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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. P $≤$ 0.05). (C) Erythrocytes were labelled with [³H]methylcholine chloride for 72 hours. Then, erythrocytes were exposed to 3.8 µM PAF16 ( ${blacktriangleup}$ ) or 3.6 µM PAF18 ( ${square}$ ) for different periods of time. Control erythrocytes ( ${bullet}$ ) 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; P $≤$ 0.05). (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; P $≤$ 0.05).

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