Characterization of the stimulatory effects of PGF2α on the release of arachidonic acid

Fibroblasts derived from a rat carrageenin granuloma were cultured in the presence of radioactive arachidonic acid, palmitic acid and linoleic acid. More than 90% of each labeled fatty acid was incorporated into a phospholipid fraction by the cells in 18 hrs. Arachidonic acid was evenly incorporated into phosphatidylcholine and phosphatidylethanolamine, while both palmitic acid and linoleic acid were almost entirely incorporated into phosphatidylcholine. The position of phosphatidylcholine where the fatty acids were incorporated was different for each fatty acid. The ratio of the amount of fatty acid incorporated into the 2-position to the amount incorporated into the 1-position of phosphatidylcholine for each fatty acid was >90% for arachidonic acid, 2:1 for palmitic acid and 5:1 for linoleic acid. In the case of phosphatidylethanolamine, most arachidonic acid (>90%) was incorporated into the 2-position. PGF_2^α caused the stimulation of arachidonic acid release but not of palmitic acid and linoleic acid from pre-labeled fibroblasts.The serum in the medium was completely replaceable by bovine serum albumin. The effect of PGF_2^α increased with an increasing concentration of bovine serum albumin, suggesting that serum only acts as a ‘trap’ for released arachidonic acid. The effect of PGF_2^α was greater than bradykinin, and no synergistic effect was seen, although an additive effect was observed.The effect of PGF_2^α depended on the concentration of calcium ions under magnesium-supplemented conditions.     

Prostaglandin F2α antagonizes thromboxane A2-induced human platelet aggregation

The effects of prostaglandin F_2α on human blood platelet function were investigated. PGF_2α at 15 μM completely blocked platelet aggregation induced by 500 μM arachidonic acid or 3 μM U46619 but had no effect on aggregatin induced by 7.5 μM ADP. A similar specificity of action was not obtained with either PGI₂ or PGE₂. Thus concentrations of PGI₂ (3 nM) or PGE₂ (20 μ M) which inhibited U46619-induced aggregation by 100% also blocked ADP-stimulated aggregation.The inhibitory properties of PGF_2α were not related to increases in platelet cAMP, since direct measurement of intracellular cAMP revealed that 15 μ M PGF_2α produced no substantial change in cAMP levels. This finding was in direct contrast to results obtained using either PGI₂ or PGE₂. Both PGI₂ (3 nM) and PGE₂ (20 μ M) induced significant increases in platelet cAMP levels.The possibility that PGF_2α directly interacts at the platelet TXA₂/PGH₂ receptor was investigated by measuring [³H]PGF_2α binding to isolated platelet membranes. It was found that [³H] PGF_2α binding reached equilibrium within 30 min at room temperature and could be 90% displaced by addition of 1000 fold excess of unlabelled PGF_2α. Furthermore, when 1000 fold excess of either the TXA₂/PGH₂ “mimetic” U46619 or the TXA₂/PGH₂ antagonist 13-azaprostanoic acid was added, specific [³H] PGF_2α binding was displaced by 95% and 85% respectively. In contrast, the same molar excess of 6-keto-PGF_1α, azo analog 1, or TXB₂, caused displacement of only 15%, 20% or 25% of the [³H] PGF_2α binding. Scatchard analysis indicated that [³H] PGF_2α has two binding sites; i.e., a high affinity binding site with an apparent Kd of 50 nM and a low affinity binding site with apparent Kd of 320 nM. These results suggest that the selective inhibition by PGF_2α of AA or U46619-induced aggregation may be mediated through interaction at the platelet TXA₂/PGH₂ receptor.     

Effect of oestradiol 17 β on prostaglandin biosynthesis by the uterus of ovariectomized rat and guinea pig

$\text{In vitro}$ prostaglandin biosynthesis by uteri of ovariectomized rats and guinea pigs treated or untreated with oestradiol 17 β, administered subsutaneously, was measured by R.I.A. of PGF_2α and PGE₂. Incubations with [1-¹⁴C] arachidonic acid were also performed and labelled metabolites were analyzed by TLC. The main metabolite in rats was 6 keto PGF_1α and in decreasing order of magniture, PGF_2α and PGE₂. In guinea pig PGF_2ga was the main product. Ovariectomy in rats completely changed the pattern of synthesized prostanoids: PGI₂ production was doubled when compared to cycling rats and PGE₂ increased 10 fold. PGF_2α walues were similar to the mean value measured during the cycle. OE₂ treatment almost completely inhibited PGI₂ synthesis and reduced PGE₂ by half. Total PG synthesis in OE₂ treated animals was decreased by 5 fold when compared to spayed rats. Endogenous PGF_sα synthesis was slightly stimulated. In the guinea pig OE₂ treatment of ovariectomized animals increased the total synthesis from 50 per cent. PGF_2α was always the main metabolite. In conclusion OE₂ regulation of uterine PG synthesis is depending on the animal species and cannot be explained by a unique effect on the cyclooxyhenase, but rather by an interplay on the various enzymes of the arachidonic acid cascade.     

Immunological and non-immunological synthesis and release of prostaglandins and thromboxanes from isolated guinea pig trachea

Specific radioimmunoassays were used to demonstrate the synthesis by the guinea pig trachea of 6-keto PGF_1α, TxB₂, and PGF_2α in addition to PGE₂. The rank order of both spontaneous and stimulated release was PGE₂ > PGF_2α > 6-keto PGF_1α = TxB₂. Ovalbumin-induced prostanoid release from sensitized tissue was antigen-specific. The release was unlikely to be a secondary consequence of tracheal contraction since incubations with calcium ionophore A23187, at a concentration which produces an equivalent magnitude of contraction of sensitized trachea, did not induce a significant PG or Tx production. In contrast, significantly higher prostanoid synthesis was induced by A23187 in unsensitized than sensitized trachea. Thus sensitization altered the profile of arachidonic acid metabolism evoked by the ionophore.     

Enhanced formation of PGI2, a potent hypotensive substance, by aortic rings and homogenates of the spontaneously hypertensive rat

Intact rings and homogenates of aorta from spontaneously hypertensive rats (SHR) contain enhanced capacity over normal rats (NR) to convert arachidonic acid into PGI₂. The PGI₂ synthetic system in SHR is stimulated to a greater extent than NR by norepinephrine. Indomethacin blocks this stimulation. PGE₂ and PGF_2α were detected in much smaller amounts in homogenates (undetected in rings) but their formation was not enhanced by the hypertensive tissue. The identity of PGI₂ was based on 1) direct pharmacological assay on the rat blood pressure. In this system identical vasodepressor responses to PGI₂ are observed after intracarotid and intrajugular administration 2) indirectly as 6-keto PGF_1α isolated after incubation of aortic homogenates with tritiated arachidonic acid and 3) indirectly by GC-MS assay of PGE₂, PGF_2α and 6-keto PGF_1α formed during incubation of aortic homogenates with excess unlabeled arachidonic acid. These results provide additional support to our recent hypothesis that PGI₂, of aortic origin, might actively participate in the regulation of systemic blood pressure. Its enhanced formation by intact hypertensive vascular tissue reflects an increase in the number of enzyme molecules immediately available to the substrate. This could probably be an adaptive response to the elevated levels of catecholamines in the circulation.     

Effects of prostaglandins E2, I2 and F2α, arachidonic acid and indomethacin on pressor responses to norepinephrine in conscious rats

The effects of prostaglandins E₂ (PGE₂), I₂ (PGI₂) and F₂α (PGF₂α), arachidonic acid and indomethacin on pressor responses to norepinephrine were examined in conscious rats. Intravenously infused PGE₂ (0.3, 1.25 μg/kg/min), PGI₂ (50, 100 ng/kg/min), PGF₂α (1.8, 5.4 μg/kg/min) and arachidonic acid (0.7, 1.4 mg/kg/min) did not change the basal blood pressure. Both PGE₂ and PGI₂ significantly attenuated pressor responses to norepinephrine, whereas PGF₂α significantly potentiated them. Arachidonic acid, a precursor of the prostaglandins (PGs), significantly attenuated pressor responses to norepinephrine. Since the attenuating effect of arachidonic acid was completely abolished by the pretreatment with indomethacin (5 mg/kg), arachidonic acid is thought to exert an effect through its conversion to PGs. On the contrary, intravenously injected indomethacin (0.2–5.0 mg/kg) facilitated pressor responses to norepinephrine in a dose-related manner without any direct effect on the basal blood pressure. These results suggest that endogenous PGs may participate in the regulation of blood pressure by modulating pressor responses to norepinephrine in conscious rats.     

Kinetic measurements of the release of prostaglandins from perfused rat lungs

Prostaglandins released from isolated, ventilated and perfused rat lungs were measured by a simple modification of the Vane technique using the rat stomach fundus as a continuous bioassay tissue. Exogeneously supplied arachidonic acid was converted mainly to PGF_2α which was determined by bioassay. A novel method for mixing a stream of inhibitors with the perfusate was used to determine PGF_2α in the presence of substrate amounts of arachidonic acid. Using this system the apparent K_m for PGF_2α production with arachidonic acid as the substrate was found to be 1.90 × 10⁻⁴M, while the K_i for aspirin was found to be 2.47 × 10⁻⁴M. These kinetic parameters are close to those reported for cell free systems and subcellular fractions suggesting that both substrate and inhibitor have ready access to the site of prostaglandin synthesis. The method appears to be generally useful to determine the effect of drugs and environmental factors on the release of prostaglandins by the lung.     

Prostaglandins and myogenic control of tension in lower esophageal sphincter in vitro

Active tension is produced by the lower esophageal sphincter (LES) of North American opossum $\text{in vitro}$ by a myogenic mechanism. Strips of LES, but not those from the esophageal body, contracted to prostaglandin (PG)F_2α, stable expoxymethano derivatives of PGH₂ and to thromboxane B₂. Stable endoperoxides were more than 500 times more potent than PGF_2α. PGI₂ and 6-keto PGF_1α were weak relaxants of LES strips. LES strips transformed arachidonic acid into contractile substances. This transformation was prevented by agents which interfere with PG synthesis by inhibiting cyclo-oxygenase [indomethacin (IDM), 5,8,11,14-eicosatetraynoic acid (ETA) or thromboxane synthetase [imidazole]. Tranylcypromine 500 μg/ml also inhibited contractions to arachidonic acid. These agents also reduced muscle tone, so that endogenous PG formation may contribute to active tension in the LES. ETA and IDM increased tone before inhibiting it, and this effect was prevented by prior treatment with ETA or imidazole. There may also be an endogenous PG which inhibits LES tone. The possibility that this may be PGI₂ is discussed.     

The effect of radiation on prostacycliln (PG2) productnio by cultured endothelial cells

The effect of ionizing irradiation on the synthesis of prostacyclin (PGI₂) by cult8erd bovine aortic endothelial cells was detemined. PGI₂ was measuured in the culture medium by a radioimmunoassay for 6-Kto PGF_1α. Two phenomena were observed following irradiation: a) Cells which suffered an immediate radiation damage (1000–5000 rads) released high quantities of PGI₂ to the culture medium. This was due to a de novo synthesis of PGI₂ stimulated by radiation induced cellular damage, since pretreatment with aspirin of the endothelial cell monolayers resulted in a marked inhibition of PGI₂ release following irradiation. b) Metabolically active cells which remained confluent and firmly attaached to the culture dish following single, low and intermediate doses (200–1200 rads) radiation, exhibited a marked decreased in their capacity to synthesize PGI₂ upon exposure to various stimuli of the archidonic acid cascade (arachidonic acid, melittin, ionophore A23187 and PGH₂. Similar results were observed with cells treated with fractionated radiation.The quantities of PGI₂ produced by the endothelial cells decreased as a function of the dose of radiation and time interval between irradiation and subsequent stimulation. Radiation had a minimal effect on the nonthrombogenic properties of the endothelial cells, as evidenced by the small increase in the platelet adherence to the endothelial cells. The effect of radiation on PGI₂ pruction by the vascular endothelium may be relevant to the development of radiation induced capillary occlusions, and the enhancement of atherosclerotic lesions in large vesses.     

Enhancement of anaphylactic mediator release from guinea-pig perfused lungs by fatty acid hydroperoxides

Fragments of chopped lung from indomethacin treated guinea-pigs had an anti-aggregating effect when added to human platelet rich plasma (PRP), probably due to the production of prostacyclin (PGI₂) since the effect was inhibited by 15-hydroperoxy arachidonic acid (15-HPAA, 10 μg ml^?1). Both 15-HPAA (1–20 μg ml^?1 min^?1) and 13-hydroperoxy linoleic acid (13-HPLA, 20 μg ml^?1 min^?1) caused a marked enhancement of the anaphylactic release of histamine, slow-reacting substance of anaphylaxis (SRS-A) and rabbit aorta contracting substance (RCS) from guinea-pig isolated perfused lungs. This enhancement was not reversed by the concomitant infusion of either PGI₂ (5 μg ml^?1 min^?1) or 6-oxo-prostaglandin F_1α (6-oxo-PGF_1α, 5 μg ml^?1 min^?1). Anaphylactic release of histamine and SRS-A from guinea-pig perfused lungs was not inhibited by PGI₂ (10 ng - 10 μg ml^?1 min^?1) but was inhibited by PGE₂ (5 and 10 μg ml^?1 min^?1). Antiserum raised to 5,6-dihydro prostacyclin (PGI₁) in rabbits, which also binds PGI₂, had no effect on the release of anaphylactic mediators. The fatty acid hydroperoxides may enhance mediator release either indirectly by augmenting thromboxane production or by a direct effect on sensitized cells. Further experiments to distinguish between these alternatives are described in the accompanying paper (27).     

Effects of superovulation, arachidonic acid or endometrium on release of prostaglandins and synthesis of proteins by bovine trophoblast

This study was conducted in vitro to examine factors that may regulate prostaglandin release by bovine trophoblast and endometrial slices. Trophoblastic tissues and endometrial slices were recovered from superovulating and normally-ovulating cattle on day 16 or 20 of pregnancy and incubated for 24 h. Release of PGF_2^α and 13,14-dihydro-15-keto-PGF_2^α (PGMF), and incorporation of [¹⁴C]-leucine into proteins were quantified and expressed per μg DNA, which gives a measure of cellular activity. Activity of trophoblastic tissue for synthesizing protein was decreased (P<.05) and for releasing PGMF was increased (P<.05) on day 20 compared to day 16 of pregnancy. Neither supercovulation nor day of pregnancy altered trophoblastic activity for releasing PGF_2^α. Supercovulation increased (P<.05) endometrial release of PGF_2^α. Endometrial release of PGF_2^α was less (P<.05) on day 20 than on day 16 of pregnancy. When arachidonic acid (0, 100, 200 or 400 μg) was added at the start of incubation, trophoblastic release of PGF_2^α changed (P<.05) quadratically with dose of arachidonic acid. When arachidonic acid was added 8 h after the start of incubation, triphoblastic release of PGF_2^α increased linearly (P<.01) with dose of arachidonic acid. Adding arachidonic acid to incubation medium did not affect trophoblastic or endometrial protein synthesis. Endometrial slices suppressed (P<.05) trophoblastic protein synthesis and release of PGF_2^α. Apparently, endometrium can modulate trophoblastic release of prostaglandins and synthesis of proteins in vitro, and trophoblastic tissue from supercovulated cattle 16 or 20 days pregnant can be used to study trophoblastic synthesis of prostaglandins and proteins.     

Prostaglandin synthesis by fetal rat bone in vitro: Evidence for a role of prostacyclin

Prostaglandin synthesis by fetal rat bones was examined by thin-layer chromatography of culture media after preincubation with labeled arachidonic acid. Cultures in rabbit complement (non-heat inactivated serum) were compared with cultures in heat-inactivated serum or cultures treated with indomethacin. The major complement-dependent products were PGE₂, PGF_2α and 6-keto-PGF_1α, the metabolite of prostacyclin (PGI₂). Since PGI₂ had not been previously identified in bone its ability to stimulate bone resorption was tested. Repeated addition of PGI₂ stimulated release of previously incorporated ⁴⁵Ca from fetal rat long bones in both short-term and long-term cultures at concentrations of 10⁻⁵ to 10⁻⁹M. Because of the short half life of PGI₂ in solution at neutral pH, we tested a sulfur analog, thiaprostacyclin (S-PGI₂) which was found to be a stimulator of bone resorption at concentrations of 10⁻⁵ to 10⁻⁶M. These studies suggest that endogenous PGI₂ production may play a role in bone metabolism. Since vessels produce PGI₂ it is possible that PGI₂ release may be responsible for the frequent association between vascular invasion and resorption of bone or calcified cartilage in physiologic remodeling and pathologic osteolysis.     

The ability of vascular tissue to produce prostacyclin decreases with age

The ability of aortae from young and mature swine to produce prostacyclin (PGI₂) has been determined. PGI₂ was measured as its hydration product, 6-keto-PGF_1α and assayed by stable isotope dilution GC-MS. There was no significant difference in 6-keto-PGF_1α production between intimal strips from young and mature aortae in the basal state. In the presence of saturating concentrations of arachidonic acid, however, intimal strips from young aortae synthesized twice as much 6-keto-PGF_1α as did older tissues. Fatty acid compositions of young and mature aortae were virtually identical, making dietary differences an unlikely explanation for the age-related decrease in PGI₂ synthesis. Both young and mature vascular tissues produced essentially only PGI₂; insignificant amounts of PGE₂ and PGF_2α were found.     

Inhibition of prostaglandin synthesis in human endothelial cells treated with metabolic inhibitors

Endothelial cell injury is often associated with increased synthesis of prostaglandin (PG)I₂. We observed, however, that endothelial cells treated with metabolic inhibitors which reduce cellular ATP content develop an injury pattern characterized by reduced PGI₂ synthesis. This study examined the relationship between cell injury, arachidonic acid metabolism and ATP content in human umbilical vein endothelial cells treated with 2-deoxyglucose (2DG), a glycolytic inhibitor, and oligomycin (OG), a respiratory chain inhibitor. Either inhibitor alone significantly reduced cellular ATP concentrations, but only OG reduced basal PG synthesis. The combination of 2DG and OG, however, was more effective than either agent alone in reducing cellular ATP content (≥ 50% of control) and inhibiting basal and agonist-stimulated PGI₂ synthesis. This reduced PGI₂ synthesis preceded ⁵¹ chromium release, lactic dehydrogenase release and was not associated with a net release of arachidonic acid from cell membranes. Histamine, A23187 and bradykinin stimulated PGI₂ synthesis in untreated but not in 2DG and OG treated cells. Exogenous arachidonic acid increased PGI₂ synthesis to a similar extent in both 2DG and OG treated and untreated cells. Therefore, reduced PG synthesis in 2DG and OG treated endothelial cells is not due to inhibition of cyclooxygenase. Furthermore, reduced PG synthesis in these cells occurs prior to cell injury and is not strictly associated with cellular ATP depletion.     

Prostacyclin production of rat aorta “in vitro” is increased by the combined action of dipyridamole plus pentoxifylline

The present study evaluates the effect of dipyridamole and pentoxifylline, individually and in combination, on PGI₂-like production and arachidonic acid metabolism of rat aorta “in vitro”. Pentoxifylline 100 μM and dipyridamole 92 and 184 μM increased PGI₂-like activity, as measured by the platelet aggregation inhibitory capacity of the aortic ring incubates, by 71%, 46% and 60% respectively; a greater increase in PGI₂-like activity was observed with the combination of the drugs than when they were used separately. This effect was observed even at the lowest doses assayed. In fact, dipyridamole 9.2 μM plus pentoxifylline 1 μM increased the PGI₂-like activity by 30% while the individual increase was 4.5% and 10.6% respectively. To obtain more information on the effect of the dipyridamole-pentoxifylline combination on arachidonic acid metabolism, arteries were incubated with (1-¹⁴C) arachidonic acid, and the 6-keto-PGF_1α and PGE₂ quantified. Dipyridamole 92 μM plus pentoxifulline 1 and 10 μM increased 6-keto-PGF_1α and PGE₂ production by about 30% and 48% respectively while combination with pentoxifylline 100 μM increased the 6-keto-PGF_1α 76.5% and the PGE₂ 50%. The possible biological effect and therapeutic implications of increased PGI₂ production by the arteries due to the dipyridamole-pentoxifylline combination remains to be ascertained.     

Production of prostaglandins by cells in vitro: radioimmunoassay measurement of the conversion of arachidonic acid to PGE2 and PGF2 alpha

A specific radioimmunoassay has been applied to the measurement of the conversion of arachidonic acid to PGE₂ and PGF_2α. PGE₂ and PGF_2α biosynthesis was linearly related to the amount of arachidonic acid added and was significantly inhibited by indomethacin in concentrations as low as 10^?10 M. Sonicated Hela, L, and HEp-2 cells synthesized 244.0, 42.3, and 22.6 ng PGE₂ per mg of protein, but made substantially less PGF_2α.     

Prostaglandin levels in BL6 melanoma cells cultured in vitro: the effect of vitamin E succinate supplementation

Malignant murine melanoma (BL6-F10) cells convert arachidonic acid primarily to PGD₂, PGF_2α, PGE₂, PGI₂ in descending order of magnitude. Supplementation with 1–10 μg/ml vitamin E succinate resulted in a significant (P ≤ 0.05) decrease in PGD₂ levels at vitamin concentrations of 3,5,7 and 10 μg/ml respectively, while PGF_2α levels were significantly decreased at 1,3,5 (P ≤ 0.05), 7 and 10 μg/ml (P ≤ 0.01) vitamin E succinate. BL6-F10 cells supplemented with 7 and 10 μg/ml vitamin E succinate showed a marked increase in PGE₂ levels with a significant increase occurring at 10 μg/ml (P ≤ 0.025). PGI₂ levels followed a similar trend to PGE₂ with a significant increase (P ≤ 0.05) occurring at 10 μg/ml.     

Amplification of arachidonic acid metabolism in rat liver cells (the C-9 cell-line) by tosyl- -phenylalanine chloromethyl ketone and phenylmethyl-sulfonyl fluoride

In the presence of the protease inhibitors phenylmethyl-sulfonyl fluoride and N-tosyl- -phenylalanine chloromethyl ketone, prostacyclin (PGI₂) production by rat liver cells treated with epidermal growth factor, platelet-activating factor, 12-O-tetradecanoylphorbol-13-acetate (TPA), and TPA-type tumor promoters (teleocidin and aplysiatoxin) or 1-oleoyl-2-acetylglycerol is amplified. The PGI2 production stimulated by thapsigargin or exogenous arachidonic acid is not amplified. N-Tosyl- -phenylalanine chloromethyl ketone also amplifies TPA's release of radioactivity from cells isotopically labeled with [³H]arachidonic acid. Indomethacin inhibits the amplification of PGI₂ production but not the release of radioactivity. The presence of the protease inhibitors is not required for the amplification of PGI₂ production. Prior incubation of the cells with these inhibitors, followed by their removal, still results in amplified PGI₂ production by cells subsequently treated with TPA, 1-oleoyl-2-acetylglycerol, or platelet-activating factor but not that stimulated by exogenous arachidonic acid. While phenylmethyl-sulfonyl fluoride's amplification of PGI₂ production by cells treated with TPA was blocked by prior incubation with TPA for 20 h, a similar block of amplification in EGF-treated cells was not observed.     

Arachidonate metabolism in bovine gallbladder muscle

Incubation of [1-¹⁴C]arachidonic acid (AA) with homogenates of bovine gallbladder muscle generated a large amount of radioactive material having the chromatographic mobility of 6-keto-PGF_1α (stable product of PGI₂) and smaller amounts of products that comigrated with PGF_2α and PGE₂. Formation of these products was inhibited by the cyclooxygenase inhibitor indomethacin. The major radioactive product identified by thin-layer chromatographic mobility and by gas chromatography - mass spectrometric analysis was found to be 6-keto-PGF_1α. The quantitative metabolic pattern of [1-¹⁴C]PGH₂ was virtually identical to that of [1-¹⁴C]AA. Incubation of arachidonic acid with slices of bovine gallbladder muscle released labile anti-aggregatory material in the medium, which was inhibited by aspirin or 15-hydroperoxy-AA.These results indicate that bovine gallbladder muscle has a considerable enzymatic capacity to produce PGI₂ from arachidonic acid.