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The production of platelet-activating factor (PAF) and the release of [3H]arachidonate were studied in human polymorphonuclear leukocytes (PMN) stimulated with thapsigargin, an inhibitor of endomembrane Ca(2+)-ATPase. Concentrations of thapsigargin as low as 10-25 nM primed PMN for both PAF production and [3H]arachidonate release in response to the chemotactic peptide (fMLP), whereas concentrations in the range 25-200 nM induced a time- and dose-dependent production of PAF, which occurred in the absence of both [3H]arachidonate release and [3H]phosphatidylethanol formation. Studies in fura-2/AM-loaded cells showed that concentrations of thapsigargin that elicited PAF production induced a protracted and long lasting elevation of cytosolic free calcium concentration ([Ca2+]i) between 200 and 700 nM. The lower concentrations primed the cells for a late [Ca2+]i elevation in response to fMLP similar to that elicited by cytochalasin B or ionomycin. PAF production showed a good correlation with the increase of [Ca2+]i (r = 0.91) irrespective of the procedure used to grade [Ca2+]i. In contrast, phorbol 12,13-dibutyrate failed to induce both PAF production and elevation of [Ca2+]i, but it was a very effective stimulator of [3H]arachidonate release and [3H]phosphatidylethanol production. These data indicate that PAF production and [3H]arachidonate release in PMN differ in both biochemical pathway and modulatory mechanisms. Whereas PAF production seems extremely sensitive to changes in [Ca2+]i, which seems to exert its modulatory effect at the lyso-PAF:acetyl-CoA acetyltransferase step, [3H]arachidonate release seems tightly modulated by protein kinase C-dependent mechanisms and is coincidental with activation of phospholipase D.

1-Alkyl-2-acetyl-sn-glycero-3-phosphocholine can be synthesized either by acetylation of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine or by transcholination of 1-alkyl-2-acetyl-sn-glycerol. Addition of opsonized zymosan to isolated human polymorphonuclear leukocytes induces a fast, transient, up to 10-fold activation of the acetyltransferase reaction without affecting the cholinephosphotransferase pathway. Based on the following results, we propose that the generation of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by human polymorphonuclear leukocytes during phagocytosis is mediated by a stimulation of the acetyltransferase reaction: 1) the time course of acetyltransferase activation agrees with it being the enzyme responsible for the synthesis of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine; and 2) a close correlation exists (r = 0.91) between the generation of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine in response to various doses of zymosan and the magnitude of acetyltransferase activation.

Human neutrophils were homogenized and fractionated on a continuous sucrose gradient to assess the subcellular location of acetyl-CoA: lyso-PAF acetyltransferase and of newly synthesized PAF (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Acetyltransferase activity showed two subcellular locations in resting neutrophils. One of them cofractionated with plasma membrane and endoplasmic reticulum markers, whereas another major location corresponded to a region of the gradient enriched in tertiary granules. No PAF was detected in resting neutrophils, but PAF synthesis was induced by cell stimulation with ionophore A23187. Most of the newly synthesized PAF was found cell-associated, showing a bimodal subcellular distribution similar to that found for acetyltransferase activity in activated cells. PAF and acetyltransferase were located in a light membrane fraction, enriched in plasma membrane and endoplasmic reticulum, and in an ill-defined region of the gradient between the specific and azurophilic granules in A23187-stimulated cells. These data support the involvement of the acetyltransferase pathway in the synthesis of PAF induced by ionophore A23187, and demonstrate the synthesis and accumulation of newly synthesized PAF in a light membrane fraction as well as in an intracellular dense organelle upon neutrophil activation.

Human polymorphonuclears release a platelet-activating factor (PAF-acether) when challenged with various stimuli. PAF-acether is a mediator that is synthesized during cell activation in a process in which a phospholipase A2 and an acetyltransferase take part. These enzymes are finely regulated and accordingly PAF-acether release may be modulated. The authors have studied some of the transductory mechanisms which are triggered during cell stimulation and the effect of their pharmacological modulation on PAF-acether release. Theophylline, methylisobutylxanthine and dipyridamole, which block phosphodiesterase of cyclic nucleotides, induce a dose-dependent inhibition of PAF-acether release without affecting phagocytic uptake. Polyamines (dansylcadaverine, rimantadine and amantadine) reduced PAF-acether release and the phagocytic process in an order of potency similar to their ability to inhibit phospholipid methylation and the cholinephosphotransferase pathway. The calmodulin antagonist trifluoperazine induced a dose-dependent inhibition of PAF-acether release and acetyltransferase at concentrations from 10(-4) to 10(-5) M. Hence it appears that modulation of PAF-acether release can be obtained by different pharmacological blockades: phosphodiesterase of cyclic nucleotides, phospholipid metabolism and calcium-calmodulin.

We have previously observed that the phagocytosis of zymosan particles coated with complement by human polymorphonuclear leucocytes is accompanied by a time- and dose-dependent inhibition of phosphatidylcholine synthesis by transmethylation [García Gil, Alonso, Sánchez Crespo & Mato (1981) Biochem. Biophys. Res. Commun.101, 740-748]. The present studies show that phosphatidylcholine synthesis by a cholinephosphotransferase reaction is enhanced, up to 3-fold, during phagocytosis by polymorphonuclear cells. This effect was tested by both measuring the incorporation of radioactivity into phosphatidylcholine in cells labelled with [Me-(14)C]choline, and by assaying the activity of CDP-choline:diacylglycerol cholinephosphotransferase. The time course of CDP-choline:diacylglycerol cholinephosphotransferase activation by zymosan mirrors the inhibition of phospholipid methyltransferase activity previously reported. The extent of incorporation of radioactivity into phosphatidylcholine induced by various doses of zymosan correlates with the physiological response of the cells to this stimulus. This effect was specific for phosphatidylcholine, and phosphatidyl-ethanolamine turnover was not affected by zymosan. The purpose of this enhanced phosphatidylcholine synthesis is not to provide phospholipid molecules rich in arachidonic acid. The present studies show that about 80% of the arachidonic acid generated in response to zymosan derives from phosphatidylinositol. A transient accumulation of arachidonoyldiacylglycerol has also been observed, which indicates that a phospholipase C is responsible, at least in part, for the generation of arachidonic acid. Finally, isobutylmethylxanthine and quinacrine, inhibitors of phosphatidylinositol turnover, inhibit both arachidonic acid generation and phagocytosis, indicating a function for this pathway during this process.

Platelet-activating factor (PAF) is a mediator of a anaphylaxis found initially in basophils and later in mouse and rat macrophages. The purpose of this paper was to determine the cellular origin of PAF released from human leucocytes and to establish if phagocytosis is a more important stimulus for PAF release than anaphylactic reactions. Phagocytic leucocytes (monocytes and PMNs) released PAF, physicochemically analogous to the PAF obtained by anaphylactic reactions in rabbits when challenged with zymosan, zymosan coated with complement, immune complexes, immunoglobulin aggregates or calcium ionophore A23187. Basophils failed to release PAF by anti-human IgE antibody, although positive degranulation and histamine liberaton were found. Pre-incubation of phagocytosing leucocytes with cytochalasin B or colchicine produced a diminution of PAF release, whereas beta-glucuronidase liberation was increased. The addition of carboxypeptidase B did not significantly modify PAF or beta-glucuronidase release. These data indicate that PAF obtained from preparations of human leucocytes comes from monocytes and polymorphonuclears; human basophils do not liberate measurable quantities of PAF, either by anaphylactic stimulus or by neutrophil cationic proteins; liberation of PAF and lysosomal content follow different mechanisms as they have different kinetics and are modified in an opposite way by drugs acting on the cytoskeleton.