The platelet activating factor receptor antagonist, RP 59227, blocks platelet activating factor receptors mediating liberation of reactive oxygen species in guinea pig macrophages and human polymorphonuclear leukocytes
Floch, A.; Tahraoui, L.; Sedivy, P.; Cavero, I.
Journal of Pharmacology and Experimental Therapeutics 258(2): 567-575
1991
ISSN/ISBN: 0022-3565 PMID: 1650834 Document Number: 380641
In elicited (mineral oil) peritoneal guinea pig macrophages, there was a specific [3H]platelet activating factor (PAF) binding which displayed concentration dependency, saturability, high affinity (Kd = 2.2 .+-. 0.2 nM, n = 15), elevated capacity (Bmax = 122,808 .+-. 10,234 sites/cell, n = 15) and irreversibility. PAF(C16) and the PAF antagonists RP 59227, Ro 19-3704 and WEB 2086 prevented the binding of [3H]PAF. RP 59227 (Ki = 3.1 .+-. 0.3 nM, n = 5) was about 5- and 8-fold more potent than Ro 19-3704 and WEB 2086, respectively. In competition studies, RP 59227 produced dextral and concentration-dependent shifts of the sigmoidal inhibition curve of [3H]PAF binding by PAF(C16). Macrophages exposed to PAF produced chemiluminescence signals, the magnitude of which was concentration related (pD2 = 8.40 .+-. 0.03, n = 13) and which were inhibited by the oxygen radical scavengers, superoxide dismutase, catalase, deferoxamine and mannitol. RP 59227 and WEB 2086 antagonized in a noncompetitive manner (pD'2 = 7.72 .+-. 0.01 and 8.15 .+-. 0.05, respectively) the control PAF concentration-response curve. By contrast, Ro 19-3704 behaved as a competitive antagonist (pA2 = 8.13 .+-. 0.11). The apparent noncompetitive effects of RP 59227 were not due to undisplaceable binding to PAF receptors because washing of macrophages exposed to RP 59227 allowed the recovery of PAF luminescence and PAF binding. This procedure was poorly effective to lessen the inhibitory activity of WEB 2086. In human polymorphonuclear leukocytes, the weak potency of PAF was enhanced by 10-fold (pD2 = 7.61 .+-. 0.06, n = 6) when polymorphonuclear leukocytes were primed for 10 min with fMLP (5 nM). In the latter preparation, RP 59227 was a competitive antagonist (pA2 = 8.20 .+-. 0.12, n = 4) of PAF-induced luminescence, 35 times more potent than Ro 19-3704. WEB 2086 behaved as a weak noncompetitive antagonist (pD'2 = 6.46 .+-. 0.06, n = 3). These studies demonstrate that specific PAF binding sites are present in guinea pig macrophages and their stimulation by PAF is accompanied by chemiluminescence responses, which are mediated by the formation of reactive oxygen species. RP 59227 is a very potent antagonist of these PAF effects in both macrophages and human polymorphonuclear leukocytes. Thus RP 59227 may represent a novel therapeutic way of preventing the deleterious cellular effects produced by oxygen-free radicals which are generated by various types of cells upon their activation by PAF, a mediator liberated during pathological conditions such as ischemia or endotoxin intoxication.