A competitive receptor binding assay for platelet-activating factor (PAF): quantification of PAF in rat brain.
A radioreceptor assay (RRA) was developed using rabbit platelet membrane preparations to quantify platelet-activating factor (PAF) and lyso-PAF, the deacylated derivative of PAF, in a variety of tissues and biological fluids. We examined PAF and lyso-PAF levels in different rat brain areas with regard to the many proven and postulated actions of PAF in brain functions. Human saliva was selected to check the validity of this RRA. The samples were extracted with methanol/chloroform/water and purified by high-performance liquid chromatography on a 5-microns Nucleosil Si column (overall recovery: 78%). Sample extracts were acetylated before chromatography to assay lyso-PAF. PAF itself was assayed in non-aceylated samples. A competitive binding assay was performed using aliquots of platelet membrane preparation and tritiated PAF. The minimum detectable amount of PAF was 144 pg per tube and the receptor was highly specific for PAF. In human saliva, we confirm the presence of PAF and lyso-PAF within the range expected. Moreover there was a good correlation between the RRA and the aggregation assay (r = 0.976). A defined cocktail of protease inhibitors allowed storage of platelet membrane preparations for at least 3 months at -20 degrees C with no change in binding properties. In the brain we observed the prevalent presence of lyso-PAF and large variations in PAF and lyso-PAF concentrations between the different brain areas analyzed. PAF was undetectable in the hypothalamus but the lyso-PAF concentration was 2.5 micrograms/g wet tissue. The PAF concentration in the cortex varied from 0 to 16 ng/g wet tissue while that of lyso-PAF was 0.7 micrograms/g wet tissue. Moreover the amount of lyso-PAF varied between the different brain areas analyzed. The hippocampus contained the highest amount (7 micrograms/g wet tissue), and relatively high levels were found in the hypothalamus, medulla oblongata and corpus striatum. The cerebellum and cortex contained the lowest levels of lyso-PAF. These findings show that PAF is present in the central nervous system mainly in its inactive form, lyso-PAF, and suggest that its effects as a modulator of brain function may be dependent on deacetylation, rather than synthesis.
Tiberghien
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INSERM U 207, Institut Pasteur URIA, Paris, France.
Laurent
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Junier
M P
MP
Dray
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F
D000818:Animals; D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism; D001923:Brain Chemistry; D002462:Cell Membrane / Q000378:metabolism; D002540:Cerebral Cortex / Q000737:chemistry; D006801:Humans; D007031:Hypothalamus / Q000737:chemistry; D008297:Male; D010972:Platelet Activating Factor / Q000031:analogs & derivatives / Q000032:analysis* / Q000378:metabolism; D011817:Rabbits; D011869:Radioligand Assay / Q000379:methods*; D051381:Rats; D011919:Rats, Inbred Strains; D012463:Saliva / Q000737:chemistry
C029271:O-deacetyl platelet activating factor; D010972:Platelet Activating Factor