Ceramide inhibits IgE-Mediated activation of phospholipase D, but not of phospholipase C, in rat basophilic leukemia (RBL-2H3) cells

Ceramide, a product of sphingomyelin hydrolysis by sphingomyelinase, elicits various cellular functions and has recently been regarded as a second messenger. To investigate the role of ceramide in rat basophilic leukemia (RBL-2H3) cells, the effects of a cell-permeable analogue, N-acetylsphingosine (C-2-ceramide), on Ag-mediated cellular responses were examined. C-2-Ceramide inhibited Ag- or PMA-induced activation of phospholipase D (PLD), whereas Ca2+ ionophore A23187-induced PLD activation was not affected. C-2-Ceramide failed to inhibit PLD activity in two different in vitro assay systems. Since PLD activity is known to be regulated by several factors, the effects of C-2-ceramide on these factors were examined. We have previously reported the possible involvement of protein tyrosine kinase in Ag-mediated PLD activation. However, C-2-ceramide had no effect on Ag-induced protein tyrosine phosphorylation, including mitogen-activated protein (MAP) kinases. In fura-2-loaded RBL-2H3 cells, C-2-ceramide suppressed Ag-induced Ca2+ influx, leaving initial Ca2+ increase and inositol phosphate production unaffected. Western blot analysis revealed that Ag caused translocation of protein kinase C (PKC) alpha, beta(1), beta(2), delta, and epsilon isozymes from cytosol to membrane fraction. Translocation of alpha, beta(1), and beta 2 isozymes was specifically prevented by C-2-ceramide. Moreover, C-2-ceramide suppressed Ag-induced serotonin release. In the absence of extracellular Ca2+, Ag-induced PLD activation and release reaction were greatly reduced. The inhibitory profile was nearly the same as that obtained in C-2-ceramide-treated cells. These results suggest that C-2-ceramide inhibits Ag-induced PLD activation and serotonin release, probably through the blockage of Ca2+ influx and translocation of Ca2+-dependent PKC isozymes in RBL-2H3 cells.