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.
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.
Department of Thoracic Medicine, Royal Brompton National Heart and Lung Institute, London, United Kingdom.
P C
PC
Ring
P M
PM
Seldon
P J
PJ
Barnes
M A
MA
Giembycz
D000818:Animals; D001381:Azepines / Q000378:metabolism* / Q000494:pharmacology; D001667:Binding, Competitive; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007700:Kinetics; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D010529:Peritoneal Cavity / Q000166:cytology; D010972:Platelet Activating Factor / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins; D011869:Radioligand Assay; D011956:Receptors, Cell Surface / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled; D016897:Respiratory Burst / Q000187:drug effects; D014230:Triazoles / Q000378:metabolism* / Q000494:pharmacology
D001381:Azepines; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D014230:Triazoles; C039351:platelet activating factor receptor; C052518:WEB 2086; C026613:1-0-octadecyl 2-0-acetyl sn-glycero-3-phosphorylcholine