Effects of prostaglandins E1, E2, and F2 alpha on N-acetyl-beta-glucosaminidase activities of human synovial cells in culture.

The acid hydrolase N-acetyl-beta-glucosaminidase (NAG) was used to examine the effects of prostaglandins E1 (PGE1), E2 (PGE2), and F2 alpha (PGF2 alpha) on the lysosomal system of human synovial cells in vitro. A spontaneous release of the enzyme occurred from control cultures, which was accelerated by each of the prostaglandins in a concentration-dependent manner, within the range of 10(-8)-10(-6) moles per litre (M). No clear order of potency could be established. The effects of the prostaglandins on release of NAG were less consistent and of smaller magnitude when human serum was replaced by bovine serum albumin in the medium. In the presence of serum small increases also occurred in intracellular NAG activity, but only the effect of PGE1 was statistically significant. The prostaglandins did not appreciably affect the previously established pattern of increased intracellular activity of NAG and reduced release produced by sucrose.     

Regulation of the production of placental protein 5 by human endometrial stromal cells; the role of prostaglandins E2 and F2 alpha

Cultured human endometrial stromal cells were found to release placental protein 5 (PP5), a glycoprotein with properties of a serine protease inhibitor. Progesterone had no effect on PP5 release, but cholera toxin and 12-O-tetradecanoylphorbol 13-acetate stimulated PP5 release in a time- and concentration-dependent fashion. Prostaglandin E2 (PGE2) caused a parallel increase in cAMP and PP5 release in a time- and concentration-dependent fashion. The lowest PGE2 concentration which increased cAMP and PP5 release was 1 X 10(-9) M. Maximal increase in cAMP (42-fold) and PP5 (25-fold) release was obtained by 10(-5) M PGE2. Stimulation of cAMP by PGE2 was detectable at 15 min and was followed by an increased PP5 release at 24 h. The concentrations of prostaglandin F2 alpha (PGF2 alpha) which stimulated cAMP and PP5 release were pharmacological suggesting that this effect is nonspecific. The results indicate that the activation of cAMP- and protein kinase C-dependent pathways in endometrial stromal cells increases the production of PP5. PGE2 could be one of the physiological ligands employing the cAMP-dependent pathway in endometrial stromal cells.     

Effect of carprofen and indomethacin on gastric function and the content of prostaglandins E2 and F2 alpha in human gastric juice

The effect of indomethacin (2 X 50 mg daily) and carprofen (2 X 150 mg daily) on gastric secretion and the generation of prostaglandins PGE2 and PGF2 alpha in gastric juice, was investigated in a single blind cross-over study in eight healthy volunteers lasting one week. We observed no statistically significant change in basal and pentagastrin-stimulated gastric secretory parameters (outputs of gastric acid, N-acetyl-neuraminic acid and pepsin) before and after treatment with indomethacin and carprofen. However, an inhibitory effect was found on the output of PGE2 and PGF2 alpha after pentagastrin stimulation. While both drugs diminished the output of PGF2 alpha to a similar extent, carprofen exerted a markedly weaker inhibitory effect on the output of PGE2 than did indomethacin. It is suggested that the gastric tolerability of non-steroidal anti-inflammatory drugs (NSAIDs) is related to their inhibitory potency on PGE2 formation, in the sense that weak inhibitors of PGE2 cause less damage to the gastric mucosa than do strong inhibitors.     

The molecular biology of RU486. Is there a role for antiprogestins in the treatment of breast cancer?

Considerable animal research and clinical trials demonstrate that progesterone antagonists could treat hormone-dependent breast cancers. Since endogenous progesterone does include mitosis in epithelial cells of the breast, in theory, exogenous progesterone can cause breast cancer. Thus administration of progesterone antagonists could block endogenous progesterone. Yet we do not know the mitosis pattern in breast cancer cells during the menstrual cycle, so research obtaining such data is needed. Ethical problems arise, however, since researchers need multiple breast tumor samples to analyze proliferative activity at various times during the cycle. A possible solution is using an aspirated tumor sample for initial mitotic analysis immediately followed by RU-486 treatment then tumor removal 24 hours later for reanalysis. Ideally well controlled studies using organ-cultured human breast tumors, human breast cancer lines, and human tumors implanted into nude mice are needed to understand the mechanisms of the mitogenic actions of progestins and progestin antagonists. Progestin antagonist may be used to treat locally advanced or metastatic cancers either as an adjuvant endocrine therapy alone or with tamoxifen. An obstacle to longterm use of RU-486 as a treatment for breast cancer is its antiglucocorticoid side effects. But the molecules of newer progesterone antagonists appear to produce maximal antiprogestin activity and minimal antiglucocorticoid activity. In addition, if RU-486 is administered with drugs that prevent adrenal steroidogenesis or peripheral aromatization of adrenal steroids to estrogens, women may take it for longterm treatment. Researchers must have the opportunity to continue basic tumor biological and molecular research to gain an understanding of the exact molecular targets and mechanisms of antagonist action. The current political climate in the US hinders such research, however.     

The effect of RU486 and progesterone on luteal function during pregnancy

In order to investigate the role of progesterone in the maintenance of pregnancy, an anti-progesterone agent, RU486 (RU) was injected subcutaneously into pregnant rats on day 12 (D12), and morphological changes of the uterus as well as endocrinological changes were observed. In all rats injected with RU, abortion occurred with macroscopic and microscopic intrauterine hemorrhage and degeneration or delivery of conceptuses. Endocrinologically, the levels of progesterone decreased rapidly 48 hours after the injection, while the levels of estradiol showed a tendency to increase. As progesterone is mainly produced by the corpus luteum but not by the placenta in rats, the decrease in progesterone is suspected to be due to luteolysis. Then in order to clarify the mechanism of luteolysis induced by RU and the effects of progesterone on this phenomenon, the dynamics of the luteotrophic factors (estradiol, LH, PRL) and specific binding capacity of the ovaries to LH/hCG were investigated in D7 pregnant rats treated with RU 1 mg/kg alone (RU group) or with both RU 1 mg/kg and progesterone 50mg/kg (RU + P group). The serum levels of progesterone in the RU group decreased significantly after 72 hours of administration, while those in the RU + P group remained within the levels of the control group. However, serum levels of luteotrophic factors in the RU group did not decrease, and some of them were even higher than those in the control group. In the RU + P group, luteotrophic factors remained within control levels. On the other hand, the specific bindings of LH/hCG to ovarian homogenates decreased significantly after 72 hours in the RU group. But in the RU + P group, the specific bindings were kept at the same levels as the controls. Scatchard analysis of these results disclosed that in the RU group, both affinity and numbers of receptors decreased compared to the controls, and that in the RU + P group only affinity decreased transiently and afterwards recovered quickly. From these results, it is concluded that deterioration of affinity and numbers of ovarian LH/hCG receptors seems to be one of the factors which induce luteolysis in pregnant rats treated with RU, and that progesterone can spare the effect of RU on the corpus luteum during pregnancy.     

Effects of RU486 on progesterone secretion by human preovulatory granulosa cells in culture

The effects of RU486 on progesterone synthesis were studied in human preovulatory granulosa cells in culture. No effect was observed at 1 and 10 micrograms/mL, but at 100 micrograms/mL, RU486 inhibited the simulation of progesterone secretion induced by LH and cAMP. It is suggested that the main target of RU486 is the cytochrome P450scc function [catalyzing the formation of pregnenolone (D5P) from cholesterol], since no accumulation of D5P or hydroxy derivatives of progesterone was observed. As RU486 is an antiglucocorticosteroid and antiprogesterone agent, the effects of dexamethasone and progesterone were also investigated. Dexamethasone did not modify progesterone secretion, but progesterone inhibited its own synthesis in both the presence and absence of LH. Thus, under these experimental conditions RU486 displayed a progesterone-like effect. However, since the effect of RU486 was observed only at a concentration around 10(-4) M, the mechanism of action may not involve a receptor pathway and may not apply to most clinical circumstances.     

Prostaglandin E and F2 alpha receptors in human uterine leiomyomas

9uman uterine leiomyomas specifically bound less (P less than 0.01) [3H]prostaglandin E1 ([3H]PGE1) and [3H] PGF2 alpha than adjacent normal myometria [leiomyomas: mean [3H]PGE1, 16.4 (range, 11.1-25.2) fmol/mg protein; mean [3H]PGF2 alpha, 4.7 (range, 0.8-12.1) fmol/mg protein; adjacent normal myometria: mean [3H]PGE1, 41.7 (range 27.1-60.7) fmol/mg protein; mean [3H]PGF2 alpha, 7.8 (range, 4.3-16.3) fmol/mg protein]. The lower binding of both [3H]PGs by leiomyomas was due to lower numbers of available high and low affinity sites. Leiomyomas and normal adjacent myometria bound 4-7 times more [3H]PGE1 than [3H]PGF2 alpha, and this appears to be due to high affinity and high numbers of low affinity PGE sites. The smooth muscle content was lower (P less than 0.01) in leiomyomas (mean, 28.0%; range, 9.7-45.5%) than that of adjacent normal myometria (mean, 58.9; range, 51.4-71.2%). In summary, this is the first demonstration of PGE and PGF2 alpha receptors in human uterine leiomyomas. Lower receptor numbers in leiomyomas appear to be due to the lower smooth muscle content of the tissue.     

The effect of intravenous infusions of prostaglandins E-2 and F-2alpha on human gastric function.

The effect of intravenous infusions of prostaglandins E-2 and F(-2alpha) at various dose levels on basal, or on maximally or submaximally pentagastrin-stimulated acid secretion, was studied in 40 male subjects. Intraluminal antral pressures were also measured. Prostaglandin F (0.08 mug kg-minus 1 min-minus 1) transiently, but significantly, inhibited submaximal acid output and increased the frequency of antral contractions. Prostaglandin E(2)(0.08 mug kg-minus 1 min-minus 1) inhibited basal acid secretion.     

The effect of intravenous prostaglandin F2 alpha and E2 on the motility of the sigmoid colon.

The effect of a 20-min intravenous infusion of prostaglandin E2 (0-08 mug kg-1 min-1) or of prostaglandin F2 alpha (0-8 mug kg-1 min-1) on the segmental pressures in the sigmoid colon was studied in 12 patients. Prostaglandin F2 alpha had no measurable effect, but prostaglandin E2 significantly inhibited sigmoid motility.     

Comparison of the effects of antiprogestins RU38486, ZK98299 and ORG31710 on periovulatory hypophysial, ovarian and adrenal hormone secretion in the rat

The antiprogestin (AP) RU38486 (RU) blocks progesterone (P) and glucocorticoid (G) actions. Administration of 4 mg RU on proestrous morning to cyclic rats dissociates LH and FSH secretion on proestrous afternoon, early estrus and on estrous afternoon. In order to ascertain which action blocked by RU is predominant in the control of periovulatory LH and FSH secretion, a study was made on the effects of: a) 1 or 4 mg of ZK98299 (ZK) (type I P antagonist; Schering), b) 2 or 8 mg of Org31710 (OR) (type II P antagonist lacking anti-G actions; Organon) or c) 1 or 4 mg of RU (type II P antagonist; Exelgyn) to 4-day cyclic rats on proestrous morning on serum concentrations of LH, FSH, inhibin-alpha (I), estradiol-17beta (E), progesterone (P) and corticosterone (B) at 18:30 h on proestrus and at 02:00 and 18:30 h on estrus. Controls, receiving 0.2 ml oil, had elevated serum concentrations of all six hormones on proestrous afternoon; at early estrus, only serum concentrations of FSH and P remained elevated, and, on estrous afternoon, all hormones but I and B, that peaked again, had reached their lowest serum levels. All AP treatments except 1 mg ZK had the same effects. On proestrous afternoon serum LH concentrations were reduced and serum FSH concentrations were suppressed whereas serum levels of I, E, P and B were unaffected. At early estrus, basal serum concentrations of LH and E increased while FSH secretion was abolished. Serum levels of I, P and B did not differ from controls. AP treatments increased basal LH concentration, hyperstimulated FSH secretion and reduced serum I concentration on the afternoon of estrus. E, P and B serum levels did not differ from controls at this stage. Treatment with 1 mg ZK was less effective in reducing serum FSH on proestrous afternoon and at early estrus, and had no effect on serum concentrations of any hormone on estrous afternoon. These results indicate that blockade of P receptor activation by P is, predominantly, the mechanism of AP action on periovulatory gonadotropin secretion in rats.     

Effects of prostaglandin F2alpha and E2 on the production of progesterone by human granulosa cells in tissue culture.

Human granulosa cells with differing steroidogenic potentials were cultured in vitro. The effects of prostaglandin F2alpha (PGF2alpha) and PGE2 on the progesterone output and viability of these cells were investigated. Prostaglandin F2alpha either alone or in combination with LH and FSH inhibited the production of progesterone over a wide range of concentrations (1-8000 ng/ml). However, the inhibitory effect of PGF2alpha was 200 times less effective when the cells were exposed to LH and FSH for 6 days before the addition of the prostaglandin. By contrast PGE2, at concentrations from 1 to 500 ng/ml, markedly stimulated the production of progesterone by granulosa cells, and this was not prevented by the addition of PGF2alpha. The degree of inhibition by PGF2alpha or stimulation by PGE2 was related to the biosynthetic capacity of the cells. These studies suggest that PGF2alpha may act directly on the adenylate cyclase system of human granulosa cells by blocking its activation by LH, and they demonstrate that functional regression of the luteal cell can be induced independently of the blood vascular system.     

Distribution of prostaglandin E2 and prostaglandin F2 alpha receptors in human myometrium

Human myometrium was studied for specific binding of PGE2 and PGF2 alpha. PGF2 alpha-binding was almost undetectable, but specific binding sites for PGE2 with high affinity (KD = 2.7 +/- 0.4 x 10(-9) M were demonstrated. The binding capacity for PGE2 exhibited a topically different distribution pattern with the highest values in the central parts and low to undetectable levels in the cervical region. Binding characteristics were analyzed by receptor kinetics, revealing a homogeneous receptor population. Binding capacity in uteri obtained from post-menopausal women was of the order of 900-940 fmol/mg protein. Oestrogen pre-treatment and pregnancy were associated with a 3-fold reduction of the PGE2-binding capacity.     

Modulation of aromatase activity in human endometrial stromal cells by steroids, tamoxifen and RU 486

The regulation of aromatase activity (AA) in human endometrial stromal cells by various steroids was studied in primary cell culture. Various progestins, but not androgens or glucocorticoids, stimulated AA. Medroxyprogesterone acetate (MPA) was the most potent progestin. Estrogen (E) alone did not change the activity but it potentiated the stimulation of AA by progestin. Biphasic regulation of AA by progestin was noted in both time- and dose-dependent manners. Endometrial AA was stimulated by MPA and reached the maximum rate between 2-5 days of incubation with subsequent decline of AA in prolonged culture. When stromal cells were treated with MPA (0.03 to 30 microM) for 3 days, AA was increased over the control at all the concentrations tested. The maximum was found at doses between 0.1-1 microM. The activities reduced steadily from the maximum stimulation to less than 50% when the concentration of MPA increased from 1-30 microM. In addition, initial treatment of stroma cells with MPA (1-3 days) resulted in further increase of activity after progestin withdrawal. The enhancement of the induction of AA by E did not alter the biphasic pattern regulated by progestin alone, i.e. E enhanced both the stimulation and the decay of AA. The time study of the effect of E showed that enhancement of AA required at least 10 h of incubation of E with MPA conditioned cells. The effect of E is dose dependent between 0.04-40 nM and shows the greatest effect in the presence of MPA between 0.01-1 microM. The optimal concentrations of E and progestin that stimulate AA in culture are similar to the plasma concentrations after pregnancy, suggesting that the physiological function of the endometrial aromatase is at the time of decidualization. The effects of antiprogestin, Ru 486, and antiestrogen, tamoxifen (TAM), on AA were studied. Ru 486 or TAM alone did not alter AA. Ru 486 inhibited the MPA stimulated AA in a dose-dependent manner suggesting that the effect of progestin may be mediated through a receptor mechanism. Enhancement, but no inhibitory effect, was observed when cells were treated with TAM + MPA and TAM + MPA + E. The effectiveness of Ru 486 to inhibit the induction of AA in endometrial cells may be of primary importance for contraception.     

Contractile effects of prostaglandins, oxytocin, and endothelin-1 in human myometrium in vitro: refractoriness of myometrial tissue of pregnant women to prostaglandins E2 and F2 alpha.

Whereas there is much evidence in support of a role for prostaglandins (PG) in the parturitional process, it has not been demonstrated unequivocally that PGs are the physiological uterotonins involved in the induction of the myometrial contractions of spontaneous labor in women. This study was conducted to evaluate the contractile responsiveness of human myometrial tissue in vitro to PGs and to compare this response with that of other uterotonins, viz. oxytocin and endothelin-1. We found that treatment of uterine smooth muscle strips obtained from nonpregnant and pregnant women with PGE2 (10(-8)-10(-6) M) caused a biphasic response characterized by an initial single contraction of increased amplitude and duration, followed by relaxation and a long period (10-15 min) of quiescence. Conversely, PGE2 acted in rat myometrium to cause a monophasic response of increased contractile frequency and force. Whereas uterine smooth muscle from nonpregnant women was responsive to PGF2 alpha, the contractile responsiveness of myometrium from pregnant women was weak. This weak response to PGF2 alpha was found in myometrium of women in labor and in myometrium of women not in labor. 15-Methyl-PGF2 alpha evoked a small response in myometrium from pregnant women. Under identical in vitro conditions, PGF2 alpha (10(-8)-10(-6) M) and 15-methyl-PGF2 alpha (10(-6) M) caused sustained contractions in human vascular smooth muscle tissues (fetal aorta and arterial smooth muscle from chorionic vessels). Similarly, oxytocin and endothelin-1 (in myometrium from pregnant women) were effective in stimulating the force and frequency of myometrial contraction in vitro. We conclude that the myometrium of pregnant women, as evaluated in vitro, is refractory to the contractile effects of PGE2 and PGF2 alpha.     

Hemodynamic, renal, and steroidogenic actions of prostaglandins E1, E2, A2, and F2 alpha in European eels

Seawater-adapted eels were implanted with both venous and arterial cannulae and catheterised. Prostaglandin in 0.9% glucose saline was given either by 10 microliters injection or by infusion (40 ng/min) while blood pressure recordings and blood samples were taken from the dorsal aorta. Glomerular filtration rates were calculated from the clearance of [3H]inulin, renal plasma flow from the clearance of PAH, and functional tubular mass from measurements of glucose reabsorption maxima (TmG). Cortisol levels were measured by radioimmunoassay. Injections of prostaglandin E1, E2, and A2 (0.01-10 ng/kg body wt) induced transient reductions in dorsal aortic blood pressure whilst concentrations of between 10 and 100 ng/kg body wt caused both prolonged vasodepression and glomerular antidiuresis, with decreased TmG and CPAH. Fractional excretion of electrolytes remained unchanged. Doses of 10-50 micrograms/kg body wt caused an initial glomerular diuresis, increased TmG and CPAH but were without effect on the fractional excretion of the filtered load. This diuretic action was followed by a longer period of antidiuresis. The vasodepression during 24 hr prostaglandin infusion became less severe after an initial 2-hr period, indicating a degree of tachyphylaxis. Prostaglandin F2 alpha in doses of 10-50 micrograms/kg body wt was slightly vasopressor but with no obvious effect on kidney function. All prostaglandins so far used, given either by infusion or injection caused a significant increase in cortisol production. These results suggest that prostaglandins may play similar roles throughout a range of vertebrates.     

RU486 antagonizes the inhibitory action of progesterone on prostacyclin and thromboxane A2 synthesis in cultured rat myometrial explants

A myometrial explant culture system was developed to investigate the effect of progesterone and the antiprogestagen RU486 on prostacyclin (PGI2) and thromboxane A2 (TXA2) synthesis by the rat myometrium. After culture, eicosanoid synthesis was stimulated with arachidonic acid (AA) and the calcium ionophore A23187 (A23187). Spontaneous release of eicosanoids was also studied. Progesterone inhibited the spontaneous release of PGI2 and TXA2 release by myometrial explants in a concentration- and time-dependent manner. Adequate inhibition of myometrial eicosanoid synthesis by physiological concentrations was achieved at 18 h of culture: all subsequent studies were carried out after an 18-h culture of explants. A23187- and AA-stimulated PGI2 and TXA2 synthesis were inhibited equipotently by progesterone. 17 beta-Estradiol alone was without effect on spontaneous AA- or A23187-stimulated PGI2 or TXA2 synthesis and was without effect on progesterone-elicited inhibition of eicosanoid synthesis in the myometrial explants. The protein synthesis inhibitors, actinomycin D and cycloheximide, did not block the inhibitory action of progesterone on A23187- or AA-stimulated eicosanoid synthesis by the myometrial explants and alone mimicked the inhibitory action of progesterone. The inhibitory action of progesterone on AA- and A23187-stimulated PGI2 and TXA2 synthesis was antagonized in a concentration-dependent manner by RU486. These data indicate that within this ex vivo system, progesterone probably inhibits myometrial cycloxygenase, that progesterone may exert this action through inhibition of a modulating or permissive protein, and that the antiprogestagen RU486 is an effective in vitro antagonist or progesterone.     

Effect of prostaglandins (F2 alpha, E1, and E2) on blood pressure and oxytocin-induced intramammary pressure responses in rats.

In lactating rats, vasoactive prostaglandin (PG) doses of F2 alpha (4 and 8 microgram/kg), E1, and E2 (2 and 4 microgram/kg each) reduced the intramammary pressure response to standard iv doses of 300 microU oxytocin by 50--80%. Adrenergic blockers, phenoxybenzamine and/or propranolol (1 mg/kg each sc) did not influence the blood pressure response to PGF2 alpha, PGE1, or PGE2. The oxytocin-antagonistic action of a single iv PGF2 alpha dose (4 microgram/kg) could not be altered by adrenergic blockers. In contrast, the oxytocin-antagonistic effects of PGE1 and PGE2 (2 microgram/kg each) were completely eliminated after alpha-receptor blockade, while the activity of oxytocin was augmented. Under beta-receptor or alpha- and beta-receptor blockade, the oxytocin-antagonistic effects of PGE1 and PGE2 were almost abolished. alpha-Receptor blockade reduced the oxytocin-antagonistic action of infused PGF2 alpha (8 microgram/kg.min for 15 min) by 38%. beta- or alpha- and beta-receptor blockade had no effect. The oxytocin-antagonistic actions of PGE1 and PGE2 (4 microgram/kg.min for 15 min each) were greatly reduced under alpha-receptor blockade. beta-Receptor blockade had no influence on the oxytocin-antagonistic activities of PGE1 or PGE2; under alpha- and beta-receptor blockade, the inhibitory actions of PGE1 and PGE2 were reduced by 60--70%. Mechanisms of PG-induced inhibition of the oxytocin response may involve mammary vasoconstriction and/or alterations in myoepithelial activity of cAMP and cGMP.