cell↗
PubMed1995

Induction of apoptosis by sphingosine in human leukemic HL-60 cells: a possible endogenous modulator of apoptotic DNA fragmentation occurring during phorbol ester-induced differentiation.

The present studies were undertaken to characterize the potential role of sphingosine in the regulation of apoptosis in HL-60 promyelocytic leukemia cells. A 6-h exposure of HL-60 cells to sphingosine or its methylated derivative, N,N-dimethylsphingosine, caused internucleosomal DNA fragmentation and stereotypical morphological changes characteristic of apoptosis (i.e., cell shrinkage, nuclear condensation, and the formation of apoptotic bodies), as well as that to pharmacological inhibitors of protein kinase C such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine and staurosporine. Apoptosis by sphingosine and N,N-dimethylsphingosine was measured using a flow cytometric method. The percentages of apoptotic cells in cultures treated with sphingosine (10 microM) and N,N-dimethylsphingosine (10 microM) for 6 h were 55.6 +/- 7.8% and 84.2 +/- 11.6%, respectively. HL-60 cells were induced to differentiate toward macrophages by treatment with 5 nM 4 beta-phorbol 12-myristate 13-acetate (PMA). Internucleosomal DNA fragmentation, which was a hallmark of apoptosis, was first detected after 10-h exposure to PMA and increased with longer treatment. Sphingosine concentrations in the cells increased concomitantly with the increasing proportion of apoptotic cells during cell differentiation. Sphingosine level in HL-60 cells differentiated by treatment with PMA for 48 h was about 3.3-fold greater than that in untreated cells. Differentiated HL-60 cells exhibited a markedly increased conversion of exogenously added [3H]ceramide to [3H]sphingosine, indicating elevation of ceramidase activity. Moreover, exposure to sphingosine resulted in down-regulation of c-myc mRNA. These observations suggest the possible role of sphingosine in induction of apoptotic DNA fragmentation during PMA-induced differentiation in myeloid leukemia cells. Sphingosine may function as an endogenous modulator mediating the apoptotic signal.

Apoptosis↗
PubMed1997

In vitro and in vivo induction of apoptosis by sphingosine and N, N-dimethylsphingosine in human epidermoid carcinoma KB-3-1 and its multidrug-resistant cells.

Sphingolipid breakdown products, including ceramide and sphingosine, regulate cell growth, cell differentiation, and apoptosis. We examined the effect of various agents, including sphingolipids, on apoptosis induction in human epidermoid carcinoma KB-3-1 and its multidrug-resistant (MDR) subclone KB-C2 cells which express P-glycoprotein. Adriamycin (ADM) induced apoptosis in KB-3-1 cells but not in KB-C2 MDR cells at the concentration of 50 microg/ml. On the other hand, 15 microM sphingosine or its methylated derivative N, N-dimethylsphingosine (DMS) induced apoptosis in both cell types in vitro. These results suggested that KB-C2 MDR cells were resistant to apoptosis induction by ADM but sensitive to that by sphingosine and DMS. Ceramide and sphingosine-1-phosphate, the initial metabolites of sphingosine, failed to induce apoptosis under the same experimental condition as sphingosine/DMS. The protein kinase C (PKC) inhibitors H7 and staurosporine did not induce apoptosis in either cell line, suggesting that PKC-independent signaling is involved in apoptosis induced by sphingosine and DMS, although both sphingosine and DMS have been shown to down-regulate PKC. Furthermore, DMS significantly inhibited the growth of KB-3-1 as well as KB-C2 MDR tumors in vivo, with evidence of increased apoptosis. The intracellular level of exogenously added [3H]sphingosine or [14C]DMS did not differ between the KB-3-1 parent cell line and its MDR subclone KB-C2, whereas that of [14C]ADM was reduced in KB-C2 MDR cells compared to KB-3-1 cells. These results suggest that P-glycoprotein acts as a transporter for ADM but not for sphingosine or DMS. Furthermore, DMS at the concentrations which induce apoptosis in KB-C2 cells did not affect the level of [14C]ADM. Because sphingosine and DMS induce apoptosis regardless of P-glycoprotein expression, they may provide a new strategy and a promising approach to the treatment of anticancer drug-resistant cancer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Formyl peptide receptor signaling in HL-60 cells through sphingosine kinase.

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.

Calcium↗
PubMed1992

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

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

Animals↗
PubMed1998

1Alpha,25-dihydroxyvitamin D3 inhibits programmed cell death in HL-60 cells by activation of sphingosine kinase.

Sphingolipid breakdown products [ceramide, sphingosine, and sphingosine-1-phosphate (SPP)] are emerging as a new class of bioactive molecules. In agreement with previous studies, treatment of human promyelocytic leukemia HL-60 cells with 1-alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] induced a transient increase of ceramide levels within 2 h, which then returned to basal levels within 8 h. In contrast, sphingosine kinase activity increased more slowly and reached maximal levels only after 20 h of exposure, leading to a concomitant increase in SPP level. Unlike treatments with cell-permeable ceramide analogues or sphingomyelinase, which induce apoptosis, 1,25-(OH)2D3 did not induce apoptosis, despite the early formation of ceramide. Moreover, prolonged treatment of HL-60 cells with 1,25-(OH)2D3 suppressed ceramide-induced apoptosis. There was a correlation between the time course and dose response of the activation of sphingosine kinase by 1,25-(OH)2D3 and the protection against apoptosis. In contrast, treatment with all-trans-retinoic acid neither stimulated sphingosine kinase activity nor protected cells from ceramide-induced apoptosis. Treatment with SPP protected HL-60 cells from ceramide-induced apoptosis, and N,N-dimethylsphingosine (DMS), a competitive inhibitor of sphingosine kinase, prevented the survival effect of 1,25-(OH)2D3. The effect of DMS was counteracted by SPP, suggesting that SPP is a critical component of the cytoprotective effect of 1,25-(OH)2D3. Chelerythrine chloride, an inhibitor of protein kinase C, markedly reduced sphingosine kinase activity and the apoptosis-sparing effect of 1,25-(OH)2D3, and conversely, the tumor promoter 12-O-tetradecanoylphorhol-13-acetate not only suppressed ceramide-induced apoptosis but also stimulated sphingosine kinase activity. Moreover, the protective effect of 12-O-tetradecanoylphorbol-13-acetate was blocked by DMS. Collectively, our observations indicate that the cytoprotective effect of 1,25-(OH)2D3 is mediated by SPP, which is formed as a consequence of activation of sphingosine kinase.

Alkaloids↗
PubMed1992

Regulation of phosphoinositide hydrolysis in cultured astrocytes by sphingosine and psychosine.

The effects of sphingosine and psychosine on phosphoinositide hydrolysis in primary cultured astrocytes were determined. Exposure to sphingosine produced a dose-dependent stimulation of phosphoinositide hydrolysis requiring the presence of external Ca++ for optimal activity. The addition of 10 microM norepinephrine resulted in a stimulation additional to that with sphingosine. The alpha 1-antagonist prazosin completely inhibited norepinephrine-induced phosphoinositide hydrolysis but had no effect on that produced by sphingosine. Psychosine (108 microM), when co-incubated with sphingosine, produced complete inhibition of sphingosine-induced phosphoinositide hydrolysis at all doses of sphingosine tested (33-668 microM). Likewise, psychosine totally inhibited norepinephrine-induced phosphoinositide hydrolysis. The protein kinase C inhibitor staurosporine (1 microM) had no effect on sphingosine-induced phosphoinositide hydrolysis. These findings suggest that lysosphingolipids such as sphingosine and psychosine may play an important role in the regulation of phosphoinositide turnover in astrocytes by a mechanism dependent on extracellular Ca++ and independent of the alpha 1-adrenergic receptor and protein kinase C.

Alkaloids↗
PubMed1988

Sphingosine and psychosine, suggested inhibitors of protein kinase C, inhibit LH effects in rat luteal cells.

The possible involvement of protein kinase C on luteinizing hormone (LH) effects in dispersed rat luteal cells was investigated using two substances that have been reported to be protein kinase C inhibitors, sphingosine and psychosine. Sphingosine efficiently inhibited protein kinase C activity both in brain and luteal cytosol fractions. Both substances inhibited LH-stimulated cyclic adenosine monophosphate (cAMP) accumulation in a dose-dependent fashion with an LD50 at 3-7 microM (sphingosine) and 40 microM (psychosine). LH-stimulated progesterone production was also inhibited with an ID50 at 6-10 microM (sphingosine) and 40-100 microM (psychosine). The inhibition was not due to an increased phosphodiesterase activity since IBMX (3-isobutyl-1-methylxanthine, 0.1 mM) and RO 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone, 0.1 mM) did not abolish the inhibitory effect of sphingosine. To study the mode of action of sphingosine, forskolin and cAMP analogues were tested. The effect of these substances on steroidogenesis was inhibited, as well as the forskolin-induced cAMP accumulation, by sphingosine. This study demonstrates a clear inhibition of LH-stimulated effects by sphingosine and psychosine. LH action in rat luteal cells is discussed in relation to protein kinase C and the possible mode of sphingosine action.

1-Methyl-3-isobutylxanthine↗
PubMed2004

Inhibition of arachidonic acid release and cytosolic phospholipase A2 alpha activity by D-erythro-sphingosine.

Sphingolipid metabolites such as sphingosine 1-phosphate (S1P) and ceramide can mediate many cellular events including apoptosis, stress responses and growth arrest. Although ceramide stimulates arachidonic acid metabolism in several cells, the effects of sphingosine and its endogenous analogs have not been established. We investigated the effects of D-erythro-sphingosine and its metabolites on arachidonic acid release in the two cells and on the activity of cytosolic phospholipase A2alpha. C2-Ceramide (N-acetyl-D-erythro-sphingosine, 100 microM) alone stimulated [3H]arachidonic acid release and enhanced the ionomycin-induced release from the prelabeled PC12 cells and L929 cells. In contrast, exogenous addition of D-erythro-sphingosine inhibited the responses in a concentration-dependent manner in the two cell lines. D-erythro-sphingosine, D-erythro-N,N-dimethylsphingosine (D-erythro-DMS) and D-erythro-dihydrosphingosine (D-erythro-DHS) significantly inhibited mastoparan-, but not Na3VO4-, stimulated arachidonic acid release in PC12 cells. D-erythro-S1P and DL-threo-DHS showed no effect on the responses. Production of prostaglandin F2alpha was also enhanced by C2-ceramide (20 microM) and suppressed by D-erythro-sphingosine (10 microM) in PC12 cells. An in vitro study revealed that D-erythro-sphingosine, D-erythro-DMS and D-erythro-DHS directly inhibited cytosolic phospholipase A2alpha activity. These findings suggest that ceramide and D-erythro-analogs of sphingosine have opposite effects on phospholipase A2 activity and thus regulate arachidonic acid release from cells.

Animals↗
PubMed2006

Normal acute and chronic inflammatory responses in sphingosine kinase 1 knockout mice.

Sphingosine-1-phosophate, generated from the phosphorylation of sphingosine by sphingosine kinase enzymes, is suggested to function as an intracellular second messenger for inflammatory mediators, including formyl peptide, C5a, and Fc. More recently, a role for sphingosine kinases during inflammation has also been proposed. Here we show that sphingosine kinase 1 knockout mice exhibit normal inflammatory cell recruitment during thioglycollate-induced peritonitis and that sphingosine kinase 1-null neutrophils respond normally to formyl peptide. In the collagen-induced arthritis model of rheumatoid arthritis, sphingosine kinase 1 knockout mice developed arthritis with normal incidence and severity. Our findings show that sphingosine kinase 1 is dispensable for inflammatory responses and support the need for more extensive studies of sphingosine kinases in inflammation.

Acute Disease↗
PubMed1993

Effects of sphingosine on peripheral membrane interactions: comparison of adriamycin, cytochrome c, and phospholipase A2.

As revealed by resonance energy transfer utilizing pyrene-labeled phosphatidylcholine donor, the mainly electrostatically controlled binding of adriamycin (Adr) and cytochrome c (cyt c) to mixed egg yolk phosphatidic acid/phosphatidylcholine (eggPA/eggPC, 15:85 molar ratio) liposomes was reversed upon the inclusion of increasing contents of sphingosine. At a [sphingosine]/[eggPA] molar ratio of approximately 2:1, the degree of fluorescence quenching by cyt c and Adr was approximately the same as when using liposomes lacking eggPA. Similarly, the increase in the surface pressure of sphingosine/eggPA monolayers on an air/water interface due to the membrane penetration of either cyt c or Adr was progressively reduced by increasing the content of sphingosine in the monolayers. The above critical [sphingosine]/[acidic phospholipid] stoichiometry yielding dissociation of the positively charged ligands Adr or cyt c from membrane acidic phospholipids was shifted from 2:1 to 1:1 upon substituting egg phosphatidylglycerol (eggPG) for eggPA. Accordingly, charge neutralization of the acidic phospholipids by sphingosine could be involved. One eggPA (having maximally two negative charges) appears to require two molecules of sphingosine whereas the maximally singly charged eggPG is neutralized by one sphingosine. For comparison we also studied the effects of sphingosine on the phospholipase A2 catalyzed hydrolysis of the pyrene-labeled acidic alkylacyl phospholipid analog 1-octacosanyl-2-[6-(pyren-1-yl)]hexanoyl-sn-glycero-3- phosphatidylmethanol (C28-O-PHPM) and the corresponding phosphatidylcholine (C28-O-PHPC). In the presence of low Ca2+ concentrations (approximately 50 nM) limiting the rate of the enzymatic reaction, sphingosine gradually inhibited the hydrolysis of phosphatidylcholine, and at 1:6 sphingosine/C28-O-PHPC a nearly complete lack of hydrolysis was evident.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1994

Sphingosine inhibits the synthesis of RNA primers by primase in vitro.

We have previously shown the presence of sphingomyelin and sphingomyelinase in cell nuclei, suggesting that they may play a role in the intranuclear production of sphingosine, a potent bioactive molecule modulating diverse cellular functions. In the present study, the direct effects of sphingosine (C18:1) on the activity of DNA replication/repair polymerases were studied in vitro. Sphingosine had no effect on DNA polymerases alpha and beta and slightly inhibited DNA polymerases gamma, delta, and epsilon. In contrast, sphingosine strongly inhibited the activity of primase in a dose-dependent manner. On the other hand, dihydrosphingosine (C18:0), glycolipids, sphingomyelin, and ceramide had no effect on primase activity. Sphingosine equally inhibited the activity of primase complexed with DNA polymerase alpha, as well as its free form, with a Ki value of 4 microM. A gel-retardation analysis showed that the binding of primase with 32P-labeled template DNA was suppressed by sphingosine. Inhibition by sphingosine was competitive with the DNA template, but not with the substrate NTPs. After product analysis, a dose-dependent decrease in the amount of RNA primer products, consisting mainly of 10- and 11-mers, was observed in the presence of sphingosine, indicating that it inhibits the synthesis of RNA primers by primase. Sphingosine, however, had no effect on T7 RNA polymerase.

Animals↗
PubMed1997

Altered ceramide and sphingosine expression during the induction phase of ischemic acute renal failure.

UNLABELLED: Recent evidence indicates that a "sphingomyelin signaling pathway" exists: in response to heterogeneous influences, sphingomyelin is hydrolyzed, liberating ceramide, and subsequently its sphingoid base, sphingosine. Ceramide and sphingosine can influence diverse cellular processes, including cell differentiation, proliferation, protein trafficking, and apoptosis. Each of these processes have important implications for post-ischemic acute renal failure (ARF). However, sphingosine and ceramide expression during the induction of ischemic/reperfusion injury have not been previously assessed. To this end, CD-1 mice were subjected to 45 minutes of unilateral renal ischemia +/- reperfusion, followed by cortical sphingosine, ceramide, and sphingomyelin assessments. Contralateral kidneys served as controls. Ischemia caused approximately 50% sphingosine and ceramide decrements. During reperfusion, sphingosine rebounded to normal values. Conversely, ceramide rose to, and was maintained at, supranormal levels (approximately 175% of controls). Subsequent studies performed with hypoxic or oxygenated isolated proximal tubules suggested that these changes: (1) had a multifactorial basis; (2) were partially simulated by enhanced PLA2 activity; (3) and were dissociated from alterations in net sphingomyelin content. To assess the potential pathogenic relevance of the documented ceramide increments, cultured human proximal tubule (HK-2) cells were subjected to ATP depletion/Ca2+ ionophore- or PLA2-induced attack with or without exogenous C2 ceramide loading. Ceramide worsened both forms of injury without exerting an independent lethal effect. Conversely, ceramide markedly attenuated arachidonic acid cytotoxicity. This occurred without any decrease in arachidonate uptake, suggesting a direct cytoprotective effect. IN CONCLUSION: (1) sphingosine and ceramide fluxes are hallmarks of early ischemic/reperfusion injury; (2) these changes occur via divergent metabolic pathways; and (3) that ceramide increments can affect divergent injury pathways, and that sphingosine and ceramide have potent cell signaling effects, suggest that the currently documented sphingosine/ ceramide fluxes could have important implications for the induction phase and evolution of post-ischemic ARF.

Acute Kidney Injury↗