Sphingosylphosphorylcholine induces a hypertrophic growth response through the mitogen-activated protein kinase signaling cascade in rat neonatal cardiac myocytes.
(65/2523)
The sphingolipid metabolites, sphingosine (SPH), SPH 1-phosphate (S1P), and sphingosylphosphorylcholine (SPC), can act as intracellular as well as extracellular signaling molecules. These compounds have been implicated in the regulation of cell growth, differentiation, and programmed cell death in nonmyocytes, but the effects of sphingolipid metabolites in cardiac myocytes are not known. Cultured neonatal rat cardiac myocytes were stimulated with SPH (1 to 10 micromol/L), S1P (1 to 10 micromol/L), or SPC (0.1 to 10 micromol/L) for 24 hours to determine the effects of sphingolipid metabolites on the rates of protein synthesis and degradation. Stimulation with SPC led to an increase in the total amount of protein, an accelerated rate of total protein synthesis, and a decrease in protein degradation in a dose-dependent manner. However, S1P had little effect and SPH had no effect on total protein synthesis. In addition, stimulation with SPC led to a 1.4-fold increase in myocardial cell size and enhanced atrial natriuretic factor gene expression. Pretreatment of the cardiac myocytes with pertussis toxin or PD98059 attenuated the SPC-induced hypertrophic growth response. Further, stimulation with SPC increased phosphorylation of mitogen-activated protein kinase (MAPK) and stimulated MAPK enzyme activity. Finally, endothelin-1 stimulated the generation of SPC in cardiac myocytes. The observation that SPC induces a hypertrophic growth response in cardiac myocytes suggests that SPC may play a critical role in the development of cardiac hypertrophy. The effects of SPC could be mediated, in part, by activation of a G protein-coupled receptor and a MAPK signaling cascade. (+info)
Analysis of natural and synthetic sphingomyelins using high-performance thin-layer chromatography.
(66/2523)
The chromatographic behaviour of molecular species of sphingomyelin on HPTLC was investigated. Sphingomyelin gave a double band pattern on HPTLC plates developed using chloroform/methanol/acetic acid/water (25 : 15 : 4 : 2, v/v) or chloroform/methanol/water (25 : 10 : 1.1, v/v). HPTLC analysis of acyl chain-defined sphingomyelins showed that the Rf values increased linearly with the length of the N-linked acyl chain. A double-banded pattern was therefore seen for natural sphingomyelins with a bimodal fatty acid composition. Racemic sphingomyelins also gave a double band pattern on HPTLC, where the lower band represented the Derythro and the upper band the Lthreo isomer. We also showed that Derythro-N-16:0-dihydrosphingomyelin migrated faster on HPTLC than Derythro-N-16:0-sphingomyelin. The upper and lower band sphingomyelins from two different cell lines (human skin fibroblasts and baby hamster kidney cells) were separately scraped off the HPTLC plates and the fatty acid and long-chain base profiles were studied using GC-MS. The lower bands contained short-chain fatty acids and most of the fatty acids in the upper bands were long. The predominant long-chain base was sphingosine, which was found in both upper and lower bands, but sphinganine was found only in the upper bands. To conclude, there are at least three possible reasons for the sphingomyelin double bands on HPTLC; acyl chain length, long-chain base composition and stereochemistry. These reasons might sometimes overlap and, therefore, HPTLC alone is insufficient for complete analysis of the molecular species of sphingomyelin. (+info)
Sphingosine 1-phosphate stimulates cell migration through a G(i)-coupled cell surface receptor. Potential involvement in angiogenesis.
(67/2523)
Sphingosine 1-phosphate (SPP) has been shown to inhibit chemotaxis of a variety of cells, in some cases through intracellular actions, while in others through receptor-mediated effects. Surprisingly, we found that low concentrations of SPP (10-100 nM) increased chemotaxis of HEK293 cells overexpressing the G protein-coupled SPP receptor EDG-1. In agreement with previous findings in human breast cancer cells (Wang, F., Nohara, K., Olivera, O., Thompson, E. W., and Spiegel, S. (1999) Exp. Cell Res. 247, 17-28), SPP, at micromolar concentrations, inhibited chemotaxis of both vector- and EDG-1-overexpressing HEK293 cells. Nanomolar concentrations of SPP also induced a marked increase in chemotaxis of human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC), which express the SPP receptors EDG-1 and EDG-3, while higher concentrations of SPP were less effective. Treatment with pertussis toxin, which ADP-ribosylates and inactivates G(i)-coupled receptors, blocked SPP-induced chemotaxis. Checkerboard analysis indicated that SPP stimulates both chemotaxis and chemokinesis. Taken together, these data suggest that SPP stimulates cell migration by binding to EDG-1. Similar to SPP, sphinganine 1-phosphate (dihydro-SPP), which also binds to this family of SPP receptors, enhanced chemotaxis; whereas, another structurally related lysophospholipid, lysophosphatidic acid, did not compete with SPP for binding nor did it have significant effects on chemotaxis of endothelial cells. Furthermore, SPP increased proliferation of HUVEC and BAEC in a pertussis toxin-sensitive manner. SPP and dihydro-SPP also stimulated tube formation of BAEC grown on collagen gels (in vitro angiogenesis), and potentiated tube formation induced by basic fibroblast growth factor. Pertussis toxin treatment blocked SPP-, but not bFGF-stimulated in vitro angiogenesis. Our results suggest that SPP may play a role in angiogenesis through binding to endothelial cell G(i)-coupled SPP receptors. (+info)
Possible involvement of cytochrome c release and sequential activation of caspases in ceramide-induced apoptosis in SK-N-MC cells.
(68/2523)
Ceramide is characterized as a second messenger of apoptosis induced by various agents such as tumor necrosis factor (TNF-alpha), Fas ligand, hydrogen peroxide, heat shock and ionizing radiation. In this study, we investigated the mechanism of ceramide-induced apoptosis using a human neuroblastoma cell line, SK-N-MC. N-Acetyl-sphingosine (C2-ceramide), a cell-permeable ceramide analogue, was able to induce apoptosis in SK-N-MC cells as estimated by DNA fragmentation and chromatin condensation. C2-ceramide-induced DNA fragmentation was blocked by caspase inhibitor (Z-Asp-CH(2)-DCB). An increase in caspase-3 (CPP32)-like protease activity was evident during C2-ceramide-induced apoptosis, suggesting that caspases are involved in this apoptosis. Moreover, enzymatic cleavage of VDVAD-AFC and LEHD-AFC (specific substrates for caspase-2 and -9, respectively) was increased by treatment with C2-ceramide. To elucidate which types of caspase are activated in C2-ceramide-treated cells, we performed Western blot analysis using antibodies against each isoform. Both proforms of caspase-2 and -3 were decreased in response to C2-ceramide in a time-dependent manner. Mitochondrial cytochrome c is also time-dependently released into the cytosol in response to treatment with C2-ceramide. Addition of cytochrome c into the S-100 fractions prepared from SK-N-MC cells could activate caspase-2 in cell-free systems. These results suggest the possibility that cytochrome c released to the cytosol can activate caspases (caspase-9, -3, and -2) during C2-ceramide-induced apoptosis of SK-N-MC cells. (+info)
Induction of apoptosis by novel synthesized acylamides of human lymphocytes.
(69/2523)
To investigate a pathway to apoptosis which may involve ceramides and to elucidate the minimum structure which leads to apoptosis, we synthesized several novel acylamides. Although the four synthesized compounds were different in structure from C2-ceramide, they caused Jurkat cells to undergo apoptosis. The most effective of them was N-myristoyl-D-alaninol (D-MA), as shown by DNA fragmentation (detected with propidium iodide) and a decrease in the mitochondrial transmembrane potential (DeltaPsi(m)) (detected with rhodamine 123). Nevertheless, peripheral blood leukocytes exhibited no change after D-MA exposure, like after C2-ceramide or anti-Fas antibody treatment. The DNA fragmentation and DeltaPsi(m) caused by D-MA were blocked by a caspase-3 specific inhibitor as in the case of anti-Fas antibody stimulation. Quantification of ceramides by metabolic labeling with [(14)C]palmitic acid and HPTLC showed no increases in the ceramide levels on stimulation with D-MA, C2-ceramide or anti-Fas antibodies. Furthermore, D-MA had an apoptosis-inducing effect on an anti-Fas-resistant subline of Jurkat cells. These data suggest that D-MA may cause apoptosis of Jurkat cells without distinct ceramide formation and that this apoptotic pathway is very comparable, i.e. not identical, to that induced by anti-Fas antibodies. (+info)
Sphingosine 1-phosphate induces arachidonic acid mobilization in A549 human lung adenocarcinoma cells.
(70/2523)
In the present paper, the effect of sphingosine 1-phosphate (Sph-1-P) on arachidonic acid mobilization in A549 human lung adenocarcinoma cells was investigated. Sph-1-P provoked a rapid and relevant release of arachidonic acid which was similar to that elicited by bradykinin, well-known pro-inflammatory agonist. The Sph-1-P-induced release of arachidonic acid involved Ca(2+)-independent phospholipase A(2) (iPLA2) activity, as suggested by the dose-dependent inhibition exerted by the rather specific inhibitor bromoenol lactone. The Sph-1-P-induced release of arachidonic acid was pertussis toxin-sensitive, pointing at a receptor-mediated mechanism, which involves heterotrimeric Gi proteins. The action of Sph-1-P was totally dependent on protein kinase C (PKC) catalytic activity and seemed to involve agonist-stimulated phospholipase D (PLD) activity. This study represents the first evidence for Sph-1-P-induced release of arachidonic acid which occurs through a specific signaling pathway involving Gi protein-coupled receptor(s), PKC, PLD and iPLA2 activities. (+info)
Convergence of Fc gamma receptor IIA and Fc gamma receptor IIIB signaling pathways in human neutrophils.
(71/2523)
Human neutrophils (PMNs) express two receptors for the Fc domain of IgG: the transmembrane FcgammaRIIA, whose cytosolic sequence contains an immunoreceptor tyrosine-based activation motif, and the GPI-anchored FcgammaRIIIB. Cross-linking of FcgammaRIIIB induces cell activation, but the mechanism is still uncertain. We have used mAbs to cross-link selectively each of the two receptors and to assess their signaling phenotypes and functional relation. Cross-linking of FcgammaRIIIB induces intracellular Ca2+ release and receptor capping. The Ca2+ response is blocked by wortmannin and by N,N-dimethylsphingosine, inhibitors of phosphatidylinositol 3-kinase and sphingosine kinase, respectively. Identical dose-response curves are obtained for the Ca2+ release stimulated by cross-linking FcgammaRIIA, implicating these two enzymes in a common signaling pathway. Wortmannin also inhibits capping of both receptors, but not receptor endocytosis. Fluorescence microscopy in double-labeled PMNs demonstrates that FcgammaRIIA colocalizes with cross-linked FcgammaRIIIB. The signaling phenotypes of the two receptors diverge only under frustrated phagocytosis conditions, where FcgammaRIIIB bound to substrate-immobilized Ab does not elicit cell spreading. We propose that FcgammaRIIIB signaling is conducted by molecules of FcgammaRIIA that are recruited to protein/lipid domains induced by clustered FcgammaRIIIB and, thus, are brought into juxtaposition for immunoreceptor tyrosine-based activation motif phosphorylation and activation of PMNs. (+info)
Nerve growth factor stimulates MAPK via the low affinity receptor p75(LNTR).
(72/2523)
Apart from its high affinity receptor TrkA, nerve growth factor (NGF) can also stimulate the low affinity receptor p75(LNTR) and induce a Trk-independent signaling cascade. We examined the possible involvement of mitogen-activated protein kinase (MAPK) in this signaling pathway in neuronal cultures of the cerebellum of P2-aged rats and PCNA cells; both cell types express p75(LNTR) but not TrkA. We found a fast and transient phosphorylation of p42- and p44-MAPK after stimulation with NGF or C(2)-ceramide which proved to be sensitive to inhibition of MAPK kinase and protein kinase A (PKA). As stimulation with NGF also activated p21Ras it can be concluded that at least part of the observed MAPK activation was effected via p21Ras and via PKA. (+info)