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doi: 10.1242/10.1242/jcs.00621

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

Arun Gidwani^1,*, H. Alex Brown^2,, David Holowka¹ and Barbara Baird^1,

¹ Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA
² Department of Molecular Medicine, Veterinary Medical Center, Cornell University, Ithaca, NY 14853, USA

View larger version (27K): [in a new window]   Fig. 3. FRET between Alexa488-IgE (donor) and Cy3-anti-Thy-1 (acceptor) is disrupted by short-chain ceramides. (A) Alexa488-IgE fluorescence monitored as a function of time, showing the effect of sequential addition of Cy3-anti-Thy-1 and 8 µM C6-cer. Steady-state donor fluorescence intensity values denoted are: ID, before acceptor addition; IDA, after acceptor addition; IDC, after ceramide addition. Typical transfer efficiency (E) between the donor and acceptor was 0.05-0.10. (B) Percent disruption of FRET between Alexa488-IgE and Cy3-anti-Thy-1 by 8 µM C6-cer (hatched bar), 32 µM C2-cer (filled bar) and 32 µM C2-dhcer (open bar). Percent disruption was calculated according to Eqn 1, and averages of at least three experiments plotted, with bars showing s.e.m.

View larger version (17K): [in a new window]   Fig. 1. Structures of ceramides used in this study.

View larger version (18K): [in a new window]   Fig. 2. Short-chain ceramides decrease lipid order as measured by fluorescence anisotropy of DPH-PC in plasma membrane vesicles from RBL cells. Ceramides were added to DPH-PC-labeled vesicles (150 µM phospholipids), and fluorescence anisotropy was measured subsequent to each addition. , C2-dhcer; , C2-cer; , C6-cer. Results shown are average values of at least three experiments for each ceramide; bars designating s.e.m. are shown where larger than the width of data points.

View larger version (31K): [in a new window]   Fig. 4. Effects of ceramides on antigen-stimulated Ca2+ mobilization in RBL cells parallel their effects on lipid order. (A-D) Ca2+ response of RBL cells treated with 32 µM C2-cer (A,D), 32 µM C2-dhcer (B) or 8 µM C16-cer (C) in the presence (A-C) or absence (D) of extracellular Ca2+. Data are representative of at least three experiments for each ceramide. Corresponding controls for all cases are shown in the same panel. IgE-sensitized and indo-1-loaded cells were triggered by the addition of 200 ng/ml DNP-BSA. (E) Percent inhibition of antigen-stimulated Ca2+ mobilization in RBL cells by various ceramides, in the presence or absence of extracellular Ca2+, were calculated according to Eqn 2, and averages for all experiments are plotted with the bars showing s.e.m. Filled bars, C2-cer; open bars, C2-dhcer; dot-filled bar, C16-cer.

View larger version (22K): [in a new window]   Fig. 5. Transphosphatidylation of PA by n-butanol inhibits antigen-stimulated Ca2+ mobilization in IgE-sensitized and indo-1-loaded RBL cells. (A) Ca2+ response of RBL cells in the presence of extracellular Ca2+: control cells (first trace), cells pretreated with 0.5% (v/v) n-butanol for 2 minutes (second trace), or pretreated with 0.5% (v/v) t-butanol for 2 minutes (third trace). (B) Percent inhibition of Ca2+ mobilization in RBL cells by n-butanol (dark bars) and t-butanol (open bars), in the presence and absence of extracellular Ca2+. IgE-sensitized and indo-1-loaded cells were evaluated for antigen-stimulated Ca2+ response with or without butanol treatment, and percent inhibition calculated according to Eqn 2. Results shown are averages of three experiments and bars show s.e.m.

View larger version (17K): [in a new window]   Fig. 6. Ceramides differentially inhibit in vitro enzyme activity of activated PLD2 (A) and activated PLD1 (B). Representative absolute activities for enzymes and control vesicles were: 50 pmole PC hydrolyzed/30 minutes at 37°C for PLD2 plus activators, and 250 pmole PC hydrolyzed/30 minutes at 37°C for PLD1 plus activators. Results are normalized with respect to control vesicles, and represent the average values for at least four experiments with error bars indicating s.e.m. (C) Percent inhibition of PLD1 activity by ceramides compared with percent inhibition of Ca2+ mobilization in RBL cells. Filled bars, C2-cer; open bars, C2-dhcer; dot-filled bars C16-cer.

View larger version (25K): [in a new window]   Fig. 7. Proposed signaling scheme shows PLD involvement in stimulated PIP2 production upstream of IP3-mediated Ca2+ release from stores in RBL cells. See text for details. Also indicated are the minimal steps required for FcRI-mediated exocytosis: PLC activation leading to sustained Ca2+ mobilization initiated by IP3-mediated Ca2+ release from stores, together with diacylglycerol (DAG)-mediated activation of PKC.