The measurement of human endometrial prostaglandin production. A comparison of two in vitro methods

Two in vitro methods for measuring human endometrial prostaglandin production were compared. Endometrial samples from eight patients were incubated over eight hours by a perifusion and a superfusion technique. The collected fractions were assayed by radioimmunoassay for PGE2 and PGF2 alpha. There was no significant difference between the perifusion and superfusion methods for the pattern and amount of PGE2 and PGF2 alpha production with time. Significantly higher production levels of PGE2 and PGF2 alpha were found in secretory phase endometria than in proliferative phase endometria. Histological examination of the tissue specimens by light and electron microscopy showed that both methods caused gross tissue damage after eight hours experimentation. The superfusion method produced more morphological damage than the perifusion method. However, no tissue damage could be detected after one hour of incubation with either method. Over an eight hour period neither the perifusion nor the superfusion technique appears to be a good indicator of in vivo endometrial prostaglandin production. Either technique used for only one to two hours may better reflect the in vivo situation.     

Prostaglandins and in vitro ovarian progestin biosynthesis

Prostaglandin F_2α (PGF_2α) did not alter the $\text{in vitro}$ biosynthesis of progesterone by slices of luteinized rat ovaries when used in concentrations from 1 to 10,000 ng/ml of incubation medium; likewise, PGF_2α did not affect the incorporation of acetate-1-¹⁴C into progestins. PGF_1α, 15-keto PGF_2α, and PGE₁ did not alter the biosynthesis of progesterone by luteinized rat ovaries; PGE₂ inhibited the production of progesterone when used at a concentration of 10 μg/ml, but not at lower doses. PGF_2α in combination with luteinizing hormone (LH) enhanced the metabolism of progesterone to 20α-hydroxypregn-4-en-3-one in luteinized rat ovaries. Interestingly, PGF_2α, at a high concentration of 10 μg/ml, did stimulate progesterone biosynthesis by slices of ovarian tissue from immature rats hormonally primed to simulate “pseudopregnancy,” suggesting a steroidogenic action of prostaglandins on the ovarian follicular or interstitial cell. PGF_2α (10 μg/ml) did not stimulate the $\text{in vitro}$ biosynthesis of progesterone or 20α-hydroxypregn-4-en-3-one by slices of rabbit corpora lutea or rabbit ovarian interstitial tissue. It is concluded that prostaglandins do not stimulate progestin biosynthesis in rat luteal tissue.     

Prostaglandin-induced enhancement of the in vitro anamnestic response

The ability of various prostaglandins (PGs) to affect the $\text{in vitro}$ 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 $\text{in vitro}$ 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.     

Comparative studies of prostaglandins F2α and E2 in late cyclic and early pregnant sheep: In vitro synthesis by endometrium and conceptus effects of in vivo indomethacin treatment on establishment of pregnancy

Endometrial concentrations of prostaglandins F₂α (PGF₂α) and E₂ (PGE₂) were measured by specific radioimmunoassay in sheep, on day 14 of estrous cycle or pregnancy, during luteolysis (Day 16 of the cycle), and after implantation (Day 23 of pregnancy) : concentrations observed on day 14 of cycle and pregnancy were similar. During luteolysis, on day 16 of cycle, a consistent drop was noticed. If luteal regression did not occur, as a consequence of the presence of an embryo, endometrial concentrations of PGF₂α on day 23, were twice those of day 14, and PGE₂ remained unchanged. $\text{In vitro}$ 2 hour incubations of endometrial caruncular tissue from 14 days cyclic or pregnant ewes resulted in de novo synthesis of PG which could be increased by Arachidonic Acid and inhibited by Indomethacin; during the first 30 min of incubation, the PGF₂α synthesis was comparable for both endometrial tissues, whereas PGE₂ synthesis was twice as great in pregnant endometrium. Fourteen and 23 day conceptuses had high PGF₂α and PGE₂ concentrations which were not due to maternal PG sequestration : $\text{de novo}$ PG synthesis which could be inhibited by Indomethacin was observed in incubated 14 day old embryos. Treatment of pregnant ewes from day 7 to day 22 after mating, either with Indomethacin (300 mg s.c. daily) or with Acetylsalicylic Acid (1 g I.V. daily) resulted in a sharp diminution of endometrial PG concentration and release, with no apparent effect on the establishment of pregnancy. These results tend to ascribe a less important role to PG during early pregnancy in sheep as compared with rodents, in terms of embryonic growth and implantation.     

Stimulatory effect of prostaglandin E1 on thyroliberin-induced α-melanotropin release from perifused neuro-intermediate lobes of frog pituitary gland

A possible direct effect of prostaglandins on α-melanotropin (α-MSH) release at the level of the intermediate lobe of the frog pituitary was investigated $\text{in vitro}$ using a perifusion system technique. The effect of prostaglandins was studied on both spontaneous and TRH-stimulated α-MSH secretion. No significant effect of PGE₁, PGE₂, PGF_1α or PGF_2α on basal release of α-MSH could be detected. Indomethacin did not alter the α-MSH release induced by TRH. Conversely a significant increase in TRH-induced α-MSH secretion was observed in the presence of 1 x 10^?6M PGE₁. This magnifying effect was directly related to the concentration of TRH for doses ranging from 1 x 10^?8M to 1 x 10^?6M.     

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 $\text{in vivo}$.     

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

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

Luteal progesterone and prostaglandin F2α production invitro during fluprostenol-induced luteolysis in the mare

Prostaglandin F₂α (PGF₂α) release $\text{in}$$\text{vitro}$ by luteal tissue from mares was quantified to determine if exogenous prostaglandin analog increased endogenous luteal PGF₂α production during induced luteolysis. On day 8 after ovulation, luteal tissue was collected by flank laparotomy and endometrium was collected by uterine biopsy. Mares were assigned to one of four treatments: (1) no intramuscular injection at 0-hr (n = 5), (2) 250 μg Fluprostenol (ICI 81008 PGF₂α analog) at 4-hr (n = 4), (3) 250 μg Fluprostenol at 12-hr (n = 5), or (4) 250 μg Fluprostenol at 28-hr (n = 5) prior to tissue collection at laparotomy. Blood was collected from a jugular vein at laparotomy. Luteal and endometrial tissues (100-mg minces) were incubated in duplicate in 5 ml of Krebs-Ringer bicarbonate buffer (pH 7.4) in an ice bath in an air atmosphere or at 37°C in an atmosphere of 95% O₂:5% CO₂. The incubation treatments consisted of: no treatment, indomethacin 1.3 × 10^?4M, 1 μg/ml of arachidonic acid, 10 μg/ml of Fluprostenol, and 100 μM dbc-AMP (Fluprostenol was not added to endometrial tissue incubations). The injection of Fluprostenol induced luteolysis in these mares as indicated by decreased plasma progesterone and luteal tissue progesterone production (P<0.01). Luteal PGF₂α production was only detectable in tissue from mares that had been injected with Fluprostenol; production reached a maximum by 12 hr post-injection and had returned to pre-treatment levels by 28 hr (P<0.01). Endometrial tissue produced PGF₂α, but this activity was not significantly affected by injection of mares with Fluprostenol. Increased production of PGF₂α by luteal tissue of mares during PGF₂α analog induced luteolysis was similar to that observed in the pig and ewe.     

The antagonism by anti-inflammatory analgesics of prostaglandin F2α-induced contractions of human and rabbit myometrium in vitro

The anti-inflammatory analgesic drugs, aspirin, indomethacin, phenylbutazone, mefenamic acid, ibuprofen and flurbiprofen are shown to inhibit in a dose-dependent manner the force of contraction of isolated human pregnant myometrial strips which have been stimulated to contract by adding prostaglandin (PG) F_2α to the tissue bath. These drugs and also flufenamic acid and salicin show a similar antagonism of the action of PGF_2α with isolated rabbit non-pregnant myometrium. The ratio of the inhibitory concentration $\text{in vitro}$ to the maximum plasma level after a normal dose $\text{in vivo}$ suggests that phenylbutazone and possibly ibuprofen may be capable of inhibiting human uterine contractions $\text{in vivo}$. Patients who were treated with aspirin during induction of abortion using PGF_2α during the second trimester of pregnancy showed no significant change in the induction-abortion interval compared with patients not taking aspirin.     

Temporal effect of prostaglandins E and F on human adrenal cAMP and cortisol output.

The $\text{in vitro}$ modulating effects of the E and F series prostaglandins upon the cAMP and cortisol output of normal human adrenal dice were evaluated with time and compared to the effects of ACTH. PGE₁ and PGE₂ significantly increased human adrenal cAMP levels; cortisol output increased in a dose related manner. Although the cortisol levels produced by E prostaglandins and ACTH were quantitatively similar, on a molar basis ACTH was greater than 10⁶ fold more effective. PGE_1α, PGF_2α, PGF_1β and PGF_2β depressed adrenal cAMP, except PGF_2α and PGF_2β at 100 μg/ml. PGF_1α and PGF_2α depressed cortisol levels at all doses. Similarly, PGF_1β and PGF_2β also depressed cortisol output, except PGF_2β at 100 μg/ml which was stimulatory. In both series of prostaglandins the temporal responses were dose related in regard to the cyclic nucleotide and steroid alterations. The findings demonstrate the E and F series prostaglandins act antagonistically in respect to cAMP and cortisol output. In addition, as the cAMP level and cortisol output are not always correlated, it appears that these prostaglandin mediated events are separable. The relationship between adrenal prostaglandins and cyclic nucleotides, therefore, invites a more sophisticated second messenger concept in terms of adrenocortical function, than currently proposed.     

Cyclic AMP formation of isolated human corpora lutea in response to HCG — Interference by PGF2α

Human corpora lutea of defined ages were excise at operation cut into pieces and incubated in the presence of HCG, PGF_2α and PGE₂ alone or in combination. Following incubation cAMP formation in tissue and medium was determined. HCG-stimulated tissue cAMP content was most pronounced at a corpus luteum age of 7–10 days after ovulation. This stimulation was antagonized by PGF_2α in corpora lutea older than 6 days. PGE₂ stimulated cAMP formation $\text{per se}$ and this effect was more pronounced when HCG and PGE₂ were combined. A possible role for PGF_2α as a luteolytic substance in the human is suggested.     

Specific changes in the in vitro production of prostanoids by the ovine cervix at parturition

A method of tissue superfusion has been used to measure $\text{in vitro}$ prostanoid production by the ovine cervix during late pregnancy and at parturition. In late pregnancy (105–135 days of gestation) cervical tissue produced relatively large amounts of prostaglandin E (PGE); in comparison, the production rates of prostaglandin F (PGF), 13, 14-dihydro-15-oxo-prostaglandin F (PGFM) and 6-oxo-prostaglandin F_1α were generally low. Thromboxane B₂ (TXB₂) production was minimal and often unmeasurable. There were significant increases in the production rates of PGE and 6-oxo-PGF_1α by cervical tissue taken immediately after delivery, when compared to late pregnancy. Mean production rates of PGE increased from 19.8 ± 4.1 to 43.8 ± 7.4 ng/g. dry wt./min; 6-oxo-PGF_1α production rates increased more than three-fold from 10.0 ± 2.7 to 34.6 ± 9.8 ng/g. dry wt./min (means ± S.E.M.). There were no significant differences in the rates of production of PGF, PGFM and TXB₂ by the two groups.     

Effects of 19-hydroxy-prostaglandins on oviductal and uterine motility

The effects of 19-hydroxy-prostaglandins (19-OH-PGs) were tested $\text{in}$$\text{vivo}$ on the rabbit oviduct and uterus and on the rhesus monkey ($\text{Macaca}$$\text{mulatta}$) uterus. The 19-OH-PGEs suppressed spontaneous oviductal and uterine activity in the rabbit. The qualitative effect on the rabbit oviduct of 19-OH-PGEs was similar to that of PGE₂. However, the typical response of the rabbit uterus to PGE₂ was an increase in muscle activity. With regard to the rabbit oviduct, 19(R)-OH-PGE₂ was as potent as PGE₂, but 19(S)-OH-PGE₂ was approximately $\text{1}\text{2}$ as potent as PGE₂. Based on the dose of 19-OH-PGEs usually required to cause a minimal suppression and the dose of PGE₂ required to cause a minimal stimulation of rabbit uterine activity, 19(R)-OH-PGE₂ was twice as potent as PGE₂ while 19(S)-OH-PGE₂ was $\text{1}\text{2}$ as potent as PGE₂. Stimulatory effects on the rabbit oviduct and uterus were observed following administration of 19-OH-PGEs and PGF_2α. The potency on the rabbit oviduct of 19(S)-OH-PGF_2α was about $\text{1}\text{5}$ to $\text{1}\text{10}$ that of PGF_2α; the potency of 19(R)-OH-PGF_2α was about $\text{1}\text{10}$ to $\text{1}\text{20}$ that of PGF_2α. Both 19-OH-PGFs were approximately $\text{1}\text{5}$ to $\text{1}\text{10}$ as potent as PGF_2α on the rabbit uterus. At the doses tested 19-OH-PGFs were inactive on the monkey uterus. Thus, these compounds are at least $\text{1}\text{5}$ as active as PGF_2α. In contrast, 19(R)-OH-PGE₂ had approximately the same potency as PGE₂ in stimulating monkey uterine activity; but 19(S)-OH-PGE₂ was approximately $\text{1}\text{3}$ as potent as PGE₂.     

Comparison of the effects of prostaglandins D2, F2α and E1 on spontaneous contractions of rabbit oviduct

The effects of PGD₂, PGF_2α and PGE₁ were studied on the circular muscle of post-ovulatory rabbit oviducts $\text{in vitro}$. PGE₁ inhibited spontaneous contractile activity. Lower concentrations of PGD₂ and PGF_2α were stimulatory and higher concentrations were inhibitory. Since PGD₂ may be produced in the oviduct, any hypothesis concerning the role of prostaglandins in the control of oviductal motility and ovum transport should include PGD₂ as well as PGFs and PGEs.     

In vitro response of relaxin-treated rat uterus to prostaglandins and oxytocin

Paired segments of rat uterus were treated $\text{in vitro}$ with relaxin (W1164-3, 150 GPU/mg) until the amplitude of contraction was reduced to at least 50% of the pre-treatment amplitude. Test segments then received 100 ng of either PGE₁, PGE₂, PGF_2α or 250 uU of oxytocin. Control segments remained untreated. There was a significant increase in contraction amplitude in response to the spasmogens (P < 0.05) but no increase was seen in controls.     

Prostaglandin endoperoxides IV. Effects on smooth muscle

The effect on smooth muscle of the endoperoxides PGG₂ and PGH₂, which are intermediates in prostaglandin biosynthesis, was studied in different systems $\text{in vitro}$ and $\text{in vivo}$. On gastrointestinal smooth muscle (gerbil colon, rat stomach) PGG₂ and PGH₂ produced contractions comparable to those of PGE₂ and PGF_2a whereas contractions elicited on vascular (rabbit aorta) and airway (guinea-pig trachea) smooth muscle were considerably greater than those of PGE₂ and PGF_2a respectively. On intravenous injection into guinea-pigs PGG₂ and PGH₂ caused a triphasic change in blood pressure and were 8–10 times more effective than PGF_2a in producing an increase in tracheal insufflation pressure. When given as aerosols the unstable endoperoxides were less effective than PGF_2a. It is concluded that the endoperoxides are potent smooth muscle stimulants and that they are more effective than their degradation products (PGD₂, PGE₂, PGF_2a) in some systems.     

Thromboxane A2 is the major arachidonic acid metabolite of human cortical hydronephrotic tissue

Human cortical hydronephrotic microsomes converted [¹⁴C] arachidonic acid to [¹⁴C] thromboxane B₂ as the major metabolic product. Using [¹⁴C] PGH₂ as substrate, similar enzymatic conversions were noted with HHT>TXB₂6KPGF_1αPGE₂PGF_2α as the major products. Inhibition of thromboxane synthetase with imidazole 5 mM reduced thromboxane B₂ production by 60% and the major product then was 6 keto PGF_1α. After addition of imidazole, the metabolic profile showed 6KPGF_1αPGE₂HHT>PGF_2α. Control experiments were carried out using normal cortical tissue obtained from kidneys removed surgically for carcinoma of kidney and rejected for transplantation secondary to fracture as a consequence of blunt trauma. These control kidneys, while they demonstrated an ability to generate thromboxane B₂$\text{in vitro}$, had much less activity than hydronephrotic kidneys and with PGH₂ as substrate PGE₂TxB₂. In addition, inhibition with imidazole produced mainly PGE₂. Thus, like the rabbit and rat, there is enhanced thromboxane and prostacyclin synthesis in human ureteral obstruction and are, therefore, potential vasoactive compounds which may in part be responsible for the hemodynamic alterations occurring in human obstructive uropathy.     

Role of prostaglandin F2α in modulation of LH-stimulated steroidogenesis in vitro in different types of rat and ewe corpora lutea

An inhibitory effect of PGF_2α at a dose of 7 × 10^?7 M on LH stimulated synthesis of progesterone was observed $\text{in vitro}$ after incubation of pseudopregnant rat ovaries for a period of 2 hours. A similar effect was seen with cyclic and gestant ewe corpora lutea at the same dose of PGF_2α. This effect was observed both in the secretion of progesterone and on the amount of progesterone present in the tissue. This inhibitory effect of PGF_2α on LH stimulated progesterone synthesis may explain the modification in the time course for gonadotropin action in luteal tissue at high and low doses.     

Potentiation of bradykinin-induced vascular permeability increase by prostaglandin E2 and arachidonic acid in rabbit skin

The activity of prostaglandins (PG) in producing vascular permeability was quantitated by dye extraction method in skin of anaesthetized rabbits. PGE₁ and PGE₂ (0.01–10 μg) produced increase in vascular permeability. Activity was approximately equal to that of histamine (Hist) and $\text{1}\text{20}$ of that of bradykinin (BK) on a weight basis. The activity of PGF_1α and PGF_2α was only $\text{1}\text{20}$ of that of PGE₁ or PGE₂.In spite of the relatively low potency of PGE₁ and PGE₂ in the rabbit, near threshold doses (0.1 or 1 μg) of PGE₂ could potentiate permeability responses to bradykinin (0.1 μg) by 10 or 100-fold, respectively. Equivalent doses (0.1 or 1 μg) of histamine could not potentiate the bradykinin responses. Arachidonic acid (AA) at 1 μg, produced a 10-fold potentiation in the permeability response to bradykinin (0.1 μg). Pretreatment of the rabbits with indomethacin (20 mg/kg, i.p.) reduced the responses of BK (0.1 μg) + AA (1 μg) down to a similar magnitude of those seen with bradykinin alone. However, indomethacin did not block responses to either, BK alone, BK + PGE₂, or BK + Hist. Various doses (1, 10, 100 and 300 μg) of arachidonic acid alone also produced increase in cutaneous vascular permeability, although its potency was only $\text{1}\text{3}$–$\text{1}\text{8}$ of that of PGE₂. This activity of arachidonic acid was attributed in part to its bioconversion to PGE₂, since its activity was significantly reduced by the prostaglandin antagonist, diphloretin phosphate (DPP) (60 mg/kg, i.v.) and by indomethacin (20 mg/kg, i.p.), which blocks conversion of arachidonic acid to prostaglandins. Arachidonic acid may owe some of its permeability increaseing effects to histamine release, since its effects were also reduced by the antihistamine, pyrilamine (2.5 mg/kg, i.v.).     

Radioimmunoassay for urinary metabolites of prostaglandin F2α

Antibodies against the main urinary metabolite of PGF_2α in the human, 5α,7α-dihydroxy-11-ketotetranorprosta-1,16-dioic acid, were raised in rabbits. The compound was coupled selectively in the ω position to bovine serum albumin prior to injection. The resulting antibodies did not distinguish between tetranor compounds varying only in structure at the ω carbon, and thus the assay could be used also for other metabolites of PGF_2α, e.g. the main urinary metabolite in the guinea pig, 5α,7α-dihydroxy-11-ketotetranorprostanoic acid. Labeled ligands for the assays were prepared either $\text{in vivo}$ by injection of |17,18-³H|-PGF_2α into humans after several days' treatment with indomethacin, or $\text{in vitro}$ by incubation of |17,18-³H|-15-keto-13,14-dihydro-PGF_2α with mitochondria from rat liver. The sensitivity of the assay was 10 pg or 4 pg with these two preparations, respectively.The assay was employed for a number of measurements: normal daily excretion in a number of humans; excretion of urinary metabolites during treatment with prostaglandin synthetase inhibitors in human subjects, or after intravenous injection of PGF_2α; excretion during human pregnancy; and prostaglandin production in the guinea pig during normal estrous cycles and pregnancies and after estrogen treatment.The results of these studies were in several cases compared to similar measurements earlier performed using mass spectrometric methods, and were found to agree well. Thus, this radioimmunoassay provides a simple and accurate method for estimating prostaglandin production, particularly suitable for long-term studies and for cases where repeated blood sampling must be avoided.