Luteolysis induced in pigtail monkeys (Macaca nemestrina) with prostaglandin f 2 alpha, ICI 80996 and ICI 81008.

Non-pregnant pigtail monkeys (M. nemestrina) were given ICI 80996 subcutaneously and ICI 81008 and PGF2alpha subcutaneously or intravaginally, once daily on days 20-30 inclusive, or two or three times on days 24 or 26 only. Doses of 50 mug/kg of ICI 80996, 100 mug/kg of ICI 81008 and approx. 1 mg/kg of PGF2alpha were used. In the majority of monkeys treated subcutaneously a rapid fall in circulating progesterone concentrations and earlier than normal menstrual bleeding occurred. When given per vaginam, ICI 81008 was as effective as when given subcutaneously, though PGF2alpha was less effective intravaginally than by the subcutaneous route.     

The effect of the prostaglandin F2α analogue ICI 81008 on uterine small arteries and on blood pressure

The effects of PGF2α and its analogue ICI 81008^* have been compared on the small arteries of the omentum uteri on the rat. The vessels measured 20–80 μm in diameter and were examined by intra-vital-microscopy. While the maximum responses of PGF2α and ICI 81008 were similar, the duration of the effect of ICI 81008 was significantly longer (P< 0.001). At 15 minutes after the administration of the drugs the effect of ICI 81008 was still almost maximal, while the PGF2α response disappeared.     

Menstrual induction with the PGF2alpha-analogue ICI 81008.

"Menstrual Induction" (MI) has been studied in 79 volunteers, using the therapeutic principle of "PG-Impact". The PGF2alpha analogue: ICI 81008 was administered under strictly aseptic precautions into the uterine cavity during the 4th week of pregnancy. The treatment catheter (inserted through the cervical canal) delivered a single dose of only 100-200 mug ICI 81008 during the pilot study with this new drug. When it was established that the side effects were acceptable, this moderately effective dose was increased at first to 200-300 mug and eventually to 400 mug. At the 400 mug dose level, 29 (76%) of the 38 study patients had complete and 8 (21%) incomplete abortions, while 1 (3%) failed to bleed. Those 9 women who had incomplete abortions or failed to abort were curetted. In comparison with PGF2alpha (428 cases) and PGE2 (114 cases), ICI 81008 (38 cases at the 400 mug level) provoked lesser side effects, excepting the transient increase in blood pressure. All patients (whose intrauterine pressure was measured) responded to the ICI 81008-impact with rapidly developing high level uterine contracture. Plasma progesterone decreased significantly if treatment was successful and insignificantly in cases of treatment failure. In current studies, the efficacy of the vaginal delivery system of ICI 81008 is examined.     

Menstrual induction with the PGF2α-analogue ICI 81008

“Menstrual Induction” (MI) has been studied in 79 volunteers, using the therapeutic principle of “PG-Impact”. The PGF2α analogue: ICI 81008 was administered under strictly aseptic precautions into the uterine cavity during the 4th week of pregnancy. The treatment catheter (inserted through the cervical canal) delivered a single dose of only 100–200 μg ICI 81008 during the pilot study with this new drug. When it was established that the side effects were acceptable, this moderately effective dose was increased at first to 200–300 μg and eventually to 400 μg.At the 400 μg dose level, 29 (76%) of the 38 study patients had complete and 8 (21%) incomplete abortions, while 1 (3%) failed to bleed. Those 9 women who had incomplete abortions or failed to abort were curetted. In comparison with PGF2α (428 cases) and PGE₂ (114 cases), ICI 81008 (38 cases at the 400 μg level) provoked lesser side effects, excepting the transient increase in blood pressure.All patients (whose intrauterine pressure was measured) responded to the ICI 81008-impact with rapidly developing high level uterine contracture. Plasma progesterone decreased significantly if treatment was successful and insignificantly in cases of treatment failure. In current studies, the efficacy of the vaginal delivery system of ICI 81008 is examined.     

Pregnancy termination in the rat “model” by a synthetic prostaglandin analogue ICI 81008

The abortifacient efficacy of the PGF2α analogue ICI 81008 had been examined in 378 Spraque-Dawley rats. Of the 3 systemic routes of administration studied: intramuscular (i.m.), subcutaneous (s.c.) and per vaginam (p.v.), the p.v. treatment had been the most effective. Complete abortions were provoked invariably by a single p.v. dose of 20 μg ICI 81008 in the broad gestational range of 6 to 10 days of pregnancy and the “abortion rate” (AbR) remained 86% at day 12. The reduction of the p.v. dose to 10 μg only decreased efficacy slightly and its increase to 40 μg improved it. The analogue ICI 81008 was highly more effective than the natural PGs: F2α and E₂, in spite of their comparable oxytocic actions on the excised uterus. This high efficacy of ICI 81008 in p.v. formulation, without apparent side effects, is a therapeutic promise which demands early clinical investigation.At day 3 the AbR decreased to 0% and at day 14 to 30%. Plotting AbR, as a function of the gestational timing of treatment, yielded a reversed U shape curve, which reflected upon the mechanism of ICI 81008 action. The pituitary, the corpus luteum and the blastocyst are equally indispensable and accessible to drugs at days 3 and 6 of gestation. Yet the efficacy of ICI 81008 increased from 0% to 100% during this 3 days period, suggesting that the primary target of ICI 81008 is the uterine environment of the implanted conceptus. Thus the earlier premise had been substantiated that in inducing abortion PGs provoke: reduction of uterine microcirculation, sustained contracture, suppression of feto-placental endocrine function (including luteotrophic support), luteolysis, progesterone (P)-withdrawal and the evolution of uterine activity.However, progesterone treatment prevented the effect of ICI 81008, completely or partly, depending on the dose of PG administered. Furthermore, extensive studies of the ICI research team showed that ICI 81008 can provoke luteolysis without pregnancy, suggesting that under certain conditions the ovary is the primary target of this drug. If this effect on the ovary also involves a microcirculatory disturbance, then the mechanism of action of ICI 81008 is retained, in a variety of species and reproductive conditions, and only the threshold of the organ serving as primary target changes.From the Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, MissouriThis work was supported by the Agency for International Development Contract AID/csd 3160 and the National Institute of Health Career Award, HD 20169-11.     

Menstrual induction by the vaginal application of ICI 81008 gel

After ～2 weeks menstrual delay (positive Pregnosticon Tests) “menstrual induction” was attempted in 75 gravidas by repeated vaginal application of a gel, containing 200 or 400 μg/ml ICI 81008. After ～10 minutes, following the 1st vaginal delivery of 400 μg ICI 81008, the uterus responded to this PGF2α analogue with sustained contracture. The highest success rate in induced bleeding (93%) and pregnancy termination (79%), without supportive therapy, was achieved when 400 μg ICI 81008 was administered 2 to 5 times at 4 hour intervals. Those gravidas (21%), who failed in induced menstruation, or stopped bleeding within 24 hours after treatment, had positive Pregnosticon Tests on day 14 and were curetted. The side effects, mostly vomiting and increased blood pressure, were transient and subjectively and medically acceptable. While the vaginal application of the drug is apparently less effective than the intrauterine (1), it has the advantage of simple delivery and the potential of self-administration.     

Luteolysis induced in pigtail monkeys (Macaca nemestrina) with prostaglandin F2α, ICI 80996 and ICI 81008

Non-pregnant pigtail monkeys (M. nemestrina) were given ICI 80996 subcutaneously and ICI 81008 and PGF_2α subcutaneously or intravaginally, once daily on days 20–30 inclusive, or two or three times on days 24 or 26 only. Doses of 50 μg/kg of ICI 80996, 100 μg/kg of ICI 81008 and approx. 1 mg/kg of PGF_2α were used.In the majority of monkeys treated subcutaneously a rapid fall in circulating progesterone concentrations and earlier than normal menstrual bleeding occurred. When given per vaginam, ICI 81008 was as effective as when given subcutaneously, though PGF_2α was less effective intravaginally than by the subcutaneous route.     

Luteolysis induced in pigtail monkeys (Macaca nemestrina) with prostaglandin F2α, ICI 80996 and ICI 81008

Non-pregnant pigtail monkeys (M. nemestrina) were given ICI 80996 subcutaneously and ICI 81008 and PGF_2α subcutaneously or intravaginally, once daily on days 20–30 inclusive, or two or three times on days 24 or 26 only. Doses of 50 μg/kg of ICI 80996, 100 μg/kg of ICI 81008 and approx. 1 mg/kg of PGF_2α were used.In the majority of monkeys treated subcutaneously a rapid fall in circulating progesterone concentrations and earlier than normal menstrual bleeding occurred. When given per vaginam, ICI 81008 was as effective as when given subcutaneously, though PGF_2α was less effective intravaginally than by the subcutaneous route.     

Progesterone withdrawal induced by ICI 81008 in pregnant rats.

Of a total of 343 pregnant rats treated with the prostaglandin F2alpha analogue ICI 81008, 137 aborted, while 83 had reduced and 123 intact litter. These biological variations depended primarily on the gestational timing of treatment and on the dose and route of administration of this synthetic PG. In comparison with the 107 controls, all experimental rats which aborted had a drastic reduction in plasma progesterone levels which was highly significant (P is less than 0.001) until day 18. In contrast, those animals which escaped suboptimal ICI 81001 treatment with a partly resorbed litter, only had a moderate reduction in progesterone which was statistically significant (P is less than 0.01) until day 16 when levels of this steroid normally begin to decrease. Ineffective treatment did not affect progesterone levels and intact pregnancy. In contrast to progesterone, there was no correlation between plasma estradiol-17beta levels and the consequences of ICI 81008 treatment.     

Phytoestrogens modulate prostaglandin production in bovine endometrium: cell type specificity and intracellular mechanisms

Prostaglandins (PGs) are known to modulate the proper cyclicity of bovine reproductive organs. The main luteolytic agent in ruminants is PGF2alpha, whereas PGE2 has luteotropic actions. Estradiol 17beta (E2) regulates uterus function by influencing PG synthesis. Phytoestrogens structurally resemble E2 and possess estrogenic activity; therefore, they may mimic the effects of E2 on PG synthesis and influence the reproductive system. Using a cell-culture system of bovine epithelial and stromal cells, we determined cell-specific effects of phytoestrogens (i.e., daidzein, genistein), their metabolites (i.e., equol and para-ethyl-phenol, respectively), and E2 on PGF2alpha and PGE2 synthesis and examined the intracellular mechanisms of their actions. Both PGs produced by stromal and epithelial cells were significantly stimulated by phytoestrogens and their metabolites. However, PGF2alpha synthesis by both kinds of cells was greater stimulated than PGE2 synthesis. Moreover, epithelial cells treated with phytoestrogens synthesized more PGF2alpha than stromal cells, increasing the PGF2alpha to PGE2 ratio. The epithelial and stromal cells were preincubated with an estrogen-receptor (ER) antagonist (i.e., ICI), a translation inhibitor (i.e., actinomycin D), a protein kinase A inhibitor (i.e., staurosporin), and a phospholipase C inhibitor (i.e., U73122) for 0.5 hrs and then stimulated with equol, para-ethyl-phenol, or E2. Although the action of E2 on PGF2alpha synthesis was blocked by all reagents, the stimulatory effect of phytoestrogens was blocked only by ICI and actinomycin D in both cell types. Moreover, in contrast to E2 action, phytoestrogens did not cause intracellular calcium mobilization in either epithelial or stromal cells. Phytoestrogens stimulate both PGF2alpha and PGE2 in both cell types of bovine endometrium via an ER-dependent genomic pathway. However, because phytoestrogens preferentially stimulated PGF2alpha synthesis in epithelial cells of bovine endometrium, they may disrupt uterus function by altering the PGF2alpha to PGE2 ratio.     

Progesterone withdrawal induced by ICI 81008 in pregnant rats

Of a total of 343 pregnant rats treated with the prostaglandin F2 analogue ICI 81008, 137 aborted, while 83 had reduced and 123 intact litter. These biological variations depended primarily on the gestational timing of treatment and on the dose and route of administration of this synthetic PG. In comparison with the 107 controls, all experimental rats which aborted had a drastic reduction in plasma progesterone levels which was highly significant (P<0.001) until day 18. In contrast, those animals which escaped suboptimal ICI 81008 treatment with a partly resorbed litter, only had a moderate reduction in progesterone which was statistically significant (P<0.01) until day 16 when the levels of this steroid normally begin to decrease. Ineffective treatment did not affect progesterone levels and intact pregnancy. In contrast to progesterone, there was no correlation between plasma estradiol-17β levels and the consequences of ICI 81008 treatment.

ICI 81008 was most effective between the 6th and 12th days of gestation, when plasma progesterone in the normal controls showed a nadir. Effective treatment reduced plasma progesterone within 6 hours. Evidently the pharmacologically provoked endocrine imbalance precedes the cessation of characteristic gestational weight gain, uterine bleeding and abortion which only manifest 24 to 48 hours after treatment.     

Effects of dibutyryl cAMP, LHRH, and aromatase inhibitor on simultaneous outputs of prostaglandin F2 alpha, and 13,14-dihydro-15-keto-prostaglandin F2 alpha by term placental explants

Explants from term human placentas were maintained in culture with daily changes of medium. Daily output of PGF2 alpha and PGFM1 decreased during the course of the incubation. Addition of 4 micrograms/ml DHEAS or 67 micrograms/ml LDL cholesterol had no effect on output of PGF2 alpha or PGFM. Addition of 1.6, 3.2, or 6.4 micrograms/ml of LHRH to the culture plates had no effect on output of PGFM or PGF2 alpha, but LHRH increased hCG output. Dibutyryl cAMP (1mM, 2mM, and 4 mM) increased output of PGF2 alpha, PGFM, and hCG. Aromatase inhibitor decreased hCG output, but it was without effect on output of PGF2 alpha, or PGFM, Significant correlations were demonstrated between progesterone, PGFM, PGF2 alpha, and hCG, suggesting that PGF2 alpha originates in the syncytiotrophoblast cell. The ability of LHRH to stimulate output of hCG but not PGF2 alpha while dbcAMP stimulated both suggests that either PGF2 alpha and hCG arise in different cells or that LHRH does not act through cAMP.     

The effect of PGF2 alpha on parathyroid hormone-stimulated cyclic AMP production in mouse osteoblastic cell, MC3T3E1.

The effect of prostaglandin F2 alpha (PGF2 alpha) was investigated in MC3T3E1 cells on the succeeding cAMP response to parathyroid hormone (PTH). PGF2 alpha increased the membrane-associated protein kinase C (PKC) activity, indicating the activation of this enzyme. The effect of PTH to increase cAMP production was enhanced by pretreatment with PGF2 alpha. Phorbol 12-myristate 13-acetate also enhanced cAMP production stimulated by PTH, and PKC inhibitor H7 attenuated the enhancement of PGF2 alpha. A23187 did not reproduce the PGF2 alpha effect, and this effect was not antagonized by the calmodulin antagonist W7. PGF2 alpha did not change the ED50 nor the maximally responsive dose of PTH in stimulating cAMP production. The effect of PGF2 alpha was not affected by pertussis toxin, and PGF2 alpha also enhanced cholera toxin- or forskolin-stimulated cAMP production. In accordance with the response of cAMP to PTH, the resorption of mouse limb bones stimulated submaximally by PTH was enhanced by the concomitant presence of PGF2 alpha. These results indicate that PGF2 alpha modulates cAMP response through the activation of PKC, the target of which might be the catalytic unit of adenylate cyclase. Such interaction between signal transduction systems may have significance in modulating the effect of PTH on bone, i.e., bone resorption.     

Prostaglandin F2alpha increases glucose transport in 3T3-L1 adipocytes through enhanced GLUT1 expression by a protein kinase C-dependent pathway

The effect of prostaglandin F2alpha (PGF2alpha) on glucose transport in differentiated 3T3-L1 adipocytes was examined. Whereas PGF2alpha had little influence on insulin-stimulated 2-deoxyglucose uptake, it increased basal glucose uptake in a time- and dose-dependent manner, reaching maximum at approximately 8 h. The long-term effect of PGF2alpha on glucose transport was inhibited by both cycloheximide and actinomycin D. In concord, while the content of GLUT4 protein was not altered, immunoblot and Northern blot analyses revealed that both GLUT1 protein and mRNA levels were increased by exposure of cells to PGF2alpha. The effect of PGF2alpha on glucose uptake was inhibited by GF109203X, a selective protein kinase C (PKC) inhibitor. In addition, in cells depleted of diacylglycerol-sensitive PKC by prolonged treatment with 4beta-phorbol 12beta-myristate 13alpha-acetate (PMA), the stimulatory effects of PGF2alpha on glucose transport and GLUT1 mRNA accumulation were both inhibited. In accord, PMA was shown to stimulate GLUT1 mRNA accumulation. To further investigate if PKC may be activated by PGF2alpha, we tested several diacylglycerol-sensitive PKC isozymes and found that PGF2alpha was able to activate PKCepsilon. Taken together, these results indicate that PGF2alpha may enhance glucose transport in 3T3-L1 adipocytes by stimulating GLUT1 expression via a PKC-dependent mechanism.     

Effects of prostaglandins on the bovine corpus luteum: granules, lipid inclusions and progesterone secretion

Corpora lutea collected at 15, 30 and 60 min after prostaglandin F2 alpha (PGF2 alpha) treatment were compared to control corpora lutea at 60 min after saline treatment. There were decreases (P less than 0.05) in the relative percentages of cytoplasm occupied by granules in large luteal cells (LLC) by 30 min and in small luteal cells (SLC) by 60 min. Differences were not observed among the groups for lipid inclusions. Luteal progesterone was decreased at all post-PGF2 alpha treatment times when compared to 60-min controls (P less than 0.05). PGF2 alpha was then compared with prostaglandin F1 alpha (PGF1 alpha), prostaglandin E1 (PGE1), and 17-phenyl-18,19,20-trinor-prostaglandin F2 alpha (17-phenyl-PGF2 alpha) in 60-min trials with plasma progesterone and luteinizing hormone (LH) determined every 5 min. LH was not affected by these treatments. Like PGF2 alpha, 17-phenyl-PGF2 alpha induced a greater loss of granules from LLC then SLC. 17-phenyl-PGF2 alpha also induced an increase in the lipid content of LLC. Treatments with PGF2 alpha and 17-phenyl-PGF2 alpha were associated with decreased concentrations of luteal progesterone but PGF1 alpha and PGE1 were without effect on this variable. In contrast to PGF1 alpha, PGE1 increased both luteal progesterone and the area occupied by cytoplasmic granules. The latter effect was greater in LLC than SLC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interferon-tau blocks the stimulatory effect of tumor necrosis factor-alpha on prostaglandin F2alpha synthesis by bovine endometrial stromal cells

Tumor necrosis factor-alpha (TNFalpha) has been shown to be a potent stimulator of prostaglandin (PG) F2alpha synthesis in bovine endometrial stromal cells. The aims of the present study were to determine the effect of interferon-tau (IFNtau) on TNFalpha-stimulated PGF2alpha synthesis and the intracellular mechanisms of TNFalpha and IFNtau action in the stromal cells. When cultured bovine stromal cells were exposed to TNFalpha (0.006-0.6 nM) for 24 h, the production of PGF2alpha and cyclooxygenase (COX)-2 gene expression were stimulated by TNFalpha (0.06-0.6 nM, P < 0.05). Moreover, a specific COX-2 inhibitor (NS-398; 5 nM) blocked the stimulatory effect of TNFalpha on PGF2alpha production (P < 0.05). Although IFNtau (0.03-30 ng/ml) did not stimulate basal PGF2alpha production in the stromal cells, it suppressed TNFalpha action in PGF2alpha production dose dependently (P < 0.05). Moreover, the stimulatory effect of TNFalpha (0.6 nM) on COX-2 gene expression was completely blocked by IFNtau (30 ng/ml; P < 0.05), although the gene expression of COX-2 was not influenced by IFNtau. The overall results indicate that the stimulatory effect of TNFalpha on PGF2alpha production is mediated by the up-regulation of COX-2 gene expression and suggest that one of the mechanisms of the inhibitory effect of IFNtau on luteolysis is the inhibition of TNFalpha action in PGF2alpha production in the stromal cells by the down-regulation of COX-2 gene expression stimulated by TNFalpha.     

Synergistic effect of prostaglandin F2alpha and cyclic AMP on glucose transport in 3T3-L1 adipocytes

The combined effect of prostaglandin F2alpha (PGF2alpha) and cAMP on glucose transport in 3T3-L1 adipocytes was examined. In cells pretreated with PGF2alpha and 8-bromo cAMP for 8 h, a synergy between these two agents on glucose uptake was found. Insulin-stimulated glucose transport, on the other hand, was only slightly affected. The synergistic effect of these two agents was suppressed in the presence of cycloheximide and actinomycin D. In concord, immunoblot and Northern blot analyses revealed that GLUT1 protein and mRNA levels were both increased in cells pretreated with both PGF2alpha and 8-bromo cAMP, greater than the additive effect of each agent alone. The synergistic action of PGF2alpha with 8-bromo cAMP to enhance glucose transport was inhibited by GF109203X, a selective protein kinase C (PKC) inhibitor. In addition, in cells depleted of diacylglycerol-sensitive PKC by prolonged treatment with 4beta-phorbol 12beta-myristate 13alpha-acetate, a PKC activator, the synergistic effects of PGF2alpha and 8-bromo cAMP on glucose transport and GLUT1 mRNA accumulation were both abolished. Taken together, these results indicate that PGF2alpha may act with cAMP in a synergistic way to increase glucose transport, probably through enhanced GLUT1 expression by a PKC-dependent mechanism.     

The antiarrhythmic action of prostacyclin (PGI2) on aconitine induced arrhythmias in rats.

Prostacyclin (PGI2) produces an antiarrhythmic effect on aconitine induced arrhythmias in rats. The ED50 of PGI2 was 0.7 microgram/kg and the maximum antiarrhythmic effect 54 per cent. The equi-effective doses of PGE2 and PGF2alpha were higher (ED50 of PGF2alpha = 1.2 microgram/kg, ED50 of PGE2 = 2.7 microgram/kg). However, PGF2alpha and PGE2 had a maximum antiarrhythmic effect of 80 per cent in this model.     

Involvement of ovarian steroids in basal and oxytocin-stimulated prostaglandin (PG) F2 alpha secretion by the bovine endometrium in vitro

It is assumed that exposure of endometrium to spontaneously secreted luteal hormones stimulates PGF2 alpha secretion and modifies oxytocin (OT) influence on the bovine uterus. At first, the time-dependent effect of endogenous luteal products on endometrial PGF2 alpha secretion was examined. Endometrial strips (100 mg) from slaughtered heifers (Days 11 to 17 of the cycle) were incubated alone or with luteal cells (1 x 10(5) cells/mL). The highest PGF2 alpha secretion by the endometrium under influence of hormones secreted from luteal cells was observed after 12 h of incubation compared with the control (P < 0.001). Then, endometrium (Days 11 to 17) was incubated with luteal cells and concomitantly with antagonists of P4 and OT. The P4 antagonist prevented the stimulatory effect of endogenous luteal hormones on PGF2 alpha secretion (P < 0.05), but the OT antagonist did not. Further, direct effects of exogenous P4, OT and estradiol (E2) on endometrial PGF2 alpha secretion (Days 11 to 17) were examined. Both OT and P4 increased PGF2 alpha secretion (P < 0.05); E2 alone had no effect on PGF2 alpha secretion, but it amplified the P4 effect (P < 0.05). Finally, we studied the effect of endogenous luteal products on OT-stimulated PGF2 alpha secretion from endometrium. When endometrium (Days 11 to 17) was incubated without luteal cells, OT stimulated PGF2 alpha secretion (P < 0.001), whereas incubation of endometrium with luteal cells abolished the stimulatory effect of OT on PGF2 alpha secretion (P < 0.001). These treatments did not affect PGF2 alpha secretion from the endometrium collected on Days 1 to 4. In conclusion, P4 stimulates PGF2 alpha secretion by the endometrium and E2 amplifies this effect. As long as the endometrium is under the influence of P4, ovarian OT does not affect PGF2 alpha secretion.