Characterization of [3H]apafant binding to PAF receptor on rabbit platelet membranes: a comparison of a microplate filtration system and a standard method.
This article describes the application of a Microplate Filtration System (MFS) to a binding assay, with the results being compared to those obtained with a conventional 24-Well Filtration Manifold (24WFM). The data reported here characterize the PAF receptor on rabbit platelet membranes using [3H]apafant. The results showed that [3H]apafant labelled a homogenous population of high-affinity binding sites in a concentration-dependent manner. Binding was very specific, saturable, reversible, and proportional to receptor concentration. [3H]Apafant interacted with membranes in an apparently competitive manner, with pseudo-Hill coefficients not significantly different from unity, thus indicating that apafant did not interact cooperatively at these binding sites. A number of PAF antagonists (apafant, lexipafant, BN-52021, SCH-37370, SR-27417, UR-12670) inhibited [3H]apafant binding with slopes near unity and with a rank order of potency in good agreement with their ability to inhibit PAF-induced rabbit platelet aggregation, suggesting that the sites labelled are functional PAF receptors. C18-PAF also competed with [3H]apafant for the receptor, but yielded biphasic inhibition curves which could be resolved into high- and low-affinity components. No significant differences were found either in the equilibrium binding parameters or in the PAF antagonists affinities obtained with the 24WFM and the MFS. The use of the latter system improved sample handling efficiency and shortened overall labor time, thus representing a more suitable way to perform receptor binding assays.
Balsa
D
D
Department of Pharmacology, J. Uriach & Cía., Barcelona, Spain.
Merlos
M
M
Giral
M
M
Ferrando
R
R
García-Rafanell
J
J
Forn
J
J
D000818:Animals; D001381:Azepines / Q000378:metabolism*; D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism*; D005374:Filtration; D008297:Male; D010972:Platelet Activating Factor / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011817:Rabbits; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D014230:Triazoles / Q000378:metabolism*
D001381:Azepines; D010972:Platelet Activating Factor; D010975:Platelet Aggregation Inhibitors; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D014230:Triazoles; C039351:platelet activating factor receptor; C052518:WEB 2086