title: An arachidonoyl (polyenoic)-specific phospholipase A2 activity regulates the synthesis of platelet-activating factor in granulocytic HL-60 cells.
abstract: Human promyelocytic leukemia cells (HL-60) were used as a cell model to determine how arachidonic acid stimulates the synthesis of platelet-activating factor (PAF) synthesized via the remodeling pathway. In these studies HL-60 cells were cultured over 30 passages in fatty acid-free medium to deplete them of arachidonic acid. Even though the phospholipid classes from these cells contained no arachidonate, they could still be differentiated into granulocytes by dimethyl sulfoxide (1.25%). When the differentiated HL-60 cells, depleted of arachidonic acid, were stimulated with calcium ionophore A23187 in the presence of Ca2+ and [3H]acetate, only minimal amounts of [3H]PAF were produced. In contrast, if the differentiated HL-60 cells were supplemented with 10 microM arachidonic acid for 24 h and then stimulated with the ionophore, there was a large amount of [3H]PAF formed. The increase in PAF synthesis depended on the length of time the cells were supplemented with arachidonic acid; only a small increase in PAF synthesis occurred during the early hours of supplementation whereas stimulation of PAF synthesis was maximal (3-5-fold) after a 24-h period of the 20:4 supplementation. Other polyenoic fatty acid supplements (20:5, 22:4, and 22:6 for 24 h) also stimulated PAF production in the ionophore-treated HL-60 cells depleted of 20:4, but the amount of PAF was significantly less than found for the supplements of 20:4 under identical experimental conditions. Also noteworthy is that undifferentiated cells supplemented with 20:4 or their unsupplemented controls could not be stimulated by the calcium ionophore to produce PAF. Addition of indomethacin (cyclooxygenase inhibitor), A63162 (5'-lipoxygenase inhibitor), or eicosatetraynoic acid (cyclooxygenase/lipoxygenase inhibitor) to the incubations caused little change in the production of [3H]PAF in the differentiated cells supplemented with 20:4 for 24 h. On the other hand, the addition of mepacrine, bromophenacyl bromide, or U26384 (phospholipase A2 inhibitors) resulted in very large decreases (80-90% lower than controls) in the amount of [3H]PAF produced under the same conditions. Analysis of the molecular species of [3H]alkylacyl-GroPCho (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, the precursor of PAF in the remodeling pathway) in 20:4-supplemented cells prelabeled with [3H]alkyl-lyso-GroPCho revealed that only the alkylarachidonoyl-GroPCho species were preferentially decreased after stimulation with the A23187 ionophore. These results demonstrate that arachidonate must be at the sn-2 position of alkylacyl-GroPCho in order for it to serve as a precursor of PAF.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Medical Sciences Division, Oak Ridge Associated Universities, Tennessee 37831-0117.
authors.forename: K
authors.initials: K
authors.lastname: Suga
authors.forename: T
authors.initials: T
authors.lastname: Kawasaki
authors.forename: M L
authors.initials: ML
authors.lastname: Blank
authors.forename: F
authors.initials: F
authors.lastname: Snyder
mesh_terms: D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D016861:Cyclooxygenase Inhibitors; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D006098:Granulocytes / Q000378:metabolism*; D006801:Humans; D016859:Lipoxygenase Inhibitors; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D010972:Platelet Activating Factor / Q000096:biosynthesis*; D014407:Tumor Cells, Cultured
chemical_list: D001095:Arachidonic Acids; D016861:Cyclooxygenase Inhibitors; D005231:Fatty Acids, Unsaturated; D016859:Lipoxygenase Inhibitors; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D010972:Platelet Activating Factor; D016718:Arachidonic Acid; D000001:Calcimycin; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D004121:Dimethyl Sulfoxide