Disruption of lipid order by short-chain ceramides correlates with inhibition of phospholipase D and downstream signaling by FcepsilonRI.
Specialized plasma membrane domains known as lipid rafts participate in signal transduction and other cellular processes, and their liquid-ordered properties appear to be important for their function. We investigated the possibility of using amphiphiles to disrupt lipid rafts and thereby inhibit IgE-FcepsilonRI signaling. We find that short-chain ceramides - C2-ceramide and C6-ceramide - decrease plasma membrane lipid order and reduce the extent of fluorescence resonance energy transfer between lipid-raft-associated molecules on intact cells; by contrast, biologically inactive C2-dihydroceramide does neither. Structural perturbations by these ceramides parallel their inhibitory effects on antigen-stimulated Ca2+ mobilization in RBL mast cells in the presence and absence of extracellular Ca2+. Similar inhibition of Ca2+ mobilization is caused by n-butanol, which prevents phosphatidic acid production by phospholipase D, but not by t-butanol, which does not prevent phosphatidic acid production. These results and previously reported effects of short-chain ceramides on phospholipase D activity prompted us to compare the effects of C2-ceramide, C2-dihydroceramide and C16-ceramide on phospholipase D1 and phospholipase D2 activities in vitro. We find that the effects of these ceramides on phospholipase D1 activity strongly correlate with their effects on antigen-stimulated Ca2+ mobilization and with their disruption of lipid order. Our results indicate that phospholipase D activity is upstream of antigen-stimulated Ca2+ mobilization in these cells, and they demonstrate that ceramides can serve as useful probes for investigating roles of plasma membrane structure and phospholipase D activity in cellular signaling.
Gidwani
Arun
A
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA. bab13@cornell.edu
Brown
H Alex
HA
Holowka
David
D
Baird
Barbara
B
D000818:Animals; D000440:Butanols / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002518:Ceramides / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D007073:Immunoglobulin E / Q000276:immunology / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism*; D008407:Mast Cells / Q000276:immunology / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism; D010712:Phosphatidic Acids / Q000096:biosynthesis; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017455:Receptors, IgE / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction
D000440:Butanols; D002518:Ceramides; D004791:Enzyme Inhibitors; D008051:Lipid Bilayers; D010712:Phosphatidic Acids; D017455:Receptors, IgE; C109343:dihydroceramide; D007073:Immunoglobulin E; D010739:Phospholipase D; C106801:phospholipase D1; D002118:Calcium