CHARACTERIZATION OF SPECIFIC BINDING-SITES OF H-3 LABELED PLATELET-ACTIVATING-FACTOR ([H-3]PAF) AND A NEW ANTAGONIST, [H-3] SR-27417, ON GUINEA-PIG TRACHEAL EPITHELIAL-CELLS

Binding of H-3-labelled platelet-activating factor ([H-3]PAF) to guinea-pig tracheal epithelial cells was time-dependent, reversible and saturable. Scatchard analysis of the saturation-binding data indicated that [H-3]PAF bound to one class of specific binding sites with high affinity (K(D) = 4.3 +/- 0.03 nM; B(max). = 0.172 +/- 0.02 fmol/10(5) cells; n = 3). Unlabelled PAF competitively and selectively inhibited the specific binding of [H-3]PAF, with 50% inhibition at 4.8 +/- 0.07 nM (n = 3). SR 27417, the first member of a newly developed PAF antagonist series, competitively displaced [H-3]PAF from its binding sites on guinea-pig tracheal epithelial cells with a K(i) of 100 +/- 3 pM (n = 3). Studies carried out in parallel demonstrated that SR 27417 was 40 times more potent than C16-PAF itself and more than 100-fold as active as the best synthetic PAF-receptor antagonist yet described. [H-3]SR 27417 displayed high-affinity, specific, reversible as well as saturable binding to a single class of binding sites on tracheal epithelial cells (K(D) = 94 +/- 7 pM; B(max.) = 0.181 +/- 0.04 fmol/10(5) cells; n = 3). C16-PAF, lyso-PAF, enantio-PAF, SR 27417 and other PAF-receptor antagonists had K(i) values which were nearly identical for both [H-3]PAF and [H-3]SR 27417, demonstrating that in guinea-pig tracheal epithelial cells they have the same binding sites. In conclusion, these data suggest that tracheal epithelial cells contain PAF-specific receptors and indicate that SR 27417 is an extremely potent PAF-receptor antagonist, as well as being a suitable radioligand for labelling PAF receptors on intact cells.