Kinetics of uptake and distribution of arachidonic acid by rat alveolar macrophages

The time course of uptake and distribution of ³H-arachidoni acid (³H-AA) into rat alveolar macrophage phospholipid pools was examined. Macrophages incubated with exogenous ³H-AA in RPMI-1640 containing 0.1% bovine serum albumin (BSA), incorporated this radiolabel into phosphatidylcholine and phosphatidylinositol (PI) with plateau reached within 2 to 4 hours, which remained relatively constant for up to 18 hours. Incorporation of ³H-AA into phospholipid pools revealed that treatment with exogenous 5 nM arachidonic acid had no effect upon pool sizes, but there was a selective incorporation if ³H-AA into PI. Cells were incubated with ³H-AA in RPMI alone or medium containing either 0.2% lactalbumin, fetal calf serum at variable concentrations, 10% Nu, Serum, or 0.1% BSA. Incubation of macrophages with ³H-AA in RPMI alone or containing 0.2% lactalbumin, resulted in approximately 70% of the radiolabel taken up by the cells being incorporated into triglyceride. The addition of BSA to RPMI-1640 medium was found to facilitate selective uptake of ³H-AA into phospholipids. Approximately 70% of incorporated ³H-AA was releseable through the action of exogenous phospholipase A₂.     

Dynamics of uptake and channeling of arachidonic acid in hamster alveolar macrophages and the effect of bleomycin on arachidonyl distribution.

The uptake and distribution of [14C]-arachidonic acid (AA) by primary culture hamster alveolar macrophages (AM) were examined. The macrophages were incubated for 2 to 18 hrs in RPMI-1640 medium containing 0.1% BSA and [14C]-AA. The uptake of [14C]-AA by AM was rapid with 71% and 83% of exogenous [14C]-AA taken up after 2 and 4 hrs of incubation, respectively. Initially, the uptaken [14C]-AA was equally distributed between phospholipids (PL) and neutral lipids (NL). However, by 8 hrs, 86% and 14% of [14C]-AA was found in PL and NL, respectively. This distribution pattern remained constant through 18 hrs. Within the PL pool, most of the [14C]-AA was initially incorporated into phosphatidylcholine (PC). However, with time, as the percent of [14C]-AA incorporated in PC declined, the percent incorporated in phosphatidylethanolamine increased. The incorporation of [14C]-AA into sphingomyelin, phosphatidylinositol, and phosphatidylserine remained constant with time. Within the NL, most of the [14C)-AA was initially found incorporated into triacylglycerols (TG). After 4 hrs, the percent of [14C]-AA found in TG decreased markedly, while the percent found in cholesterol esters markedly increased. Incubation of AM prelabeled with [14C]-AA with bleomycin at 0.5, 5, 50 and 500 microM for 2 and 8 hrs failed to have any effect on the distribution of [14C]-AA in PL and NL pools.     

Compartmentation of phosphorylated precursors of phospholipid biosynthesis in cultured neuroblastoma cells

The continuous turnover of membrane phospholipids requires a steady supply of biosynthetic precursors. We evaluated the effects of decreasing extracellular Na+ concentration on phospholipid metabolism in cultured neuroblastoma (N1E 115) cells. Incubating cultures with 145 to 0 mM NaCl caused a concentration-dependent inhibition of [32P]phosphate uptake into the water-soluble intracellular pool and incorporation into phospholipid. Phospholipid classes were differentially affected; [32P]phosphate incorporated into phosphati-dylethanolamine (PE) and phosphatidylcholine (PC) was consistently less than into phosphatidylinositol (PI) and phosphatidylserine (PS). This could not be attributed to decreased phospholipid synthesis since under identical conditions, there was no effect on arachidonic acid or ethanolamine incorporation, and choline utilization for PC synthesis was increased. The effect of Na+ was highly specific since reducing phosphate uptake to a similar extent by incubating cultures in a phosphate-deficient medium containing Na+ did not alter the relative distribution of [32P]phosphate in phospholipid. Of several cations tested only Li+ could partially (50%) replace Na+. Incubation in the presence of ouabain or amiloride had no effect on [32P]phosphate incorporation into phospholipid. The differential effects of low Na+ on [32P]phosphate incorporation into PI relative to PC and PE suggests preferential compartmentation of [32P]phosphate into ATP in pools used for phosphatidic acid synthesis and relatively less in ATP pools used for synthesis of phosphocholine and phosphoethanolamine, precursors of PC and PE, respectively. This suggestion of heterogeneous and distinct pools of ATP for phospholipid biosynthesis, and of potential modulation by Na+ ion, has important implications for understanding intracellular regulation of metabolism.     

A new human cholangiocellular carcinoma cell line (HuCC-T1) producing carbohydrate antigen 19/9 in serum-free medium

Summary A human cholangiocellular carcinoma cell line, HuCC-T1, was established in vitro from the malignant cells of ascites of a 56-yr-old patient. Histologic findings of the primary liver tumor revealed a moderately differentiated adenocarcinoma. Tumor cells from the ascites have been cultured with RPMI 1640 medium containing 0.2% lactalbumin hydrolysate and the cultured cells grew as monolayers with a population doubling time of 74 h during exponential growth at Passage 25. They had an epithelial-like morphology and were positive for mucine staining. Ultrastructural studies revealed the presence of microvilli on the cell surface and poorly developed organelles in the cytoplasm. The HuCC-T1 cell was tumorigenic in nude mice. The number of chromosomes in HuCC-T1 ranged from 61 to 80. These human cholangiocellular carcinoma cells in serum-free medium secreted several tumor markers, including carbohydrate antigen 19/9, carbohydrate antigen 125, carcinoembryonic antigen, and tissue polypeptide antigen. The carbohydrate antigen 19/9 secretion level of HuCC-T1 cells cultured in PRMI 1640 medium with 1% fetal bovine serum was sixfold higher than that with 0.2% lactalbumin hydrolysate. These findings suggest that HuCC-T1 will provide useful information to clarify the mechanism of tumor marker secretion and tumor cell growth in the human cholangiocellular carcinoma.     

DNA transfection of mouse lymphoid cells by the combination of DEAE-dextran-mediated DNA uptake and osmotic shock procedure

Several mouse lymphoid cell lines were efficiently transfected with plasmid DNA by a novel method combining DEAE-dextran-mediated DNA uptake and osmotic shock procedure. The cells were first incubated with DNA-DEAE-dextran complex, treated with hypertonic Tris-HCl buffer containing 0.5 M sucrose and 10% poly(ethylene glycol), and then exposed to hypotonic RPMI 1640 medium. This transfection protocol exhibited maximal frequencies of 0.3% and 3.10(-5) for transient gene expression and stable transformation in P3-NSI/1-Ag4-1 cells, respectively.     

Uptake and utilization of arachidonic acid in infective larvae of Angiostrongylus cantonensis

Infective larvae of Angiostrongylus cantonensis may take up and incorporate exogenous arachidonic acid into their lipid pool. By scintillation counting, uptake and incorporation were determined to be time dependent. Arachidonic acid was mainly incorporated into phospholipid (56.8%) and neutral lipid (22.4%) pools. In the neutral lipids, 64.0% was diglyceride and 36.0% triglyceride. Phosphatidylcholine was the predominant fatty acid in the phospholipid pool. In addition to the release of leukotriene B4, the parasite was found to generate radiolabelled CO2 after incubation with [U-14C]arachidonate. Moreover, enzymatic analysis of crude extracts revealed the presence of acyl-CoA dehydrogenase (short and long chain), thiolase, enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase. These findings suggest that infective larvae of A. cantonensis not only take up and incorporate exogenous arachidonic acid into their lipid pool, but may also utilize the fatty acid through a functional β-oxidation pathway.     

不同氨基酸对克隆的胰岛β细胞株INS-1E细胞作用的比较

INS-1E细胞经传代培养2 d后,在Krebs-Ringer缓冲液中37℃培养箱预培养30 min,再用含16.7 mmol.L-1葡萄糖和不同AA的改良Krebs-Ringer缓冲液培养60 min,然后留取上清液进行INS测定。结果:L-亮氨酸、L-谷氨酰胺未能显示促进16.7 mmol.L-1葡萄糖诱导的INS-1E细胞的INS分泌,其余4种AA均促进葡萄糖诱导的INS-1E细胞的INS分泌。本研究显示L-脯氨酸、L-丙氨酸、L-赖氨酸、L-精氨酸均能增加葡萄糖诱导的INS-1E细胞分泌INS。     

Uptake of [H]-arachidonic acid by human endometrium. Differences between normal and menorrhagic tissue

Human proliferative and secretory endometrium from normal women and from menorrhagic patients was maintained in culture for up to a 24 h in the presence of [³H]-arachidonic acid (³H-AA). This prostaglandin (PG) precursor was incorporated into endometrial neutral lipids and phospholipids in a time-dependent manner. Uptake of ³H-AA into phospholipids was significantly higher in normal secretory endometrium than in normal proliferative endometrium. However, this increased uptake of ³H-AA into phospholipids between the 2 phases of the cycle did not occur in menorrhagic endometrium. In contrast, uptake of ³H-AA into neutral lipids (especially triglyceride) was approximately 2-fold higher in menorrhagic endometrium compared to normal endometrium at both stages of the cycle, particularly during the proliferative phase. Abnormalities apparently exist in menorrhagic endometrium in the uptake processes which control arachidonic acid (AA) turnover. These abnormalities may be responsible, in part for abnormal PG production by menorrhagic endometrium.     

Uptake of [3H]-arachidonic acid by human endometrium. Differences between normal and menorrhagic tissue

Human proliferative and secretory endometrium from normal women and from menorrhagic patients was maintained in culture for up to 24 h in the presence of [3H]-arachidonic acid (3H-AA). This prostaglandin (PG) precursor was incorporated into endometrial neutral lipids and phospholipids in a time-dependent manner. Uptake of 3H-AA into phospholipids was significantly higher in normal secretory endometrium than in normal proliferative endometrium. However, this increased uptake of 3H-AA into phospholipids between the 2 phases of the cycle did not occur in menorrhagic endometrium. In contrast, uptake of 3H-AA into neutral lipids (especially triglyceride) was approximately 2-fold higher in menorrhagic endometrium compared to normal endometrium at both stages of the cycle, particularly during the proliferative phase. Abnormalities apparently exist in menorrhagic endometrium in the uptake processes which control arachidonic acid (AA) turnover. These abnormalities may be responsible, in part for abnormal PG production by menorrhagic endometrium.     

Differential turnover of polyunsaturated fatty acids in plasmalogen and diacyl glycerophospholipids of isolated cardiac myocytes

To investigate the relative turnover of esterified polyunsaturated fatty acids in diacylglycerophospholipids and plasmalogens in isolated cardiac myocytes, we characterized the phospholipid composition and distribution of radiolabel in different phospholipid classes and in individual molecular species of diradyl choline (CGP) and ethanolamine (EGP) glycerophospholipids after incubation of isolated cardiac myocytes with [3H]arachidonate or [14C]linoleate. Plasmalogens in CGP (55%) and EGP (42%) quantitatively accounted for the total plasmalogen content (39%) of cardiac myocyte phospholipids. Plasmalogens comprised 86% and 51% of total arachidonylated CGP and EGP mass, respectively, and [3H]arachidonate was primarily incorporated into plasmalogens in both CGP (65%) and EGP (61%) classes. The specificity activity of [3H]arachidonylated diacyl-CGP was approximately 2- to 5-fold greater than that of [3H]arachidonylated choline plasmalogen, whereas comparable specific activities were found in the [3H]arachidonate-labeled ethanolamine plasmalogen and diacyl-EGP pools. Of the total linoleate-containing CGP and EGP mass, 54% and 57%, respectively, was esterified to plasmalogen molecular species. However, [14C]linoleate was almost exclusively incorporated into diacyl-CGP (96%) and diacyl-EGP (86%). The specific activities of [14C]linoleate-labeled diacyl-CGP and diacyl-EGP were 5- to 20-fold greater than that of the [14C]linoleate-labeled plasmalogen pools. The differential incorporation of polyunsaturated fatty acids in plasmalogens and diacylglycerophospholipids demonstrates that the metabolism of the sn-2 fatty acyl moiety in these phospholipid subclasses is differentially regulated, possibly fulfilling separate and distinct physiologic roles.     

The role of phosphatidylinositol turnover in initiation of prostaglandin biosynthesis in concanavalin A-induced activation of rat peritoneal macrophages

When macrophages were stimulated by concanavalin A (50 micrograms/ml) in the presence of 32Pi and 3H-arachidonic acid (AA), rapid incorporation of these radiolabeled substances into phosphatidylinositol (PI) and phosphatidic acid (PA) were induced within 10 min. The pool of PI and PA incorporating 3H-AA was the same as that of PI and PA which incorporated 32P. However, significant increase in incorporation of 32P and 3H-AA did not occur in phosphatidylcholine, phosphatidylethanolamine or phosphatidylserine within this time interval as compared with control levels. These results indicate that PI turnover might play a role in the initiation of prostaglandin biosynthesis in the early stages of macrophage activation induced by concanavalin A.     

Micromethod for chromosome preparations from Chinese hamster lymphocytes.

Chromosome preparations from Chinese hamster lymphocytes were made by culturing for 2 to 6 days 50,000 to 800,000 lymphocytes in flat-bottomed Cooke microtiter plates in 0.1 or 0.2 ml Roswell Park Memorial Institute-1640 medium (RPMI-1640) supplemented with 5--40 microliters PHA/ml, 20% fetal calf serum (FCS), and 40 microns 2-mercaptoethanol. Depending on blood volume and cell concentrations used, it was possible to obtain up to 1400 well-spread metaphases from one venepuncture.     

Bovine seminal plasma phospholipid-binding proteins stimulate phospholipid efflux from epididymal sperm.

Several studies have shown that sperm capacitation was accompanied by a change in the lipid composition of the sperm membrane. In cattle, the major proteins of (bovine)seminal plasma (BSP proteins: BSP-A1/A2, BSP-A3, and BSP-30-kDa) potentiate sperm capacitation induced by high-density lipoprotein (HDL). Our recent studies indicate that these proteins and HDL stimulate sperm cholesterol efflux during capacitation. In order to gain more insight into the mechanisms of BSP-mediated sperm capacitation, we studied whether or not BSP proteins induce phospholipid efflux from epididymal sperm membrane. By direct determination of choline phospholipids on unlabeled epididymal sperm, the results show that sperm incubated in the presence of BSP-A1/A2 protein lost 34.4% of their choline phospholipids compared with the control (11.5%). Similar results were obtained using labeled epididymal sperm. Labeling was carried out by incubating washed epididymal sperm for 1 h with medium containing [(3)H]palmitic acid. The majority of the label was incorporated into sperm phosphatidylcholine. Studies of sperm phospholipid efflux were done by incubating the labeled sperm with purified BSP proteins, delipidated BSA, or bovine seminal ribonuclease (RNase, control protein). When labeled ([(3)H]phospholipid) epididymal sperm were incubated with BSP proteins (20-120 microg/ml) for 8 h, the sperm lost [(3)H]phospholipid in a dose-dependent manner (maximum efflux of approximately 30%). After the incubation with BSP proteins, the efflux particles were fractionated by size-exclusion chromatography. Analysis of the fractions obtained showed that the [(3)H]phospholipid was associated with BSP proteins. BSA (6 mg/ml) stimulated a specific phospholipid efflux of approximately 22%. In contrast, bovine RNase (120 microg/ml) did not stimulate phospholipid efflux. These results indicate that BSP proteins participate in the sperm cholesterol and phospholipid efflux that occurs during capacitation.     

A vinblastine sensitive high affinity choline uptake system

1. The Limulus cardiac ganglion high affinity choline uptake system (HAChUS) was inhibited 40, 51 and 64% following pre-exposure to 10, 100 and 500 microM vinblastine, respectively. 2. In contrast, high affinity uptake of choline in the Limulus corpora pedunculata and abdominal ganglia, tissues in which a cholinergic function has been described, were unaffected. 3. In pulse-chase experiments, the cardiac ganglion was incubated in 0.1 microM [3H]choline for 60 min and then switched to an incubation medium containing 1 mM unlabelled choline for varying periods of time. 4. Under these conditions, a 3-fold increase of radiolabel above basal level was measured in the pellet fraction within 2 hr of post-labelling incubation. 5. Prior exposure of the ganglion to 500 microM vinblastine completely eliminated this increase of radioactivity in the pellet fraction. 6. Treatment of the radiolabelled pellet fraction with phospholipase C resulted in the solubilization of 72% of the radiolabel. 7. Ten (10) microM 5-hydroxytryptamine (5-HT), a concentration previously shown to inhibit spontaneous electrical activity within the cardiac ganglion, resulted in a 40% decrease in high affinity choline uptake in this tissue selectively. 8. These results are consistent with the view that a probable role of the Limulus cardiac ganglion HAChUS is the supply of choline subserving the synthesis of membrane phospholipid. 9. It is further speculated that this membrane phospholipid synthesis may be associated with synaptic vesicle turnover.     

Uptake of [3H]-arachidonic acid by human endometrium. Differences between normal and menorrhagic tissue

Human proliferative and secretory endometrium from normal women and from menorrhagic patients was maintained in culture for up to a 24 h in the presence of [³H]-arachidonic acid (³H-AA). This prostaglandin (PG) precursor was incorporated into endometrial neutral lipids and phospholipids in a time-dependent manner. Uptake of ³H-AA into phospholipids was significantly higher in normal secretory endometrium than in normal proliferative endometrium. However, this increased uptake of ³H-AA into phospholipids between the 2 phases of the cycle did not occur in menorrhagic endometrium. In contrast, uptake of ³H-AA into neutral lipids (especially triglyceride) was approximately 2-fold higher in menorrhagic endometrium compared to normal endometrium at both stages of the cycle, particularly during the proliferative phase. Abnormalities apparently exist in menorrhagic endometrium in the uptake processes which control arachidonic acid (AA) turnover. These abnormalities may be responsible, in part for abnormal PG production by menorrhagic endometrium.     

Arachidonic acid turnover in peritoneal macrophages is altered in endotoxin-tolerant rats

Peritoneal macrophages from endotoxin-tolerant rats have been found to exhibit depressed metabolism of arachidonic acid (AA) to prostaglandins and thromboxane in response to endotoxin. The effect of endotoxin tolerance on AA turnover in peritoneal macrophages was investigated by measuring [14C]AA incorporation and release from membrane phospholipids. Endotoxin tolerance did not affect the amount of [14C]AA incorporated into macrophages (30 min-24 h). However, the temporal incorporation of [14C]AA into individual phospholipid pools (15 min-24 h) was altered. In endotoxin-tolerant macrophages, [14C]AA incorporation into phosphatidylcholine (PC) (2, 4, 24 h) and phosphatidylethanolamine (PE) (8 h) was increased, while the incorporation into phosphatidylserine (PS) (2-24 h) was reduced (P less than 0.005) compared to control macrophages. There was no change in [14C]AA incorporation into phosphatidylinositol (PI). Following 2 or 24 h of incorporation of [14C]AA, macrophages were incubated (3 h) with endotoxin (50 micrograms/ml) or A23187 (1 microM), and [14C]AA release was measured. Endotoxin-tolerant macrophages released decreased (P less than 0.05) amounts of [14C]AA in response to both endotoxin and the calcium ionophore A23187 compared to controls. Control macrophages in response to endotoxin released [14C]AA from PC, PI and PE. In contrast, tolerant cells released [14C]AA only from PC (P less than 0.05). A23187 released [14C]AA from all four pools in the control cells, but only from PC and PE in the tolerant cells. These data demonstrate that endotoxin tolerance alters the uptake and release of AA from specific macrophage phospholipid pools. These results suggest that changes in AA turnover and/or storage are associated with endotoxin tolerance.     

Glucose-induced phospholipid hydrolysis in isolated pancreatic islets: quantitative effects on the phospholipid content of arachidonate and other fatty acids

Our recent findings indicate that glucose-induced insulin secretion from isolated pancreatic islets is temporally associated with accumulation of substantial amounts of free arachidonic acid and that arachidonate may serve as a second messenger for intracellular calcium mobilization in islets. In an effort to determine the source of this released arachidonate, the endogenous fatty acid composition of phospholipids from islets has been determined by thin-layer chromatographic separation of the phospholipids, methanolysis to the fatty acid methyl esters, and quantitative gas chromatographic analyses. The relative abundance of phospholipids in islets as judged by their fatty acid content was phosphatidylcholine (PC), 0.63; phosphatidylethanolamine (PE), 0.23; phosphatidylinositol (PI), 0.067; phosphatidylserine (PS), 0.049. Arachidonate constituted 17% of the total islet fatty acid content, and PC contained 43% of total islet arachidonate. Islets incubated with [3H]arachidonate in the presence of 28 mM D-glucose incorporated radiolabel into PC with a considerably higher specific activity than that of PE, PS or PI. The total fatty acid content of PC from islets incubated with 28 mM glucose for 30 min was significantly lower than that of islets incubated with 3 mM glucose, and smaller effects were observed with PE, PS and PI. The molar decrement in PC arachidonate was 3.2 pmol/islet under these conditions, which is sufficient to account for the previously observed accumulation of free arachidonate (2 pmol/islet). A sensitive method involving negative ion-chemical ionization-mass spectrometric analyses of the pentafluorobenzyl esters of fatty acids derived from trace amounts of lysophosphatidylcholine (lyso-PC) was developed, and glucose-stimulation was found to reduce islet lyso-PC content by about 10-fold. These findings indicate that the insulin secretagogue D-glucose induces phospholipid hydrolysis in islets and suggest that PC may be the major source of free arachidonate which accumulates in glucose-stimulated islets.     

Arachidonic acid-induced swelling in incubated rat brain cortical slices

The influence of bovine serum albumin (BSA) on the rat brain cortical swelling induced by sodium arachidonate and polyunsaturated fatty acids has been studied. Coincubation of arachidonate with BSA at a molar ratio of 5 (arachidonate/BSA) or less greatly inhibited the arachidonate-induced swelling. As the molar ratio of arachidonate/BSA increased, the degree of swelling increased. The swelling was not reversed by BSA, although the BSA released 46% of the previously incorporated [³H]arachidonic acid from the cortical slices. The entry of [³H]arachidonate into the slice was completely abolished by 0.1 mM BSA or partially inhibited by exogenous arachidonate. It is concluded that the induction of brain swelling by arachidonate requires the intracellular transport of exogenous arachidonate.     

Acetate and arachidonate incorporations into lipids of ovine chorioallantoic tissue at day 24 of pregnancy

Incorporation of acetate and arachidonic acid into lipid classes was examined in chorioallantoic membranes obtained from sheep at Day 24 of pregnancy. Conceptus tissues were incubated in vitro with 5 mM acetate, 0.042 mM arachidonate, 0.45 muCi [1-14C]acetate, and 5.0 muCi [5,6,8,9,11,12,14,15-3H]arachidonate for 3 and 6 h. After incubation, tissue lipid fractions were extracted, isolated, and examined for radiolabel incorporations. Medium was extracted and analyzed for radiolabeled metabolites. Metabolic pathways commonly associated with fatty acid metabolism were confirmed to be present. Acetate was utilized for de novo synthesis of free cholesterol and free fatty acid. Fatty acids containing radiolabel from both acetate and arachidonate were mainly esterified in phospholipid and triglyceride, major lipid classes found in chorioallantoic tissue. Labeled metabolites of acetate were not sufficient for analytical measurement in medium. Metabolites of arachidonic acid from lipoxygenase and cyclooxygenase pathways were determined in medium after incubation. Results suggest that, within Day 24 ovine chorioallantoic tissue, utilization of exogenous arachidonate and de novo lipogenesis from acetate function in a parallel and anabolic mode appropriate for membrane expansion.     

Phospholipid and arachidonic acid metabolism in zymosan-stimulated human monocytes: modulation by cAMP

Receptor-ligand interaction in mononuclear phagocytes is intimately linked to alterations in membrane phospholipids and release of arachidonic acid (AA). In addition, synthesis of bioactive lipids from released AA can result in further modification of cell responses. Upon challenge with opsonized zymosan, [3H]-arachidonic acid ([3H]-AA)-labeled human monocytes released 25 +/- 2% of their incorporated radiolabel within 30 min. Pretreatment of the monocytes with 5 X 10(-4) M isobutylmethylxanthine (IBMX) or 1 X 10(-3) M dibutyryl cyclic AMP (d-cAMP) inhibited total [3H]-AA release in the presence of zymosan by 47% and 42%, respectively. Analysis of incorporated [3H]-AA in cellular phospholipid pools indicated that significant amounts of label were lost from both phosphatidylcholine (PC) and phosphatidylinositol (PI) during zymosan stimulation. Treatment with d-cAMP substantially inhibited the loss of label from PC, but had no affect on PI. HPLC analysis of cell supernatants from zymosan-treated cells indicated that 5-HETE was the predominant metabolite generated from [3H]-AA, and its production was depressed during treatment with d-cAMP. Phospholipase activity in human monocyte homogenates was not effected by d-cAMP or IBMX at the highest concentrations used, whether these were added directly to the homogenate or by pretreatment of whole cells, demonstrating that inhibition required an intact cell. These results suggest that human monocytes exposed to opsonized zymosan release AA via two mechanisms and that modulation by cAMP is indirectly effecting a phospholipase directed towards PC.