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.