Decreased prostaglandin production by cholesterol-rich macrophages

The regulation of prostaglandin production by macrophages enriched in cholesterol was examined. Mouse peritoneal macrophages were incubated for 18 h with 25 micrograms/ml of human acetyl-LDL (low density lipoprotein) and trace amounts of labeled arachidonic acid. After cholesterol enrichment, the cells were incubated with phorbol 12-myristate 13-acetate (PMA), calcium ionophore, or zymosan to stimulate endogenous arachidonic acid metabolism. A high performance liquid chromatography profile of the eicosanoids released revealed no qualitative differences between unmodified and modified macrophages. Cholesterol-rich cells, however, released less prostacyclin (PGI2) and prostaglandin E2 (PGE2) compared to unmodified cells, and products from the lipoxygenase pathway became the predominant metabolites. A decrease in the synthesis of PGI2 and PGE2 by cholesterol-rich macrophages was confirmed by radioimmunoassay and radiolabeled experiments. The activity of prostaglandin synthetase was modestly increased in the cholesterol-modified macrophages compared to controls. As an estimation of phospholipase activity, the release of labeled arachidonic acid from membrane phospholipids, however, was significantly decreased in cholesterol-rich macrophages. The phosphatidylinositol fraction was particularly resistant to arachidonate release in response to calcium ionophore and PMA in the modified cells. The measurement of membrane phospholipid fatty acid composition before and after calcium ionophore supported the observation that less arachidonate was released by cholesterol-enriched cells in response to the ionophore. Based on these observations, we propose that prostaglandin synthesis from endogenous arachidonate stores is decreased in the cholesterol-rich macrophage. A decrease in agonist-induced activation of the phospholipase activity is proposed as a mechanism for this effect.     

Ca2+ requirement of prostanoid but not of superoxide production by rat Kupffer cells

The release of the prostanoids prostaglandin D2 (PGD2), prostaglandin E2 (PGE2) and thromboxane induced by zymosan and phorbol ester in cultured rat Kupffer cells was found to depend on the extracellular concentration of Ca2+ to some extent. Prostanoid formation following the addition of the calcium ionophore A 23187 was totally inhibited when calcium ions were withdrawn from the medium whereas the prostanoid synthesis from added arachidonic acid was independent of Ca2+. A half-maximal rate of PGE2 release by cells treated with zymosan, phorbol ester or A23187 was obtained at 0.6-0.7 microM free extracellular Ca2+ and greater than or equal to 100 microM free Ca2+ was required to stimulate PGE2 formation maximally. The calmodulin antagonist R24571 partially inhibited the release of PGE2 elicited by zymosan and A23187 but not by phorbol ester or arachidonic acid. Verapamil and nifedipine, two calcium channel blockers, had no effect on the formation of PGE2 irrespective of the stimulus. TMB 8 [3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester] an intracellular calcium antagonist, inhibited the synthesis of PGE2 induced by zymosan and phorbol ester. The superoxide formation following the addition of zymosan and phorbol ester was not influenced by removal of calcium ions from the medium or by addition of the various calcium antagonists. The data presented here suggest that Ca2+-dependent reactions are involved in the synthesis of prostanoids induced by zymosan and phorbol ester and that both extracellular Ca2+ and mobilization of Ca2+ from intracellular stores are needed to induce maximally the production of prostanoids in cultured rat Kupffer cells.     

Synthesis of prostanoids and cyclic nucleotides by phagocytosing rat Kupffer cells

Rat Kupffer cells in monolayer culture were allowed to phagocytose unopsonized zymosan granules. They responded with a strongly stimulated synthesis and release of prostanoids, mainly the immunologically determined prostaglandins PGE2 and PGF2 alpha. The same response could be obtained by treatment with the calcium ionophore A23187. The effects of the ionophore and the zymosan particles were of the same magnitude but not additive. The rapid uptake of Ca2+ after contact with phagocytosable material recently described by us [(1983) Eur. J. Biochem. 131, 539-543] appears to mediate the enhanced prostaglandin synthesis. That response was suppressed not only by indomethacin but also by trifluoperazine which does not inhibit Ca2+ entry in the Kupffer cells. Similar effects by R24571 and 4-bromophenacyl bromide support the participation of calcium-calmodulin and of phospholipase A2. The calcium channel blocker Verapamil did not influence the zymosan-provoked production of prostaglandin PGE2 nor were any indications obtained for a feedback inhibition by PGE1 or PGE2. Contact with zymosan resulted in a rapid but transient rise of the intracellular levels of cAMP and cGMP: 10 nM indomethacin completely blocked the increase of both cyclic nucleotides while trifluoperazine elicited different responses in the cAMP and cGMP levels. The stimulated release of prostaglandin E2 was inhibited in a dose-dependent manner by nordihydroguaiaretic acid, an inhibitor of 5-lipoxygenase and by FPL 55712, known as a receptor antagonist for some leukotrienes. This suggests a regulatory role for its metabolites on prostaglandin synthesis.     

Leukotriene B4 biosynthesis by alveolar macrophages

Resting alveolar macrophages in culture synthesized small amount of leukotriene B₄. This synthesis was increased 2.5 fold following phagocytic stimulation by zymosan, and was increased 12.6 fold after stimulation with calcium and calcium ionophore A23187. The leukotriene B₄ synthesis could be completely inhibited by nordihydroguaiaretic acid (10^?5M). Phorbol myristate acetate, a membrane perturbant, has no effect on leukotriene B₄ production by macrophages.     

Ultrastructural localization of arachidonic acid in stimulated macrophages

The distribution of 3[H] arachidonic acid incorporated into cultured mouse peritoneal macrophages was assessed upon stimulation of the cells with either the calcium ionophore A23187 or zymosan. After a labeling time of 24 h, cells were stimulated and processed for light and electron microscopic autoradiography. Grains were primarily localized over the plasma membrane and lipid-containing vesicles of both control and stimulated cells. In macrophages stimulated with ionophore, a decreased labeling density was evident in both of these cell compartments. Similar alterations in labeling pattern were observed in zymosan treated cells, although a larger decline in grain density occurred from the plasma membrane compartment. Immunocytochemical localization of PGE2, a major eicosanoid product released upon ionophore stimulation, revealed the presence of the prostaglandin in clear vesicular structures, many of which appear to be continuous with the plasma membrane. These results provide morphological evidence that different cellular pools of arachidonic acid may be differentially mobilized for eicosanoid production as a function of the mode of stimulation.     

Intracellular calcium does not appear to be essential for arachidonic acid release from stimulated macrophages as shown by studies with Quin-2

The present investigation was undertaken to study the potential role of intracellular calcium on the release of arachidonic acid from mouse peritoneal macrophages activated by inflammatory stimuli. The intracellular calcium concentration, as measured using fluorescent probe Quin-2, was 112 +/- 8.4 nM. The chelation of intracellular calcium with Quin-2 did not affect the release of arachidonic acid from macrophages upon stimulation with phorbol myristate acetate, opsonized zymosan or calcium ionophore A23187. However, the removal of calcium from the extracellular medium resulted in a 30-50% decrease in arachidonic acid release from phorbol myristate acetate- and zymosan-stimulated macrophages and also the stimulation of arachidonic acid release from calcium ionophore-stimulated cells were nullified. These studies indicated the existence of calcium-dependent and independent mechanisms modulating the release of arachidonic acid from macrophages subjected to inflammatory stimuli.     

Phospholipase A2-mediated release of arachidonic acid in stimulated guinea pig alveolar macrophages: interaction with lipid mediators and cyclic AMP

The stimulation of cultured guinea pig alveolar macrophages by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine, or by the phospholipid inflammatory mediator platelet activating factor (PAF) induced an increase in arachidonic acid release and its cyclooxygenase products. This release, which was mimicked by the association of threshold concentrations of the calcium ionophore A 23187 and of the protein kinase C activator tetradecanoyl phorbol acetate arose mainly from diacyl- and alkyl-acyl-phosphatidylcholine and phosphatidylinositol. Using [1-14C]arachidonic acid-labeled membranes as an endogenous substrate as well as dioleoyl-phosphatidyl [14C]ethanolamine as an exogenous substrate, we showed that phospholipase A2 activity of stimulated macrophages increases upon stimulation. Treatment of macrophages by prostaglandin E2 decreased the arachidonic acid release elicited by the chemotactic peptide and PAF. Furthermore, prostaglandin E2 increased and PAF decreased the cellular content in cyclic AMP. From these results we suggest that an initial stimulation of alveolar macrophages by a bacterial signal initiates the sequential activation of a phospholipase C and of phospholipase A2, leading to the release of PAF and eicosanoids. These mediators may in turn modulate the cell response by increasing or decreasing cyclic AMP, Ca2+, or diacyglycerol macrophage content.     

Release of arachidonic acid from 1-alkyl-2-acyl-sn-glycero-3-phosphocholine, a precursor of platelet-activating factor, in rat alveolar macrophages

Platelet activating factor and the bioactive metabolites of arachidonic acid are secreted by alveolar macrophages in response to stimulation by phagocytic agents or calcium ionophore. We have previously shown a deacylation-acetylation sequence in the formation of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) from alkylacyl-(long chain)-GPC (Albert, D.H. and Snyder, F. (1983) J. Biol. Chem. 258, 97-102). This sequence may be an important source of 20:4 during inflammatory reactions since, in alveolar macrophages, the ether lipid precursor of PAF represents 35% of the choline glycerophospholipids and has a much higher content (35%) of 20:4 in the sn-2 position than does diacyl-GPC (17%). Alveolar macrophages prelabeled with 14C-labeled fatty acids (16:0, 18:1, 18:2 and 20:4) and [1-3H]alkyllyso-GPC were used to study the release of fatty acids from ether-linked and diacyl phospholipids. Each of these fatty acids was incorporated primarily into the choline glycerophospholipids of alveolar macrophages. The release of 20:4 from macrophage phospholipids was increased by treatment of the labeled cells with the calcium ionophore A23187 (2 microM) or zymosan (1 mg/ml), whereas the release of 16:0, 18:1 and 18:2 was not increased above control levels by either stimuli. Although more of the labeled 20:4 is released from the diacyl-GPC (50% of the total released), substantial amounts (44%) of 20:4 are derived from alkylacyl-GPC after incubating the stimulated cells for 60 min. The loss of 20:4 continued from the diacyl species throughout the incubation period studied, whereas a slower net release of 20:4 lost from the alkylacyl-GPC fraction was evident after 2 h. We conclude that the deacylation-reacylation cycle is an important aspect of the metabolism of 20:4 and alkylacyl-GPC during inflammatory stimulation of alveolar macrophages and that the deacylation of this ether-linked phospholipid (which is the first step in the formation of PAF) is responsible for a significant amount of the 20:4 released.     

Epidermal growth factor (EGF), tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) and calcium ionophore A23187 increase cytoplasmic free calcium and stimulate arachidonic acid release and PGE2/6-keto PGF1 alpha production in cultured porcine thyroid cells

Epidermal growth factor (EGF), 12-O-tetradecanoylphorbol 13-acetate (TPA) and calcium ionophore A23187 increase cytoplasmic free calcium ([Ca2+]i) and stimulate arachidonic acid release and production of PGE2 and 6-keto PGF1 alpha, an end metabolite of PGI2, in cultured porcine thyroid cells. Addition of EGF, TPA or A23187 to the cells loaded with fura-2, a fluorescent Ca2+ indicator, causes an immediate increase in [Ca2+]i, which is the earliest event after mitogen stimulation. This [Ca2+]i response occurs immediately, reaching a maximum within several seconds. EGF, TPA and A23187 stimulate arachidonic acid release and PGE2 and 6-keto PGF1 alpha production; the maximum effects are obtained after 2-4 h incubation. EGF, TPA and A23187 increase [Ca2+]i and then stimulate arachidonic acid release and PG production.     

Release of arachidonic acid metabolites from isolated human alveolar type II cells.

Human alveolar type II cells are thought to play a role in the pathogenesis of lung injury. Patterns of mediator release of arachidonic acid metabolism by type II cells were therefore studied after challenge with calcium ionophore A23187, opsonized zymosan and hydrogen peroxide. A time- and concentration dependent release of cyclooxygenase products was observed, with release of PGE2 greater than 6-keto-PGF1 alpha greater than TxB2. Addition of glutathione or bicarbonate further increased the production of PGE2. N-ethylmaleimide, a sulfhydryl (SH) reactant, induced a dose-dependent increase in the release of TxB2 and 6-keto-PGF1 alpha, but not of PGE2. This relates most likely to the SH-dependency and glutathione requirement of the PGE2 isomerase and SH-independence of thromboxane and prostacyclin isomerase.     

Leukotriene C4 formation by enriched human basophil preparations from normal and asthmatic subjects.

Numbers of circulating basophils are increased in asthmatic subjects, compared to normal subjects. Basophil enriched cell preparations from normal and asthmatic subjects were challenged in vitro with the calcium ionophore A23187, anti-IgE, or opsonized zymosan to study leukotriene C4 formation, histamine release, and prostaglandin D2 formation. No prostaglandin D2 formation by basophils was observed. Furthermore, opsonized zymosan was not capable of inducing any mediator formation or release from basophils. At optimal stimulation conditions no differences were found between basophils from normal and asthmatic subjects concerning A23187 or anti-IgE induced leukotriene C4 formation or histamine release. A23187 and anti-IgE induced leukotriene C4 formation were in the range of 1-20 and 0.6-4.8 pmol/10(6) basophils respectively.     

Calcium ionophore and phorbol myristate acetate synergistically inhibited proteoglycan biosynthesis in articular chondrocytes by prostaglandin independent mechanism

In rabbit articular chondrocytes, phorbol myristate acetate (PMA), 1,2-dioctanoyl-sn-glycerol (DG) and calcium ionophore (A23187), reduced the proteoglycan synthesis, in a dose-dependent manner. The combined treatment by PMA and A23187 resulted in an enhanced inhibition of proteoglycan production, indicating a synergistic effect. In presence of PMA or A23187, the release of prostaglandin E2 (PGE2) was dramatically increased. The addition of indomethacin and BW755c to chondrocytes stimulated by PMA or A23187, suppressed the liberation of PGE2, but did not stop the decrease of proteoglycan synthesis.     

Release of leukotrienes C4 and B4 and prostaglandin E2 from human monocytes stimulated with aggregated IgG, IgA, and IgE

Purified human peripheral blood monocytes were stimulated with aggregated human myeloma proteins of different classes or the calcium ionophore A23187 and the release of leukotrienes C4 and B4 (LTC4, LTB4), and prostaglandin E2 (PGE2) into the supernatant was determined. The ionophore induced release of 10 +/- 5 ng LTC4/10(6) cells and 25 +/- 8 ng LTB4/10(6) cells. Aggregated IgG, IgA, and IgE, but not IgM or monomeric immunoglobulins (Ig), induced release of LTC4 and LTB4 that was approximately 10 to 20% of that induced by ionophore. In addition, IgG, IgA, and IgE, but not IgM, induced release of PGE2 (range 0.015 to 0.22 ng/10(6) cells). Aggregated Ig induced LTC4, LTB4, and PGE2 release in a dose-dependent manner; maximal leukotriene (LT) release was observed by 30 min, in contrast to PG release, which continued to increase up to 2.5 hr. Both ionophore- and Ig-induced LTC4 and LTB4 release were completely inhibited by removal of calcium from the media and by preincubation of cells with nordihydroguaiaretic acid. Indomethacin inhibited Ig-induced PGE2 release by 80%. Phagocytosis of the Ig aggregates was not required for LT or PGE2 release, since release was not inhibited by cytochalasin B. Release of LTC4, LTB4, and PGE2 induced by IgG, IgA, and IgE, but not IgM, correlated with the presence or absence of monocyte Fc receptors (FcR) as determined by rosette assays. The data suggest that IgG, IgA, and IgE immune complexes mostly likely induce monocyte arachidonic acid metabolism via cross-linking of FcR. The ability of monocytes to release eicosanoids in the absence of phagocytosis suggests that interaction of monocytes with immobilized immune complexes, such as those deposited in blood vessel walls or glomerular basement membranes, could initiate metabolism of arachidonic acid by monocytes. Such a mechanism could contribute to inflammatory reactions characterized by mononuclear cell infiltrates.     

In SHR aorta, calcium ionophore A-23187 releases prostacyclin and thromboxane A2 as endothelium-derived contracting factors

In mature spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY), acetylcholine and the calcium ionophore A-23187 release endothelium-derived contracting factors (EDCFs), cyclooxygenase derivatives that activate thromboxane-endoperoxide (TP) receptors on vascular smooth muscle. The EDCFs released by acetylcholine are most likely prostacyclin and prostaglandin (PG)H(2), whereas those released by A-23187 remain to be identified. Isometric tension and the release of PGs were measured in rings of isolated aortas of WKY and SHR. A-23187 evoked the endothelium-dependent release of prostacyclin, thromboxane A(2), PGF(2alpha), PGE(2), and possibly PGH(2) (PGI(2) > thromboxane A(2) = PGF(2alpha) = PGE(2)). In SHR aortas, the release of prostacyclin and thromboxane A(2) was significantly larger in response to A-23187 than to acetylcholine. In response to the calcium ionophore, the release of thromboxane A(2) was significantly larger in aortas of SHR than in those of WKY. In both strains of rat, the inhibition of cyclooxygenase-1 prevented the release of PGs and the occurrence of endothelium-dependent contractions. Dazoxiben, the thromboxane synthase inhibitor, abolished the A-23187-dependent production of thromboxane A(2) and inhibited by approximately one-half the endothelium-dependent contractions. U-51605, an inhibitor of PGI synthase, reduced the release of prostacyclin elicited by A-23187 but induced a parallel increase in the production of PGE(2) and PGF(2alpha), suggestive of a PGH(2) spillover, which was associated with the enhancement of the endothelium-dependent contractions. These results indicate that in the aorta of SHR and WKY, the endothelium-dependent contractions elicited by A-23187 involve the release of thromboxane A(2) and prostacyclin with a most likely concomitant contribution of PGH(2).     

Differential alteration of lipoxygenase and cyclooxygenase metabolism by rat peritoneal macrophages induced by endotoxin tolerance

Altered macrophage arachidonic acid metabolism may play a role in endotoxic shock and the phenomenon of endotoxin tolerance induced by repeated injections of endotoxin. Studies were initiated to characterize both lipoxygenase and cyclooxygenase metabolite formation by endotoxin tolerant and non-tolerant macrophages in response to 4 different stimuli, i.e. endotoxin, glucan, zymosan, and the calcium ionophore A23187. In contrast to previous reports of decreased prostaglandin synthesis by tolerant macrophages, A23187-stimulated immunoreactive (i) leukotriene (LT)C4/D4 and prostaglandin (PG)E2 production by tolerant cells was greater than that by non-tolerant controls (p less than 0.001). However, A23187-stimulated i-6-keto-PGF1 alpha levels were lower in tolerant macrophages compared to controls. Stimulation of prostaglandin and thromboxane (Tx)B2 synthesis by endotoxin or glucan was significantly less in tolerant macrophages compared to controls (p less than 0.05). iLTC4/D4 production was not significantly stimulated by endotoxin or glucan, but was stimulated by zymosan in the non-tolerant cells. Synthesis of iLTB4 by control macrophages was stimulated by endotoxin (p less than 0.01). These results demonstrate that arachidonic acid metabolism via the lipoxygenase and cyclooxygenase pathways in macrophages is differentially altered by endotoxin tolerance.     

Inhibition of phospholipase A2 activity of guinea-pig alveolar macrophages by lipocortin-like proteins purified from mice lung

Guinea-pig alveolar macrophages were harvested by bronchoalveolar lavage and purified by differential adhesion. They were labeled with 14C-Arachidonic acid and then exposed to platelet-activating factor or to the calcium ionophore A23187. The activity of cellular phospholipase A2 was considered as the release of free 14C-Arachidonic acid in the cell supernatant. The pretreatment of guinea-pig alveolar macrophages with two lipocortin-like proteins (36 kDa and 40 kDa) purified from mice lung induced a significant inhibition of their phospholipase A2 activity upon platelet-activating factor and calcium ionophore stimulation. These results indicate that lipocortin-like proteins can modulate the phospholipase A2 activity of isolated cells in vitro.     

A selective defect in arachidonic acid release from macrophage membranes in high potassium media

Murine peritoneal macrophages cultured in minimal essential medium (alpha-MEM; 118 mM Na+, 5 mM K+) released arachidonic acid (20:4) from phospholipids on encountering a phagocytic stimulus of unopsonized zymosan. In high concentrations of extracellular K+ (118 mM), 3H release from cells prelabeled with [3H]20:4 was inhibited 80% with minimal reduction (18%) in phagocytosis. The inhibitory effect of K+ on 20:4 release was fully reversed on returning cells to medium containing Na+ (118 mM). Preingestion of zymosan particles by macrophages maintained in high K+ medium resulted in cells being "primed" for 20:4 release, which was only effected (without the further addition of particles) by changing the medium to one containing Na+. In contrast, 20:4 release from cells stimulated with the calcium ionophore A23187 was unimpaired by the elevated K+ medium, suggesting no direct effect of high K+ on the phospholipase. Macrophages stimulated with zymosan in alpha-MEM metabolized the released 20:4 to prostacyclin, prostaglandin E2 (PGE2), and leukotriene C (LTC). The smaller quantity of released 20:4 in high K+ medium was recovered as 6-Keto-PGF1 alpha, the breakdown product of prostacyclin, and PGE2. No LTC was synthesized. In high K+, resting (no zymosan) macrophages synthesized hydroxyeicosatetraenoic acids from exogeneously supplied 20:4 in proportions similar to cells maintained in alpha-MEM. These findings and the similarity of products (including LTC) produced by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 stimulated cells in alpha-MEM and high K+ medium indicated that the cyclooxygenase and lipoxygenase pathway enzymes were not directly inhibited by high extracellular K+. We conclude that high concentrations of extracellular K+ uncouple phagocytosis of unopsonized zymosan from the induction of the phospholipase responsible for the 20:4 cascade and suggest that the lesion is at the level of signal transduction between the receptor-ligand complex and the phospholipase.     

LPS-stimulated release of prostaglandin E and thromboxane B2 from the U937 cell line

Human monocytes are known to metabolize arachidonic acid (AA) and to release prostaglandins upon stimulation. Previous data indicate that in vitro maturation and differentiation of monocytes result in alteration of this property with greatly diminished response to stimulators of release of prostaglandin E (PGE) and thromboxane B2 (TxB2) occurring after cells have been cultured. To further study the effects of differentiation on human monocyte AA metabolism, a model system was established based upon the human histiocytic cell line U937. Among tested stimulants, which included opsonized zymosan, complement fragment C3b, phorbol myristate acetate (PMA), calcium ionophore A23187, and concanavalin A, it was found that Escherichia coli lipopolysaccharide (LPS) was unique in that it stimulated increased release of TxB2 from U937 cells. The effect of the phorbol ester PMA, a compound commonly used to induce differentiation of U937, on the ability of U937 to respond to LPS was examined. Following 48 hr of treatment with PMA, U937 became capable of releasing both PGE and TxB2 in response to small doses of LPS. As previously observed for human monocytes, the release of PGE was delayed for several hours following stimulation and failed to reach maximal cumulative levels in culture until 24-48 hr following stimulation. In contrast to human monocytes, PMA-induced U937 were capable of maintaining their responsiveness to LPS for several days. Thus, the U937 cell line provides a useful model for study of the effects of differentiation of human mononuclear phagocytes on their ability to metabolize AA, and for the effects of LPS on histiocytic tumor cell prostaglandin release.     

Biosynthesis of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) from 1-alkyl-2-acyl-sn-glycero-3-phosphocholine by rat alveolar macrophages. Phospholipase A2 and acetyltransferase activities during phagocytosis and ionophore stimulation

1-Alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-acyl-GPC) comprises 11% of the total phospholipids of rat alveolar macrophages. This endogenous pool of alkylacyl-GPC was prelabeled by incubating the macrophages with [1,2-3H]alkyllyso-GPC (54 Ci/mmol), which enters the cells and is acylated. The effect of various stimuli on the synthesis and release into the media of labeled alkylacetyl-GPC (platelet-activating factor) from the cells was used to establish the role of inactive alkylacyl-GPC as a precursor of the biologically active derivative. A phagocytic agent (zymosan, 100 micrograms/ml) and an ionophore (A23187, 2 microM) stimulated the release of both alkylacetyl-GPC and alkyllyso-GPC into the media at the expense of cellular alkylacyl-GPC. Phospholipase A2 activity (at pH 4.5 and in 1 mM EDTA) was also increased in the media. The stimulatory effect of zymosan and the ionophore on alkylacetyl-GPC release was prevented by mepacrine (0.1 mM), an agent that inhibits the release of fatty acids from phospholipids. These data indicate that phospholipase activity is required for the biosynthesis of alkylacetyl-GPC. However, since the inhibitory effect of mepacrine was not apparent when acetate was present, it appears that the acetylation step is rate limiting. Exposure of alveolar macrophages in culture to zymosan or A23187 stimulated acetyltransferase activity 250-300%. In contrast, phorbol myristate acetate (1.6 microM), which stimulated the accumulation of lysophospholipids but not the level of alkylacetyl-GPC in the media, did not substantially increase acetyltransferase activity. We conclude that alkylacyl-GPC serves as a precursor of alkylacetyl-GPC and that the production of this potent mediator by rat alveolar macrophages can be stimulated by agents that affect phospholipase A2 and acetyltransferase activities. The latter enzyme appears to have a regulatory function in the biosynthesis of alkylacetyl-GPC.