Metabolism of prostacyclin. III. Urinary metabolite profile of 6-keto PGF1α in rat

The in vivo metabolism of 6-keto PGF_1α was investigated in rats. Following continuous intravenous infusion for 14 days the urinary metabolites were isolated and identified. A substantial amount of unchanged 6-keto PGF_1α was recovered in the urine. The metabolic pattern very closely resembles that of PGI₂ in rats. Metabolites were found which represented 15-dehydrogenation, β-oxidation, ω and ω-1-hydroxylation and oxidation.Previous work showed that 6-keto PGF_1α is very poorly oxidized by 15-PGDH. We administered 15-[H³]-PGI₂ and 15-[H³]-6-keto PGF_1α to rats and measured urinary tritiated water as an index for in vivo 15-PGDH activity. The results showed that PGI₂ and 6-keto PGF_1α were both oxidized to the 15-keto product, although the rate of oxidation of PGI₂ was greater than that of 6-keto PGF_1α. We concluded that the administered PGI₂ was oxidized by 15-PGDH before hydrolysis to 6-keto PGF_1α. A portion of the dose is probably hydrolyzed before 15-dehydrogenation.     

Oxytocin-stimulated production of prostaglandin F2α by isolated endometrium of rabbit: Modulation by ovarian steroids

To test the hypothesis that ovarian steroid hormones modulate oxytocin-induced release of prostaglandin F_2α (PGF_2α) from uterine endometrium, 2 ovariectomized rabbits were pretreated with progesterone (5 mg/day for 10 days), 2 with estradiol-17β (25 μg/day for 10 days), 2 with both steroids, and one with sesame oil only. On the last day of treatment, endometrial fragments were excised and incubated in vitro with or without oxytocin (100 μU/ml). Although endometrium from rabbits pretreated with combined steroids released more PGF_2α immediately after excision than did tissue from animals pretreated with either steroid by itself, endometrium from animals pretreated with estrabdiol-17β alone released the most PGF_2α during sustained incubation in vitro. Moreover, only this tissue exhibited significant oxytocin-dependent release of PGF_2α. At the dosages used, progesterone completely antagonized both of these effects of estradiol-17β. The results support the hypothesis that ovarian steroid hormones regulate oxytocindependent release of PGF_2α from endometrial cells. A possible mechanism of action is suggested.     

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

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

2-(aminomethyl)-2-decarboxyprostaglandin F2α type analogs

The 2-(aminomethyl)-2-decarboxy analogs of prostaglandin F₂α (PGF₂α), (15S)-15-methyl-PGF₂α, 16-phenoxy-ω-tetranor-PGF₂α and 16,16-dimethyl-PGF₂α were synthesized. The amino analogs closely resemble the parent PGF₂α compounds as antifertility agents in the hamster.     

Synthesis and antifertility activity of ω-chain phenyl- and 16-phenoxy-analogues of (±)-11-deoxyprostaglandin F1α

Some ω-chain phenyl- and 16-phenoxy- analogues of (±)-11-deoxyprostaglandin F_1α have been synthesized and evaluated for anti-fertility activity in the hamster. 11-Deoxy-16-phenoxy-17,18,19,20-tetranor-PGF_1α was the most active member of the series with an ED₅₀ equal to that of PGF_2α. 11-Deoxy-17-phenyl-18,19,20-trinor-PGF_1α, which was one third as active as PGF_2α, was more potent than the corresponding 16- and 18-phenyl compounds. Aryl ring substitution was found to lower activity, except that with the 16-phenyl compound, p-bromo and m-trifluoromethyl substitution increased the potency.The antifertility activity of the phenoxy compounds, which were poor substrates for 15-hydroxyprostaglandin dehydrogenase, was shown to correlate well with the binding affinity for the bovine corpus luteum PGF_2α receptor. Some quantitative structure-activity data supporting this finding are presented.     

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

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

Effects of PGF2α and PGF2α, 1–15 lactone on the corpus luteum and on early pregnancy in the rhesus monkey

The effects of prostaglandin (PG)F_2α and PGF_2α, 1–15 lactone were compared in luteal phase, non-pregnant and in early pregnant rhesus monkeys. Animals treated with either PG after pretreatment with human chorionic gonadotropin (hCG) had peripheral plasma progesterone concentrations that were not statistically different from those in animals treated with hCG and vehicle. However, menstrual cycle lengths in monkeys treated with PGF_2α, 1–15 lactone were significantly (P <0.02) shorter than those in vehicle treated animals. In the absence of hCG pretreatment, plasma progesterone concentrations were significantly (P <0.008) lower by the second day after the initial treatment with either PGF_2α or PGF_2α, 1–15 lactone than in vehicle treated monkeys. Menstrual cycle lengths in monkeys treated with either PG were significantly (P <0.04) shorter than those in animals treated with vehicle. There were no changes in plasma progesterone concentrations in early pregnant monkeys treated with PGF_2α, and pregnancy was not interrupted. In contrast, plasma progesterone declined and pregnancy was terminated in 5 of 6 early pregnant monkeys treated with PGF_2α, 1–15 lactone. These data indicate that PGF_2α, 1–15 lactone decreases menstrual cycle lengths in non-pregnant rhesus monkeys. More importantly, PGF_2α, 1–15 lactone terminates early pregnancy in the monkey at a dose which is less than an ineffective dose of PGF_2α.     

Plasma 13, 14-dihydro-15-keto-PGF2 (PGFM) in pregnant and nonpregnant heifers prior to and during surgery and following intrauterine injection of PGF2

On day 17 postestrus or postmating, heifers were given intrauterine injections of saline (2 pregnant, 2 non-pregnant) or 200 μg PGF₂α (7 pregnant, 6 nonpregnant) through cannulae installed surgically into the uterine horn ipsilateral to the corpus luteum bearing ovary. Jugular blood samples were collected prior to the laparotomy at which the cannulae were installed during surgery, and for 90 min following the intrauterine injection. Plasma was assayed for progesterone and 13,14-dihyro-15-keto-PGF₂α )PGFM). Laparotomies were reopened to confirm proper cannula placement and to determine if blastocysts were present in mated heifers. Concentrations of PGFM were higher in pregnant compared to nonpregnant heifers during the presurgery (68 26 24 26 pg/ml; P < 0.25) and surgery (186 47 65 17 pg/ml; P < .05) periods. Pregnancy status did not alter the mean concentrations of PGFM (pregnant, 554 70 pg/ml; nonpregnant, 422 81 pg/ml) or the half-life of its decline in concentration (18 min) following intrauterine injection of PGF₂α. Pregnancy at 17 days in cattle does not appear to influence PGF₂α transport from the uterine lumen or its metabolism in the uterus or elsewhere in response to an acute intrauterine injection.     

The effects of prostaglandin F2α on sperm output in boars

The effect of prostaglandin F_2α (PGF_2α) on the sperm output of six boars was investigated in two studies. Although PGF_2α did not significantly affect sperm numbers in the ejaculate, a significantly longer (P < 0.05) ejaculation of the sperm rich fraction occurred following injection of PGF_2α. In the second study it was found that PGF_2α produced a 49% increase (P < 0.05) in the number of sperm in the sperm rich fraction of the ejaculate. The implications of these results on artificial breeding are discussed.     

Increased sperm output of rabbits and bulls treated with prostaglandin F2α

Seven rabbits were ejaculated four times once weekly, and saline or 2.5 mg PGF_2α tromethamine salt was injected sc at 4 and 2 hours or at 8 and 4 hours before ejaculation. First ejacula taken at 2 hours after the second injection of PGF_2α contained 150% more (P.07) sperm than those after injections of saline. The comparable difference (60%) at 4 hours after PGF_2α was not significant. PGF_2α did not influence sperm output in second, third or fourth ejacula. After 28 daily sc injections of 5 mg PGF_2α in another experiment, the fertility of four treated rabbits was as high as that for four controls. Without sexual preparation in seven bulls, im injections of 40 or 80 mg PGF_2α 30 minutes before ejaculation resulted in 33% greater (P<.05) sperm output than that after injection of 0, 7 or 20 mg PGF_2α, but the highest sperm output after PGF_2α was 30% less (P<.05) than that after sexual preparation in the same bulls. We conclude that injections of PGF_2α result in increased sperm output in ejacula taken without sexual preparation within 2 hours in rabbits and in bulls.     

Luteolysis, estrus and ovulation, and blood prostaglandin F after intramuscular administration of 15, 30 or 60 mg prostaglandin F2α

During diestrus in three consecutive estrous cycles, each of six heifers was given (im) 30 mg, 15 mg (twice at 6-hr intervals) and 60 mg prostaglandin F_2α (PGF_2α) tham salt. Neither the decline in blood progesterone, the increase in blood estradiol, the duration or the peak of the LH surge, the interval to onset of estrus, nor the interval to ovulation was affected significantly by dose of PGF_2α. Thus, relative to that after 30 mg PGF_2α im, two injections of 15 mg at 6-hr intervals or 60 mg PGF_2α did not hasten luteolysis. Thirty mg was an ample im dose of PGF_2α to cause luteolysis. Regardless of im dose of PGF_2α, blood PGF peaked at about 6.0 ng/ml within 10 minutes and returned to basal values (<1.0 ng/ml) within 90 minutes. In another trial, after a single iv injection of 5 mg PGF_2α, blood PGF peaked (25 ng/ml) within 5 minutes and returned to basal values within 15 minutes. During a 30-minute infusion (0.5 mg/minute) of PGF_2α, blood PGF plateaued at 29.5 ng/ml with a metabolic clearance rate of 17.0 liters per minute.     

Contractility of rat testicular seminiferous tubules in vitro: Prostaglandin F1α and indomethacin

The frequency of spontaneous in vitro contractions of seminiferous tubules of the rat appeared to be increased in a dose-dependent manner by prostaglandin F_1α. PGF_1α treatment increased the tonus of the smooth muscle cells in the wall of the tubules as indicated by a reduction in the diameter of the tubules. When the tubules were rinsed successively with fresh Tyrode's solution, the contractile frequency was diminished. Returning the original bathing medium to the tubules restored their contractile frequency, as did treatment of the rinsed tubules with PGF_1α (10⁻⁷ M). Pre-injecting the rats with indomethacin tended to reduce the contractile frequency of the extirpated tubules. Treating the tubules with a solution of indomethacin for 90 min. in vitro was more effective than pretreatment in vivo in reducing contractile frequency, but a combination of these two procedures produced the greatest inhibition. PGF_1α restored the contractile frequency of the indomethacin-treated tubules. Our results indicate that PGs modulate the in vitro contractility of the tubules.     

Effects of dibuturyl cAMP, LHRH, and aromatase inhibitor on simultaneous outputs of prostaglandin F2α, and 13,14-dihydro-15-keto-prostaglandin F2α by term placental explants

Explants from term human placentas were maintained in culture with daily changes of medium. Daily output of PGF_2α and PGFM¹ decreased during the course of the incubation. Addition of 4 μg/ml DHEAS or 67 μg/ml LDL cholesterol had no effect on output of PGF_2α or PGFM. Addition of 1.6, 3.2, or 6.4 μg/ml of LHRH to the culture plates had no effect on output of PGFM or PGF_2α, but LHRH increased hCG output. Dibutyryl cAMP (1mM, 2mM, and 4mM) increased output of PGF_2α, PGFM, and hCG. Aromatase inhibitor decreased hCG output, but it was without effect on output of PGF_2α, or PGFM. Significant correlations were demonstrated between progesterone, PGFM, PGF_2α, and hCG, suggesting that PGF_2α originates in the syncytiotrophoblast cell. The ability of LHRH to stimulate output of hCG but not PGF_2α while dbcAMP stimulated both suggests that either PGF_2α and hCG arise in different cells or that LHRH does not act through cAMP.     

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 in vitro to the maximum plasma level after a normal dose in vivo suggests that phenylbutazone and possibly ibuprofen may be capable of inhibiting human uterine contractions 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.     

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.     

Prostaglandin-induced enhancement of the in vitro anamenstic response

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

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.     

Effect of in vivo prostaglandin treatment on H-PGF2α uptake in hamster corpora lutea

Pregnant hamsters were administered (SC) prostaglandin or vehicle on the morning of the 4th day of pregnancy. Serum progesterone was significantly depressed (p<.01) at 0.5, 2, and 6 hours after treatment with 100 μg PGF_2α. Serum progesterone levels were unchanged 2 hours and 6 hours after treatment with 100 μg PGF_2β and 2 hours after treatment with 1 mg PGF_2β. Progesterone levels were depressed to less than 1 ng/ml 6 hours after treatment with 1 mg PGF_2β. The specific uptake of ³H-PGF_2α in whole hamster corpora lutea was significantly depressed 2 hours and 6 hours following 100 μg PGF_2α treatment. A 15% depression in specific uptake occurred 0.5 hour post-treatment. Treatment with 100 μg PGF_2β resulted in no change. Administration of 1 mg PGF_2β resulted in depressed ³H-PGF_2α uptake at both 2 and 6 hours post-treatment.Prostacyclin (PGI₂) treatment resulted in no change in either ³H-PGF_2α specific uptake or serum progesterone 2 hours after 100 μg treatment SC. These parameters were both reduced approximately 30% 6 hours post-treatment. Treatment with 6-keto-PGF_1α resulted in a complete lack of measurable ³H-PGF_2α uptake and serum progesterone levels less than 1 ng/ml at both 2 and 6 hours after treatment with 1 mg SC.     

Open-pulled straw vitrification differentiates cryotolerance of in vitro cultured rabbit embryos at the eight-cell stage

The objective was to determine cryotolerance of in vitro cultured rabbit embryos to the open-pulled straw (OPS) method. Overall, 844 rabbit embryos at pronuclear, 2- to 4-cell, 8-cell, and morula/blastocyst stages were vitrified, and ≥ 1 mo later, were sequentially warmed, rehydrated, and subjected to continuous culture (n = 691) or embryo transfer (ET, n = 153). Embryos vitrified at the 8-cell stage or beyond had greater survival, expanded blastocyst and hatched blastocyst rates in vitro, and better term development than those vitrified at earlier stages. The 8-cell group had 70.1% expanded blastocysts, 63.7% hatched blastocysts, and 25.7% term development, as compared to 1.5-17.7%, 1.5-4.3% and 2.8-3.7% in the pronuclear, 2-cell and 4-cell embryos, respectively (P < 0.05). The expanded and hatched blastocyst rates in vitrified morula/blastocyst post-warming were higher than that in the 8-cell group; however, their term development after ET was similar (8-cell vs morula/blastocyst: 25.7 vs 19.4%, P > 0.05). Development after ET was comparable between vitrified-warmed embryos and fresh controls at 8-cell and morula/blastocyst stages (19.4-25.7 vs 13.7-26.6%, P > 0.05). For embryos at pronuclear or 2- to 4-cell stages, however, term rates were lower in the vitrified-warmed (2.8-3.7%) than in fresh controls (28.6-35.6%, P < 0.05). Therefore, cultured rabbit embryos at various developmental stages had differential crytolerance. Under the present experimental conditions, the 8-cell stage appeared to be the critical point for acquiring cryotolerance. We inferred that for this OPS cryopreservation protocol, rabbit embryos should be vitrified no earlier than the 8-cell stage, and stage-specific protocols may be needed to maximize embryo survival after vitrification and re-warming.     

A comparison of the contractile activity fo PGD2 and PGF2α on human isolated bronchus

Prostaglandins may be implicated in the bronchoconstriction which occurs in asthma. Prostaglandins F_2α (PGF_2α and D₂ (PGD₂) have been reported to produce bronchoconstriction in asthmatic subjects in vivo and PGF_2α cotnracts human isolated airway smooth muscle. We examined the relative efficacy and potency of PGF_2α and PGD₂ on human bronchial spiral strips taken from 6 patients at thoracotomy. PGF_2α had greater efficacy than PGD₂. The mean % T_max (percentage of maximal contractile response) ± s.e. mean were 84 ± 7 and 54 ±7 respectively (P < 0.05). PGF_2α (mean pD₂ ± s.e. mean = 6.39 ± 0.6) tended to be more potent than PGD₂ (5.68 ± 0.2). Since, in vivo, PGD₂ has greater efficacy and potency than PGF_2α, our results suggest that the in vivo effect of these prostaglandins does not result solely from an action on airway muscle.