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 (C2-ceramide), on Ag-mediated cellular responses were examined. C2-Ceramide inhibited Ag- or PMA-induced activation of phospholipase D (PLD), whereas Ca2+ ionophore A23187-induced PLD activation was not affected. C2-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 C2-ceramide on these factors were examined. We have previously reported the possible involvement of protein tyrosine kinase in Ag-mediated PLD activation. However, C2-ceramide had no effect on Ag-induced protein tyrosine phosphorylation, including mitogen-activated protein (MAP) kinases. In fura-2-loaded RBL-2H3 cells, C2-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 C2-ceramide. Moreover, C2-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 C2-ceramide-treated cells. These results suggest that C2-ceramide inhibits Ag-induced PLD activation and serotonin release, probably through the blockage of Ca2+ influx and translocation of Ca(2+)-dependent PKC isozymes in RBL-2H3 cells.
Nakamura
Y
Y
Department of Otolaryngology, Gifu University School of Medicine, Japan.
Nakashima
S
S
Ojio
K
K
Banno
Y
Y
Miyata
H
H
Nozawa
Y
Y
D000818:Animals; D000941:Antigens / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000276:immunology; D004790:Enzyme Induction / Q000187:drug effects / Q000276:immunology; D005110:Extracellular Space / Q000201:enzymology; D006868:Hydrolysis / Q000187:drug effects; D007073:Immunoglobulin E / Q000187:drug effects* / Q000502:physiology*; D015471:Leukemia, Basophilic, Acute / Q000201:enzymology* / Q000276:immunology; D010716:Phosphatidylinositols / Q000378:metabolism; D010739:Phospholipase D / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000187:drug effects; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000187:drug effects*
D000941:Antigens; D010716:Phosphatidylinositols; D007073:Immunoglobulin E; D000001:Calcimycin; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D010738:Type C Phospholipases; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium