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Biosynthesis of platelet-activating factor (PAF)acether). III. Formation of PAF-acether from synthetic substrates by
Abstract:
Rat adherent macrophages (M phi) isolated from 2.5 x 10(6) peritoneal cells release 5.7 +/- 1.3 ng/ml platelet-activating factor (PAF-acether: 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) when stimulated by zymosan (Z). The yield of PAF-acether was increased to 13.8 +/- 1.8 ng/ml when stimulation occurred in the presence of 0.1 mM acetyl-CoA. That M phi used the acetyl moiety of acetyl-CoA to increase PAF-acether biosynthesis was demonstrated by the following criteria 1) as opposed to sodium acetate, malonyl-CoA, butyryl-CoA, palmitoyl-CoA, and CoA were inefficient in increasing the yield of PAF-acether; 2) radiolabeled PAF-acether was obtained after exposure of Z-stimulated M phi to 3H acetyl-CoA; actually, both radioactivity and biologic activity were eluted from HPLC with the same retention time as synthetic or natural PAF-acether; 3) after treatment with phospholipase A2, both radioactivity and biologic activity disappeared from these fractions. The yield of PAF-acether was further increased to 23.6 +/- 2.9 ng/ml by adding 0.5 microM totally synthetic 2-lyso PAF-acether (1-O-octadecyl-sn-glyceryl-3-phosphorylcholine) to Z-stimulated M phi cultured in the presence of acetyl-CoA. Again, using 3H 2-lyso PAF-acether, the formation of radiolabeled PAF-acether could be demonstrated. These results indicate that an acetyl-transferase is capable of synthesizing PAF-acether from 2-lyso PAF-acether and acetyl-CoA in intact M phi.
Insights
Rat macrophages synthesize more platelet-activating factor (PAF) when provided with acetyl-CoA. This process involves an acetyl-transferase enzyme utilizing acetyl-CoA and 2-lyso PAF to produce PAF.
Area of Science:
- Biochemistry
- Immunology
- Cell Biology
Background:
- Platelet-activating factor (PAF) is a potent lipid mediator involved in inflammatory responses.
- Macrophages are key immune cells that produce and respond to PAF.
Purpose of the Study:
- To investigate the role of acetyl-CoA in PAF biosynthesis by macrophages.
- To identify the enzymatic mechanism for PAF production in macrophages.
Main Methods:
- Isolation of rat adherent macrophages.
- Stimulation of macrophages with zymosan and acetyl-CoA.
- Detection and quantification of PAF using High-Performance Liquid Chromatography (HPLC) and radiolabeling.
- Enzymatic assays using radiolabeled precursors.
Main Results:
- Zymosan-stimulated macrophages released 5.7 ng/ml of PAF, which increased to 13.8 ng/ml with acetyl-CoA.
- Acetyl-CoA, but not other acyl-CoAs or acetate, significantly enhanced PAF production.
- Radiolabeled acetyl-CoA confirmed the incorporation of the acetyl group into PAF.
- Addition of 2-lyso PAF further increased PAF yield to 23.6 ng/ml, with radiolabeled 2-lyso PAF also incorporated.
Conclusions:
- Macrophages utilize acetyl-CoA for PAF biosynthesis.
- An acetyl-transferase enzyme in macrophages synthesizes PAF from 2-lyso PAF and acetyl-CoA.