Conversion of alkylacetylglycerol to platelet-activating factor in HL-60 cells and subcellular localization of the mediator.
A human promyelocytic leukemia (HL-60) cell line was used to investigate the conversion of 1-alkyl-2-acetyl-sn-glycerol (alkylacetyl-G) to platelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by intact cells and in subcellular fractions in order to examine the fate of PAF synthesized de novo. Lipid extracts obtained from undifferentiated HL-60 cells incubated with [3H]alkylacetyl-G contained 2-4% of the label as [3H]PAF; several related metabolites were also detected. The yield of [3H]PAF could be dramatically increased by pretreating the cells with either oleic acid, an activator of CTP:phosphocholine cytidylyltransferase, or phenylmethylsulfonyl fluoride, an inhibitor of PAF acetylhydrolase. These results, together with a kinetic study of [3H]alkylacetyl-G metabolism, indicate the sequential participation of a cholinephosphotransferase for the conversion of [3H]-alkylacetyl-G to PAF and acetylhydrolase and transacylase activities in the remodeling pathway that metabolize the newly formed [3H]PAF to 1-[3H]alkyl-2-acyl(long chain)-sn-glycero-3-phosphocholine. The dithiothreitol-insensitive cholinephosphotransferase activity capable of converting alkylacetyl-G to PAF was localized in subcellular fractions that contain CDP-choline:1,2-dioleoyl-sn-glycerol cholinephosphotransferase (dithiothreitol-sensitive), as well as marker enzyme activities for the endoplasmic reticulum and Golgi membranes. Subcellular localization analyses also indicated that the majority of newly formed [3H]PAF and a large portion of its deacetylated metabolite were associated with the plasma membrane-containing fractions, whereas most of the 1-[3H]alkyl-2-acyl(long chain)-sn-glycero-3- phosphocholine was present in the intracellular organelles. Incubations of HL-60 cells with exogenous [3H]PAF produced a similar subcellular distribution of metabolites. Very little (less than 10%) of the [3H]PAF produced from [3H]alkylacetyl-G was released from intact cells under a variety of incubation conditions but 50% of the de novo-derived mediator was recovered in the medium of cells that were permeabilized with saponin. Our results indicate that PAF is rapidly translocated from its intracellular site of enzymatic synthesis to the plasma membrane where it is apparently sequestered in a pool that is not accessible to extracellular acceptors in contact with intact cells.
Vallari
D S
DS
Medical Sciences Division, Oak Ridge Associated Universities, Tennessee. 37831-0117.
Record
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Snyder
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D002460:Cell Line; D002798:Diacylglycerol Cholinephosphotransferase / Q000378:metabolism; D005995:Glyceryl Ethers / Q000378:metabolism*; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute; D010743:Phospholipids / Q000302:isolation & purification / Q000378:metabolism; D010972:Platelet Activating Factor / Q000096:biosynthesis* / Q000302:isolation & purification; D013347:Subcellular Fractions / Q000378:metabolism; D014316:Tritium
D005995:Glyceryl Ethers; D010743:Phospholipids; D010972:Platelet Activating Factor; D014316:Tritium; C055394:1-palmityl-2-acetylglycerol; D002798:Diacylglycerol Cholinephosphotransferase