Characterization of platelet-activating factor binding sites on uterine membranes from pregnant rabbits

G. B. Kudolo, M. J.K. Harper

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Abstract

One of the earliest signs of endometrial preparation for blastocyst implantation is a localized increase in capillary permeability, an event that is essentially inflammatory in character and thought to be a prerequisite for subsequent decidual tissue formation. Platelet-activating factor (PAF), chemically identified as 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine, is a very potent vasoactive compound that recently has been implicated in the implantation process. In the present study, PAF binding sites are characterized in the rabbit uterus. A specific, reversible, saturable, and thermally labile binding of [3H]PAF to uterine membranes has been demonstrated, exhibiting multiple binding sites. The equilibrium dissociation constant (K(d)) of the higher affinity binding site (type 1) was 3.6 ± 0.4 nM (mean ± SD) with a binding capacity (B(max)) of 3.4 ± 1.6 pmol/mg protein. The second (lower affinity) binding site (type 2) had an apparent K(d) of 114.6 ± 13.5 nM and a B(max) of 164.3 ± 17.6 pmol/mg membrane protein, under the conditions of maximal [3H]PAF binding, 25°C, 150 min. Incubations at 4°C for up to 3 h yielded only 30% of the B(max) observed at 25°C. In crude and purified endometrial membrane preparations in which the PAF binding was predominantly located, the affinity of the binding for PAF was significantly higher than for the whole uterus, giving K(d)s of 1.5 ± 0.8 and 0.8 ± 0.5 nM; these latter values were not significantly different. However, the B(max) values of 3.9 ± 0.9 pmol/mg protein and 376.8 ± 163.3 fmol/mg protein for the two endometrial preparations, respectively, did differ significantly. Kinetic analysis at 25°C resulted in a calculated K(d) of 3.28 ± 1.14 nM, which did not differ from the value for the whole uterus at the same temperature, but was greater than for the endometrial preparations. Using 4 nM [3H]PAF to selectively label only the type 1 binding sites, the relative potencies of PAF and its antagonists in displacing [3H]PAF were lyso-PAF>CV3988>PAF>U66985>A02405>BN52021>U66982. The antagonists SRI 63,441 and L652,731 were ineffective in displacing [3H]PAF at up to 5000-fold molar excess of [3H]PAF. [3H]Lyso-PAF binding at 4 nM was displaceable by PAF. All cations tested, i.e. Ca2+, Mg2+, K+, Na+, and Li+, inhibited [3H]PAF binding. Serine hydrolase inhibitors, diisopropylfluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF), inhibited binding, but bacitracin, leupeptin, and antipain stabilized it. Bacitracin was best and more consistent in combination with other enzyme inhibitors, allowing up to 20% more binding to be measured. Under the conditions of maximum [3H]PAF binding, the label was metabolized to lyso-PAF (17%) and alkylacyl-glyceryl ether phosphorylcholine (GPC) (43%), with only 36% remaining intact. In the presence of the phospholipase A2 inhibitors, dibromoacetophenone (2 μM) and quinacrine (10 μM), and the receptor-stabilizing antibiotic bacitracin (100 μM), metabolism of the [3H]PAF was substantially controlled so that 60-70% of the added [3H]PAF remained unchanged, the level of conversion to alkylacyl-GPC was reduced to less than 6%, and the amount converted to lyso-PAF remained at less than 20%. Using both [3H]PAF and [3H]lyso-PAF to trace the metabolic pathways, we found that [3H]PAF was metabolized first to lyso-PAF during the deacetylation step and then further degraded to alkylacyl-GPC. There was no evidence that the alkylacyl-GPC or lyso-PAF was converted back to PAF to any significant degree under these experimental conditions. These data suggest that the type 1 binding sites are the PAF receptors. The reversible nature of the binding between lyso-PAF, a biologically inactive metabolite, and PAF at the receptor site may serve under physiological conditions to desensitize the uterus during lyso-PAF occupation. The ability of PAF antagonists to bind to different degrees may help to elucidate the molecular characteristics of the PAF receptor sites and permit synthesis of antagonists that work specifically in the uterus.

Original languageEnglish (US)
Pages (from-to)587-603
Number of pages17
JournalBiology of reproduction
Volume41
Issue number4
DOIs
Publication statusPublished - Dec 1 1989

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ASJC Scopus subject areas

  • Reproductive Medicine
  • Cell Biology

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