H. Alex Brown
Principle Investigator; Professor of Pharmacology, Chemistry, and Biochemistry; Associate Director, VICB;
Last active: 2/12/2015

Disruption of lipid order by short-chain ceramides correlates with inhibition of phospholipase D and downstream signaling by FcepsilonRI.

Gidwani A, Brown HA, Holowka D, Baird B
J Cell Sci. 2003 116 (Pt 15): 3177-87

PMID: 12829737 · DOI:10.1242/jcs.00621

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.

MeSH Terms (15)

Animals Butanols Calcium Cells, Cultured Ceramides Enzyme Inhibitors Fluorescence Resonance Energy Transfer Immunoglobulin E Lipid Bilayers Mast Cells Membrane Microdomains Phosphatidic Acids Phospholipase D Receptors, IgE Signal Transduction

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