Receptor-dependent Metabolism of Platelet-activating Factor in Murine Macrophages

Abstract

Degradation of platelet-activating factor (PAF) was examined by incubating PAF with macrophages from PAF receptor-deficient mice. The degradation rate was halved as compared with wild-type mice. The reduction of the rate was comparable with the presence of a PAF antagonist WEB 2086 in wild-type cells. PAF was internalized rapidly (t1 ⁄2 Ϸ 1 min) into wild-type macrophages. The PAF internalization was inhibited by the treatment of 0.45 M sucrose but was not affected by phorbol 12-myristate 13-acetate, suggesting that PAF internalizes into macrophages with its receptor in a clathrindependent manner. Internalized PAF was degraded into lyso-PAF with a half-life of 20 min. Treatment of concanavalin A inhibited the conversion of PAF into lyso-PAF, suggesting that uptake of PAF enhances PAF degradation. Lyso-PAF was subsequently metabolized into 1-alkyl-2-acyl-phosphatidylcholine. In addition, release of PAF acetylhydrolase from macrophages was enhanced when wild-type macrophages were stimulated with PAF but not from macrophages of PAF receptordeficient mice. Thus, the PAF stimulation of macrophages leads to its degradation through both intracellular and extracellular mechanisms.

Anti-apoptotic Actions of the Platelet-activating Factor Acetylhydrolase I α2 Catalytic Subunit

“…Fluorescent intensity was quantified by comparison to resolved fluorescent standards. Cytosolic and extracellular lipids were fractionated from B-PAF-treated cultures with an acidified methanol wash separating phospholipids bound to proteins on plasma membrane from internalized lipids (36). We found that 1 M B-PAF was stable in cell-free, serum-free treatment media at 37°C for at least 60 min; no degradation to B-lyso-PAF was observed in the absence of cells (Fig.…”

“…Passive PAF internalization is regulated by transbilayer movement (flipping) across the plasma membrane occurring as a result of physicochemical changes in membrane properties accompanying cellular activation (15). It has been suggested that PAF internalization in the absence of PAFR is not rapid enough to elicit acute biological activity in hematopoietic cells (36,49). In this study, we present evidence that PAFR-independent PAF internalization by nonhematopoietic cells is indeed sufficient to trigger apoptotic cell loss.…”

“…In fact, internalization of PAF mediated by PAFR endocytosis (36) may protect cells from this cell death cascade. Endocytosis of the PAF⅐PAFR complex triggers release of plasma PAF-AH from macrophages thereby reducing extracellular ligand concentrations (36).…”

Protection against Oxidative Stress-induced Hepatic Injury by Intracellular Type II Platelet-activating Factor Acetylhydrolase by Metabolism of Oxidized Phospholipids in Vivo

“…Murine peritoneal macrophages were previously shown to metabolize PAF when cells were incubated with [acetyl-3 H]PAF, and the aqueous degradation products of PAF gradually accumulated in the culture medium in a time-dependent manner (47). Using this system we investigated the effect of deficiency of PAF-AH (II) on PAF degradation by murine peritoneal macrophages.…”

“…PAF Degradation Assay-PAF degradation by cultured peritoneal macrophages was measured as described (47). Peritoneal exudate macrophages were obtained by washing the peritoneal cavity with 5 ml of ice-cold phosphate-buffered saline 3 days after intraperitoneal injection of 2 ml of sterile 4% thioglycollate.…”