Retinoic acid enhances the initiation of DNA synthesis stimulated by prostaglandin F2α in Swiss 3T3 cells

Retinoic acid at concentrations of 10⁻⁸–10⁻⁶M increases the initiation of DNA synthesis stimulated by prostaglandin F_2α (PGF_2α), alone or with insulin, in confluent resting Swiss mouse 3T3 cells. Analogues of retinoic acid had a similar effect. Adding retinoic acid, alone or with insulin, did not stimulate DNA synthesis. The synergistic effect of PGE₁ or PGE₂ with PGF_2α and insulin was not further enhanced by retinoic acid. Neither the synergistic effect of retinoic acid nor that of PGE₁ or PGE₂ with PGF_2α was affected by indomethacin, an inhibitor of prostaglandin synthesis. The results suggest that the synergy of retinoic acid with PGF_2α is not mediated through an increase of prostaglandin synthesis and that retinoic acid stimulates event(s) in common with those of PGE₁ and PGE₂ leading to an increase in the initiation of DNA synthesis.     

Prostaglandin synthesis by the cochlea

The exogenous and endogenous syntheses of prostaglandins (PG's) by the cochlea of adult mongolian gerbils were studied . After incubation of the whole membraneous cochlea with [³H]-arachidonic acid (AA), syntheses of PGF_2α, 6-keto PGF_1α, PGE₂, thromboxane (TX) B₂ and PGD₂ were evidenced in this order. The synthesis of radioactive PG's was almost completely inhibited by incubation with 10⁻⁵ M indomethacin. No significant amounts of those PG's were detected by radioimmunoassay (RIA) in the cochlea obtained from animals killed by microwave irradiation at 5.0 kw for 0.8 sec. However, when the homogenate of the whole membraneous cochlea obtained from animals without microwave irradiation was incubated at 37°C for 0–15 min, PGD₂, PGE₂, PGF₂α and 6-keto PGF₁α were found to be formed from endogenous AA in the cochlea by RIA. PG's were formed already at 0 time to considerable level (PGD₂, PGF₂α and 6-keto PGF₁α, 90–120 pg/cochlea; PGE₂, 370 pg/cochlea), reached to the maximum level (PGD₂, PGF₂α and 6-keto PGF₁α, 170–200 pg/cochlea; PGE₂, 500 pg/cochlea) at a 5-min incubation, and then gradually decreased. On the other hand, the amount of TXB₂ was lower than the detection limit by RIA (<50 pg/cochlea) even after the incubation. The cochlea was dissected into three parts: organ of Corti + modiolus (OC + M), lateral wall (LW), and cochlear nerve (CN), and then PG's formed by these tissues were determined after a 5-min incubation of the homogenates. In the CN and OC + M, PGE₂ was the major PG (100 and 160 pg/tissue, respectively), and the amounts of PGD₂, PGF₂α and 6-keto PGF₁α were about of those of PGE₂. In the LW, the amounts of PGD₂, PGE₂, PGF₂α and 6-keto PGF₁α were about the same level (70–100 pg/LW).     

Catechol oestrogens stimulate and direct prostaglandin synthesis

We have tested the action of a catechol oestrogen -2,3,17β- trihydroxy oestra-1,3,5 (10)-triene (2-OH oestradiol) in stimulating prostaglandin (PG) production by an homogenate of rat uterus. Marked and dose dependent stimulation was observed in PGF_2α and PGE₂ production using 20–250 μM concentrations of catechol oestrogen; a concentration of 250 μM 2-OH oestradiol resulted in a 23 fold increase in PGF_2α production with a 50% reduction in the synthesis of 6-keto PGF_1α. Tryptophan, catechol and glutathione were without effect on PGF_2α and PGE₂ production whereas adrenalin stimulated the production of all PGs, although the increase was less than that seen with 2-OH oestradiol. Oestradiol had a slight stimulatory action on PGF_2α production which reached a maximum at around 40 μM but had a more marked stimulation of 6-keto PGF_1α formation. Stimulation of prostaglandin production by oestradiol and 2-OH oestradiol showed no variation at different stages of the rat oestrous cycle. The use of 5 to 100 mg of tissue/ml gave similar product distribution although the effect of catechol oestrogen both in terms of stimulation of E and F formation (expressed per mg of tissue) and in its action on product distribution was more marked at lower concentrations of tissue.     

Bone resorption and prostaglandin production by mouse calvaria in vitro: Response to exogenous prostaglandins and their precursor fatty acids

Mouse calvaria were maintained in organ culture for 96 h and endogenous prostaglandin production and active bone resorption (⁴⁵ Ca release) measured. After a lag phase of 12 h, active resorption increased over the 96 h period. The amounts of prostaglandins released into the culture medium (measured by radioimmunoassay) were highest in the first 24 h of culture. Unless these were removed by preculturing for 24 h, or suppressed by indomethacin, no response to exogenous PGE₂, PGF_2α or prostaglandin precursors could be demonstrated. Bone resorption was stimulated after preculture by both PGE₂ and PGF_2α in a dose-dependent manner (10^?18M – 10^?5M), with PGE₂ being the more potent. Collagen synthesis was unaffected by PGF_2α, whereas PGE₂ (10^?5M) had an inhibitory effect. Eicosatrienoic acid did not stimulate bone resorption at lower concentrations (10^?7M – 10^?5M_, but was inhibitory at 10^?4M. Arachidonic acid also inhibited resorption at 10^?4M, but at lower concentrations (10^?7M – 10^?5M0 increased active resorption. This was concomitant with a rise in PGE₂ and PGF_2α levels, PGE₂ production being significantly higher than PGF_2α. The effects of PGE₂ (10^?8M) and PGF_2α (10^∞M appeared additive: there was no evidence of synergistic or antagonistic effects when varying ratios of PGE₂ : PGF_2α were employed.     

Prostaglandin synthesis and binding is increased in regressing NMU mammary carcinoma

Mammary tumors induced in Buffalo rats by treatment with nitrosomethyl urea will regress after oophorectomy. Their ability to synthesize and bind prostaglandins E and F₂α was studied in the growing and regressing states. Prostaglandins present in suspensions of 100,000 xg tumor membranes after 2 hr incubation at 37°C ± 5×10⁻⁴M indomethacin were partially purified by silica gel column chromatography before assay by specific PG RIA. The amounts of PGE and F₂α synthesized rose from 0.13 and 10.5 ng/mg protein in the growing tumors to a maximum of 1.2 and 26.5 ng/mg protein 5 days after oophorectomy. Specific binding of ³H-PGE₂ and ³H-PGF₂α to 15,000 xg tumor membranes was achieved during a 45 min incubation at 23°C ± excess unlabelled PG. Free and bound prostaglandins were separated by filtration. Binding reached equilibrium after 30 min, was saturable and reversible. Scatchard analysis revealed high affinity binding of PGF₂α but only low affinity PGE₂ binding in membranes obtained from growing tumors. A 2–3-fold increase in specific binding of PGE₂ and PGF₂α was noted at 4 days after oophorectomy which represented an increase in the number of PGF₂α receptors. PGE₂ binding retained a low affinity character. The elevated PGF₂α synthesis rates observed in the regressing tumors coupled with a regression-associated increase in receptor number suggests that PGF₂α-plays a significant role in hormone-dependent mammary tumor regression.     

The in vitro effect of indomethacin on basal bone resorption, on prostaglandin production and on the response to added prostaglandins

Mouse calvaria were maintained in organ culture without serum additives. Basal active resorption, as measured by ⁴⁵Ca and hydroxyproline release, was significantly inhibited to 74% control levels by indomethacin (1.4 × 10⁻⁷ M). Prostaglandin F and prostaglandin E₂ production, determined by radioimmunoassay, were both significantly lowered by this concentration of indomethacin. DNA, protein and hydroxyproline synthesis, as indices of cell toxicity, were unaffected by low concentrations of indomethacin, while concentrations of 1.4 × 10⁻⁶M inhibited protein synthesis (p<0.005). In the presence of indomethacin (1.4 × 10⁻⁷M) both PGE₂ and PGF_2α stimulated resorption in a dose-dependent manner, with PGE₂ being the more potent. Neither prostaglandin affected hydroxyproline synthesis at low concentrations, but PGE₂ had a marked inhibitory action at a higher concentration (10⁻⁶M). In combination, the effects of PGE₂ and PGF_2α showed no evidence of synergism or any antagonistic action. The study shows that in vitro calcium and hydroxyproline resorption in the unstimulated mouse calvaria are inhibited by indomethacin at concentrations measured in serum during human therapy. The decreased PGF and PGE₂ production associated with this decreased bone resorption in the presence of non-toxic concentrations of indomethacin would suggest a role for these prostaglandins in maintaining the basal resorption of cultured bone.     

Myocardial and coronary effects of exogenous arachidonic acid on the isolated and perfused heart preparation and its metabolism in the heart of trout (oncorhynchus mykiss)

1. The effects of arachidonate (AA) on myocardial and coronary function, and the ability to metabolize AA have been explored for the first time in the heart of rainbow trout, Oncorhynchus mykiss.2. On the isolated and perfused working heart exogenous AA (10⁻⁷ and 10⁻⁵ M) elicits a significant reduction of cardiac output and power output while heart rate is unaffected.3. The negative inotropic effect is abolished in presence of 10⁻⁵ M indomethacin. At the same AA concentration a pronounced increase in coronary resistance (175% change from baseline values) is apparent which is reduced but not abolished in the presence of 10⁻⁵ M indomethacin.4. ¹⁴C-arachidonate is metabolized by trout ventricle homogenate into PGs E₂, F_2α, D₂, and in lesser amount into TXB and 6-keto-PGF_1α. Ca-ionophore A23187 enhances the production of both PGE₂ and PGF_2α. The lipoxygenase products assayed as the hydroxy acids (HETEs) appear to be less actively synthesized than prostanoids.     

The presence of discrete receptors for prostaglandin F2 alpha in the cell membranes of bovine corpora lutea.

The cell membranes isolated from bovine corpora lutea bound ³H-prostaglandin (PG) F₂α with high affinity and specificity. The specific binding of ³H-PGF₂α was detectable at 10^?10M added ³H-PGF₂α and reached saturation at 10^?7M to 10^?6M. Unlabeled PGF₂α, as low as 10^?9M, inhibited the binding of ³H-PGF₂α with complete inhibition occurring at 10^?6M. The Scatchard analysis of equilibrium binding data revealed that the PGF₂α receptors are heterogeneous: Kd₁?5.1 × 10^?9M, n?289 fmoles/mg protein; Kd₂?1.8 × 10^?8M, n?780 fmoles/mg protein. The relative affinities of various other PGs for binding to PGF₂α receptors were (PGF₂α?100%): PGF₁α?17.5; PGE₁?0.8; PGE₂?22.4; PGA₁?0.007; PGB₁?0.01. The specificity and affinity of ³H-PGF₂α binding is consistent with the possibility that this receptor interaction may reflect an initial event in the action of PGF₂α as a luteolytic agent.     

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.     

Measurement of prostaglandins in embryonic tissue using radioimmunoassay

Although prostaglandins appear to play an important role in numerous physiological processes in the adult, neonate, and fetus, very little is known about the role of these compounds in the embryo. This study demonstrates that rat embryo homogenates synthesized 6-oxo-PGF_1α; PGE and PGF in markedly different amounts from endogenous substrate. Synthesis was inhibited by indomethacin (10 μM) in varying degrees (70–89%) depending on the prostaglandin. The metabolite of PGF_2α, 13,14-dihydro-15-keto PGF_2α (PGF_2α-M), was produced in limited amounts in the absence of exogenous NAD. In the presence of exogenous NAD and PGF however, embryonic homogenates produced PGF_2α-M. The potential role of prostaglandins during embryogenesis is discussed.     

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.     

The in vitro production of prostanoids by cultured bovine articular chondrocytes

Bovine articular chondrocytes, cultured as cell suspensions and monolayers, produced prostaglandin (PG) E₂ and PGI₂ (assayed as 6 keto PGF₁α), rather less PGF₂α and irregular quantities of thromboxane (Tx) B₂. Addition of foetal calf serum to the medium greatly stimulated PG production (a sixfold increase in PGE₂ and a twofold increase in 6 keto PGF₁α).Prostanoid production by cell suspension grown in serum-free medium generally plateaued after 24 hours. In the presence of 20% foetal calf serum, prostanoid production in long-term monolayer cultures increased during the first 6 days of culture. Levels of PGE₂α levels remained high. Indomethacin (10-⁶M) inhibited chondrocyte PG production both in the presence and absence of added arachidonic acid (10-⁴M). Prostanoids produced by chondrocytes may play a role in the modulation of cartilage metabolism $\text{in vivo}$.     

Biosynthesis of prostaglandins from arachidonic acid by the rat ovary

Biosynthesis of prostaglandins (PGs) from ¹⁴C-arachidonic acid was studied using homogenates of the ovaries from immature rats. In ascending order of metabolizing potency were, the ovaries from untreated rats, from rats treated with pregnant mare's serum gonadotropin (PMS), and from PMS-human chorionic gonadotropin (hCG) treated rats. Among the radioactive metabolites extracted, PGE₂ and 6-keto PGF_1∝ were purified and identified by silicic acid column-, thin layer-, reversed phase partition chromatographies, and radiogaschromatography. Production of PGE₂ and 6-keto PGF_1∝ was observed in homogenates of the ovaries of intact and PMS-hCG treated rats at conversion rates of 0.72; 0.43% and 7.62; 2.31%, but not by FMS treated rat ovaries. Treatment with PMS-hCG activated metabolism of arachidonic acid into radioactive metabolites including PGE2 and 6-keto PGF_1α to a large extent. Accordingly, it is concluded that luteinizing hormone and hCG play a significant role in the biosynthesis of PGs by the rat ovarian follicle.     

Mechanical stimulation of skeletal muscle cells mitigates glucocorticoid-induced decreases in prostaglandin production and prostaglandin synthase activity

The glucocorticoid dexamethasone (Dex) induces a decline in protein synthesis and protein content in tissue cultured, avlan skeletal muscle cells, and this atrophy is attenuated by repetitive mechanical stretch. Since the prostaglandin synthesis inhibitor indomethacin mitigated this stretch attenuation of muscle atrophy, the effects of Dex and mechanical stretch on prostaglandin production and prostaglandin H synthase (PGHS) activity were examined. In static cultures, 10^?8 M Dex reduced PGF_2α production 55–65% and PGE₂ production 84–90% after 24–72 h of incubation. Repetitive 10% stretch-relaxations of non-Dex-treated cultures increased PGF_2α efflux 41% at 24 h and 276% at 72 h, and increased PGE₂ production 51% at 24 h and 236% at 72 h. Mechanical stimulation of Dex-treated cultures increased PGF_2α production 162% after 24 h, returning PGF_2α efflux to the level of non-Dex-treated cultures. At 72 h, stretch increased PGF_2α efflux 65% in Dex-treated cultures. Mechanical stimulation of Dex-treated cultures also increased PGE₂ production at 24 h, but not at 72 h. Dex reduced PGHS activity in the muscle cultures by 70% after 8–24 h of incubation, and mechanical stimulation of the Dex-treated cultures increased PGHS activity by 98% after 24 h. Repetitive mechanical stimulation attenuates the catabolic effects of Dex on cultured skeletal muscle cells in part by mitigating the Dex-induced declines in PGHS activity and prostaglandin production. © 1994 wiley-Liss, Inc.     

Influence of angiotensins (I,II & III) on prostaglandin production by minced rat renal medulla

Minced rat renal medulla was incubated for 30 min at 37 °C in the presence of angiotensin, I, II or III (100 ng/ml) to determine the existence of a direct stimulating effect on prostaglandin (PG) production. PGE₂, PGF_2α, 6-keto PGF_1α and Thromboxane B₂ (TXB₂)_were determined by radioimmunoassay.For analysis of data variance, the results were separated according to whether the net output of PGE₂ was above or below 1.5 ng PGE₂ equivalent/mg tissue/30 min. Under low-output conditions, angiotensin I, II or III stimulated PGE₂ production significantly (p<0.02) and tended to augment PGF_2α production, while under high-output conditions no effect on PGE₂ or PGF_2α production was observed.Under either output condition, angiotensin I, II or III had no effect on 6-keto PGF_1α and TXB₂.     

Leukotriene synthesis inhibition and anti-ige challenge of human lung parenchyma

The leukotriene (LT) synthesis inhibitors BAY x1005 and MK-886 were evaluated in human lung parenchyma challenged with an anti-IgE. The anti-IgE-induced LTE₄ release was time- and dose-dependent. Treatment of the parenchyma with indomethacin (3 μM) prior to anti- IgE challenge inhibited the 6-keto prostaglandin F_1α (6-keto PGF_1α) release and enhanced (36%) the quantities of LTE₄ detected during IgE-stimulations. BAY x1005 and MK-886 were assessed in the presence of indomethacin (3 μM) and the IC₅₀ values for both inhibitors were similar (0.13 μM). BAY x1005 (1 μM) produced the same percent of inhibition of anti-IgE-induced LTE₄ release in the presence or absence of indomethacin. BAY x1005 (1 μM) did not alter the 6-keto PGF_1α release during anti-IgE challenge. The results indicate that BAY x1005 and MK-886 are potent inhibitors of LT synthesis when human lung parenchyma were stimulated by an anti-IgE.     

Inhibitory effects of aspirin and indomethacin on the biosynthesis of PGE2 and PGF2α

A gas-liquid chromatography system has been used to study the effects of indomethacin and aspirin on the biosynthesis of PGE₂ and PGF_2α by the prostaglandin synthetase system of bovine seminal vesicle. Both compounds were found to inhibit the production of PGE₂ and PGF_2α. However, based on statistical analyses, the inhibitory effect of indomethacin was found to be non-selective while aspirin produced statistically significant preferential inhibition of PGE₂ over PGF_2α.     

Contractile responses of longitudinal and circular smooth muscle of the canine stomach to prostaglandins E and F2α

Muscle strips were excised from the circular and longitudinal layers of the fundus, corpus and antrum, and from the inner portion of the pyloric ring. In general prostaglandin(PG)F₂α as well as PGE₁ and PGE₂ stimulated the longitudinal muscle. However, there were remarkable regional differences. The sensitivity to PGs was greatest in the fundus and corpus (threshold near 10⁻¹⁰ mol/1) and only weak in antrum (threshold 5·10⁻⁸ to 10⁻⁷ mol/1). In longitudinal antrum strips acetylcholine induced a combined phasic-tonic response, whereas PGs produced purely phasic responses. The effects of PGF_2α and PGE on the circular layer were complex. PGF_2α produced excitatory responses in circular fundus and corpus similar to those in the longitudinal layer of the same regions. PGE produced dual responses in circular fundus (excitation at low concentration and strong inhibition at concentration of 10⁻⁷ mol/1). In circular corpus PGE induced pure inhibition (threshold near 10⁻⁹ mol/1). In circular antrum and inner pylorus both PGE and PGF_2α produced inhibition of the phasic activity (threshold 10⁻⁸ to 10⁻⁷ for antrum and 10⁻⁹ mol/1 for inner pylorus). These effects of PGs appeared in the presence of adrenergic and cholinergic blocking agents as well as of tetrodotoxin and were therefore, direct effects on smooth muscle. Indomethacin (10⁻⁷–10⁻⁶ mol/1) suppressed spontaneous tone of the fundus and corpus and increased phasic activity of inner pylorus. This indicates that endogenous PG synthesis may be involved in the control of spontaneous activity in gastric muscle.     

Prostaglandin levels in isolated brain microvessels and in normal and norepinephrine-stimulated cat brain homogenates

The levels of PGD₂, PGE₂, PGF_2α and 6-keto-PGF_1α (6KF_1α) produced from endogenous archidonic acid (AA) were quantitated in cat cerebral cortical homogenates and microvessels isolated from cat cerebral cortex using gas chromatography/mass spectrometry (GC/MS). There was a six-fold enrichment of 6KF_1α levels in isolated microvessels, compared to homogenates, suggesting that 6KF_1α is of vascular, rather than neuronal origin. In order to further understand any possible role that norepinephrine (NE)_might have on modulation of PG synthesis, we studied the effects of 0.5 mM NE on PG synthesis from endogenous AA and from ³H-PGG₂, the endoperoxide precursor of PGs. In cat cortical homogenates NE induced a 74% increase in PGD₂ and PGF_2α, a 62% increase in PGE₂, and a 36% increase in 6KF_1α, as measured by GC/MS. NE caused a twofold increase in the conversion of ³H-PGG₂ to ³h-PGG_2α, with a concomitant decrease in ³H-PGE₂ and ³H-6KF_1α formation, and no change in ³H-PGD₂ synthesis. NE had no effect on the total conversiob of ³H-PGG₂ to ³H-PGs, nor on the breakdown of ³H-PGG₂ in the absence of brain tissue. We conclude that NE stimulates extravascular synthesis of PGD₂, PGE₂ and PGF_2α by stimulation of the prostaglandin synthetase complex, in addition to NE's stimulatory effect on the conversion of PGG₂ to PGF_2α, and that the lack of effect of NE on 6KF_1α synthesis reflects either a failure to achieve an adequate concentration at the vascular tissue, or an absence of the mechanism whereby NE stimulates PG synthetase.