Interactions of prostaglandins with subpopulations of ovine luteal cells. II. Inhibitory effects of PGF2α and protection by PGE2

Purified preparations of ovine large luteal cells were utilized in a series of experiments to test the effects of prostaglandins (PG) E₂ abd F₂α on cell morphology, viability and secretion of progesterone. Luteal cells were allowed to attach to culture dishes overnight before experiments. In the first series of experiments incubation of large steroidogenic cells with PGF₂α for 6 hr resulted in morphological changes including a retraction of the cell cytoplasm and apparent extrusion of cytoplasmic components which became more pronounced after 12 hr. In a second series of experiments, PGF₂α decreased and PGE₂ increased progesterone accumulation in media after 6 hr when media were not replaced during the incubation period, while progesterone accumulation was not different than that observed in control dishes when both prostaglandins were present. Hourly replacement of the media negated the inhibitory effects of PGF₂α but had no effect on the stimulated secretion of progesterone induced by PGE₂. Finally, in incubations without media replacement, PGF₂α induced a dose-dependent decrease in progesterone accumulation while PGE₂ elicited a biphasic response with progesterone secretion increasing from 0.1 ng/ml to maximal levels at 10 ng/ml followed by a dose-dependent decrease at 100 and 1000 ng/ml. These data are compatible with the hypotheses that: 1) luteolysis is initiated, at least in part, by an action of PGF₂α on large luteal cells; and 2) the embryonic signal from the pregnant uterus which rescues the ovine corpus luteum may be PGE₂.     

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 induced shape changes in fibroblasts grown in cell culture

PGE2 induced shape changes in porcine adventitial fibroblasts grown on glass in low density monolayer cell cultures. Incubation of the cells with PGE2 at concentrations of 100 ng/ml and 1000 ng/ml induced rounding of flat fibroblasts within one hours. The rounded cells had a small rim of cytoplasm around the nucleus and from one to several long thin arborizing cytoplasmic processes extending outward along the substratum. Removal of the PGE2 resulted in transient blebbing of the cell membrane of both the cell body and the processes as the cells returned to their flat normal morphology within one hour. The effect could be inhibited by 1% fetal calf serum. PGF2 alpha did not however induce similar changes. This difference between PGE2 and PGF2 alpha is similar to a report on spreading and migration of mouse peritoneal macrophages, and suggests that under certain conditions PGE2 may have the ability to induce shape changes in cells.     

Prostaglandin formation in bacteria

We report here that intact Gram positive and Gram negative bacteria, in the presence of exogenous arachidonic acid, produce and release PGE₂ and PGF_2α into the medium as measured by radioimmunoassay. The seven bacterial strains so far studied release 6.5–50.9 ng PGE₂ and less than 0.02–0.51 ng PGF_2α per mg bacterial protein during a 1 hour incubation, quantities of the same order of magnitude as those observed in mammalian systems. PGE₂ and PGF_2α formation in bacteria are inhibited by indomethacin.     

Primary colonic epithelial cell culture of the rabbit producing prostaglandins

We have established primary colonic epithelial cell culture from adult rabbits and examined effects of anti-inflammatory drugs on prostaglandin (PG) E₂ production. Colonic epithelium of adult rabbits was scraped and minced into small pieces. They were incubated for isolation in Hanks' balanced salt solution with 0.35 % collagenase and Earle's solution with 1 mM EDTA. Isolated cells were cultured in Coon's modified Ham's F-12 medium with 10 % fetal bovine serum and antibiotics on collagen coated cell wells. The medium was refed twice a week. The production of PGs was assessed by high pressure liquid chromatography (HPLC). PGE₂ and PGF_2α were measured by radioimmunoassay. Within 24 hours after inoculation, the cell clumps attached to the surface of the wells and cells began to spread out and grow. Monolayer cultures became confluent in 4 days. Phase contrast microscopy showed that these cells consisted of a homogeneous population of epithelial cells with large oval nuclei, polyhedral shape, and organized sheet-like growth pattern. HPLC profile showed synthesis of 6-keto-PGF_1α, thromboxane B₂, PGF_2α, PGE₂, and PGD₂ by cultured cells. Quantitatively, 117±7 ng/mg-protein/hour PGE₂ by 7.4±0.7 ng/mg-protein/hour PGF_2α were produced. While hydrocortisone (10⁻⁴-10⁻² M) did not show a significant effect on PGE₂ production, indomethacin (10⁻⁸-10⁻⁶ M), and 5-aminosalicylic acid (2×10⁻⁴-5×10^{−3 M}) inhibited PGE₂ production. We have established relatively convenient procedure for primary culture of colonic epithelial cells from adult rabbits. Different actions of anti-inflammatory drugs on PGE₂ synthesis suggest that these cultured cells might be a good tool for the various cellular functional studies of normal colonic epithelial cells.     

Sperm output and serum testosterone in rabbits given prostaglandin F2α or E2

Two experiments were conducted, the first to compare sperm output and the second to determine serum testosterone in rabbits given PGF₂α or PGE₂. In the first, six rabbits were ejaculated twice each Monday, Wednesday and Friday for 5 weeks. Each rabbit was given subcutaneously (sc) each of the following treatments five times: 1) saline, 2) 5 mg PGF₂α and 3) 5 mg PGE₂. Treatments were given, half at 4 hr and half at 2 hr before first ejaculations. Both PGF₂α and PGE₂ caused increased (50% and 84%) sperm content of first ejacula, without significantly altering characteristics of second ejacula. The extra sperm in first ejacula was a function of increased sperm density, because seminal volume was unaltered.In the second experiment, 15 rabbits were bled at 0.5-hr intervals for 9 hr and given (sc): 1) saline at 1 and 3 hr (n=4), 2) 2.5 mg PGF₂α at 1 and 3 hr (n=4), 3) 2.5 mg PGE₂ at 1 and 3 hr (n=4) or 4) 5 mg PGF₂α at 1 hr after the onset of blood sampling. In saline-treated controls, episodic surges of testosterone occurred on the average every 5 hours. After the injection of 2.5 or 5.0 mg PGF₂α, serum testosterone began to rise at 0.5 hr, peaked (8 to 13 ng/ml) at 1 hr and approached a nadir (0.5 ng/ml) within 4 hours. The second injection of 2.5 mg PGF₂α failed to significantly affect serum testosterone. PGE₂ treatment was followed by significantly depressed serum testosterone; only 1 of these 4 rabbits had any surge of testosterone for the 8 hr after treatment. In conclusion, PGF₂α and PGE₂ both increased sperm output, but PGF₂α increased serum testosterone while PGE₂ depressed serum testosterone. Thus, the sperm output effect of these prostaglandins probably is independent of the acute changes in testosterone secretion.     

Dose-related action of estradiol on placental prostanoid prediction

Prostanoids play an important role throughout all of pregnancy and during the initiation and progress of labor. The human placenta at term produces large quantities of prostanoids, yet little is known of the factors that regulate their biosynthesis. Herein, we report the effect of estradiol or estradiol and progesterone on the basal release of placental prostanoids from fresh human term placental explants using a perifusion system.The basal release of prostaglandin E₂ (PGE₂, prostaglandin F_2α (PGF_2α), thromboxane (TxB₂) and 6-keto-prostaglandin F_1α (6-keto-PGF_1α) increased about 50% from the fifth to the ninth hour in culture, while the release of 13, 14-dihydro-15-keto-PGF (PGFM) remained constant and hCG release decreased. The dose-related effect of estradiol (20–2,000 ng/ml) in the perifusing medium starting at the fifth hour of perifusiOn (i.e., the zero treatment time) effected no change in the release of TxB₂, PGF_2α, PGFM or hCG. A biphasic action on the release of 6-keto-PGF,. was observed, i.e. it was significantly decreased when incubated with 20 ng/ml of estradiol, but effected an increase after exposure to 200 ng/ml. The concomitant addition of progesterone (2,000 ng/ml) with estradiol (200 ng/ml) significantly inhibited the stimulatory action of estradiol at this dose. The release of PGE₂ was inhibited in a dose-related fashion with increasing dose of estradiol. The addition of progesterone with estradiol (2,000 and 200 ng/ml, respectively) reversed the inhibition of PGE₂ by estradiol alone.These data demonstrate that physiologic levels of estradiol affect 6-keto-PGF_α and PGE₂ release from the human term placenta, but do not significantly alter production of TxB₂, PGFM or hCG under these conditions.     

Prostaglandin E2 enhances uterine stromal cell alkaline phosphatase activity

Prostaglandins have been implicated in the process of uterine decidualization , but sites of action are uncertain. Since one of the earliest changes in endometrial stroma following induction of decidualization is an increase in alkaline phosphataseactivity, we have investigated the effects of PGs on stromal cell alkaline phosphatase activity . Immature rats were pretreated with hormones to sensitize their uteri for the decidual cell reaction. Endometrial stromal cells were isolated and cultured for up to 4 days with PGE₂ (0–10 μg/ml) or PGF₂ (0–10 μg/ml) Analysis of variance revealed a highly significant interaction between day of culture and concentration of PGE₂ in medium (P<0.01). Stromal cell alkaline phosphatase activity decreased significantly with increasing culture duration (P<0.01). In the presence of PGE₂, alkaline phosphatase activity was significantly higher (P<0.01) regardless of day of culture. In contrast, PGF_2α had only a small and inconsistent effect. These data indicate that PGs, and in particular PGE₂, can act directly upon stromal cells.     

The excretion of prostaglandin F2α in milk of cows

Prostaglandin F_2α (PGF_2α) was measured by immunoassay in plasma and milk of four cows (six experiments). After 30 mg PGF_2α im, plasma PGF_2α peaked at 15 minutes (2.4 ± 0.7 ng/ml) and declined toward basal values by 3 hours; maximum milk PGF_2α (0.91 ± 0.12 ng/ml) occurred at 1 hour. The average excretion rate in milk was 2.9 μg/day 0.9 μg (0.003%) of which was due to the 30 mg PGF_2α injected. In six non-pregnant control cows, daily changes of milk PGF_2α and progesterone were not consistently related.     

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.     

The effect of adrenergic stimulation on urinary prostaglandin E2 and 6 keto PGF1α in man

To evaluate the details of the adrenergic stimulation of urinary prostaglandins in man, ten normal volunteers were given various agonists and antagonists. The effect of 4 hour IV infusions of norepinephrine (NE), NE + phentolamine (PHT), NE + phenoxybenzamine (PHB), NE + prazosin (PZ), isoproterenol (ISO), and PHT alone on urinary PGE₂ and PGI₂ (6 keto PGF_1α) were determined. PGE₂ and 6 keto PGF_1α were measured by radioimmunoassay from 4 hour urine samples. NE stimulated both PGE₂ (196±40 to 370±84 ng/4 hrs/g creatinine and 6 keto PGF_1α(184±30 to 326±36), both p<0.01. In contrast, ISO had no effect on either PGE₂ or 6 keto PGF_1α excretion. Alpha blockade with PHT. PHB, or PZ inhibited the NE induced systemic pressor effect. However, the effect of the alpha blockers on the NE induced stimulation of PGE₂ and 6 keto PGF_1α varied. PHT did not alter the NE stimulated PGE₂ or 6 keto PGF_1α release (370±84 vs. 381±80) PGE₂ and (326±50 vs. 315±40) 6 keto PGF_1α, both p>0.2). PHT alone stimulated only 6 keto PGF_1α. PHB and the specific α₁ antagonist PZ similarly eliminated the NE induced prostaglandin release. These results suggest that adrenergically mediated urinary prostaglandin release in man is via an alpha receptor with α₁ characteristics.     

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.     

Effects of prostaglandins on the spreading, adhesion and migration of mouse peritoneal macrophages

The effects of prostaglandins on the properties of mouse peritoneal macrophages namely spreading, adhesion and migration were investigated. PGE₁ and PGE₂ inhibit the spreading and adhesion of complete Freund's Adjuvant induced peritoneal macrophages significantly at concentrations of 1 ng per ml and above whereas they enhance the migration of these cells at concentrations of 100 ng per ml and above. PGA₂ and PGB₂ are less potent as they inhibit spreading and adhesion only at a concentration of 1 μg per ml. At this concentration PGB₂ enhances migration whereas PGA₂ has no effect. PGF_2α has no effect on the spreading, adhesion and migration of macrophages in the concentration range of 0.1 ng to 1,000 ng per ml.     

Determination of prostaglandin F2α, E2, D2 and 6-keto-F1α in human cerebrospinal fluid

Prostaglandin (PG)F_2α, E₂, D₂ and 6-keto-F_1α were determined in human cerebrospinal fluid by a mass spectrometric technique. The samples were obtained from 12 patients with suspected intracranial disease. A 64 fold variation in PG levels was observed. The major PG was 6-keto-F_1α (0.12–15 ng/ml). PGF_2α and PGE₂ were present in lower concentrations PGD₂ was below the level of detection (0.05 ng/ml) except in one patient with extremely high total levels of PGs.     

Effect of PGF2α on progesterone secretion and adenylate cyclase activity in ovine luteal tissue

Ovine luteal slices were used to study the effects of prostaglandins (PG) F₂α on luteinizing hormone (LH)-stimulated secretion of progesterone and adenylate cyclase activity. The accumulation of progesterone in incubation medium and adenylate cyclase activity was similar after incubation of luteal slices with Medium 199 alone or Medium 199 containing PGF₂α (250 ng/ml) for 3 hr. Addition of luteinizing hormone (LH; 100 ng/ml) resulted in a 2–3 fold increase in both the rate of progesterone accumulation and adenylate eyclase activity by 3 hr. When luteal slices were incubated in the presence of both LH and PGF₂α the rates of progesterone accumulation and adenylate cyclase activity were identical to those in flasks containing LH alone after 1 hr; however, after 3 hr both LH stimulated progesterone accumulation and adenylate cyclase activity were inhibited to levels similar to those observed in control slices.In a second experiment, after 60–120 min of exposure to PGF₂α the rate of progesterone accumulation in the medium was not different from that in untreated control slices. In addition, after this experiment the luteal slices were homogenized and the basal, sodium fluoride, LH, isoproterenol (ISO) and PGE₂ sensitive adenylate cyclase activities were determined to evaluate the hormonal specificity of the negative effect of the pretreatment with PGF₂α. Both LH and ISO stimulated adenylate cyclase activities were reduced after PGF₂α pretreatment. However, fluoride ion stimulated adenylate cyclase activity was not significantly effected by PGF₂α pretreatment and PGE₂ sensitive adenylate cyclase was effected only slightly.     

Effects of prostaglandins on leukocyte migration

Artificially synthetized prostaglandins (PGE₁, PGE₂ PGF_1α, and PGF_2α) were found, using Boyden's chamber, to induce significant migration of polymorphonuclear leukocytes (PMNs) of the rabbit; PGF_2α had greater effects than PGE₁ or E₂. A typical dose dependent relationship was found between the PMNs migration and PGF_2α concentrations. Indomethacin pretreatments of rabbits did not significantly alter the PMNs migration indicating that PGs synthetized in vivo was not involved in the migration.PGF_2α was placed in the lower compartment opposite to PMNs and also in the upper compartment together with PMNs. No significant difference was found in the number of migrated PMNs between the two experimental conditions. PGs diffusion occurred across the millipore filter separating the two compartments where the concentrations were almost equal at the end of 3 hours incubation. It was thus concluded that PGs effects are to induce random PMNs movements rather than to initiate chemotactic directional migration.     

In vitro stimulation of prostaglandin synthesis in the rat pancreas by carbamylcholine, caerulein and secretin

Rat pancreas pieces spontaneously released PGE₂ (2.3 ng/100 mg × 45 min) and PGF_2α (7.6 ng/100 mg × 45 min). This release corresponds probably to a neo-synthesis since it was abolished by indomethacin. Carbamylcholine (≥ 10 μM), caerulein (≥ 10 nM) and secretin (≥ 10 nM) stimulated the release of PGE₂ and PGF_2α : the concentrations of stimulators required to increase PGs release were thus much higher than those which trigger enzyme secretion. Atropine specifically inhibited the cholinergic stimulation, whereas indomethacin blocked the stimulatory effects of all secretagogues. Stimulation of PGE₂ and PGF_2α release was reduced in a Ca⁺⁺-free medium, abolished by EGTA and mimicked by the ionophore A23187, underscoring the crucial role of Ca⁺⁺ in the regulation of PGs synthesis by the pancreas. Neither PGE₂ nor PGF_2α stimulated enzyme secretion in this system and indomethacin did not inhibit the secretory effect of carbamylcholine. Increased synthesis of prostaglandins in response to pancreatic secretagogues does not appear to be involved in the process of enzyme secretion.     

Chemotactic activity of thromboxane B2, prostaglandins and their metabolites for polymorphonuclear leucocytes

(1) The chemotactic activities of thromboxane B₂ (TxB₂, PGE₂, PGF_2α, the 15-oxo, 15-oxo-13,14-dihydro and 13,14-dihydro metabolites of PGE₂, PGF_2α, and a metabolite of TxB₂ for polymorphonuclear leucocytes (PMN) have been investigated.(2) Thromboxane B₂ increased the directional migrationm of rat peritoneal PMN at a concentration of 2.0 μg/ml and of human peripheral neutrophils at a concentration of 0.5 μg/ml.(3) Neither PGE₂, PGF_2α nor their metabolites showed chemotactic activity for rat peritoneal PMN.(4) PGF_2α and 15-oxo-13,14-dihydro-thromboxane B₂ showed no chemotactic activity for human peripheral PMN.(5) The possible role of thromboxane B₂ in inflammation is discussed.     

Reversal of indomethacin blockade of ovulation in gilts by prostaglandins

Prepubertal gilts given 750 IU pregnant mares′ serum gonadotropin (PMSG) followed 72 h later by 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation fail to ovulate when 10 mg/kg indomethacin (INDO) is injected 24 h after hCG administration. This study examines the effects of administration of exogenous prostaglandins F_2α and E₂ (PGF_2α and PGE₂) alone or in combination, and at various times prior to the expected time of ovulation, on the INDO blockade of ovulation in PMSG/hCG-treated gilts. Occurrence of ovulation was determined by visual observation at laparotomy 48 h after hCG. When 5 mg or 10 mg PGF_2α was injected at each of 38, 40 and 42 h after hCG injection, 63% and 79%, respectively, of preovulatory follicles ovulated. In contrast, injection of 5 mg PGE₂ or 5 mg PGE₂ plus 5 mg PGF_2α induced ovulation in 0% and 24% of preovulatory follicles, respectively. In control groups, 100% of folicles in PMSG/hCG-treated gilts ovulated whereas none did so in PMSG/hCG/INDO-treated animals. These results indicate that administration of PGF_2α can induce ovulation in the PMSG/hCG/INDO-treated prepubertal gilt and suggest that PGE₂ is ineffective and may be antagonistic to PGF_2α in overcoming the ovulation blocking effect of INDO.     

The effects of prostaglandins PGE1, PGE2, PGF1a, and PGF2a on canine synovial perfusion

In these experiments we have examined the effects of PGE₁, PGE₂, PGF_1α and PGF_2α on synovial perfusion in the normal canine synovial microcirculation. The effects of the drugs on synovial perfusion were determined indirectly from the changes produced in the rate of clearance of ¹³³Xenon from the joint by their intra-articular injection. Prostaglandins PGE₁ and PGE₂ were found to be strongly vasodilator with PGE₁ being the more active. PGF_1α appeared to have little or no vasoactive properties in doses up to 1 ugm. (2.8 × 10^−5M) in our I preparation while PGF_2α was vasodilator at this high dosage only. Neither SC19920 nor diphloretin phosphate antagonised the effects of PGE₁ in these experiments.