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J A Shayman

Publications and source records attributed to J A Shayman.

At least 37 records · Page 2Linked to original sources
PubMed1998

Purification and characterization of 1-O-acylceramide synthase, a novel phospholipase A2 with transacylase activity.

A novel pathway for ceramide metabolism, 1-O-acylceramide formation, was previously reported (Abe, A., Shayman, J. A., and Radin, N. S. (1996) J. Biol. Chem. 271, 14383-14389). In this pathway a fatty acid in the sn-2 position of phosphatidylethanolamine or phosphatidylcholine is transferred to the 1-hydroxyl position of ceramide. An enzyme that catalyzes the esterification of N-acetylsphingosine was purified from the postmitochondrial supernatant of calf brain through consecutive steps, including ammonium sulfate fractionation, DEAE-Sephacel, phenyl-Sepharose, S-Sepharose, Sephadex G-75, concanavalin A-agarose, and heparin-Sepharose chromatography. The molecular mass of the enzyme was determined to be 40 kDa by gel filtration on Sephadex G-75. The enzyme bound to concanavalin A-agarose column was eluted with the buffer containing 500 mM alpha-methyl-D-mannopyranoside. Further purification by heparin-Sepharose chromatography resulted in separation of two peaks of enzyme activity. Coincidence between the transacylase activity and a stained protein of a molecular mass of 40 kDa was observed, as determined by SDS-polyacrylamide gel electrophoresis and recovery after separation over an acidic native gel. The second peak of activity from the heparin-Sepharose chromatography represented a purification of 193,000-fold. These results are consistent with the enzyme being a glycoprotein of a molecular mass of about 40 kDa with a single polypeptide chain. The purified enzyme had a pH optimum at pH 4.5. The divalent cations Ca2+ and Mg2+ enhanced but were not essential for the transacylase activity. Neither activation nor inactivation of the enzyme activity was observed in the presence of 2 mM ATP or 2 mM dithiothreitol. Preincubation of the enzyme with 1 mM N-ethylmaleimide, 1 mM phenylmethylsulfonyl fluoride, or 3.1 microM bromoenol lactone, a potent inhibitor of cytosolic Ca2+-independent phospholipase A2, had no significant effect on the enzyme activity. The enzyme activity was completely abolished in the presence of greater than 773 microM Triton X-100. Partial inhibition of the enzyme activity was observed in the presence of 10-100 microg/ml heparin. In the absence of N-acetylsphingosine, the enzyme acted as a phospholipase A2. These results strongly suggest that 1-O-acylceramide synthase is both a transacylase and a novel phospholipase A2.

Acetyltransferases↗
PubMed1998

Effect of an inhibitor of glucosylceramide synthesis on cultured human keratinocytes.

Glucosylceramide (GlcCer) is a major glycosphingolipid component of epidermis, which is thought to be related to the barrier function of skin permeability. However, the role of glycosphingolipids in keratinocyte growth and differentiation has not been fully clarified. It has been reported that D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an inhibitor of GlcCer synthase (EC 2.4.1.80), depletes cells of glycosphingolipids. This inhibitor has been used as a tool for elucidating their functions. In the present study, the effect of PDMP on cultured normal human keratinocytes was investigated. The cells were treated with various concentrations of PDMP. Forty-eight hours later cell growth, thymidine incorporation, and lipid content were studied. The cell growth and the incorporation of thymidine into cells were inhibited by PDMP in a dose dependent manner. The synthesis of GlcCer was strongly inhibited by PDMP treatment, whereas no significant changes in ceramide level were observed. We concluded that GlcCer in epidermis may play an important role in regulating epidermal growth and suggested that PDMP may be beneficial for treating proliferative skin disorders in the future.

Adult↗
PubMed1997

Mitogen-activated protein kinase activation during IgG-dependent phagocytosis in human neutrophils: inhibition by ceramide.

In FMLP-activated polymorphonuclear leukocytes (PMNs) challenged with IgG-opsonized erythrocytes (EIgG), the termination of phagocytosis correlates with an accumulation of ceramide, a product of sphingolipid metabolism. Furthermore, the exogenous addition of short chain ceramides inhibits EIgG-mediated phagocytosis. In the present study, we identified p42 and p44 mitogen-actived protein (MAP) kinases, referred to as extracellular signal-regulated kinases ERK2 and ERK1, respectively, as intracellular targets of ceramide action during Fc gammaR-mediated phagocytosis. The tyrosine phosphorylation of ERK1 and ERK2 increased within 30 s of addition of EIgG, with maximal phosphorylation by 1 to 5 min. By 30 min, ERK1 and ERK2 were almost completely dephosphorylated. The kinetics of ERK1 and ERK2 tyrosine phosphorylation indicated that MAP kinase activation preceded target ingestion. N-Acetylsphingosine (C2-ceramide) inhibited phagocytosis, reduced ERK1 and ERK2 phosphorylation to basal levels, and reduced ERK1 and ERK2 activity by 85 to 90% and 70 to 80%, respectively. In contrast, N-acetyldihydrosphingosine (dihydro-C2-ceramide) had no effect on either tyrosine phosphorylation or activity of ERK1 and ERK2. In the presence of the MAP kinase kinase (MEK) inhibitor, PD 098059, phagocytosis was reduced by approximately 50%, while ERK1 and ERK2 activity was reduced by 85 to 90%. Thus, engagement of Fc gammaRs led to ERK1 and ERK2 phosphorylation and activation, and the activation of these enzymes was critical for phagocytosis. Furthermore, the inhibition of phagocytosis by C2-ceramide correlated with the inhibition of tyrosine phosphorylation and activation of ERK1 and ERK2. These results suggest that ceramides generated during phagocytosis act on the MAP kinase signaling pathway, ultimately "turning off" the phagocytic response.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

Ceramide inhibits IgG-dependent phagocytosis in human polymorphonuclear leukocytes.

Ceramide is a product of agonist-induced sphingolipid metabolism in several cell types, including polymorphonuclear leukocytes (PMNs). In adherent PMNs, the kinetics of ceramide production correspond with the termination of fMLP-stimulated H2O2 release. Furthermore, short chain ceramides inhibit fMLP-mediated H2O2 release in adherent PMNs. In the present study, we investigated the effects of short chain ceramides and sphingoid bases on phagocytosis of IgG-opsonized erythrocytes (EIgG) by suspended PMNs activated with fMLP. N-Acetylsphingosine, N-acetylphytosphingosine, phytosphingosine, sphingosine, and dihydrosphingosine, but not N-acetyldihydrosphingosine, inhibited phagocytosis of EIgG. In contrast, these same lipids did not inhibit fMLP-mediated chemotaxis. Endogenous ceramide levels increased within the first few minutes of phagocytosis, with a significant (P < .05) accumulation by 30 minutes, the time by which phagocytosis was terminated. Neutral sphingomyelinase activity paralleled the increase in ceramide, consistent with the generation of ceramide by the hydrolysis of sphingomyelin. The N-acetyl-conjugated sphingols (C2 ceramides) blocked phosphatidylethanol formation indicating that phospholipase D (PLD) is an intracellular target of ceramide action. These data suggest that ceramides, generated through activation of the sphingomyelin cycle, act as negative regulators of Fc(gamma)R-mediated phagocytosis.

Ceramides↗
PubMed1997

Sphingolipids--the enigmatic lipid class: biochemistry, physiology, and pathophysiology.

The "sphingosin" backbone of sphingolipids was so named by J. L. W. Thudichum in 1884 for its enigmatic ("Sphinx-like") properties. Although still an elusive class of lipids, research on the involvement of sphingolipids in the signal transduction pathways that mediate cell growth, differentiation, multiple cell functions, and cell death has been rapidly expanding our understanding of these compounds. In addition to the newly discovered role of ceramide as an intracellular second messenger for tumor necrosis factor-alpha, IL-1beta, and other cytokines, sphingosine, sphingosine-1-phosphate, and other sphingolipid metabolites have recently been demonstrated to modulate cellular calcium homeostasis and cell proliferation. Perturbation of sphingolipid metabolism using synthetic and naturally occurring inhibitors of key enzymes of the biosynthetic pathways is aiding the characterization of these processes; for examples, inhibition of cerebroside synthase has indicated a role for ceramide in cellular stress responses including heat shock, and inhibition of ceramide synthase (by fumonisins) has revealed the role of disruption of sphingolipid metabolism in several animal diseases. Fumonisins are currently the focus of a FDA long-term tumor study. This review summarizes recent research on (i) the role of sphingolipids as important components of the diet, (ii) the role of sphingoid base metabolites and the ceramide cycle in expression of genes regulating cell growth, differentiation, and apoptosis, (iii) the use of cerebroside synthase inhibitors as tools for understanding the role of sphingolipids as mediators of cell cycle progression, renal disease, and stress responses, and (iv) the involvement of disrupted sphingolipid metabolism in animal disease and cellular deregulation associated with exposure to inhibitors of ceramide synthase and serine palmitoyltransferase, key enzymes in de novo sphingolipid biosynthesis. These findings illustrate how an understanding of the function of sphingolipids can help solve questions in toxicology and this is undoubtedly only the beginning of this story.

Amidohydrolases↗
PubMed1996

A novel enzyme that catalyzes the esterification of N-acetylsphingosine. Metabolism of C2-ceramides.

A unique transacylase that catalyzes esterification of a short chain ceramide, N-acetylsphingosine, was found in Madin-Darby canine kidney cell and mouse tissue homogenates. It esterified the hydroxyl group at the carbon-1 position of the ceramide. The enzyme has a pH optimum of 4.2 and a Km of 9.4 microM for N-acetylsphingosine at pH 4.5. The transacylase activity is independent of free fatty acid or acyl-CoA and instead uses the 2-acyl group of phosphatidylethanolamine or phosphatidylcholine. The transacylase activity in the homogenate was present in the 100,000 x g supernatant, and the lipid extracted from the membranous fraction could function as a donor of the acyl group. When liposomes consisting of dioleoylphosphatidylcholine:1-palmitoyl-2-[14C]arachidonoyl-phosphati dylethanolamine:sulfatide (70:0.2:30) were incubated with the supernatant and N-acetylsphingosine, the formation of free arachidonic acid and O-arachidonoyl-N-acetylsphingosine was observed. The ratio of the two products depended on the concentration of ceramide; only the free acid was formed if the truncated ceramide was absent. Both deacylase and transacylase activities were inhibited 50-60% by 20 microM D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of several glucosphingolipid synthases. Neither activity was inhibited by nonadecyltetraenyl trifluoromethyl ketone, a potent inhibitor of cytosolic phospholipase A2. N-Acetyldihydrosphingosine and N-octanoylsphingosine were only 55 and 10%, respectively, as effective as N-acetylsphingosine as acyl acceptors. Oleoylsphingosine was only slightly reactive. An esterase that releases the truncated ceramide from its ester linkage appears to be membrane bound. Lecithin was less effective than phosphatidylethanolamine as an acyl donor in the transacylation. Madin-Darby canine kidney cell cultures treated with N-acetyl-[3-3H]sphingosine formed radioactive polar sphingolipids, long chain ceramide, free sphingosine, and O-acyl-N-acetylsphingosine. This suggests that the deacylation and transacylation reactions observed in vitro occur in growing cells as well.

Acyltransferases↗
PubMed1996

(1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol as an inhibitor of ceramidase.

In this study, we have examined the cellular and biochemical activities of the ceramide analog (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (D-erythro-MAPP). Addition of 5 microM D-e-MAPP to HL-60 human promyelocytic leukemia cells resulted in a concentration- and time-dependent growth suppression accompanied by an arrest in the G0/G1 phase of the cell cycle; thus mimicking the action of exogenous ceramides. Its enantiomer L-e-MAPP was without effect. Two lines of evidence suggested that D-e-MAPP may not function as a direct analog of ceramide. First, D-e-MAPP possesses a stereochemical configuration opposite to that of D-erythro-ceramide. Second, D-e-MAPP failed to activate ceramide-activated protein phosphatase in vitro. Therefore, we examined if D-e-MAPP functioned indirectly by modulating endogenous ceramide levels. The addition of D-e-MAPP to cells, but not L-e-MAPP, caused a time- and concentration-dependent elevation in endogenous ceramide levels reaching greater than 3-fold over baseline following 24 h of treatment. Both D-e-MAPP and L-e-MAPP underwent similar uptake by HL-60 cells. D-e-MAPP was poorly metabolized, and remained intact in cells, whereas L-e-MAPP underwent a time- and concentration-dependent metabolism; primarily through N-deacylation. In vitro, L-e-MAPP was metabolized by alkaline ceremidase to an extent similar to that seen with C16-ceramide. D-e-MAPP was not metabolized. Instead, D-e-MAPP inhibited alkaline ceramidase activity in vitro with an IC50 of 1-5 microM. D-e-MAPP did not modulate the activity of other ceramide metabolizing enzymes in vitro or in cells, and it was a poor inhibitor of acid ceramidase (IC50>500 microM). Finally, D-e-MAPP inhibited the metabolism of L-e-MAPP in cells. These studies demonstrate that D-e-MAPP functions as an inhibitor of alkaline ceramidase in vitro and in cells resulting in elevation in endogenous levels of ceramide with the consequent biologic effects of growth suppression and cell cycle arrest. These studies point to an important role for ceramidases in the regulation of endogenous levels of ceramide.

Acid Ceramidase↗
PubMed1996

Induction of glucosylceramide synthase by synthase inhibitors and ceramide.

Glucosylceramide (GlcCer) synthase acts on the sphingolipid, ceramide, to transer a glucose moiety from UDP-glc, thus forming the first member of a large family of glucosphingolipids. Two inhibitors of the enzyme, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-threo-PDMP) and N-butyldeoxynojirimycin (NBDN), have been found to induce an elevated level of the synthase in MDCK cells. In cells treated with 20 muM PDMP, then assayed for synthase activity under conditions in which the absorbed PDMP was partially diluted out, the assay showed that the enzyme's specific activity had risen considerably in only 1 h and reached a maximum of about three times the control activity within 6 h. Both cycloheximide and actinomycin D, inhibitors of translational and transcriptional protein synthesis, caused much of the synthase activity to disappear in 6 h, presumably because of normal catabolic destruction. However, simultaneous inclusion of PDMP or NBDN in the cell medium slowed the rate of synthase disappearance. L-Cycloserine, which blocked the synthesis of ceramide, nevertheless allowed PDMP to elevate the synthase activity. Thus the inductive effect appears to be due, in part at least, to resistance of the enzyme-inhibitor complex to the normal process of enzyme degradation. Two other inhibitors of GlcCer synthase, more active than PDMP, did not produce detectable induction because they could not be dissociated from the enzyme during the cell washing and diluting steps. Agents that produced a large increase in endogenous cell ceramide level (DL-erythro-PDMP,N-acetylsphingosine, and bacterial sphingomyelinase) also induced an elevated level of GlcCer synthase. The latter two agents did not protect the synthase from catabolism in the presence of cycloheximide. These findings suggest the existence of a second mechanism of enzyme induction, enhanced synthesis of the enzyme due to the increased availability of the enzyme's lipoidal substrate. The possibility is raised that events involving ceramide in cell signalling may be mediated in part by changes in glucosphingolipid levels.

1-Deoxynojirimycin↗
PubMed1996

Glycosphingolipid synthesis and proliferation in a renal cell line grown in high glucose.

We evaluated the role of sphingolipids as potential mediators of the renal epithelial growth response to growth in high-glucose media. The mouse cortical tubule (MCT) cell line was studied under high-glucose (450 mg/dl) and normal glucose (100 mg/dl) conditions. In cells plated at low-density, high-glucose media stimulated cell proliferation as measured by DNA, protein, and cell number and [3H]thymidine incorporation with a corresponding increase in glucosylceramide (GlcCer). The GlcCer synthase inhibitor, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), blocked the proliferative response to high glucose in association with a decrease in endogenous GlcCer and an increase in ceramide concentrations. Addition of N-acetylsphingosine, a short-chain homologue of natural ceramides, increased the levels of endogenous ceramides and inhibited proliferation. In contrast, the beta-glucosidase inhibitor conduritol B epoxide resulted in increased cell GlcCer but did not increase proliferation. We conclude that MCT cells proliferate when grown in the presence of high glucose. GlcCer levels increase and ceramide levels decrease in concert with the proliferative response. Pharmacologically increasing endogenous levels of ceramide inhibits the proliferative response to high glucose, but increasing endogenous levels of GlcCer does not stimulate proliferation.

Animals↗
PubMed1996

Sphingolipids: their role in intracellular signaling and renal growth.

Glycerophospholipids have long been recognized as substrates for the formation of important intracellular second messengers. Recently, another class of membrane-associated lipids, sphingolipids, has been identified as potentially important in cell-signaling events. Ceramides, long-chain bases, and sphingosine derivatives may be mediators of cellular growth, differentiation, and programmed death responses. These lipids have been proposed to act through the regulation of cell kinases, phosphatases, and gene transcription. Observations of sphingolipid abnormalities in two examples of aberrant renal growth, diabetes mellitus and autosomal recessive kidney disease, suggest that sphingolipids may play a role in the pathophysiology of common renal disorders.

Animals↗
PubMed1995

Ceramide formation during heat shock: a potential mediator of alpha B-crystallin transcription.

Ceramide has been identified as a potential second messenger that may mediate cell differentiation and apoptosis after exposure to hormonal agonists such as 1 alpha, 25-dihydroxyvitamin D3, tumor necrosis factor alpha, or gamma-interferon. The secondary cellular events that follow ceramide generation remain undefined. We report that in NIH WT-3T3 cells, ceramide induces an enhancement of gene transcription of alpha B-crystallin, a small heat shock protein. The levels of alpha B-crystallin, as measured by Northern blot and immunoblot analyses, were increased by the addition of an exogenous short-chain ceramide, N-acetylsphingosine, or by increasing endogenous intracellular ceramide by inhibition of glucosylceramide synthase. Similar effects were not seen in the expression of the closely related gene, Hsp25. To ascertain whether ceramide-mediated gene transcription was a feature of the heat shock response, cell ceramide was measured in heat shocked cells and observed to be elevated 2-fold immediately upon the return of cells to 37 degrees C. Thus ceramide formed after heat shock treatment of 3T3 cells may mediate the transcription events associated with the cell stress response.

3T3 Cells↗
PubMed1995

Bcl-x and Bcl-2 inhibit TNF and Fas-induced apoptosis and activation of phospholipase A2 in breast carcinoma cells.

Tumor necrosis factor (TNF) induces cel death in several tumor cell lines by undefined mechanisms. Using a cDNA expression cloning strategy we identified two cDNAs that completely inhibit the TNF-induced death pathway in MCF7 breast carcinoma cells. These cDNAs encoded for Bcl-2 and Bcl-x. To compare the cytotoxic signal transduction pathway induced by the TNF receptor versus that induced by Fas, we transfected MCF7 cells with a Fas expression construct. The resulting cell line, MCF-Fas, was highly sensitive to cytotoxicity induced by TNF or anti-Fas. Expression of either bcl-2 or bcl-x in these cells rendered them completely resistant to lysis induced by either TNF or Fas. Interestingly, exposure of MCF-Fas cells to anti-Fas or TNF induced activation of phospholipase A2 (PLA2), while only TNF activated NF-kappa B. Activation of PLA2 was completely blocked whereas activation of NF-kappa B was unaffected by overexpression of either bcl-x or bcl-2. Moreover, PLA2-inhibitors, quinacrine and dexamethasone, partially inhibited cytotoxicity induced by either TNF or anti-Fas. These data suggest an involvement of PLA2 in both TNF- and Fas-mediated cytotoxicity and a novel mechanism of action for bcl-2 and bcl-x, i.e. inhibition of arachidonic acid metabolism, by which they may, in addition of apoptosis, modulate inflammation.

Antigens, Surface↗
PubMed1995

Cell cycle arrest induced by an inhibitor of glucosylceramide synthase. Correlation with cyclin-dependent kinases.

In an attempt to define the basis for sphingolipid regulation of cell proliferation, we studied the effects of glucosylceramide (GlcCer) synthase inhibition by threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on NIH 3T3 cells overexpressing insulin-like growth factor-1 (IGF-1) receptor. PDMP treatment resulted in a time-dependent decrease in GlcCer levels and an increase in cellular ceramide levels. PDMP abolished serum and IGF-1-stimulated cell proliferation, as measured by a reduction in [3H]thymidine incorporation, protein, and DNA levels. However it did not affect IGF-1-mediated early signaling events, including receptor tyrosine kinase, MAP kinase, and phosphatidylinositol 3-kinase activities. Two-color flow cytometry with propidium iodide and 5-bromo-2'-deoxyuridine monophosphate labeling revealed an arrest of the cell cycle at G1/S and G2/M transitions in an asynchronous population of cells. These changes were time dependent, with maximal effects seen by 12-24 h. Removal of PDMP from the cell medium resulted in reversal of the cell cycle changes, with cells re-entering the S phase. The cell cycle arrest at the G1/S and G2/M transitions was confirmed in cells synchronized by pretreatment with nocodazole, aphidicolin, or hydroxyurea, and released from blockade in the presence of PDMP. A decrease in the activities of two cyclin-dependent kinases, p34cdc2 kinase and cdk2 kinase, was observed with PDMP treatment. When cell ceramide levels were increased by N-acetylsphingosine, comparable changes in the cell cycle distribution were seen. However, sphingomyelinase treatment was without effect. Therefore, it appears that ceramide mediates in part the inhibitory effect of GlcCer synthase inhibition on IGF-1-induced cell proliferation in 3T3 cells. The rapid production of decreased cyclin-dependent kinase activities by PDMP suggests that one of the crucial sites of action of the inhibitor lies in this area.

3T3 Cells↗
PubMed1995

Dexamethasone modulates rat renal brush border membrane phosphate transporter mRNA and protein abundance and glycosphingolipid composition.

Glucocorticoids are important regulators of renal phosphate transport. This study investigates the role of alterations in renal brush border membrane (BBM) sodium gradient-dependent phosphate transport (Na-Pi cotransporter) mRNA and protein abundance in the dexamethasone induced inhibition of Na-Pi cotransport in the rat. Dexamethasone administration for 4 d caused a 1.5-fold increase in the Vmax of Na-Pi cotransport (1785 +/- 119 vs. 2759 +/- 375 pmol/5 s per mg BBM protein in control, P < 0.01), which was paralleled by a 2.5-fold decrease in the abundance of Na-Pi mRNA and Na-Pi protein. There was also a 1.7-fold increase in BBM glucosylceramide content (528 +/- 63 vs. 312 +/- 41 ng/mg BBM protein in control, P < 0.02). To determine whether the alteration in glucosylceramide content per se played a functional role in the decrease in Na-Pi cotransport, control rats were treated with the glucosylceramide synthase inhibitor, D-threo-1-phenyl-2-decanoyl-amino-3-morpholino-1-propanol (PDMP). The resultant 1.5-fold decrease in BBM glucosylceramide content (199 +/- 19 vs. 312 +/- 41 ng/mg BBM protein in control, P < 0.02) was associated with a 1.4-fold increase in Na-Pi cotransport activity (1422 +/- 73 vs. 1048 +/- 85 pmol/5 s per mg BBM protein in control, P < 0.01), and a 1.5-fold increase in BBM Na-Pi protein abundance. Thus, dexamethasone-induced inhibition of Na-Pi cotransport is associated with a decrease in BBM Na-Pi cotransporter abundance, and an increase in glucosylceramide. Since primary alteration in BBM glucosylceramide content per se directly and selectively modulates BBM Na-Pi cotransport activity and Na-Pi protein abundance, we propose that the increase in BBM glucosylceramide content plays an important role in mediating the inhibitory effect of dexamethasone on Na-Pi cotransport activity.

Animals↗
PubMed1995

Structural and stereochemical studies of potent inhibitors of glucosylceramide synthase and tumor cell growth.

Analogs and homologs of PDMP were synthesized, based on its structure (D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol). This compound had previously been found to block the synthesis of GlcCer (glucosylceramide). Increasing the acyl chain length from 10 to 16 carbon atoms greatly enhanced the efficacy of the enzyme inhibitor, as did the use of a less polar cyclic amine, especially a pyrrolidine instead of a morpholine ring. Replacement of the phenyl ring by a chain corresponding to sphingosine also yielded a strongly inhibitory material. By using a chiral synthetic route, we showed that the isomers active against GlcCer synthase had the R,R-(D-threo)-configuration. However, strong inhibition of the growth of human cancer cells in plastico was produced by both the threo and erythro racemic compounds, showing involvement of an additional factor (beyond simple depletion of cell glycosphingolipids by blockage of GlcCer synthesis). The growth arresting effects could be correlated with increases in cellular ceramide and diglyceride levels. The aliphatic pyrrolidino compound was strongly inhibitory toward the glucosyltransferase and produced almost complete depletion of glycolipids, but did not inhibit growth or cause an accumulation of ceramide. Attempts were made to see whether the differences in growth effects could be attributed to the influence of the inhibitors on related enzymes (ceramide and sphingomyelin synthase and ceramidase and sphingomyelinase). While some stimulation of enzyme activity was noted, particularly at high inhibitor concentrations (50 microM), these findings did not explain the differing effects of the different inhibitors. The best inhibitors of GlcCer synthase compared favorably in efficacy with some cancer chemotherapeutic drugs in current use when tested with a battery of human cancer cells.

Animals↗
PubMed1994

Ceramide regulates oxidant release in adherent human neutrophils.

We investigated the role of sphingolipids in regulating oxidant release in adherent human neutrophils. Stimulation of adherent neutrophils with formyl-Met-Leu-Phe (fMLP) resulted in the accumulation of ceramide at a time when H2O2 release is terminated. H2O2 release in fMLP-stimulated neutrophils was suppressed in a concentration-dependent manner by the exogenous addition of several free sphingoid amines and short chain ceramides. Sphingosine, dihydrosphingosine, phytosphingosine, N-acetylsphingosine, and N-acetylphytosphingosine, but not N-acetyldihydrosphingosine, inhibited formyl peptide-stimulated oxidant release. The half-maximal inhibitory concentrations of N-acetylsphingosine and N-acetylphytosphingosine were 0.51 and 0.38 microM, respectively. Sphingosine, dihydrosphingosine, and phytosphingosine were less potent inhibitors with half-maximal inhibitory concentrations of 1.78, 15.4, and 1.48 microM, respectively. The 4 beta-phorbol 12 beta-myristate 13 alpha-acetate-induced respiratory burst was inhibited by 5 microM of sphingosine but not by 5 microM of N-acetylsphingosine. The effects of N-acetyl-conjugated sphingols (C2 ceramides) on phosphatidylcholine-specific phospholipase D and phosphatidic acid phosphohydrolase were markedly different from the effects of the related sphingoid bases. Both C2 ceramides and sphingoid bases partially inhibited the diradylglycerol formation by the phosphatidylcholine-specific phospholipase D pathway. Under the same conditions, however, N-acetyldihydrosphingosine and dihydrosphingosine failed to suppress H2O2 release in fMLP-stimulated neutrophils. These findings demonstrate that C2 ceramides inhibit H2O2 generation in fMLP-stimulated neutrophils via protein kinase C- or sphingoid base-independent mechanisms. The effect of ceramide in inhibiting the respiratory burst is structurally specific, because either a 4,5-trans double bond or 4-hydroxyl group is required for the inhibition. Therefore, ceramides may regulate oxidant release in adherent neutrophils.

Cell Adhesion↗
PubMed1994

Phospholipase D-mediated diradylglycerol formation coincides with H2O2 and lactoferrin release in adherent human neutrophils.

Polymorphonuclear leukocytes (PMNs) adherent to fibrinogen exhibit a delay in the onset of the respiratory burst in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP). Previously, we demonstrated that H2O2 release in adherent PMNs coincides with the exocytosis of lactoferrin-containing specific granules. Since diradylglycerol (DRG) has been implicated in PMN secretion and oxidant release, we measured DRG formation during PMN adhesion to fibrinogen. PMNs were added to fibrinogen-coated plastic in the presence of fMLP, and H2O2 release, lactoferrin release, and DRG formation measured over a time course of 120 min. H2O2 and lactoferrin release were not apparent until 45-60 min, reaching maximal levels by 120 min. In contrast, DRG concentration increased by 15-30 min, from 275 +/- 27 pmol/mg of protein in resting cells to 600 +/- 173 pmol/mg protein in cells exposed to fMLP. DRG levels returned to base line by 30-45 min (383 +/- 32 pmol/mg of protein) before increasing again between 60 and 120 min (944 +/- 230 pmol/mg of protein and 1632 +/- 351 pmol/mg of protein, respectively). Propranolol, an inhibitor of phosphatidate phosphohydrolase, caused a dose-dependent inhibition of both H2O2 and lactoferrin release, with maximal inhibition at 50-100 microM. Propranolol also inhibited the second, but not the first phase of DRG formation. Similarly, ethanol treatment completely blocked H2O2 and lactoferrin release, and the second phase of DRG formation. In the presence of ethanol, phospholipase D (PLD)-mediated formation of [3H]phosphatidylethanol from 3H-O-alkyl-phosphatidylcholine corresponded to the second, but not the first, phase of DRG formation (23,169 +/- 2,017 cpm/mg protein, ethanol versus 2,696 +/- 261 cpm/mg protein, control). These data indicate that DRG, generated through the activation of PLD, plays an important role in degranulation and oxidant release in adherent PMNs.

Cell Adhesion↗