Hypoxia enhances prostaglandin synthesis in renal mesangial cell cultures

In view of recent findings which suggest that renal prostaglandins mediate the effect of hypoxia on erythropoietin production, we have studied whether hypoxia is a stimulus for in vitro prostaglandin synthesis. Studies were carried out in rat renal mesangial cell cultures which produce erythropoietin in an oxygen-dependent manner. Production rates of PGE₂ and in specified samples also of 6-keto-PGF_1α, as a measure of PGI₂, and PGF_2α were determined by radioimmunoassay after incubation at either 20% O₂ (normoxic) or 2% O₂ (hypoxic) in gas permeable dishes for 24 hrs. Considerable variation in PGE₂ production was noted among independent cell lines. PGE₂ production appeared to be inversely correlated to the cellular density of the cultures. In addition, PGE₂ production was enhanced in hypoxic cell cultures. The mean increase was 50 to 60%. PGF_2α and 6-keto-PGF_1α increased by about the same rate. These results indicate that hypoxia is a stimulus for in vitro prostaglandin production.     

Prostaglandin-induced enhancement of the in vitro anamenstic response

The ability of various prostaglandins (PGs) to affect the in vitro anamnestic immune response of keyhole limpet hemocyanin (KLH)-primed rabbit popliteal lymph node cells was investigated. Of the four PGs studied (PGA₁, PGE₂ and PGF_2α), PGE₁ was found to have a stimulatory effect, whereas PGA₁, PGE₂ and PGF_2α were ineffective in stimulating or inhibiting the in vitro anamnestic response. Under the conditions studied, a 3.5-fold increase in antibody production was obtained in PGE₁-treated, KLH-stimulated cultures. Maximum enhancement was obtained when 0.2 μg of PGE₁ were added at the time of culture initiation and were allowed to remain in contact with the lymph node cells for 24 hours.     

Prostaglandin E1 and F2α specific binding in bovine corpora lutea: Comparison with luteolytic effects

Preliminary characterization indicated the presence of separate prostaglandin (PG)E₁ and (PG)F_2α binding sites in membrane fractions prepared from bovine corpora lutea. These differ in the rate and temperature dependence of the specific binding. Equilibrium binding data indicate the apparent dissociation constants as 1.32 × 10⁻⁹M and 2.1 × 10⁻⁸M for PGE₁ and PGF_2α, respectively. Competition of several natural prostaglandins for the PGE₁ and PGF_2α bovine luteal specific binding sites indicates specificity for the 9-keto or 9α-hydroxyl moiety, respectively. Differences in relative ability to inhibit ³H-PG binding were found due to sensitivity to the absence or presence of the 5,6-cis-double bond as well.Bovine luteal function was affected following treatment of heifers with 25 mg PGF_2α as measured by reduced estrous cycle length, decreased corpus luteum size and significantly decreased plasma progesterone levels. In contrast, treatment with 25 mg PGE₁ resulted in cycle lengths comparable to those of non-treated herdmates with no apparent modification in corpus luteum size. However, plasma progesterone levels were increased significantly following PGE₁ treatment compared to pretreatment values. In so far as data obtained in vitro on PGF_2α relative binding affinity to the bovine CL can be compared to data obtained independently in vitro on PGF_2α induced luteolysis in the bovine, PGF_2α relative binding to the CL and luteolysis appeared to be associated. By similar reasoning, there was no apparent relationship between PGE₁ relative binding affinity in the luteal fractions and luteolysis in estrous cyclic cattle.     

α-Adrenergic stimulants, prostaglandins and catecholamine release from the adrenal gland in vitro

In rat adrenal glands incubated in Locke's solution in vitro norepinephrine and phenylephrine inhibited the release of epinephrine. PGE₂ and PGE₁ also inhibited the release of catecholamines but PGF_α1 had no effect on the adrenal. Thus, catecholamine release from adrenal cells may be regulated by the same mechanisms as in adrenergic nerve endings.     

Stimulatory effect of prostaglandins on the production of hexosamine-containing substances by cultured fibroblasts (4) adenosine 3′:5′-cyclic monophosphate independent stimulation by prostaglandin F2α

The mechanism of the stimulatory effect of prostaglandin (PG) F_2α on the production of hexosamine-containing substances by cultured fibroblasts was studied with special reference to adenosine 3′:5′- cyclic monophosphate (cAMP). At the stationary phase, the cells were exposed for 6 hrs to PGF_2α, E₁, cAMP or dibutyryl-cAMP in a wide range of concentrations. cAMP itself showed a slight stimulation on the production of hexosamine-containing substances, and the effect was enhanced by using the dibutyryl derivative. PGF_2α had much a greater capacity than either the exogeneous cAMP or the dibutyryl-cAMP for enhancing the production of hexosamine-containing substances. To know whether cAMP is involved in the stimulatory effect of PGF_2α, intracellular cAMP level was concomitantly measured in both PGF_2α and PGE₁ treated cultures. Although the cellular cAMP level in PGE₁ treated cultures was much higher than that in the PGF_2α treated cultures, the stimulatory effect on the production of hexosamine-containing substances in PGE₁ treated cultures was always much smaller than that in the PGF_2α treated cultures. Moreover, PGF_2α had a significant stimulatory effect on the production of hexosamine-containing substances even at a low concentration as 100 pg/ml, which is small enough not to increase any cellular cAMP level. From these results, it was concluded that the stimulatory effect of PGF_2α on the production of hexosamine-containing substances by cultured fibroblasts is not mediated by cAMP and is caused by a mechanism different from that caused by cAMP.     

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

Prostaglandin biosynthesis by the rat uterus during the oestrus cycle, temporal correlation with plasma oestradiol and progesterone concentrations, identification of 6 keto PGF1α as the major metabolite

Prostaglandin biosynthesis was studied in the rat uterus during the oestrous cycle. Uterine homogenates were incubated for 20 minutes in the presence of exogenous substrate (2.10⁻⁵M). PGF_2α and PGE₂ were measured by R.I.A.. A sharp peak PGF_2α and a smaller peak of PGE₂ were observed at prooestrus, 20 h. Another small PGE₂ peak occurred at dioestrus II, 15 h. The lowest values of both PGs were found on dioestrus, 15 h. Plasma oestradiol concentration were highest at proestrus, 15 h and 20 h. A sharp progesterone peak occurred at prooestrus, 20 h. The PGF_2α peak is next to the oestradiol peak and is superimposable or lags slightly beyond the progesterone peak.Incubation with ¹⁴C arachidonic acid and subsequent analysis of extracts by TLC and scanning showed that the major metabolite is PGI₂, identified as 6 keto PGF_1α. The conversion rate of arachidonic acid into 6 keto PGF_1α is 5 times higher than into PGF_2α. 6 keto PGF_1α was further identified by GC/MS. No significant difference was observed between 6 keto PGF_1α production during oestrus and dioestrus.     

PGE2 attenuates PGF2α-induced increases in free intracellular calcium in ovine large luteal cells

When ovine large luteal cells are placed in culture and exposed to PGF_2α, there is a rapid and sustained increase in the concentration of free intracellular calcium which is believed to play a major role in the luteolytic and cytotoxic effects of PGF_2α. Since administration of exogenous PGE₂ can prevent spontaneous and PGF_2α-induced luteolysis in vivo, and the cytotoxic effects of PGF_2α on large luteal cells in vitro, the objective of this study was to determine if one mechanism by which PGE₂ acts is to attenuate increases in free intracellular calcium induced by PGF_2α. At concentrations of 10 nM or greater, PGF_2α caused a significant and sustained increase in free intracellular calcium in large luteal cells. Similarly, PGE₂ also induced increases in free intracellular calcium but required doses 20-fold greater than PGF_2α. When PGE₂ (1, 10 or 100 nM) was incubated with PGF_2α (100 nM) increases in free intracellular calcium induced by PGF_2α were attenuated (P<0.05) when measured 5 min, but not at 30 min, after initiation of treatment. The observed decrease in the concentration of free intracellular calcium at 5 min in response to PGF_2α was the result of fewer cells responding to PGF_2α. In addition, the concentrations of free intracellular calcium attained in the cells that did respond was reduced 25% compared to cells treated with PGF_2α alone. Thus, part of the luteal protective actions of PGE₂ appears to involve an inhibition of the early (5 min) increase in free intracellular calcium induced by PGF_2α.     

Prostaglandin (PG) analysis in urine of humans and rats by different radioimmunoassays: Effect on PG-excretion by PG-synthetase inhibitors, laparotomy and furosemide

Thw radioimmunological (RIA) determination of prostaglandin (PG) E₂ and of PGF_2α in urine humans and rats is described in detail. After extraction and chromatography PGE₂ was determined by using a PGE specific antibody or by using either PGB or PGF_2α specific antibodies after the respective conversion procedures. The three different RIA procedures were compared to each other. PGF_2α was determined by a specific antibody to PGF_2α. Basal excretion of PGE₂ and of PGF_2α in healthy women on free diet was 9.3 ng/hour ± 0.96 and 18.3 ng/hour ± 2.5 respectively. Furosemide increased the excretion of PGE₂ and of PGF_2α in humans significantly, while PG-excretion rates decreased on indomethacin. In rat urine PGE₂ and PGE_2α increased markedly from 46.2 pg/min ± 9.3 and 27 ± 3.4 to 253.8 ± 43.3 and 108 ± 12.6 pg/min (per one kidney) in the anesthetized-laparotomized animal. This increase was abolished after giving two different PG synthetase inhibitors.     

Prostaglandin E2: Midterm abortion in rats using Silastic-PVP tubes

The results of the present study establish that 1.5 mg PGE₂ (lyophilized sodium salt) incorporated in one cm long open-ended Silastic-polyvinylpyrrolidone (PVP) tube when inserted into 10 day pregnant rats induced abortion within 70–72 hours in all the treated rats. A combined treatment of PGE₂ and 17β-estradiol failed to increase the abortion inducing effect of a Silastic-PVP-PGE₂ tube. It is observed that PGE₂ is about 4 times less potent than PGF_2α in inducing midterm abortion in rats. It is suggested that either PGE₂ exerts luteolytic effect after being converted to PGF_2α, although how it occurs is not clear; or PGE₂ causes expulsion of the fetuses by its uterine stimulating property. 17β- estradiol increases the uterine synthesis of PGF_2α as described earlier but seems not affecting the production of PGE₂ by the uterus. The release rate of ³H-PGE₂ from Silastic-PVP tube and is also described.     

Prostaglandins and oxytocin: Their effects on uterine smooth muscle

A subcellular fraction was isolated from uteri of non-pregnant and pregnant cows. ATP-dependent calcium binding was shown to take place in this fraction. This calcium binding was inhibited in a dose related fashion when increasing amounts of prostaglandin (PG) E₂ or F_2α were added to the in vitro experimental medium. The physiologically inactive PGF_1β had no inhibitory effect. Oxytocin caused inhibition of calcium binding in preparations from both pregnant and non-pregnant cows. The response to PGE₂ and PGF_2α was somewhat greater in preparations from pregnant uteri than from non-pregnant uteri. The response to oxytocin was very much greater in pregnant uteri. Because of the high PG sensitivity of calcium binding in preparations from the non-pregnant uterus, it is concluded that the PGs may be the more suitable agent in the control of reproduction.     

Radioimmunological and biological measurement of prostaglandins in rabbit urine : Decrease of PGE2 excretion at high NaCl intake

The estimation of prostaglandin (PG) E₂ and of PGF_2α by radioimmunoassay is described in detail. PGE₂ was measured after conversion to either PGB₂ or PGF_2α and the results compared to bioassay. The methods were used to follow the excretion of PGE₂ and PGF_2α after salt loading in rabbits. A marked reduction of PGE₂ levels was observed at high NaCl intake, while PGF_2α excretion remained unchanged.     

Identification and quantitative determination of prostaglandins by high pressure liquid chromatography

High pressure liquid chromatography (HPLC) using 4′ × 1/8″ columns of a pellicular silica support (Corasil-II) allows identification of prostaglandins diastereomers based on their characteristic retention relative to a standard, PGE₂ in this study. Surprisingly this simple method allows separation of PGE₂, PGE₁, and PGE_o (dihydro-PGE₁) or PGF₂α, PGF₁α, and PGF_oα without resort to silver nitrate impregnated stationary phases. Even more subtle distinctions such as that between 13,14-dihydro-PGF₂α, PGF₁α and 5,6- -PGF₂α are possible by HPLC. The differential refractometer detector used throughout can also be used for quantitation. This is illustrated by a study of the relative rates of degradation of natural PGF₂α (an oil at the temperature employed, 41°C) and racemic PGF₂α (mp. 63°) on exposure to air.     

Stereospecificity of enzymatic reduction of prostaglandin E2 to F2α

Antibodies directed toward PGF_2β were prepared in rabbits. The serologic specificity of the immune reaction was determined by inhibition of sodium borohydride-reduced (³H) PGE₂ anti-PGF_2β binding by several prostaglandins. The antibodies to PGF_2β recognize the β-hydroxyl configuration in the cyclopentane ring of PGF_2β. With the use of both anti-PGF_2α and anti-PGF_2β, the product of PGE₂ reduction by 9-ketoreductase purified from chicken heart was identified as PGF_2α. Guinea pig liver and kidney homogenates were examined for PGE 9-ketoreductase activity. Although enzyme activity was present, no evidence of PGF_2β production was found.     

Effect of mifepristone and antiestrogens on uterine PGF2α and PGE2 concentrations in ovariectomized and pregnant rats

Four antiestrogens (anordiol, tamoxifen, RU 39411, ICI 182780) and the antiprogestin, mifepristone (RU 486), were administered to the following three animal models: (1) ovariectomized rats, (2) mated rats treated post-coitally; and (3) pregnant rats treated post-implantation. The antiestrogens were administered alone or in combination with mifepristone at doses effective in preventing and/or terminating pregnancy in rats. The objective of the study was to determine whether these drugs influenced uterine concentrations of prostaglandins (PGF_2α and PGE₂).Antiestrogens administered alone to ovariectomized rats did not effect uterine PGE₂ or PGF_2α concentrations; whereas the combination of anordiol/mifepristone increased uterine PGF_2α concentration, resulting in an increase in the PGF_2α/PGE₂ ratio.Mated rats were treated post-coitally for three consecutive days with anordiol, tamoxifen, estradiol and mifepristone alone and with the combination of anordiol/mifepristone and tamoxifen/mifepristone. An increase in uterine PGF_2α concentrations and in the PGF_2α/PGE₂ ratio occurred only in anordiol/mifepristone treated group. A decrease in uterine PGE₂ concentrations occurred in animals treated with anordiol, tamoxifen and estradiol, resulting in an increase in the PGF_2α/PGE₂ ratio.Anordiol (5.0 mg/kg/day) and mifepristone (4.0 mg/kg/day) alone and the combination of anordiol/mifepristone (2.5/1.0 mg/kg/day) administered to pregnant rats on days 7, 8 and 9 of pregnancy induced an increase in PGF_2α levels without affecting uterine PGE₂ concentration. The changes in uterine PGF_2α concentrations induced by anordiol and the combination of anordiol/mifepristone resulted in an increase in the PGF_2α/PGE₂ ratio.The antiestrogens tested except for ICI 182780 possessed agonist activity when assayed by measuring their capacity to increase the uterine weights in ovariectomized rats. Also, ICI 182789 was the only antiestrogen that did not influence uterine PG concentrations. It can be concluded that ICI 182780 is the only “pure” antiestrogen among those tested.The present results show that antiestrogens and the combination of mifepristone plus anordiol at doses preventing implantation and terminating pregnancy increase uterine PGF_2α and/or decrease PGE₂ concentrations, resulting in an alteration of PGF_2α/PGE₂ ratio. These findings suggest that there exists a critical balance of PGF_2α to PGE₂ concentrations in the uterus required for the normal passage of fertilized ova through the oviduct, initiating implantation of the blastocysts, development of embryos, and maintenance of pregnancy.     

Effect of prostaglandins on in vitro synthesis of progesterone and oestradiol-17β in placenta from early human pregnancy

In vitro synthesis of progesterone and estradiol-17β from endogenous precursors was studied in the placenta from women in early stage of gestation (< 7 weeks). Radioimmunoassay techniques were used to measure progesterone and estradiol-17β.It was shown that placental tissue from as early as six weeks of gestation can synthesize both progesterone and estradiol-17β in vitro. Prostaglandins F_2α and E₂ in concentration of 100 μg/ml of the incubation media did not have any significant effect on the in vitro synthesis of progesterone and estradiol-17β in the placental tissue.It seems unlikely that the abortifacient effect of natural prostaglandins PGE₂ and PGF_2α is due to their direct action on the synthesis of progesterone and estradiol-17β in the placenta.     

Production of cyclooxygenase products and superoxide anion by macrophages in response to chemotactic factors

Mononuclear phagocytes are knwon to play a key role in various phlogistic reactions by synthesizing and releasing products that may potentiate or inhibit inflammatory processes. The expression of these products appears to be dependent on the source of the macrophage population as well as the stimulus employed. We have studied superoxide anion (O₂⁻) production as well as the generation of PGE₂, PGF_2α, and TXB₂ from resident, oil-elicited and thiogylcollate-induced peritoneal macrophages in mice in the presence and absence of chemotactic peptides. Production of O₂⁻, occurred only in elicited macrophages stimulated with high concentrations of FMLP or C5a; resident cells stimulated with either of the chemotactic peptides were completely unresponsive. Although resident peritoneal macrophages incubated with chemotactic peptides did not generate O₂⁻, these cells did secrete significant levels of PGE₂, PGF_2α, and TXB₂ in response to C5a. FMLP had no stimulatory effect. Elicited macrophages generated increased levels of PGE₂ and PGF_2α when incubated with C5a. However, production of TXB₂ was not stimulated. FMLP was inactive in stimulating PGE₂, PGF_2α, and TXB₂ in all types of macrophages studied. These studies indicate a heterogeneity in the production of inflammatory mediators from various macrophage populations in response to chemotactic factors.     

Pharmacological modulation of prostaglandin production by phagocytic cells of the thymic reticulum in relation to immunoregulation

We cultured phagocytic cells derived from the thymic reticulum in order to study the regulation of prostaglandin (PG) production by antiinflammatory or immunostimulating agents. The kinetics of PGE₂, 6-keto-PGF_1α and PGF_2α production were measured by specific radioimmunoassays of the supernatants harvested from cells treated with dexamethasone, a steroidal antiinflammatory drug and by two non steroidal inhibitors (indomethacin and sulindac) or by various immunostimulating agents, one of them, RU 41740 is currently being used in humans. Our results revealed that ech of these drugs exerts a differential effect on the PG production, with a striking action on PGE₂ synthesis, a lesser effect on 6-keto-PGF_1α production and almost no effect on PGF_2α synthesis. The possible mechanisms responsible for this complex regulation of PG production are discussed.     

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).     

On the mechanism of action of 15-methyl-PGF2α as an abortifacient

Several hours following administration of long acting vaginal suppositories containing 3.0 mg of 15-methyl-PGF_2α for interruption of second trimester pregnancies there is an up to 10-fold increase in endogenous production of PGE₂ and PGF_2α before abortion as reflected by gas chromatographic-mass spectrometric determination of the major plasma metabolites of PGE₂ and PGF_2α. The data suggest that this increased formation of endogenous prostaglandins contributes to the induced uterine activity during the latter part of the abortion process.