Pharmacological characterization of a receptor for platelet-activating factor on guinea pig peritoneal macrophages using [3H]apafant, a selective and competitive platelet-activating factor antagonist: evidence that the noncompetitive behavior of apafant in functional studies relates to slow kinetics of dissociation

PC Ring, PM Seldon, PJ Barnes and MA Giembycz

Department of Thoracic Medicine, Royal Brompton National Heart and Lung Institute, London, United Kingdom.

In this paper we report the characterization of a receptor for platelet- activating factor (PAF) on guinea pig peritoneal macrophages, using a radiolabeled hydrophilic PAF antagonist, [3H] apafant. [3H]Apafant bound to intact macrophages in a concentration-dependent manner that was specific, saturable, reversible, and inhibited competitively by C18- PAF (1-O-octadecyl-2-O-acetyl-sn-glyceryl- 3-phosphocholine). Scatchard transformation and Hill analysis of these data revealed that [3H]apafant identified a homogeneous population of noninteracting sites with a pKd of 8.22 nM and a Bmax of 31,600 sites/cell. The rate at which [3H]apafant associated with (Kon = 2.9 x 10(6) M-1.min-1) and dissociated from (Koff = 0.043 min-1) intact macrophages was slow, with t1/2 values of 15 and 50 min, respectively; the kinetically derived pKd was 8.3. In competition studies C18-PAF inhibited in a biphasic manner the binding of [3H]apafant to intact macrophages, which could be resolved into high (pKi = 8.27; 60%) and low (pKi = 6.06; 40%) affinity components. In macrophage membranes, the affinity of C18-PAF (pKi = 8.48) determined from competition studies with [3H]apafant was significantly reduced (pKi = 6.95) by guanosine-5'-O-(3- thio)triphosphate, whereas the mean slope of the inhibition curves was increased from 0.470 to 0.700. Functionally, C18-PAF (10 nM to 10 microM) evoked concentration-dependent .O2- generation that was biphasic in nature. Pretreatment of macrophages with apafant antagonized in a noncompetitive manner the first phase of C18-PAF (< 100 nM)-induced respiratory burst, whereas the second component (> 1 microM C18-PAF) of this response was unaffected. It is concluded that guinea pig peritoneal macrophages express receptors for PAF for which apafant has high affinity. The biphasic competition curves obtained with C18-PAF in binding experiments and the effect of guanosine-5'-O-(3- thio)triphosphate are consistent with the hypothesis that these apafant- sensitive PAF receptors are coupled to guanine nucleotide-binding proteins and can exist in at least two guanine nucleotide-regulated conformational states. It is also suggested that the noncompetitive antagonism of PAF-induced .O2- generation by apafant may be a consequence of the slow rate at which this antagonist dissociates from PAF receptors on intact macrophages.

Volume 43, Issue 2, pp. 302-312, 02/01/1993
Copyright © 1993 by American Society for Pharmacology and Experimental Therapeutics

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