A comparative study of thromboxane and prostacyclin release from ex vivo cultured bovine vascular endothelium

Thromboxane B₂, 6-keto-Prostaglandin F_1α, and Prostaglandin E₂ release have been quantitated from cultured adult by bovine endothelial cell monolayers and from $\text{ex Vivo}$ vascular segments employing specific radioimmunoassay and thin layer chromatography. Release of all three prostaglandins was demonstrable from both endothelial cell systems under basal conditions and following exposure to the ionophore A23187 and arachidonic acid. In culture, the quantity of 6-keto-PGF_1α released was diminished compared to amounts released from the vessel segments while thromboxane B₂ and prostaglandin E₂ release were similar in the two endothelial model systems. However, the amount of thromboxane B₂ assayed was small and the quantity of thromboxane A₂ it represents is probably of little $\text{in Vivo}$ significance to prostacyclin.     

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.     

Prostaglandin E2, prostaglandin E1 and thromboxane B2 release from human monocytes treated with C3b or bacterial lipopolysaccharide

C3b or lipopolysaccharide treatment of human peripheral blood monocytes in culture stimulates an early release of thromboxane B₂ and a delayed release of prostaglandin E into culture supernatants. Immunoreactive thromboxane B₂ release is maximal from 2–8 h, whereas prostaglandin E release is maximal from 16–24 h after stimulation of monocytes in culture. We further examined this process by comparing the time course of labelled prostaglandin E₂, prostaglandin E₁ and thromboxane B₂ release from human monocytes which were pulse or continuously labelled with [³H]arachidonic acid and [¹⁴C]eicosatrienoic acid. The release of labelled eicosanoids was compared with the release of immunoreactive prostaglandin E and thromboxane B₂. The time course of prostaglandin E₂ release was virtually identical to the release of prostaglandin E₁ in all culture supernatants regardless of labelling conditions. However, release of immunoreactive prostaglandin E paralleled the release of labelled prostaglandin E₁ and E₂ only for continuously labelled cultures. The release of labelled prostaglandin E₁ and E₂ from pulse labelled cultures paralleled the release of thromboxane B₂ and not immunoreactive prostaglandin. In contrast, labelled and immunoreactive thromboxane B₂, quantitated in the same culture supernatants, demonstrated similar release patterns regardless of labelling conditions. These findings indicate that the differential pattern of prostaglandin E and thromboxane B₂ release from human monocytes is not related to a time-dependent shift in the release of prostaglandin E₁ relative to prostaglandin E₂. Because thromboxane B₂ and prostaglandin E₂ are produced through cyclooxygenase mediated conversion of arachidonic acid, these results further suggest that prostaglandin E₂ and thromboxane B₂ are independently metabolized in human monocyte populations.     

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.     

Cyclic stretch induces cyclooxygenase-2 gene expression in vascular endothelial cells via activation of nuclear factor kappa-β

Vascular endothelial cells respond to biomechanical forces, such as cyclic stretch and shear stress, by altering gene expression. Since endothelial-derived prostanoids, such as prostacyclin and thromboxane A₂, are key mediators of endothelial function, we investigated the effects of cyclic stretch on the expression of genes in human umbilical vein endothelial cells controlling prostanoid synthesis: cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), prostacyclin synthase (PGIS) and thromboxane A₂ synthase (TXAS). COX-2 and TXAS mRNAs were upregulated by cyclic stretch for 24 h. In contrast, PGIS mRNA was decreased and stretch had no effect on COX-1 mRNA expression. We further show that stretch-induced upregulation of COX-2 is mediated by activation of the NF-κβ signaling pathway.     

Theophylline infusion modulates prostaglandin and leukotriene production in man

Although theophylline has been used in the treatment of asthma for decades, it is not a first line choice any more. It is a well-known bronchodilator, but was recently discovered also to be an anti-inflammatory, immunomodulatory and bronchoprotective agent. Therefore we wanted to establish the role of theophylline on prostaglandin and leukotriene production, which plays a part in the pathogenesis of asthma. Theophylline was infused (bolus 5 mg/kg in 15 min and infusion 0.4 mg/kg/h for 1 h 45 min) into healthy volunteers. Thromboxane B₂, prostaglandin E₂ and leukotriene E₄ were measured from the A23187-stimulated whole blood samples and stable metabolites of thromboxane A₂; prostacyclin and leukotriene E₄ were measured from urine. Theophylline increased prostaglandin E₂ production and decreased leukotriene E₄ production ex vivo in whole blood, thus increasing the prostanoid/leukotriene ratio. It did not change thromboxane B₂ production stimulated by either spontaneous clotting or A23187 in the whole blood. Theophylline had hardly any effect on in vivo thromboxane, prostacyclin and leukotriene E₄ production measured as urinary metabolites, 11-dehydro-thromboxane B₂, 2,3-dinor-6-keto-prostaglandin F_1α and leukotriene E₄, respectively. Serum theophylline concentrations were at the lower level of normal therapeutic range during the infusion. The increase in PGE₂ and the decrease in LTE₄ synthesis ex vivo may offer a new explanation for the mode of antiasthmatic action of theophylline. It is notable that this phenomenon occurs at low serum theophylline concentrations. These results confirm the idea that theophylline has an anti-inflammatory and bronchoprotective action and support the use of theophylline as a therapeutic agent in asthmatic patients.     

A comparison of imidazole and 9,11-azoprosta-5,13-dienoic acid Two selective thromboxane synthetase inhibitors

Two selective thromboxane A₂ synthetase inhibitors, imidazole and 9,11-azoprosta-5,13-dienoic acid (azo analog I) were compared to determine their effects on the quantitative formation of thromboxane B₂ and prostaglandin E₂ accompanying human platelet aggregation. Azo analog I was at least 200 times more potent, on a molar basis, than imidazole in suppressing thromboxane B₂ formation in either platelet-rich plasma or washed platelet suspensions aggregated with arachidonic acid or prostaglandin H₂. The inhibitors differed in their effect on the aggregation response itself. Azo analog I selectively suppressed thromboxane A₂ formation with an accompanying, parallel, suppression of the platelet aggregation.Imidazole selectively suppressed thromboxane A₂ formation, but only suppressed the accompanying aggregation in platelet rich plasma, and not washed platelet suspensions. The results indicate that azo analog I functions by competitive inhibition of prostaglandin H₂ on the thromboxane synthetase, and that imidazole, while it suppresses thromboxane A₂ formation, may have an associated agonist activity that enhances platelet aggregation. The data presented support this hypothesis, and they emphasize the importance of thromboxane A₂ in arachidonate mediated platelet aggregation.     

An antiserum to 5,6-dihydro prostacyclin (PGI1) which also binds prostacyclin

An antiserum was raised in rabbits using 5,6-dihydro prostacyclin, a stable analogue of prostacyclin, as the hapten, conjugated to bovine serum albumin. When added to platelet rich plasma the antiserum neutralised the inhibitory activity of prostacyclin, prostaglandin E₁ and D₂. The amount of antiserum required to neutralise completely a dose of prostacyclin giving 90–95% inhibition of ADP induced aggregation was 10–30 times less than that required for the other two prostaglandins. Small amounts of antiserum prevented the inhibitory activity of prostacyclin generated from endothelial cells in platelet rich plasma.     

Differential separation of thromboxanes from prostaglandins by one and two-dimensional thin layer chromatography

[¹⁴C]-labelled thromboxane B₂ and hydroxy fatty acids were isolated using thin layer and gas chromatographic procedures from human platelets incubated with [1-¹⁴C]-arachidonic acid. A number of TLC solvent systems were evaluated for differential separation of thromboxanes and hydroxy fatty acids from prostaglandins E₂, A₂, D₂ and F_2α. Chromatographic properties in nine different solvent systems are tabulated. Two dimensional TLC procedures suitable for complete resolution of mixtures of these compounds on a single plate were developed. The systems were used to demonstrate conversion of [1-¹⁴C]-arachidonic acid to thromboxane B₂ and prostaglandin E₂ by human lung fibroblasts in tissue culture.     

Effects of epoxymethano analogues of prostaglandin endoperoxides on aggregation,on release of 5-hydroxytryptamine and on the metabolism of 3′,5′-cyclic AMP and cyclic GMP in human platelets

The effects on human platelets of two synthetic analogues of prostaglandin endoperoxides were examined in order to explore the relationship between aggregation and prostaglandin and cyclic nucleotide metabolism, and to help elucidate the role of the natural endoperoxide intermediates in regulating platelet function.Both analogues (Compound I, (15S)-hydroxy-9α,11α-(epoxymethano)-prosta-(5Z,13E)-dienoic acid, and Compound II, (15S)-hydroxy-11α,9α-(epoxymethano)-prosta-(5Z,13E)-dienoic acid) caused platelets to aggregate, an effect which could be inhibited by prostaglandin E₁ but not by indomethacin. Compound II produced primary, reversible aggregation at concentrations which did not induce release of 5-hydroxytryptamine. Production of thromboxane B₂ and malonyldialdehyde was monitored as an index of endogenous production of prostaglandin endoperoxides and thromboxane A₂ and were increased after incubation of human platelets with thrombin, collagen or arachidonic acid. However, neither malonydialdehyde nor thromboxane B₂ levels were significantly influenced by the endoperoxide analogues. Both analogues produced a small elevation of adenylate cyclase activity in platelet membranes and of cyclic AMP content in intact platelets, but neither had any modifying effect on the much greater stimulation of adenylate cyclase and cyclic AMP levels by prostaglandin E₁. Of all the aggregating agents tested, only arachidonic acid produced any significant increase in platelet cyclic GMP levels.These results suggest that the epoxymethano analogues of prostaglandin endoperoxides induce platelet aggregation independently of thromboxane biosynthesis and without inhibiting adenylate cyclase or lowerin platelet cyclic AMP levels. They therefore differ from better known aggregating agents such as ADP, epinephrine and collagen, which increase thromboxane A₂ production and reduce cyclic AMP levels, at least in platelets previously exposed to prostaglandin E₁.     

Kinetic studies on the conversion of prostaglandin endoperoxide PGH2 by thromboxane synthase

We have investigated the time course of formation of thromboxane A₂, thromboxane B₂, and the C-17 hydroxy fatty acid, HHT, from arachidonic acid in a washed human platelet suspension. Our results indicate that HHT is not a breakdown product of thromboxane A₂, but rather thromboxane A₂ decomposes exclusively into thromboxane B₂. The kinetics of formation of thromboxane B₂ from the endoperoxide prostaglandin H₂ in human platelet microsomes was examined. Our data suggest that a bimolecular reaction is involved in the formation of thromboxane A₂ from prostaglandin H₂ and that thromboxane synthase is not an isomerase, but may be acting via a dismutase-type reaction. One possibility is that thromboxane and HHT are produced simultaneously from two molecules of prostaglandin H₂.     

Respiratory movements alter the generation of prostacyclin and thromboxane A2 in isolated rat lungs: The influence of arachidonic acid-pathway inhibitors on the ratio between pulmonary prostacyclin and thromboxane A2

The influence of hyperventilation on the spontaneous generation of prostacyclin and thromboxane A₂ by isolated rat lungs was studied. Both prostacyclin and thromboxane A₂, as measured by RIA of their stable end-products, 6-oxo-PGF_1α and TXB₂ respectively, were continuously released into the perfusate. However, the concentration of prostacyclin in the perfusate was higher than thromboxane A₂. Under normal ventilation at a rate 40–50 breaths/min, the ratio between these two compounds was 5:1. Increasing the rate of respiration to 100 breaths/min preferentially stimulated the release of prostacyclin. During hyperventilation, the ratio between 6-oxo-PGF_1α and TXB₂ was 12:1. Aspirin and indomethacin suppressed both basal and hyperventilation-stimulated release of prostacyclin and thromboxane A₂. Hydroperoxy-fatty acids and tranylcypromine inhibited only the release of prostacyclin but did not affect the generation of thromboxane A₂. Our findings confirm that the lung generates prostacyclin predominantly, and provide direct evidence that respiratory movements are involved in generation of pulmonary prostacyclin and thromboxane A₂.     

Differential of sex steroids of prostaglandin secretion by male and female cultured piglet endothelial cells

The effect of sex steroids, 17β-estradiol and testosterone, on the production of 6-keto-prostaglandin F_1α, prostaglandin F_2α and prostaglandin E₂ was studied in cultures of piglet aorta endothelial cells. In cells isolated from female animals both steroids stimulated the secretion of prostaglandins. In contrast, sex steroids did not affect prostaglandin synthesis by endothelial cells taken from male animals. In addition, female endothelial cells convert testosterone into Estriol, estrone and estradiol. estradiol-induced stimulation of prostacyclin production may explain in part the beneficial role generally attributed to naturally occuring estrogens in cardiovascular diseases.     

Properties of endothelial cell and smooth muscle cell cultured in ambient pressure

Summary Cultures of umbilical vein endothelial cells and smooth muscle cells were studied in a constant pressure chamber. The following results were obtained: (a) Endothelial cell growth was maximal at 80 mmHg and minimal at 0 mmHg (atmospheric pressure) for the first 2 d of incubation. However, these growth rates were reversed during the following 6 d because of steady increase in growth at 0 mm Hg and a decrease in growth at higher pressures. A degeneration of endothelial cells began at 120 mmHg and marked degeneration was noted at 160 mmHg. Growth of Smooth muscle cells was not influenced by ambient pressure and a steady increase in labeled nuclei continued throughout the period of culture. (b) Elastin, stainable with tannic acid, was noted electronmicroscopically in both endothelial and smooth muscle cells. (c) Production of prostacyclin by endothelial cells was maximal at 0 mmHg and minimal at 80 mmHg, in contrast to the growth pattern of these cells. Production of thromboxane B₂ by endothelial cells and prostacyclin and thromboxane B₂ by smooth muscle cells was very slight and not significantly different. Although it is not known at present what mechanism acts on the vascular cells when cultured in ambient pressure, these results may indicate a new concept of the behavioral relationship between endothelial cell, smooth muscle cell, and blood pressure in vivo.     

Regulatory role of cyclic adenosine 3′,5′-monophosphate on the plattelet cyclooxygenase and platelet function

Thromboxane A₂ plays and important role in arachidonic acid- and prostaglandin H₂-induced platelet aggregation. Agents that stimulate platelet adenylate cyclase (prostaglandin I₂, prostaglandin I₁, and prostaglandin E₁) and dibutyryl cyclic AMP inhibit both thromboxane A₂ formation and arachidonate-induced aggregation platelet-rich plasma. Despite complete suppression of aggregation with agents that elevate cyclic AMP, considerable thromboxane A₂ is still formed. Prostaglandin H₂-induced aggregations which bypass the cyclooxygenase regulatory step are also inhibited by agents that elevate cyclic AMP without any measurable effect on thromboxane A₂ production. These data demonstrate that cyclic AMP can inhibit platelet aggregation by a mechanism independent of its ability to suppress the cycyooxygenase enzyme. Parallel experiments with washed platelet preparations suggest that they may be an inadequate mode for studying relationship between the platelet cyclooxygenase and platelet function.     

Prostaglandins and thromboxanes in amniotic fluid during rivanol-induced abortion and labour

Abortion or delivery were induced by extra-amniotic instillation of Rivanol during the second trimester in twelve patients and during the third trimester in two patients with fetal death and one patient with fetal acrania. Serial sampling of amniotic fluid was performed through a transabdominal catheter and the levels of free arachidonic acid (AA), prostaglandin F₂α (PGF₂α), prostaglandin E₂ (PGE₂), 6-keto-prostaglandin F₁α (6-keto-PGF₁α) and thromboxane B₂ (TXB₂) were determined. The levels of AA, PGF₂α, PGE₂, 6-keto-PGF₁α and TXB₂ in amniotic fluid increased significantly during induction with the exception of AA in fetal death which was high and remained constant during induction. Furthermore, PGF₂α, 6-keto-PGF₁α and TXB₂ were all significantly correlated to AA.These observations suggested that free AA is released during Rivanol-induction of abortion and labour giving an increased synthesis of PGF₂α, PGE₂ prostacyclin and thromboxane A₂ in the fetal membranes and the decidua but not in the fetus. This increase might be relevant for the initiation and progress of abortion and labour in these patients.     

An in vitro system to screen for diarrheagenic chemicals

We examined an in vitro system to screen for diarrheagenic chemicals using an established intestinal cell line (T₈₄ human colonic carcinoma). The cells were grown on Millicell-PCF (polycarbonate membrane) wells. The cells were seeded at approximately 5 × 10₆ cells/30mm well and incubated for 9–11 days in a 5% C0₂ incubator saturated with water at 37°C. The culture medium was a 1:1 mixture of Ham's F12 and Dulbecco's MEM with 5% fetal bovine serum and 25 pglml gentamicin sulfate. The well containing cells was removed from the incubator and mounted in a modified Ussing chamber for measurement of shortcircuit current (I_sc). Chemical-induced increases in Psc are usually indicative of electrogenic epithelial Cl^– secretion, which is associated with diarrheagenic effects in animals and humans. T₈₄ cells grown on Millicell-PCF membrane responded with an increase in Isc after basolateral addition of the cholinergic (muscarznic) agonist carbachol, prostaglandin E₂, 16,16-dimethylprostaglandin E₂, and forskolin, while non-diarrheagenic prostaglandin D₂ did not affect I_sc. Based on our results, this in vitro system has the potential to be adapted as a rapid screen for detecting diarrheagenic chemicals.Abbreviations dmPGE₂ 16,16-dimethylPGE₂ - EC₅₀ 50% of maximum effective concentration - EDTA ethylenediaminetetraacetate - I_SC short-circuit current - PGD₂ prostaglandin D₂ - PGE₂ prostaglandin E₂ - PD potential difference - R_T transepithelial resistance     

Effects of prostaglandin E1 and isoproterenol on cyclic AMP content of human fibroblasts modified by time and cell density in subculture

In confluent cultures of “young” (< 30 generations) human fibroblasts, maximally effective concentrations of prostaglandin E₁ (5.6 μM) and isoproterenol (2 μM) increased cyclic AMP content several hundred-fold and approximately 30-fold, respectively. On the first day after initiation of cultures at either low (approx. 3 · 10⁵ cells) or high (approx. s · 10⁶ cells) cell density the magnitude of the isoproterenol effect was similar to that in confluent cultures. It increased during the next few days, reaching a maximum around day 2–3, and then declined. On any day during the period of subculture, the magnitude of the isoproterenol effect was inversely related to cell density. Alterations in response to prostaglandin E₁ as a function of time in subculture or cell density were less dramatic. The effects of prostaglandin E₁ were, however, smaller at some point during the first few days of subculture than after day 7, and when effects of prostaglandin E₁ were minimal, those of isoproterenol were maximal and approached those of prostaglandin E₁. On any day of subculture, cells in cultures of higher density tended to accumulate more cyclic AMP in response to prostaglandin E₁ than did those in low density cultures. The effects of prostaglandin E₁ and isoproterenol on cyclic AMP content were qualitatively similar in “young” and in “old” (> 60 generations in culture) human fibroblasts although the changes associated with duration of subculture and cell density tended to be less marked with “old” cells. In the “young” fibroblasts responsiveness to isoproterenol and prostaglandin E₁ appears to correlate with cell morphology and with the fractional rate of growth in subcultures. It is suggested that the capacities of the fibroblasts to respond to these two agents may be altered independently during growth of human fibroblasts.     

The effect of acute hypoxia on prostaglandin release in perfused human fetal placenta

The release of prostaglandin E₂ and F_2α, thromboxane B₂ and 6-keto-prostaglandin F_1α was measured in isolated human placental cotyledons perfused under high- and low-oxygen conditions. Also the effect of reoxygenation on prostaglandin production was studied. During the high-oxygen period, prostaglandin E₂ accounted for 44 % and 6-keto-prostaglandin F_1α for 28 % of all prostaglandin release, and the rank order of prostaglandin release was E₂ > 6-keto-prostaglandin F_1α > thromboxane B₂ > prostaglandin F_2α. Hypoxia had no significant effect on quantitative prostaglandin release, but the ration of prostaglandin E₂ to prostaglandin F_2α was significantly increased. After the hypoxic period during reoxygenation the release of 6-keto-prostaglandin F_1α was significantly decreased, as was the ratio of 6-keto-prostaglandin F_1α to thromboxane B₂. Also the ratio of the vasodilating prostaglandins (E₂, 6-keto-prostaglandin F_1α) to the vasocontricting prostaglandins (thromboxane B₂, prostaglandin F_2α) was decreased during reoxygenation period. With the constant flow rate, the perfusion pressure increased during hypoxia in six and was unchanged in three preparation. The results indicate that changes in the tissue oxygenation in the placenta affect prostaglandin release in the fetal placental circulation. This may also have circulatory consequences.     

Stimulation of arachidonic acid metabolism by CCl4 in isolated rat hepatocytes

Arachidonic acid metabolism was evaluated in isolated rat hepatocytes after CCl₄ exposure. CCl₄ induced dose-dependently the synthesis and release of prostacyclin (PGI₂) and thromboxane (TXB₂). Treatment with prostaglandin E₂ (PGE₂) 30 min after exposure to CCl₄, significantly reduced the cell damage as well as the release of TXB₂ from the cells.