Prostaglandins and enzyme secretion from dispersed rat pancreatic acinar cells

The role of prostaglandins in exocrine pancreatic enzyme secretion was studied. The effects of three inhibitors of prostaglandin and thromboxane syntheses, were evaluated on release of amylase from dispersed rat pancreatic acinar cells. Mepacrine inhibited, while indomethacin and imidazole had no effect on basal or carbachol or cholecystokinin stimulated enzyme release. Exogenous arachidonic acid or various prostaglandins (E₁, E₂, F_2α, I₂), also did not affect the secretory process. Acinar cells actively incorporated radioactive arachidonic acid, principally into phospholipids (especially phosphatidylcholine), however release of the free fatty acid and subsequent synthesis of radioactive endogenous prostaglandins was not stimulated by the presence of different pancreatic stimulants. Pancreatic microsomes were found to be lacking in cyclo-oxygenase, an enzyme involved in endegenous synthesis of prostaglandins. The data suggest that prostaglandins are not involved directly in excitation-secretion coupling in the exocrine pancreas.     

Metabolism of linoleic and arachidonic acids in VX2 carcinoma tissue: Identification of monohydroxy octadecadienoic acids and monohydroxy eicosatetraenoic acids

The metabolism of arachidonic and linoleic acids by VX₂ carcinoma tissue was determined. Prostaglandin E₂ was the major metabolic product of arachidonic acid in the neoplastic tissue. Minor products accounting for 3– 8% of arachidonic acid metabolism were 11-hydroxy-5, 8, 12, 14-eicosatetraenoic acid (11-HETE) and 15-hydroxy-5, 8, 11, 13-eicosatetraenoic acid (15-HETE). Linoleic acid was converted to a mixture of 9-hydroxy-10, 12-octadecadienoic acid (9-HODD) and 13-hydroxy-9, 11-octadecadienoic acid (13-HODD). The conversion of linoleic acid to monohydroxy C-18 fatty acids varied from 40–80% 9-HODD and 20–60% 13-HODD in tumor tissue harvested from different animals. The quantity of monohydroxy C-18 fatty acids biosynthesized by VX₂ carcinoma tissue from endogenous linoleic acid equals or exceeds that of prostaglandin E₂ biosynthesis from endogenous arachidonic acid. The presence of a hydroxyl group adjacent to a conjugated diene suggest that the monohydroxy C-18 and monohydroxy C-20 fatty acids were formed via the action of lipoxygenase-like enzymes. These lipoxygenase-like reactions are inhibited by indomethacin in a concentration-dependent fashion similar to the inhibition of prostaglandin E₂ biosynthesis. The enzymes catalyzing the lipoxygenase-like reactions of linoleic and arachidonic acids are localized in the microsomal fraction of VX₂ carcinoma tissue. These data suggest that the lipoxygenase-like reactions are catalyzed by fatty acid cyclooxygenase and that there are two major pathways of fatty acid cyclooxygenase metabolism of polyenoic fatty acids in the neoplastic tissue. One pathway involves the formation of prostaglandin E₂ via cyclic endoperoxy intermediates. The second pathway involves the formation of monohydroxy C-18 fatty acids from linoleic acid via lipoxygenase-like reactions.     

The mechanisms of preterm labour; the interaction between amnion cells and leukocytes in the metabolism of arachidonic acid

Chorioamnionitis is frequently associated with preterm labour. We have used a cell culture model system to examine the effects of leukocytes upon the metabolism of endogenous arachidonic acid from within amnion cells. We have demonstrated that activated leukocytes release substances which increase the overall release and metabolism of endogenous arachidonic acid within amnion cells causing an increase in prostaglandin E₂ production as well as a smaller increase in non-cyclooxygenase metabolism. When amnion cells and leukocytes are cultured together, in addition to prostaglandin E₂ production by amnion cells, arachidonic acid released by the amnion cells appears to be metabolised by leucocytes to prostaglandin F_2α, prostacyclin and thromboxane A₂. Prostaglandins E₂ and F_2α are the principal cyclo-oxygenase products of this interaction.We postulate that chorioamnionitis stimulates preterm labour not only by causing an increase in prostaglandin E2 synthesis by amnion cells but by metabolism of amnion derived arachidonic acid to the powerfully oxytocic prostaglandin F_2α by leukocytes.     

Inhibitors of protein synthesis,puromycin and emetine,inhibit prostaglandin E2 release by skeletal muscle

Addition of 1μM puromycin or 1 μM emetine to rat soleus muscle in vitro decreases muscle prostaglandin E₂ release by 51–77%. This inhibition appears to be caused by decreased availability of endogenous arachidonic acid for prostaglandin E₂ synthesis, because neither puromycin nor emetine inhibits muscle prostaglandin E₂ production from arachidonic acid added into the incubation medium.     

The antihypertensive action of arachidonic acid in the spontaneous hypertensive rat and its antagonism by anti-inflammatory agents

Changes in arterial blood pressure and heart rate were observed in the spontaneous hypertensive (SH) rat following the intravenous administration of arachidonic acid, the precursor of prostaglandin E₂ (PGE₂). The pronounced fall in blood pressure and the increase in heart rate induced by arachidonic acid were also observed in SH rats receiving either prostaglandin E₁ (PGE₁) or PGE₂. In SH rats receiving various anti-inflammatory agents the cardiovascular responses to arachidonic acid were inhibited, but the blood pressure responses to the E-type prostaglandins were not altered. The data are interpreted to suggest that cardiovascular changes induced by arachidonic acid are mediated via its conversion to PGE₂.     

Combinations of palytoxin or 12-O-tetradecanoylphorbol-13-acetate and recombinant human insulin growth factor-I or insulin synergistically stimulate prostaglandin production in cultured rat liver cells and squirrel monkey aorta smooth muscle cells

In the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) or the non-TPA-type tumor promoter, palytoxin, recombinant human insulin growth factor-I (IGF-I) and insulin synergistically stimulate prostaglandin production in rat liver cells (the C-9 cell line). Combinations fo palytoxin or TPA with recombinant human IGF-I or insulin also synergistically stimulate deesterification of c ellular lipids in C-9 cells prelabelled with [³H]arachidonic acid. With both types of stimulations, prostaglandin production or deesterification, the synergistic response of the IGF-I and insulin is greater with palytoxin than with TPA. Production of prostaglandin E₂ and F_2α by squirrel monkey smooth muscle cells incubated in the presence of TPA and insulin also is greater than the sum of the effects taken independently.     

Beta-adrenergic stimulation of prostaglandin production by human amnion tissue

Arachidonic acid is released from specific glycerophospholipids in human amnion and is used to synthesize prostaglandins that are involved in parturition. In an investigation of the regulation of prostaglandin production in amnion, the effects of isoproterenol on discs of amnion tissue maintained were examined. Isoproterenol caused a large but transitory increase in the amount of cyclic AMP in amnion discs and this was accompanied by a sustained stimulation of the release of arachidonic acid (but not palmitic acid or stearic acid) and prostaglandin E₂. The dependencies of cyclic AMP accumulation, arachidonic acid mobilization and prostaglandin E₂ release on the concentration of isoproterenol were similar, each response was maximal at 10⁻⁶ M isoproterenol and was inhibited by propranolol. Dibutyryl cyclic AMP stimulated the release of prostaglandin E₂ from amnion discs. Although prostaglandin E₂, when added to amnion discs caused an accumulation of cyclic AMP, it did not appear to mediate isoproterenol-induced accumulation of cyclic AMP since the latter effect was insensitive to indomethacin in concentrations at which prostaglandin production was inhibited greatly. These data support the proposition that catecholamines, found in increasing amounts in amniotic fluid during late gestation, my be regulators of prostaglandin production by the amnion.     

Synthesis of prostaglandin E2 and prostaglandin F2α by toadfish red blood cells

Saline washed red blood cells of the toadfish convert [1-¹⁴C] arachidonic acid to products that cochromatograph with prostaglandin E₂ and prostaglandin F_2α. This synthesis is inhibited by indomethacin (10 μg/ml). Conversion of arachidonic acid to prostaglandin E₂ was confirmed by mass spectrometry. When saline washed toadfish red blood cells were incubated with a mixture of [1-¹⁴C]-arachidonic acid and [5,6,8,9,11,12,14,15,-³H]-arachidonic acid, comparison of the isotope ratios of the radioactive products indicated that prostaglandin F_2α was produced by reduction of prostaglandin E₂. The capacity of toadfish red blood cells to reduce prostaglandin E₂ to prostaglandin F_2α was confirmed by incubation of the cells with [1-¹⁴C] prostaglandin E₂.     

Prostaglandin F2α produced by rabbit renal slices is not a metabolite of prostaglandin E2

Slices of rabbit renal medulla and rabbit renal papilla were incubated with a mixture of [1-¹⁴C]-arachidonic acid and [5,6,8,9,11,12,14,15-³H]-arachidonic acid. In both tissues, comparison of the isotope ratios of the radioactive products with the isotope ratio of the added arachidonic indicated that: (a) there was no discernable isotope effect in the biosynthesis of prostaglandin E₂; (b) prostaglandin F₂α was formed by reduction of prostaglandin H₂ and not by reduction of prostaglandin E₂; and (c) most of the radioactive product arose from arachidonic acid that had been incorporated into the tissue and not from the direct action of cyclooxygenase on arachidonic acid in the medium.     

Localization of cyclo-oxygenase and prostaglandin E2 in the secretory granule of the mast cell

The application of anti-cyclo-oxygenase and anti-prostaglandin E2 immunoglobulins to A23187-stimulated rat connective tissue mast cells has permitted the localization of cyclooxygenase activity (prostaglandin H2 synthetase) and the site of prostaglandin E2 (PGE2) formation in the secretory granules. Because binding was carried out after stimulation but before dehydration and embedding, we have limited the loss of these antigens due to normal degradation and to aqueous and solvent washes. As this method permits labeling of exposed cell surfaces, only granules that have been exteriorized can be labeled. Contrary to what might have been expected, no labeling was associated with plasma membranes or with any portion of damaged cells. Antibodies to PGE2 were bound evenly over the surface of the granule matrix, whereas antibodies to cyclo-oxygenase appeared to be bound to strands of proteo-heparin projecting from the surface of the granule matrix. Where granule matrix had become unraveled and dispersed, label appeared to adhere throughout the ribbon-like proteo-heparin strands. These results support our previous conclusion that the secretory granule is the site of the arachidonic acid cascade during exocytosis.     

Inhibition of arachidonate- and escherichia coli-induced enteropooling in the rat by acetylsalicylic acid and similar drugs

Some foods and laxatives stimulate prostanoid biosynthesis and this effect is inhibited by acetylsalicylate (1); prostanoid administration causes diarrhoea and other symptoms of gut dysfunction (2,3,4). We therefore studied the effects of arachidonic acid, prostaglandins E₁ and E₂, endotoxin, laxatives and cyclooxugenase inhibitors in the rat ‘enteropooling’ test (5). All drugs were given orally. Prostaglandin E₂ (0.2mg/ kg), prostaglandin E₁ (0.74mg/kg), arachidonic acid (129mg/kg), castor oil (0.42ml/kg), magnesium sulphate (37mg/kg) and endotoxin (39.5mg/kg) doubled intestinal fluid volume. Cyclooxygenase inhibitors reduced arachidonate-induced enteropooling (indomethacin > acetylsalicylic acid > paracetamol > sodium salicylate > bismuth subsalicylate). Acetylsalicylic acid inhibited endotoxin-, castor oil-, but not prostaglandin E₂-or magnesium sulphate-induced enteropooling. Because acetylsalicylic acid was unexpectedly active in this test, we suggest that it may prove useful for the treatment of ‘travellers’ diarrhoea.     

Synthesis of prostaglandins and eicosanoids by the mast cell secretory granule

The identification of a non-bilayer phospholipid storage in the secretory granule and the linking of the eicosanoid production with the release of histamine have prompted us to examine whether the secretory granule may also serve as both the source as well as the site of prostaglandin synthesis during exocytosis. By exposing the contents of purified granules to exogenous arachidonic acid at neutral pH, we observed the rapid formation of many eicosanoids. The presence of prostaglandins E2, D2 and F2a were identified. The kinetics of E2 formation was also followed. The localization of the arachidonic acid cascade to the secretory granule explains why the production of eicosanoids is so intimately tied to the process of granule exocytosis.     

Effect of acetaminophen on prostaglandin E2 and prostaglandin F2α synthesis in the renal inner medulla of rat

Effects of acetaminophen on the renal inner medullary production of prostaglandin E₂ and F_2α were compared with the well-known effects of aspirin on this process. Acetaminophen was found to elicit a dose-dependent inhibition of both prostaglandin E₂ and F_2α accumulation in media with a K_i of 100–200 μM. This inhibition could not be accounted for by increased accumulation of prostaglandins within slices. Acetaminophen inhibition was reversed by removal of acetaminophen during the incubation or by addition of arachidonic acid. Similar manipulations did not reverse aspirin or indomethacin-mediated inhibition of prostaglandin synthesis. Thin-layer and gas chromatographic analysis of acetaminophen following incubation with slices demonstrated that this material was identical to authentic acetaminophen. This, in addition to the lack of an effect of glutathione on inhibition, suggests that acetaminophen does not have to be metabolized to exert this inhibition. Arachidonic acid did not alter the metabolism or increase the efflux of acetaminophen. Lower levels of prostaglandin E₂ observed with 5 mM acetaminophen and 1 mM aspirin caused a corresponding decrease in cyclic AMP content. Removal of acetaminophen from the second incubation or addition of arachidonic acid caused increases in both prostaglandin E₂ and cyclic AMP. Aspirin inhibition of cyclic AMP content was not reversed by similar manipulations. In vivo inhibition of inner medullary prostaglandin E₂ and prostaglandin F_2α synthesis was observed 2 h after a 375 mg/kg, intraperitoneal injection of acetaminophen. These data suggest that acetaminophen, like aspirin, is capable of reducing tissue prostaglandin synthesis. However, the mechanisms by which these two analgesic and antipyretic agents elicit their inhibition of prostaglandin synthesis are quite different.     

Separation and quntification of prostaglandins E1 and E2 as their panacyl derivatives using reverse phase high pressure liquid chromatography

Separation and quatification of prostaglandin E₁ (PGE₁) and prostaglandin E₂ (PGE₂) were achieved using reverse phase high performance liquid chromatography (HPLC). Panacyl bromide (p-(9-anthroyloxy)phenacyl bromide) (PAB) derivatives of PGE₂ and PGE₁ were prepared. Reverse phase HPLC using a linear gradient of 56% to 80% acetonitrile in water containing 0.10% acetic acid gave baseline resolution of the two derivatives. A 3 um diameter particle, C18 column provided good resolution and reproducible recoveries. Human synovial tissue cells were incubated with the precursor fatty acids for PGE₁ or PGE₂ and stimulated with a crude Interleukin 1 (IL-1) preparation. Cells grown in the presence of dihomogammalinolenic acid (DGLA), the precursor for PGE₁, made significantly more PGE₁ than cells grown in control medium or in the presence of arachidonic acid, precursor for PGE₂. PGE₂ synthesis was reduced when DGLA was added to cells (resting or IL-1-stimulated).     

Characterization of prostaglandin formation by human amnion cells in monolayer culture

In the present investigation, we found that among the prostanoids that human amnion cells, which are maintained in monolayer culture, secrete into the culture medium, prostaglandin E₂ is by far the predominant one. In the presence of inhibitors of prostaglandin synthase, the production of prostaglandin E₂ by these cells is abolished. Amnion cells maintained in the presence of fetal calf serum produce greater quantities of prostaglandin E₂ than do cells maintained in serumless medium. In the amnion cells, there is little or no metabolism of prostaglandin E₂; this also is true of amnion tissue. The unique characteristics of prostaglandin biosynthesis and metabolism by human amnion cells in monolayer culture are identical with those of human amnion tissue. Hence, we suggest that amnion cells in culture constitute an excellent model for investigations of the regulation of prostaglandin E₂ biosynthesis in this tissue.     

Enzymic coupling of acylhydrolase and prostaglandin synthase activities in subcellular fractions from rabbit renal medulla

We have recently shown that mitochondrial and plasma-membrane fractions from kidney medulla possess Ca²⁺-stimulated acylhydrolase and prostaglandin synthase activities. The nature of the enzymic coupling between the Ca²⁺-stimulated arachidonic acid release and its subsequent conversion into prostaglandins was investigated in subcellular fractions from rabbit kidney medulla. Plasma-membrane, mitochondrial and microsomal fractions were found to have similar apparent K_m values for conversion of added exogenous arachidonate into prostaglandins. The rate of prostaglandin biosynthesis (V_max.) from added arachidonic acid in the microsomal fraction was approx. 2-fold higher than in the other subcellular fractions. In contrast, prostaglandin E₂ synthesis from endogenous arachidonate in plasma-membrane and mitochondrial fractions was 3–4-fold higher than in microsomes. Furthermore, Ca²⁺ stimulated endogenous arachidonate deacylation and prostaglandin E₂ generation in the former two fractions but not in microsomes. In mitochondrial or crude plasma-membrane fractions, in which prostaglandin biosynthesis was inhibited with aspirin, arachidonate released from these fractions was converted into prostaglandins by the microsomal prostaglandin synthase. Thus an intracellular prostaglandin generation process that involves inter-fraction transfer of arachidonic acid can operate. Prostaglandin generation by such an inter-fraction process is, however, less efficient than by an intra-fraction process, where arachidonic acid released by mitochondria or crude plasma membranes is converted into prostaglandins by prostaglandin synthase present in the same fraction. This demonstrates the presence of a tight intra-fraction enzymic coupling between Ca²⁺-stimulated acylhydrolase and prostaglandin synthase enzyme systems in both mitochondrial and plasma-membrane fractions.     

Endogenous formation of the prostaglandin endoperoxine metabolite,thromboxane B2, by brain tissue

Thromboxane B₂ was formed from endogenous precursors during short incubations of guinea pig and rat cerebral cortex. The amount formed by guinea pig brain tissue was 5–6 times the formation of prostaglandin F_2α and E₂. Noradrenalin stimulated and indomethacin and mercaptoethanol inhibited thromboxane B₂ formation. The mass spectrum of the brain compound was identical to thromboxane B₂ formed from arachidonic acid by guinea pig lung and human platelets.     

Inhibition of prostaglandin synthesis by lysolecithin

Exogenous lysolecithin inhibits prostaglandin E₂ synthesis from arachidonic acid in bovine seminal vesicle microsomes at plausible physiological levels (lysolecithin-to-protein ratios ? 0.03 [w/w]) by inhibiting fatty acid cyclo-oxygenase activity. Structurally defined lysolecithins with varying fatty acid chain length exhibit varying effectiveness as inhibitors. Addition of equimolar quantities of free fatty acid lowers the lysolecithin concetration required for inhibition. Exogenous lysolecithin inhibits unstimulated and thrombin-stimulated prostaglandin E₂ synthesis from endogenous substrate in SVBalb/3T3 cells. Serum treatment of SVBalb/3T3 cells, which generates endogenous lysolecithin and free fatty acids, decreases the efficiency of conversion of free arachidonic acid to prostaglandins. These results suggest a possible role for the products of phospholipase A2 action in the regulation of prostaglandin synthesis.     

A bioluminescent enzyme immunoassay for prostaglandin E2 using Cypridina luciferase

Prostaglandin E₂ is one of the major cyclooxygenase metabolites of arachidonic acid. We developed a competitive immunosorbent assay for prostaglandin E₂ utilizing a bioluminescent enzyme Cypridina luciferase. The prostaglandin E₂ amount could be quantified over the concentration ranging from 7.8 to 500 pg/mL. The amount of unlabeled prostaglandin E₂ required to displace 50% of the maximal binding of Cypridina luciferase‐labeled prostaglandin E₂ (B/B₀) was approximately 35 pg/mL. The results show a great potential of Cypridina luciferase as a new labeling enzyme for enzyme‐linked immunosorbent assay. Copyright © 2009 John Wiley & Sons, Ltd.