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First published online 1 March 2005
doi: 10.1242/jcs.01730

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Stimulation of erythrocyte ceramide formation by platelet-activating factor

¹ Department of Physiology, University of Tübingen, Germany
² Department of Radiation Oncology, University of Tübingen, Germany
³ Department of Biochemistry and Molecular Biology, University of Tokyo, Japan
⁴ Department of Anesthesiology and Transfusion Medicine, University of Tübingen, Germany

View larger version (55K): [in a new window]   Fig. 1. Expression of the PAF receptor in erythrocytes. (A) Ponceau-Red protein-staining of a western blot of protein extracts from whole blood, diluted whole blood (1:20), purified erythrocytes (RBCs) or diluted platelets (1:20). Fifty microgramms or 2.5 µg (1:20) of protein were loaded per lane. The purified erythrocyte preparation contained 2.9% platelets and 4.7% white blood cells of the original whole blood sample. The molecular mass marker (size marker) is shown in the middle of the blot. (B) Erythrocyte protein extracts were prepared as described in A. Expression of the PAF receptor was then analysed by western blot with a polyclonal goat anti-PAF-receptor antibody in the presence (left) or absence (right) of specific blocking peptide. Positions of molecular mass markers are indicated in the middle. The arrow indicates the position of the PAF receptor. The asterisk indicates a non-specific band in whole blood extracts. (C) Erythrocyte protein extracts from the blood of four different donors (# 1-4) were prepared and 50 µg of protein were loaded per lane. Expression of the PAF receptor was then analysed by western blot with a polyclonal goat anti-PAF-receptor antibody (left blot). Additionally, 50 µg of the same erythrocyte extracts were loaded per lane and a platelet extract was included as positive control (Platelets). Expression of the platelet marker CD61 was analysed by western blot with a monoclonal mouse anti-CD61 antibody (right blot). Positions of molecular mass markers are indicated at the left of each blot. The arrows indicate the positions of PAF receptor (left blot) and CD61 (right blot). Representative immunoblots of three independent experiments are shown.

View larger version (25K): [in a new window]   Fig. 2. Increase of PAF by hyperosmotic shock of erythrocytes. (A) Calibration curve of PAF using the [3H] SPA system. The normalised % of bound PAF (% B/B0) is plotted as a function of the log10 PAF concentration. Data points represent the mean of two determinations with an error less than 5% by using purified PAF as standard (20-1280 pg/tube). (B) Erythrocytes were treated with isotonic Ringer or hypertonic solution (850 mOsm) for 1 or 2 hours. Lipids were then extracted and PAF in the samples was determined with the [3H] SPA system. Arithmetic means ± s.e.m. (n=8-12) of PAF concentrations in erythrocytes exposed to isotonic (control, white bars) or hypertonic (850 mOsm, black bars) extracellular fluid are given in pg/108 cells. * indicates significant difference from control (two-tailed t-test. P0.05). (C) Erythrocytes were treated with isotonic Ringer or hypertonic solution (850 mOsm) for 1 hour in the presence or absence of 25 µM quinacrine. Arithmetic means ± s.e.m. (n=4) of PAF concentrations in erythrocytes exposed to vehicle-containing isotonic (control) or vehicle-containing hypertonic (850 mOsm) extracellular fluid, to isotonic extracellular fluid in the presence of 25 µM quinacrine (Quin.) or to hypertonic extracellular fluid in the presence of 25 µM quinacrine (850 mOsm + Quin.). Values are given in pg/108 cells; # indicates significant difference from control (ANOVA, using Dunnett's test as post hoc-test. P0.05).

View larger version (32K): [in a new window]   Fig. 3. Stimulation of erythrocyte annexin-binding by PAF and influence of PAF on cell number. (A) Histograms of FITC-annexin-V-binding as obtained by FACS analysis of a representative experiment of erythrocytes incubated for 24 hours in Ringer solution containing 0.3% ethanol as control (control), in Ringer solution + PAF16 (1 µM PAF16; 3.8 µM PAF16) and in Ringer solution + PAF18 (1 µM PAF18; 3.6 µM PAF18). Numbers depict the percentage of annexin-positive cells. (B) FITC-annexin-V-binding in % of the total population as obtained by FACS analysis of erythrocytes after a 24-hour treatment with Ringer solution containing 0.3% ethanol as control (control) or after incubation with 3.8 µM PAF16 or 3.6 µM PAF18 (arithmetic means±s.e.m., n=8). * indicates significant difference from control (ANOVA, using Dunnett's test as post-hoc test; P0.05). (C) FITC-annexin-V-binding of erythrocytes in % of the total population as obtained by FACS analysis (arithmetic means±s.e.m., n=6-8), treated for 24 hours with different concentrations (0.45-3.8 µM) of PAF16 () or PAF18 (). Controls (no PAF) contained appropriate amounts of ethanol. Additionally, erythrocytes were preincubated with 25 µM quinacrine for 3 hours, and PAF16 () was diluted to the appropriate concentrations (125–2000 nM) from 2 mM DMSO stock solutions. FITC-annexin-V-binding in % of the total population as obtained by FACS analysis (arithmetic means ± s.e.m., n=3-5) is depicted in the insert. * indicates significant difference from controls (ANOVA, using Dunnett's test as post-hoc test; P0.05). (D) Number of erythrocytes seeded at 0.3% haematocrit and treated for 24 hours with different concentrations (0.45-7.6 µM) of PAF16 () or PAF18 (). The number of erythrocytes (arithmetic means ± s.e.m., n=6-8) after treatment is given in 106 cells/ml. Controls (no PAF) contained appropriate amounts of DMSO vehicle. * indicates significant difference from controls (ANOVA, using Dunnett's test as post-hoc test; P0.05).

View larger version (33K): [in a new window]   Fig. 4. Effect of PAF on erythrocyte cell volume. (A) Histograms of forward-scatter in FACS analysis of a representative experiment of erythrocytes incubated for 24 hours in Ringer solution containing 0.3% ethanol as a control (control), in Ringer solution + PAF16 (1 µM PAF16; 3.8 µM PAF16) and in Ringer solution + PAF18 (1 µM PAF18; 3.6 µM PAF18). Numbers depict the geometric mean of the forward scatter of the cell population. (B) Erythrocyte forward scatter in FACS analysis after a 24-hour treatment with Ringer solution containing 0.3% ethanol (control) and after incubation with 3.8 µM PAF16 or 3.6 µM of PAF18 (means ± s.e.m., n=8). * indicates significant difference from control (ANOVA, using Dunnett's test as post-hoc test; P0.05). (C) Forward scatter in FACS analysis of erythrocytes (means ± s.e.m., n=6-8), treated for 24 hours with different concentrations (0.45-3.8 µM) of PAF16 () or PAF18 (). Controls (0 µM PAF) contained appropriate amounts of ethanol. Additionally, erythrocytes were preincubated with 25 µM quinacrine for 3 hours and PAF16 () was diluted to the appropriate concentrations (125-2000 nM) from 2 mM DMSO stock solutions. Forward scatter in FACS analysis (means ± s.e.m., n=3-5) is depicted in the insert and is given in arbitrary units. * indicates significant difference from controls (ANOVA, using Dunnett's test as post-hoc test; P0.05).

View larger version (18K): [in a new window]   Fig. 5. Influence of Triton X-100 on cell number and erythrocyte-annexin-binding. (A) Number of erythrocytes seeded at 0.2% haematocrit and treated for 24 hours with different concentrations (0.0003%-0.03%) of Triton X-100 (). The number of erythrocytes after treatment is given in 106 cells/ml (arithmetic means ± s.e.m., n=6). Controls (0% Triton X-100) were treated with Ringer solution. * indicates significant difference from controls (ANOVA, using Dunnett's test as post-hoc test; P0.05). (B) Number of FITC-Annexin-V-binding cells per 20,000 cells as obtained by FACS analysis of erythrocytes after a 24-hour treatment with Ringer solution or after incubation with 0.0003% or 0.001% Triton X-100 (arithmetic means ± s.e.m., n=6). As a positive control, erythrocytes were treated with 1 µM PAF16 diluted from a 2 mM DMSO stock solution. * indicates significant difference from vehicle-treated control (ANOVA using Dunnett's test as post hoc test; P0.05).

View larger version (21K): [in a new window]   Fig. 6. Knockout of PAF-receptor abrogates and treatment with the PAF-receptor antagonist ABT491 blunts PAF-induced stimulation of erythrocyte-annexin-binding. (A) Histograms of FITC-Annexin-V-binding as obtained by FACS analysis in a representative experiment of murine erythrocytes from a wild-type mouse (PAF-R+/+; upper histograms) or a PAF receptor knockout mouse (PAF-R–/–; lower histograms) incubated for 3 hours in Ringer solution containing DMSO vehicle (left histograms, control) or in Ringer solution containing 0.5 µM PAF16 (right histograms). Numbers give the percentage of annexin-positive cells. (B) FITC-Annexin-V-binding in % of control as obtained by FACS analysis of murine erythrocytes from PAF-R+/+ (black bars) or PAF-R–/– mice (white bars) after a 3-hour treatment with Ringer solution containing DMSO vehicle (0 µM), or after incubation with 0.5 µM PAF16 (arithmetic means ± s.e.m., n=10). * indicates significant difference from the respective control erythrocytes; # indicates significant difference from PAF-treated erythrocytes of PAF-R+/+ mice (ANOVA, using Tukey's test as post-hoc test; P0.05). (C) FITC-Annexin-V-binding in % of control as obtained by FACS analysis of human erythrocytes (arithmetic means ± s.e.m., n=8), treated for 24 hours with Ringer solution containing DMSO vehicle (0 µM) or 0.5 µM PAF16 in the absence (black bars) or presence (white bars) of 1 µM ABT491. * indicates significant difference from the respective control erythrocytes; # indicates significant difference from PAF-treated erythrocytes in the absence of ABT491 (ANOVA, using Tukey's test as post-hoc test; P0.05).

View larger version (17K): [in a new window]   Fig. 7. Intracellular Ca2+-activity and annexin-binding of erythrocytes following treatment with PAF and ionomycin. Erythrocytes were loaded with Fluo-3/AM and intracellular Ca2+ was determined by FACS analysis, as described in Materials and Methods, after a 30-minute treatment with 3.8 µM PAF16 or 1 µM ionomycin. Additionally, annexin-binding was determined in parallel by FACS analysis. (A) Representative histograms of Ca2+-dependent fluorescence in FL1 of vehicle-treated erythrocytes (black line), of PAF16-treated erythrocytes (red line) or of ionomycin-treated erythrocytes (red line). Numbers give the percentage of Fluo-3 positive cells. (B) Arithmetic means ± s.e.m. (n=4) of Fluo-3-positive cells in % of the total population of vehicle-treated erythrocytes (control), PAF-treated erythrocytes (PAF16) or ionomycin-treated erythrocytes (ionomycin). * indicates significant difference from control erythrocytes (ANOVA, using Dunnett's test as post-hoc test; P0.05). (C) Arithmetic means ± s.e.m. (n=4) of FITC-Annexin-V-binding in % of the total population of vehicle-treated erythrocytes (control), PAF-treated erythrocytes (PAF16) or ionomycin-treated erythrocytes (ionomycin). * indicates significant difference from control erythrocytes (ANOVA, using Dunnett's test as post-hoc test; P0.05).

View larger version (29K): [in a new window]   Fig. 8. Induction of ceramide formation and sphingomyelin breakdown in erythrocytes following PAF treatment and inhibition of PAF-induced annexin-binding of erythrocytes by urea. (A) Histograms of anti-ceramide FITC-coupled-antibody-binding as obtained by FACS analysis in a representative experiment of erythrocytes incubated for 24 hours in Ringer solution containing 0.3% ethanol (all histograms; black lines), in Ringer solution + 3.8 µM PAF16 (left histogram; red line), in Ringer solution + 3.6 µM PAF18 (middle histogram; red line) or in Ringer solution + 1 U/ml SMase (right histogram; red line). (B) Fluorescence intensity of anti-ceramide FITC-coupled antibody (in arbitrary units) as obtained by FACS analysis of erythrocytes after a 24-hour treatment with Ringer solution containing appropriate amounts of ethanol (control) or after incubation with 3.8 µM PAF 16, 3.6 µM PAF18 or after 1 U/ml SMase (means ± s.e.m., n=8). * indicates significant difference from control (ANOVA, using Dunnett's test as post-hoc test. P0.05). (C) Erythrocytes were labelled with [3H]methylcholine chloride for 72 hours. Then, erythrocytes were exposed to 3.8 µM PAF16 () or 3.6 µM PAF18 () for different periods of time. Control erythrocytes () were treated in parallel with Ringer solution containing 0.3% ethanol. After incubation, sphingomyelin was determined with bacterial sphingomyelinase. Sphingomyelin levels are given in % of control (means ± s.e.m., n=3). * indicates significant difference from control (ANOVA, using Dunnett's test as post-hoc test; P0.05). (D) FITC-Annexin-V-binding in % of the total population as obtained by FACS analysis of erythrocytes after a 24-hour treatment with Ringer solution containing 0.3% ethanol vehicle (control) or after incubation with 3.8 µM PAF16 in the absence (black bars) or presence (white bars) of 600 mM urea (arithmetic means ± s.e.m., n=8). * indicates significant difference from the respective control; # indicates significant difference from PAF16-treated erythrocytes in the absence of urea (ANOVA, using Tukey's test as post-hoc test; P0.05).