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Detection of Receptor and Synthesis Antagonists of Platelet Activating Factor in Human Whole Blood and Neutrophils using Luminol- Dependent Chemiluminescence.
Van Dyke-K; Castranova-V
New Horizons In Platelet Activating Factor Research 1987:181-187
The effects of receptor and synthesis antagonists of platelet activating factor (PAF) in human blood and in neutrophils were determined using chemiluminescence. Granulocytes were purified from blood by dextran sedimentation and centrifugal elutriation. Neutrophils were isolated by density centrifugation. Chemiluminescence was measured in a liquid scintillation counter on suspensions of 1,000,000 granulocytes in medium containing 100,000,000 molar luminol. Transmembrane potential was measured fluorometrically. In the presence of extracellular sodium, PAF induced a rapid but transient depolarization of the granulocyte plasma membrane, which peaked within 15 nanoseconds in response to a maximal dose of PAF and returned to the reference level within 90 seconds. Depolarization induced by PAF was unaffected by removal of extracellular calcium, suggesting that the change resulted from a transient increase in sodium permeability but not calcium permeability. Chemiluminescence was found to be a simple and rapid method of monitoring the effects of PAF on granulocytes, including granulocytes in whole blood. The luminescence generated from purified granulocytes exhibited a time course similar to that of whole blood, although the magnitude of the response was larger with purified cells because of quenching of light by red cells in whole blood. A PAF receptor antagonist, 63-072, caused an 88 percent decrease in chemiluminescence induced by 0.00001 molar PAF in neutrophils. Kadsurenone, another antagonist, caused a 70 percent inhibition. Lyso-PAF, the inactive metabolite of PAF, induced a small and delayed luminescence, suggesting that lyso-PAF may be used by the cells as a substrate to synthesize PAF and that the latter induces the chemiluminescence. The authors conclude that PAF is not only a secretory product of granulocytes but is also an activator of these cells, and that the chemiluminescence assay has proved to be useful for screening inhibitors of activation and PAF receptor antagonists.
Blood-cells; Leukocytes; Platelets; Plasma-membrane; Calcium-compounds; Cell-wall-permeability; Photochemical-reactions; Light-emission;
New Horizons In Platelet Activating Factor Research
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