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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.
Mol Pharmacol. 1993 Feb; 43(2):302-12.MP

Abstract

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.

Authors+Show Affiliations

Department of Thoracic Medicine, Royal Brompton National Heart and Lung Institute, London, United Kingdom.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

8381515

Citation

Ring, P C., et al. "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." Molecular Pharmacology, vol. 43, no. 2, 1993, pp. 302-12.
Ring PC, Seldon PM, Barnes PJ, et al. 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. Mol Pharmacol. 1993;43(2):302-12.
Ring, P. C., Seldon, P. M., Barnes, P. J., & Giembycz, M. A. (1993). 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. Molecular Pharmacology, 43(2), 302-12.
Ring PC, et al. 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. Mol Pharmacol. 1993;43(2):302-12. PubMed PMID: 8381515.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Ring,P C, AU - Seldon,P M, AU - Barnes,P J, AU - Giembycz,M A, PY - 1993/2/1/pubmed PY - 1993/2/1/medline PY - 1993/2/1/entrez SP - 302 EP - 12 JF - Molecular pharmacology JO - Mol Pharmacol VL - 43 IS - 2 N2 - 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. SN - 0026-895X UR - https://www.unboundmedicine.com/medline/citation/8381515/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_ L2 - http://molpharm.aspetjournals.org/cgi/pmidlookup?view=long&amp;pmid=8381515 DB - PRIME DP - Unbound Medicine ER -