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 microM) and dibromoacetophenone (2 microM), and 0.25% BSA, 87.4 +/- 3.2% of added [3H]PAF C18:0 remained intact after incubation at 25 degrees 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 degrees 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 Kd 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 Kd 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 Kd 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: Kds 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.(ABSTRACT TRUNCATED AT 400 WORDS)
Department of Obstetrics and Gynecology, University of Texas Health Science Center, San Antonio 78284-7836.
G B
GB
Kudolo
M J
MJ
Harper
D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins; D002855:Chromatography, Thin Layer; D004717:Endometrium / Q000378:metabolism*; D005260:Female; D005615:Freezing; D008024:Ligands; D010980:Platelet Membrane Glycoproteins; D011247:Pregnancy; D011270:Pregnancy, Animal / Q000378:metabolism*; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled; D012710:Serum Albumin, Bovine / Q000494:pharmacology*; D005719:gamma-Globulins / Q000494:pharmacology
D002352:Carrier Proteins; D008024:Ligands; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D005719:gamma-Globulins; C039351:platelet activating factor receptor; D012710:Serum Albumin, Bovine