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.     

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.     

Effect of incubation of guinea-pig taenia coli in potassium-free media on arachidonate release and lipid metabolism

We studied the effects of immersion of guinea-pig taenia coli strips in potassium-free media on arachidonate stores and other lipid fractions. Control studies obtained with the strips in Krebs solution showed that greater than 97% of arachidonate was found esterified in phospholipid with the following distribution: phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylserine plus phosphatidylinositol. 30 min incubation of the strips with [3H]arachidonate complexed to albumin resulted in incorporation of this isotope into phospholipid and neutral lipid fractions, phosphatidylcholine greater than neutral lipid greater than phosphatidylserine plus phosphatidylinositol greater than phosphatidylethanolamine. 30 min incubations with 32PO4(2-)-resulted in an isotope incorporation into phospholipids, phosphatidylcholine greater than phosphatidylserine plus phosphatidylinositol greater than phosphatidylethanolamine. After 'loading' with [3H]arachidonate and 32P, placing the strips in potassium-free media caused the following: there was an increased release of [3H]arachidonate from the tissue into the bathing solution. [3H]Arachidonate and 32P radioactivity in phosphatidylinositol fell without a change in phosphatidylinositol content. [3H]Arachidonate and 32P radioactivity in other phospholipid fractions was unchanged. Arachidonate specific activity fell and arachidonate content increased in the phosphatidylserine plus phosphatidylinositol fraction. [3]Arachidonate in neutral lipid did not change significantly. We conclude that exposure of taenia coli to potassium-free media activates turnover of phosphatidylinositol, which results in release of arachidonate.     

[3H]Arachidonic acid metabolism in rat brain minces: Effects of nucleotide triphosphates,CDPcholine and CMP

Rat brain minces were used to investigate the effects of nucleotides on the metabolism of arachidonic acid in nerve tissue. Brain free fatty acids, neutral lipids and phospholipids, were radiolabeled in vivo following intracerebral injection of [³H]arachidonic acid. Minces were prepared from the radiolabeled cerebra and were incubated in a modified Krebs-Ringer buffer with and without various nucleotides. The incubation-induced accumulation of unesterified [³H]arachidonate was reduced in the presence of CDPcholine, ATP, CTP, GTP, and UTP. These nucleotides inhibited choline and inositol glycerophospholipid hydrolysis. They also reduced the amount of labeled diglycerides. However, CDPethanolamine had no effect on arachidonic acid metabolism in the mince preparation and CMP appeared to stimulate further hydrolysis of choline glycerophospholipids, resulting in increased accumulation of [³H]arachidonic acid and labeled diglycerides. We suggest that the production of unesterified [³H]arachidonate and labeled diglycerides is due to the involvement of more than one catabolic reaction, since the high energy nucleotides had similar effects on fatty acid accumulation, but different effects on phospholipid labeling.     

The influence of exogenous PMS and HCG on the arachidonic acid content of the immature rat ovary.

The influence of exogenous PMS and/or HCG, on the arachidonic acid (C 20:4omega6) content of the immature rat ovary was examined. Changes in ovarian arachidonate content associated with hormone administration were assessed in total lipid extracts, and in several neutral and phospholipid fractions. Both relative percentage and absolute amounts of arachidonic acid in several lipids were measured as well as uptake of radioactivity into total lipid resulting from the administration of 3H-labeled arachidonic acid in vivo. On the basis of these studies, we conclude (1) PMS, with or without HCG promotes increased uptake of exogenous arachidonic acid into ovarian total lipids; (2) Arachidonic acid is a mojor fatty acid constituent from noncholine containing phosphatides at the onset of normal estrous (ca. 38 days) even in the animals which received no PMS or HCG; (3) Changes in ovarian arachidonic acid levels following gonadotropin administration are more striking in the two phospholipid fractions than in the two neutral lips examined; (4) PMS is associated with a rapid outpouring of ovarian lipid, accompanied by a high turnover of arachidonic acid which is enhanced or modified temporally by added HCG in vivo. These results provide the first quantitative evidence that gonadotropins may regulate prostaglandin biosynthesis in the ovary by their effects on the uptake, storage, or release of arachidonic acid, a major PG precursor, from specific ovarian lipids. While the data strongly suggest that the regulation of one or more ovarian esterases (cholesterol esterase, lipase, phospholipase) is the mechanism by which gonadotropins regulate PG biosynthesis, a direct action on PG synthetase is not ruled out.     

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.     

Manoalide,a Phospholipase A2 Inhibitor,Inhibits Arachidonate Incorporation and Turnover in Brain Phospholipids of the Awake Rat

The Fatty Acid method was used to determine whether incorporation of plasma radiolabeled arachidonic acid into brain phospholipids is controlled by phospholipase A₂. Awake rats received an i.v. injection of a phospholipase A₂ inhibitor, manoalide (10 mg/kg), and then were infused i.v. with [1-¹⁴C]arachidonate or [³H]arachidonate. Animals were killed after infusion by microwave irradiation, and tracer distribution was analyzed in brain phospholipid, neutral lipid and acyl-CoA pools. Calcium-independent phospholipase A₂ activity in brain homogenate was reduced by manoalide, whereas phospholipase C activity was unaffected. At 60 min but not at 20 or 40 min after its injection, manoalide had significantly decreased by 50% incorporation of unesterified arachidonate into and turnover within brain phospholipids, taking into account dilution of the brain arachidonoyl-CoA pool by recycled arachidonate. Manoalide also increased by 100% the net rate of unesterified arachidonate incorporation into brain triacylglycerol. This study indicates that manoalide can be used to inhibit brain phospholipase A₂ in vivo, and that phospholipase A₂ plays a critical role in arachidonate turnover in brain phospholipids and neutral lipids.     

Stimulated production and natural occurrence of 1,2-diarachidonoylglycerophosphocholine in human neutrophils

Incorporation of arachidonic acid into phospholipid molecular species of the human neutrophil was found to be dependent, to a large extent, upon the concentration of arachidonate used during the in vitro incubations. When high concentrations of [3H] arachidonate were employed, only two glycerolipids incorporated label. One glycerolipid was a unique glycerophospholipid characterized by HPLC retention time and fast atom bombardment mass spectrometry as 1,2-diarachidonoyl-sn-glycero-3-phosphocholine. The second and most highly labeled glycerolipid was found to be arachidonoyl triacylglycerol species. Human neutrophils isolated from normal individuals and not previously exposed to arachidonic acid in vitro were found to contain a small but measurable amount of diarachidonoyl-GPC. The dose-dependent increase of diarachidonoyl-GPC and arachidonoyl-labeled triacylglycerol when cells were exposed to increasing concentrations of arachidonic acid implied that these lipid molecular species have the capacity to expand their pools, perhaps in manner regulating levels of endogenous arachidonic acid for further metabolism. These observations point to the importance of the concentration of arachidonic acid employed during in vitro labeling studies.     

Bradykinin-stimulated release of [3H]arachidonic acid from phospholipids of HSDM1C1 cells: comparison of diacyl phospholipids and plasmalogens as sources of prostaglandin precursors

Ethanolamine plasmalogens (1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamines) of many tissues contain high levels of arachidonate at their 2-position, and in certain tissues have been implicated as possible donors of arachidonate required in the synthesis of prostaglandins and thromboxanes. In the present study, [3H]arachidonate-labeled phospholipids of HSDM1C1 cells, a cell line derived from a mouse fibrosarcoma, were examined to determine the donor of the arachidonic acid released upon bradykinin stimulation of the synthesis of PGE2. HSDM1C1 cells labeled with [3H]arachidonic acid for 24 hr in serum-free medium were used in most of the experiments and had the following distribution of label among the cellular lipids; phosphatidylcholine (33%), phosphatidylinositol (20%), diacyl-sn-glycero-3-phosphoethanolamine (15%), ethanolamine plasmalogen (15%), and less polar lipids )16%). Bradykinin treatment stimulated a rapid hydrolysis of [3H]arachidonate from the cellular lipids and conversion of the released acid to PGE2, which was secreted into the medium. The label was released predominantly from phosphatidylinositol and possibly from phosphatidylcholine with no detectable change in the labeling of diacyl- or 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. The ethanolamine plasmalogens, therefore, do not appear to be involved in the stimulated release of arachidonate in the HSDM1C1 cells. Indomethacin blocked the bradykinin-stimulated synthesis of PGE2 and to a lesser degree inhibited the release of [3H]arachidonate from the cellular lipids into the medium.     

Heterogeneity of arachidonate and paf-acether precursor pools in mast cells.

In mammalian cells, arachidonate release and paf-acether formation are frequently associated. The alkyl-acyl-GPC has been proposed as an important source for released arachidonic acid and arachidonate-containing alkylacyl-GPC species as unique precursor for paf-acether. However, the specificity of precursor pools either concerning arachidonic acid or paf-acether is still a matter of controversy. We studied the relationship between the precursor pools for both autacoids in antigenically-stimulated cultured mast cells. We took advantage of the particular arachidonate turnover rate in each phospholipid to investigate the role of alkyl-arachidonyl-GPC in the supply of arachidonic acid by using newly and previously [14C]arachidonate-labeled cells. The specific activity of the released arachidonate was reduced 2-fold following overnight cell incubation, whereas labeling in alkyl-arachidonoyl-GPC was only slightly modified and never corresponded to that of released arachidonate when newly or previously labeled cells were triggered with the antigen. These results are not in favor of a major role for alkyl-arachidonoyl-GPC in supplying arachidonate. In contrast, by using previously labeled cells, we demonstrated that all arachidonate-containing phospholipids were involved in the release of arachidonic acid. The pattern of alkyl chains in alkyl-arachidonoyl-GPC, as well as in total alkylacyl-GPC, is unique since it consists mainly of 18:1 (more than 55%), whereas the 16:0 represents only about 30% of total alkyl chains. Therefore, we analyzed paf-acether molecular composition in order to compare it to the alkyl composition of the precursor pools. The content in 18:1 species of paf-acether, as measured by bioassay (aggregation of rabbit platelets), was always lower than that of 16:0 species and then did not correspond to the alkyl composition of the precursor. These data suggest that the enzymes involved in paf synthesis might be specific for 16:0 alkyl chains of precursor pool.     

Inhibition of arachidonic acid incorporation into erythrocyte phospholipids by peracetic acid and other peroxides. Role of arachidonoyl-CoA: 1-palmitoyl-sn-glycero-3-phosphocholine acyl transferase

To explore possible mechanisms of the arachidonic acid deficiency of the red blood cell membrane in alcoholics, we compared the effect of ethanol and its oxidized products, acetaldehyde and peracetic acid, with other peroxides on the accumulation of [14C]arachidonate into RBC membrane lipids in vitro. Incubation of erythrocytes with 50 mM ethanol or 3 mM acetaldehyde had no effect on arachidonate incorporation. Pretreatment of erythrocytes with 10 mM hydrogen peroxide, 0.1 mM cumene hydroperoxide or 0.1 mM t-butyl hydroperoxide had little effect on [14C]arachidonate incorporation in the absence of azide. However, pretreatment of cells with N-ethylmaleimide, 0.1 mM peracetic acid or performic acid, with or without azide, inhibited arachidonate incorporation into phospholipids but not neutral lipids. In chase experiments, peracetate also inhibited transfer of arachidonate from neutral lipids to phospholipids. To investigate a possible site of this inhibition of arachidonate transfer into phospholipids by percarboxylic acids, we assayed a repair enzyme, arachidonoyl CoA: 1-palmitoyl-sn-glycero-3-phosphocholine acyl transferase (EC 2.3.1.23). As in intact cells, phospholipid biosynthesis was inhibited more by N-ethylmalemide and peracetic acid than by hydrogen peroxide, cumene hydroperoxide, and t-butyl hydroperoxide. Peracetic acid was the only active inhibitor among ethanol and its oxidized products studied and may deserve further examination in ethanol toxicity.     

Are blastocyst prostaglandins produced endogenously?

This study was undertaken to determine whether preimplantation mouse and rabbit blastocysts possess cyclooxygenase activity and therefore are able to metabolize arachidonic acid (AA) to prostaglandins (PGs) and thromboxane B2. Single rabbit blastocysts or groups of 100 mouse blastocysts were incubated for 4 h in the presence of 5 muCi/ml [3H] AA (sp. ac. 61 Ci/mmol) or for 24 h and 18 h with 0.2 and 0.1 muCi/ml [14C] AA (sp. ac. 56.5 Ci/mmol), respectively. Incubated blastocysts were subsequently exposed for 10 min to 24 h to the Ca2+ specific ionophore, X-537A (10 microM), to stimulate release of radiolabeled AA from the phospholipid pool. After incubation, incorporation of AA into the phospholipid and neutral lipid pools, and metabolism of the fatty acid to PGs and thromboxane B2, were determined using thin-layer chromatographic (TLC) and liquid scintillation spectroscopic techniques. In addition, spent incubation media were analyzed for radiolabeled PG content. In blastocysts of both species, incorporation of AA into phospholipids was greater than that into neutral lipids (mouse: 1.0 vs. 0.7 pmol/blastocyst; rabbit: 159.7 vs. 56.9 pmol/blastocyst). The ionophore stimulated the release of AA from the phospholipid, and to a lesser extent, from the neutral lipid pool, of both blastocysts. No newly synthesized PGs were detected in either mouse blastocysts or their spent incubation media after stimulation with X-537A. Radiolabeled PGs (PGE2, PGF2 alpha, PGD2, PGA2) and thromboxane B2 were present in the media of rabbit blastocyst incubations, however, but were undetectable in the tissue extracts. Increases in metabolism of AA into each compound were observed with an increase in time of exposure to ionophore, and meclofenamic acid (2 microM) partially inhibited the synthesis of all compounds during a 24-h incubation. The results are discussed with regard to the role of blastocyst PGs in the events of implantation.     

Enrichment of endothelial cell arachidonate by lipid transfer from high density lipoproteins: relationship to prostaglandin I2 synthesis

We have previously shown that plasma high density lipoproteins (HDL) stimulate release of prostacyclin, measured as its stable metabolite, 6-keto-PGF1 alpha, by cultured porcine aortic endothelial cells. The present experiments were designed to elucidate the contribution of HDL lipids to endothelial cellular phospholipid pools and to prostacyclin synthesis. In experiments with reconstituted HDL, both the lipid and protein moieties were required to stimulate prostacyclin release in amounts equivalent to the native HDL particle. Endothelial cells incorporated label from reconstituted HDL containing cholesteryl [1-14C]arachidonate into the cellular neutral and phospholipid pools as well as into 6-keto-PGF1 alpha and PGE2. Labeled arachidonate incorporated into endothelial cell lipids from reconstituted HDL containing cholesteryl [1-14C]arachidonate was also metabolized to prostaglandins after the cells were exposed to the calcium ionophore, A-23187. Both rat and human HDL which stimulated 6-keto-PGF1 alpha release (rat greater than human) increased the weight percentage of arachidonate in endothelial cell phospholipids; phospholipid arachidonate in the enriched cells fell after exposure to the phospholipase activator, A-23187, with release of 6-keto-PGF1 alpha which was greater than in control cells. Rat HDL that was depleted of cholesteryl arachidonate (achieved by incubation with human low density lipoproteins (LDL) in the presence of cholesteryl ester transfer protein) stimulated 6-keto-PGF1 alpha release less than native rat HDL. LDL enriched in cholesteryl arachidonate stimulated 6-keto-PGF1 alpha release more than native LDL. ApoE-depleted HDL also stimulated 6-keto-PGF1 alpha release more than apoE-rich HDL suggesting the apoE receptor was not involved in the response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of postdecapitative ischemia on arachidonate release from brain synaptosomes

Synaptosomal phosphoglycerides were labeled after incubation with [1-¹⁴C]arachidonic acid, ATP, Mg²⁺, CoASH, and a small amount of 1-acylglycerophosphocholines. Under this incubation system, radioactivity was directed largely to diacylglycerophosphocholines but diacylglycerophosphoinositols were also labeled to a lesser extent. Synaptosomes obtained after a 5-min ischemic treatment indicated a decrease (10–20%) in incorporation of radioactivity into the phospholipids. The ischemic synaptosomes also tended to retain a larger portion of the labeled arachidonate during the wash with bovine serum albumin. Upon incubation of the prelabeled synaptosomes in a sucrose-Tris (pH 7.4) medium at 37°C, a time-dependent release of labeled arachidonate from the phospholipids was observed in both control and ischemic samples. Arachidonate release from the prelabeled synaptosomes was not affected by EDTA (1 mM) or taurocholate (0.4%) but was stimulated by Ca²⁺ (2.5 mM) or Ca²⁺ (3.5 mM) together with EDTA (1 mM). After incubation at 37°C for 1 hr without added factors, the phospholipid degradation, as well as the appearance of free fatty acids, were higher in the ischemic samples (especially after 1 min of treament) as compared to controls.     

Metabolism of bis(monoacylglycero)phosphate in macrophages

To further elucidate the role of bis(monoacylglycero)phosphate in lysosomes, its metabolism was assessed by incubation of intact and disrupted macrophages in the presence of labeled lipid precursors. In rabbit pulmonary macrophages bis(monoacylglycero)P accounted for 17.9% and acylphosphatidylglycerol for 2.6% of phospholipid phosphorus. Major fatty acids in bis(monoacylglycero)P were oleic (47%), linoleic (29%), and arachidonic (6.4%); those in acylphosphatidylglycerol were of similar distribution except for a high content of palmitic acid (20%). When homogenates of rabbit pulmonary and peritoneal macrophages, rat pulmonary macrophages, and human blood leukocytes were incubated with sn[(14)C]glycerol-3-phosphate and CDP-diacylglycerol at pH 7.4, there was labeling of bis(monoacylglycero)P and acylphosphatidylglycerol that correlated with content of bis(monoacylglycero)P. When intact rabbit pulmonary macrophages were incubated for 60 min with [(3)H]glucose and [(32)P]orthophosphate, small amounts of label appeared in bis(monoacylglycero)P and only traces in acylphosphatidylglycerol. In contrast, incubation of intact cells with the (14)C-labeled fatty acid precursors palmitic, oleic, and arachidonic acids resulted in much greater labeling of the two lipids. Labeling of phospholipids was greatest with arachidonate as precursor and least with palmitate; after 60 min, labeling of bis(monoacylglycero)P with arachidonate was 10- and 50-fold greater than with oleate and palmitate, respectively, and was exceeded only by that of phosphatidylcholine. Calculated ratios of labeling of fatty acid to P, particularly those for arachidonate, were much greater for bis(monoacylglycero)P and for acylphosphatidylglycerol than for other phospholipids. This suggests a uniquely high turnover of fatty acids in bis(monoacylglycero)P and acylphosphatidylglycerol and thus a more specific role for these compounds in metabolism of complex lipids in the lysosome.-Huterer, S., and J. Wherrett. Metabolism of bis(monoacylglycero)phosphate in macrophages.     

Prostaglandin release from in vitro guinea-pig gallbladder

In order to study prostaglandin release from guinea pig gallbladder, full thickness tissue sections were incubated for one hour in Krebs solution. Extraction and two dimensional chromatography of incubation media obtained in the presence of radio-labelled arachidonic acid demonstrated the presence of PGE2, PGF2 alpha, 6-keto-PGF1 alpha and thromboxane B2. These results were supported by radioimmunoassay of incubations conducted in the absence of exogenous arachidonate and in the presence of varying concentrations of unlabelled exogenous arachidonate. The previously reported predominance of PGE2 was only seen at high concentrations of exogenous arachidonate.     

Brugia malayi: arachidonic acid uptake into lipid bodies of adult parasites

Lipid bodies are non-membrane bound intracellular organelles, which have been recognized morphologically in a diversity of mammalian and nonmammalian cells, but are of uncertain function. In mammalian cells, in addition to serving as a storage site of cholesterol and triglyceride, lipid bodies can be a repository of esterified arachidonic acid. Adult worms of the human filarial parasite Brugia malayi have been found to esterify exogenous [3H]arachidonic acid into parasite phospholipids and neutral lipids. Electron microscopic autoradiography demonstrated that [3H]arachidonate was preferentially incorporated into filarial lipid bodies. The dominant incorporation of arachidonate into lipid bodies of a nematode establishes that lipid bodies are a site of arachidonic acid accumulation in nonmammalian, as well as mammalian, cells.     

Tocopherol analogs suppress arachidonic acid metabolism via phospholipase inhibition.

alpha-Tocopherol and three derivatives in which the phytol chain is modified or deleted were examined for their effect on cultured keratinocyte arachidonic acid metabolism. 2,2,5,7,8-Pentamethyl-6-hydroxychromane (PMC), in which the phytol chain is replaced by a methyl group, inhibited basal, bradykinin (BK)- and A23187-stimulated prostaglandin E2 (PGE2) synthesis with an apparent Ki of 1.3 microM. The Ki of the analogue with six carbon atoms in the side chain (C6) was 5 microM while that of the C11 analogue was 10 microM. No effect of alpha-tocopherol was observed. The mechanism of inhibition was studied using PMC. The effect of PMC on phospholipase and cyclooxygenase activity was assayed using stable isotope mass measurements of PGE2 formation, which assesses arachidonate release and cyclooxygenase metabolism simultaneously. BK-stimulated formation of PGE2, derived from endogenous phospholipid, was decreased 60% by 5 microM PMC and eliminated by 50 microM PMC, compared with controls. No difference in PGE2 formed from exogenous arachidonic acid was observed, indicating no effect of PMC on cyclooxygenase activity. In contrast, no effect of 5 microM PMC was observed on BK-stimulated [3H]arachidonic acid release from prelabeled cultures. The capacity of PMC to inhibit phospholipase activity in vitro was also assessed. PMC inhibited hydrolysis of phospholipid substrate by up to 60%. These results suggest that alpha-tocopherol analogues with alterations in the phytol chain inhibit eicosanoid synthesis by preferential inhibition of phospholipase.     

Analysis of prostaglandins formed from endogenous and exogenous arachidonic acid in homogenates of human reproductive tissues

Isotope-labelled arachidonic acid has been used to study in vitro formation of prostaglandins and other products in mammalian tissue. Quantitative conclusions about cyclooxygenase activity have been drawn from such studies. However, arachidonic acid is present in all tissues, free and esterified, and therefore it can be expected that endogenous arachidonate would interfere with transformation of the radioactive exogenous substrate. (1-14C)-labelled arachidonate was, therefore, incubated with homogenates of various human tissues (amnion, chrorion, placenta and myometrium), and the two molecular forms, 12C and 14C, of arachidonic acid as well as of prostaglandin E2 and prostaglandin F2 alpha were quantitated, before and after 30 min of incubation, using gas chromatography-mass spectrometry with multiple ion detection. The results demonstrate a substantial release of arachidonic acid into the medium during incubation. There was no correlation between either the initial concentration of [12C]arachidonic acid and initial concentration of [12C]prostaglandin E2 or the percent increase of those compounds during incubation. The net formation of [12C]prostaglandin E2 and [14C]prostaglandin E2 from endogenous and exogenous precursor, respectively, were also very different. The study shows that by simply incubating (1-14C)-labelled arachidonic acid in tissue homogenates and measuring the amount of radioactivity transformed into various prostaglandins only qualitative conclusions can be drawn.     

Eicosapentaenoic acid metabolism in brain microvessel endothelium: effect on prostaglandin formation

Mouse brain microvessel endothelial cells convert eicosapentaenoic acid (EPA) to prostaglandin (PG) E3, PGI3, and several hydroxy fatty acid derivatives. Similar types of products are formed by these microvessel endothelial cells from arachidonic acid. The formation of PGI2 and PGE2 is reduced, however, when the brain microvessel endothelial cultures are incubated initially with EPA. Exposure to linolenic or docosahexaenoic acid also decreased the capacity of these microvessel endothelial cells to form PGI2 and PGE2, but the reductions were smaller than those produced by EPA. Like the endothelial cultures, intact mouse brain microvessels convert EPA into eicosanoids, and incubation with EPA reduces the subsequent capacity of the microvessels to produce PGI2 and PGE2. Brain microvessel endothelial cells took up less EPA than arachidonic acid, primarily due to lesser incorporation into the inositol, ethanolamine, and serine glycerophospholipids. By contrast, considerably more EPA than arachidonic acid was incorporated into triglycerides. These findings suggest that the microvessel endothelium may be a site of conversion of EPA to eicosanoids in the brain and that EPA availability can influence the amount of dienoic prostaglandins released by the brain microvasculature. Furthermore, the substantial incorporation of EPA into triglyceride suggests that this neutral lipid may play an important role in the processing and metabolism of EPA in brain microvessels.