N-Acetylsphingosine (C2--ceramide) Inhibited Neutrophil Superoxide Formation and Calcium Influx

Abstract

Ceramide, a product arising from sphingomyelinase activity, has been shown to act as an intracellular second messenger in effecting growth inhibition, cellular differentiation, and apoptosis. In the present study, the relative effects of cell-permeable ceramides, N-acetylsphingosine (C2-ceramide) and N-hexanoylsphingosine (C6-ceramide), on neutrophil responses were measured. When cells were activated with fMet-Leu-Phe, C2-ceramide both potentiated (< 1 microM) and inhibited (> 1 microM) superoxide generation. C2- and C6-ceramide inhibited phorbol ester-induced superoxide release from neutrophils at IC50 values of 5 and 120 microM, respectively. C2-ceramide had no effect on semipurified protein kinase C activity. Neither ceramide affected significantly the general level of phosphorylated proteins in phorbol ester-treated cells. C2-ceramide (1-20 microM) alone did not change cytosolic free Ca2+ levels but inhibited Ca2+ and Mn2+ influx in fMet-Leu-Phe-activated neutrophils. In contrast, sphingosine enhanced Ca2+ entry; thus, ceramide conversion to sphingosine was not significant. Unlike C2-ceramide, C2-dihydroceramide failed to block superoxide generation or Ca2+ influx. Preincubation of cells with 10 nM okadaic acid reversed slightly the effects of C2-ceramide. Calyculin A, tautomycin, and much higher concentrations of okadaic acid inhibited agonist-induced Ca2+ influx. We postulate that C2-ceramide may inhibit neutrophil superoxide release by activation of type 2A protein phosphatases. Results suggest that protein phosphatase type 1 up-regulates Ca2+ entry, whereas type 2A (or a ceramide-activated subtype) forestalls Ca2+ entry by inactivating a calcium influx factor.

Conversion of short‐chain ceramides to short‐chain ceramide GM3 in B16 melanoma cells

“…Several lines of evidence have indicated that short-chain Cer, which could mimic natural Cer, evoke various physiological effects in different types of cell [1][2][3][4][5]. The most important finding of this study was that short-chain Cer could be converted to short-chain gangliosides GM3 in intact cells.…”

“…For example, C2-and C6-Cer have been shown to induce cell differentiation [1] and programmed cell death [2], to mediate EGF receptor phosphorylation [3] and endocytosis [4], and to inhibit neutrophil superoxide formation and calcium influx [5]. However, there have been very few reports about the intracellular metabolism of these synthetic short-chain Cer.…”

Direct Effect of Ceramide on the Mitochondrial Electron Transport Chain Leads to Generation of Reactive Oxygen Species

“…The mechanism underlying this behavior is unclear, although similar effects have also been seen in other cell types. Hence, studies describing the regulation of fMLP-induced superoxide anion generated by neutrophils found that C 2 at concentrations below 1 M potentiated the generation of this reactive species, whereas at concentrations greater than 1 M inhibited its production (40). In addition, the ability of ceramide to activate and phosphorylate protein kinase C , an atypical protein kinase C isoform, has been shown to be bifunctional (33).…”

The Formation of Ceramide-1-phosphate during Neutrophil Phagocytosis and Its Role in Liposome Fusion

“…Direct evidence for a role of ceramides in signal transduction has come from studies using exogenous sphingomyelinase and cell-permeable ceramides to duplicate receptor-mediated activation. Sphingomyelinase and short chain ceramides have been used to implicate ceramides in such diverse processes as apoptosis, Ca 2ϩ influx, superoxide generation, proliferation, and cell cycle regulation (12)(13)(14)(15)(16).…”