Estimation of platelet-activating factor receptors in the endometrium of the pregnant rabbit: Regulation of ligand availability and catabolism by bovine serum albumin

High affinity receptors have been demonstrated for the potent phospholipid autacoid, platelet-activating factor (PAF C18:0; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) in a variety of tissues, including the endometrium. Because of the relative instability of PAF and our previous demonstration that lyso-PAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphorylcholine), the major metabolite of PAF, displaced [³H]PAF from endometrial PAF receptor sites, we have examined the ability of bovine serum albumin (BSA) to prevent degradation of PAF and have characterized PAF and lyso-PAF binding sites in purified rabbit endometrial membranes isolated on Day 6 of pregnancy. In buffer containing the phospholipase A₂ inhibitors, quinacrine (10 μM) and dibromoacetophenone (2 μM), and 0.25% BSA, 87.4 ± 3.2% of added [³H]PAF C18:0 remained intact after incubation at 25°C for 150 min. The metabolic products, lyso-PAF and 1-O-alkyl-2-acyl-sn-glycero-3-phosphorylcholine (alkylacyl-GPC), only amounted to 5.2 ± 3.2 and 3.3 ± 1.1, respectively. At the same concentration, rabbit serum albumin (RSA) also significantly protected [³H]PAF C18:0 from metabolism, but bovine gamma globulin (BGG) was ineffective. The presence of 0.25% BSA, however, did not protect [³H]lyso-PAF C18:0 from extensive catabolism: the major product formed was [³H]alkylacyl-GPC. Insignificant amounts of [³H]PAF were formed. Under the same conditions (25°C, 150 min) in the presence of 0.25% BSA, saturation analysis revealed the presence of two types of PAF C18:0 receptors in the endometrial membranes. Type 1 sites had a K(d) of 0.42 ± 0.03 nM (mean ± SD; n = 3) and binding capacity of 0.11 ± 0.01 pmol/mg protein. Type 2 receptor sites had a K(d) of 5.96 ± 0.35 nM and a binding capacity of 1.59 ± 0.22 pmol/mg protein. Thus, in the presence of BSA, the binding capacities of the two classes of receptors were markedly reduced compared to values generated previously in its absence. The K(d) of the Type 1 sites was not significantly changed by the presence of BSA. A single class of saturable high-affinity binding sites was demonstrable for lyso-PAF C18:0 K(d)s ranged from 0.76 ± 0.58 to 11.1 ± 0.62 nM, depending on which method of analysis was used (Eadie-Hofstee, Scatchard-Rosenthal, or the Lundon nonlinear method). The binding capacities were equally varied, ranging from 0.15 ± 0.08 to 15.17 ± 4.95 pmol/mg protein. These results confirm the ability of BSA to bind PAF C18:0, but not lyso-PAF C18:0, with such high affinity that BSA-PAF complexes, but not lyso-BAF-BSA complexes, are resistant to enzymatic degradation. Because of this high affinity interaction, free [³H]PAF C18:0 was reduced and exchange was restricted to PAF receptor sites, which had even greater affinities for PAF, thus reducing the population of labeled membrane sites compared to values generated in the absence of BSA.