6-Keto-prostaglandin F1 alpha is not added to urine during passage through the rat urinary bladder in vivo

Studies were conducted to determine whether prostaglandins are added to the urine during its passage through the rat urinary bladder in vivo. Control rats and rats with chronic streptozotocin-induced diabetes were anesthetized with Inactin, 100 mg/kg i.p., and urine was collected simultaneously from both kidneys. Urine from the left kidney was collected directly from the renal pelvis via a ureteral cannula, while urine from the right kidney was collected via a cannula in the urinary bladder. Prostaglandins in the urine were measured by radioimmunoassay. No difference in urinary concentration or rate of excretion of 6-keto-PGF1 alpha or PGE2 was seen between ureteral urine and bladder urine from either normal or diabetic rats. The results of this study indicate that in vivo there is no intralumenal addition of either 6-keto-PGF1 alpha or PGE2 to the urine by the ureteral bladder of rats.     

Changes in urinary 6-keto-prostaglandin F1α excretion during pregnancy and labor

Urinary excretion of 6-keto-PGF_1α was measured by high pressure liquid chromatography and radioimmunoassay at various stages of pregnancy and labor. In the first trimester of pregnancy, urinary 6-keto-PGF_1α concentrations were nor different from those measured before pregnancy, but they showed a significant increase in the second trimester of pregnancy (p <0.001). The levels rose further in the third trimester, although this increase was not statistically significant when compared to levels obtained in the second trimester. There was no evidence for a significance change in 6-keto-PGF_1α excretion with the onset of labor. During well-established, progressive labor mean values of 6-keto-PGF_1α excretion were about twice as high as before the onset of labor, but the range of values during labor was so wide that there was no statistical difference with values obtained in the second half of pregnancy.It is concluded that the increase in the urinary excretion of 6-keto-PGF_1α occurs later in pregnancy than the increase in TXB₂ excretion and that labor at term is not associated with marked changes in 6-keto-PGF_1α excretion.     

Analysis of 6-keto-prostaglandin F1α in human urine: Age-specific differences

A radioimmunoassay (RIA) for the estimation of 6-keto-PGF_1α in human urine is described in detail. The RIA method was validated by direct comparison to gas chromatography-mass spectrometry. In adults and in one year old children basal excretion of 6-keto-PGF_1α was found to be lower than that reported for PGE₂ or PGF_2α. However, during the first week of life, significantly more 6-keto-PGF_1α was excreted. The very high levels of 6-keto-PGF_1α in urine seen on the third day of life seemed already to decrease during the first week of life. It is concluded that prostacyclin may have a major role for kidney function in the newborn, possibly by protecting the immature kidney from high levels of angiotensin II.     

A radioimmunoassay for 6-keto-prostaglandin F1α utilizing an antiserum against 6-methoxime-prostaglandin F1α: Application to study renal synthesis of prostacyclin

PGI₂ and 6-keto-PGF_1α were converted to 6-methoxime-PGF_1α (6-MeON-PGF_1α) by treatment with methoxyamine HCl in acetate buffer. The formed 6-MeON-PGF_1α was measured by radioimmunoassay. Antisera were raised in rabbits after immunization against 6-MeON-PGF_1α-BSA conjugate. Diluted 1:20.000 to bind 50% of the tracer (³H-6-MeON-PGF_1α, 100 Ci/mmol), the antiserum cross reacted 0.8% with PGE₂, 1% with PGF_2α and less than 0.2% with PGD₂, PGF_1α, PGF_2β and TXB₂. The radioimmunoassay was used to estimate release of PGI₂ and 6-keto-PGF_1α from chopped rabbit renal medulla and cortex incubated in Krebs-Ringer bicarbonate buffer (37°C, 30 min). The 6-keto-PGf_1α radioimmunoassay was validated in biological samples by mass fragmentography. The chopped medulla (n=5) released 38±9 ng/g/min and the cortex (n=5) 4.7±2.0 ng/g/min, while the release of immunoreactive PGE₂ (iPGE₂) and iPGF_2α was 171±26 and 74±13 ng/g/min from the medulla and 4.3±1.3 and 2.7±0.3 ng/g/min from the cortex, respectively. The results confirm previous findings, which indicate that in the renal medulla prostaglandin endoperoxides are mainly transformed to prostaglandins, while in the cortex transformation to PGI₂ seems to be of greater importance.     

Effects of indomethacin, prostaglandin E2, prostaglandin F2α and 6-keto-prostaglandin F1α on hatching of mouse blastocysts

The present study has been performed to investigate how PGs would participate the hatching process. Effects of indomethacin, an antagonist to PGs biosynthesis, on the hatching of mouse blastocysts were examined in vitro. Furthermore, it was studied that prostaglandin E₂ (PGE₂), prostaglandin F_2α (PGF_2α) or 6-keto-prostaglandin F_1α (6-keto-PGF_1α) were added to the culture media with indomethacin. (1) The hatching was inhibited by indomethacin yet the inhibition was reversible. (2) In the groups with indomethacin and PGE₂, no improvement was seen in the inhibition of hatching and the inhibition was irreversible. (3) In the groups with indomethacin and PGF_2α, inhibition of hatching was improved in comparison with the group with indomethacin. (4) In the groups with indomethacin and 6-keto-PGF_1α, no improvement was seen. The above results indicated that PGF_2α possibly had an accelerating effect on hatching and a high concentration of PGE₂ would exert cytotoxic effect on blastocysts.     

The origin of circulating 13,14-dihydro-15-keto-prostaglandin F2α during delivery

All uterine tissues as well as the fetal membranes and the placenta can form prostaglandins from endogenous precursors but it is not clear which of the tissues is the main site for the increase in PGF_2α production during human parturition. To examine this question, we measured plasma prostaglandin levels before and at intervals after expulsion of the fetus, placenta, and membranes. The concentration of PGFM at the beginning of the second stage of labor was significantly higher than before the onset of labor. Five minutes after the birth of the infant, the concentration had doubled. Thirty minutes after the expulsion of placenta and membranes, plasma PGFM had fallen to the levels at full dilatation; two hours postpartum it was still significantly raised over levels before labor. Since the halflife of PGFM in the circulation is about 7 minutes, these findings indicate that the uterine tissues are important sources of PGFM during labor. In contrast, endogenous oxytocin levels, which were significantly raised over control levels at the second stage of labor, did not change during the third stage, and decline postpartum to control levels. Oxytocin infusion did not influence PGFM levels at 5 and at 30 minutes postpartum, but raised them at 2 hours.     

Radioimmunoassays of prostaglandin F2α and prostaglandin F2α-main urinary metabolite with prostaglandin-I-tyrosine methylester amide

Radioimmunoassays for measuring prostaglandin F_2α (PGF_2α) and 5α, 7α-dihydroxy-11-keto tetranorprosta-1,16-dioic acid, PGF_2α-main urinary metabolite (PGF_2α-MUM), with ¹²⁵I-tyrosine methylester amide (TMA) of PGF_2α and PGF_2α-MUM were developed.Antibody to PGF_2α was produced in rabbits immunized with conjugates of PGF_2α coupled to bovine serum albumine. Antibody to PGF_2α-MUM was also produced in rabbits immunized with conjugates of PGF_2α-MUM coupled to bovine serum albumin.PGF_2α-¹²⁵I-TMA had an affinity to antiserum to PGF_2α. PGF_2α-MUM-¹²⁵I-TMA also responded to antiserum to PGF_2α-MUM.     

6 keto prostaglandin F1α plasma levels in aminonucleoside nephrosis in the rat

Levels of 6 keto Prostaglandin F_1α were measured by radioimmunoassay in rats with nephrotic syndrome induced by the intraperitoneal administration of aminonucleoside of puromycin. The plasma levels were found to be significantly elevated in nephrotic as compared to control rats (p < 0.01).     

Release of 6-keto-prostaglandin F1α and thromboxane B2 from mouse peritoneal macrophages during their adhesion and spreading on a glass surface

The release of 6-keto-prostaglandin F_1α (6KF_1α)_and of thromboxane B₂ (TXB₂) from cells were investigated using mouse peritoneal exudate cells (PECs) and non-cultured peritoneal macrophages. They were prepared by adhesion to glass dishes and incubated for 1 hr at 37°C in 5% Co₂ in air. Both the percentage of spreading macrophages and the release of 6KF_1α and TXb₂ increased in proportion to the incubation time. 6KF_1α and TXB₂ were released from the macrophages, not from the non-adherent cells. When PECs were incubated in silicon-coated glass dishes, the spreading of macrophages was hardly detected and lower amounts of 6KF_1α and TXB₂ were released from these cells compared with cells incubated in non-treated glass dishes. These findings suggest that adhesion with the correlated spreading of macrophages on glass dishes serve as a considerable physical factor for the release of 6KF_1α and TXB₂.     

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

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

Cardiovascular actions of prostaglandins F2α; 15-me-F2α; F1α; F2β and F1β in the cat

Prostaglandin F_2α (5μg/kg, i.v.) causes an increase in pulmonary arterial pressure, decrease in systemic arterial pressure, and reflex bradycardia in the anesthetized cat. The same dose of the 15-methyl analogue of PGF_2α produces the same triad of effects but of greater magnitude and duration. Although prostaglandins F_1α, F_2β and F_1β also cause the same cardiovascular effects as F_2α, there is a decrease in potency for all parameters measured, with PGF_2α>PGF_1α>PGF_2β>PGF_1β. When compared to the actions of PGF_2α in producing an increase in pulmonary arterial pressure, PGs F_1α, F_2β and F_1β were less potent by approximately 10, 100, and 1000 fold respectively.     

Synthesis of thromboxane B2 and 6-keto-prostaglandin F1α by bovine gastric mucosal and muscle microsomes

Bovine gastric mucosal and muscle microsomes synthesize prostaglandins and thromboxane B₂ (TXB₂) from arachidonic acid (AA). TXB₂ and 6-keto-prostaglandin F₁α (6-keto-PGF₁α) were the major products synthesized by pylorus, body, and cardiac region of the gastric mucosa. Gastric muscle mainly synthesized 6-keto-PGF₁α. TXB₂ and 6-keto-PGF₁α synthesis occurs at an appreciable rate from endogenous precursors but more rapidly with added arachidonate. Prostaglandins E₂, F₂α and D₂ were synthesized in smaller amounts under the conditions studied.     

Release of prostaglandin F2α during the bovine peripartal period

Progesterone, estrone and 15-keto-13,14-dihydro-PGF_2α levels were determined in the peripheral blood circulation during the peripartal period in 12 cows. Plasma concentrations of progesterone showed a gradual and continuous decrease during the last 60 days before parturition. This gradual decrease was followed by an abrupt decline in the progesterone concentration occurring 24–48 hours before delivery. The plasma levels of estrone started to increase about 30 days prior to parturition with high concentrations attained during the last days of pregnancy. After delivery the estrone content decreased to baseline levels. Increased levels of the PGF_2α metabolite were recorded 24–48 hours before parturition. These increased PGF_2α metabolite levels occurred before or in conjunction with prepartum luteolysis. Prostaglandin metabolite levels remained high during parturition and returned to baseline 10–20 days after delivery.     

Prostaglandin F2α (PGF2α) and prolactin secretion in rats

Plasma prolactin and F-prostaglandins (PGF) were measured in anesthetized male Sprague-Dawley rats before and at 15, 30, 45 and 60 minutes following i.v. injection of either PGF_2α (4 mg/kg), chlorpromazine, 1 mg/kg or chlorpromazine (1 mg/kg) after pretreatment with i.p. indomethacin (2 mg/kg). Following PGF_2α administration, plasma prolactin levels increased significantly only at 15 and 30 minutes in spite of extremely high PGF levels throughout 60 minutes. Besides the expected rise in plasma prolactin, chlorpromazine caused a transient but statistically significant increase in PGF. Indomethacin blocked the chlorpromazine-induced PGF rise but not prolactin increase. Animals stressed with ether anesthesia showed elevation of plasma prolactin, which was not blocked by indomethacin although PGF concentration fell. These results indicate that PGF_2α can stimulate prolactin release. This effect does not appear to be physiologic since very high PGF levels are required. Furthermore, blockade of prostaglandin synthesis by indomethacin does not prevent the release of prolactin in response to chlorpromazine or stress. Our findings do not support a possible role of PGFs as intermediaries in prolactin release. However, it is possible that PGFs may work through other mechanisms not investigated in our study.     

Disappearance of prostaglandins F2α and 15-methyl F2α from amniotic fluid as measured by radioimmunoassay

A sensitive and relatively specific radioimmunoassay for 15 (S) 15 methyl prostaglandin F_2α was used to determine the levels of the drug in amniotic fluid after it had been injected intra-amniotically for termination of second trimester pregnancy. The disappearance of the free acid (tham salt) and methyl ester of the prostaglandin analogue were similar. The results of this preliminary study suggest that the drug rapidly equilibrates in the fluid and this is followed by a slow removal from the amniotic sac. A comparison with a similar study with PGF_2α, revealed that the analogue had a longer half-life in the amniotic fluid.     

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.     

Effect of prostaglandin F3α on gastric mucosal injury by ethanol in rats: Comparison with prostaglandin F2α

In humans eicosapentaenoic acid can be converted to 3-series prostaglandins (PGF_3α, PGI₃, and PGE₃). Whether 3-series prostaglandins can protect the gastric mucosa from injury as effectively as their 2-series analogs is unknown. Therefore, we compared the protective effects of PGF_3α and PGF_2α against gross and microscopic gastric mucosal injury in rats. Animals received a subcutaneous injection of either PGF_3α or PGF_2α in doses raning from 0 (vehicle) to 16.8 μmol/kg and 30 min later they received intragastric administration of 1 ml of absolute ethanol. Whether mucosal injury was assessed 60 min or 5 min after ethanol, PGF_3α was significantly less protective against ethanol-induced damage than PGF_2α. These findings indicate that the presence of a third double bond in the prostaglandin F molecule between carbons 17 and 18 markedly reduces the protective effects of this prostaglandin on the gastric mucosa.     

Cross-Reactivity of Δ17-6-Keto-PGF1α with 6-Keto-PGF1α antibodies

The cross-reactivity of the PGI₃ metabolite, Δ17-6-keto-PGF_1α, with antibodies against 6-keto-PGF_1α for radioimmunoassays (RIA) has been investigated. Δ17-6-keto-PGF_1α was obtained either from commercial sources or after its purification from endothelial cells. In the latter case, primary cultured bovine aortic endothelial cells were incubated for 20 min at 37°C with 10 μM eicosapentaenoic acid (EPA) in the presence of 2 μM 13-hydroperoxy-octadecadienoic acid, an activator of the EPA cyclooxygenation, and the 6-keto-PGF_1α and Δ17-6keto-PGF_1α produced were separated by RP-HPLC. Then, cross-reactivities of the commercial and purified Δ17-6-keto-PGF_1α with 6-keto-PGF_1α antibodies were determined and found not to exceed 10%. In addition, the amounts of prostacyclin-related compounds detected by direct measurements in media of cells loaded with EPA were compared with those obtained after purification of 6-keto-PGF_1α. In accordance with the cross-reactivity data, we found that RIA in media mainly measured 6-keto-PGF_1α, the Δ17-6-keto-PGF_1α formed being undetected at 90%. It is concluded that 6-keto-PGF_1α antibodies generally used for RIA of 6-keto-PGF_1α are highly specific since they can discriminate a metabolite bearing an additional double bond such as the PGI₃ metabolite Δ17-6-keto-PGF_1α.     

Metabolism of prostaglandin F1α in the pulmonary circulation

³H_5,6-Prostaglandin F_1α is largely eliminated from the circulation during a single passage through the pulmonary vascular bed. Its elimination requires cellular uptake. The volume of distribution and the mean transit time of the radioactivity are greater than those of an intravascular marker, blue dextran. The lungs retain 25–30% of the radioactivity, most in the form of a metabolite less polar than PGF_1α itself. The pulmonary venous effluent contains the same metabolite along with some unmetabolized PGF_1α. The metabolite can be distinguished from prostaglandins of the A, B, D, E and F series. It is reduced on reaction with borohydride, but reduction does not yield PGF_1α. Na0H has no effect on the metabolite, a finding that rules out the presence of a β-hydroxy-ketone configuration of the ring structure. The chromatographic behavior of the metabolite and its reaction with borohydride are those expected of 9,11-hydroxy-15-ketoprostanoic acid.     

Metabolism of prostaglandin F1α in the pulmonary circulation

³H_5,6-Prostaglandin F_1α is largely eliminated from the circulation during a single passage through the pulmonary vascular bed. Its elimination requires cellular uptake. The volume of distribution and the mean transit time of the radioactivity are greater than those of an intravascular marker, blue dextran. The lungs retain 25–30% of the radioactivity, most in the form of a metabolite less polar than PGF_1α itself. The pulmonary venous effluent contains the same metabolite along with some unmetabolized PGF_1α. The metabolite can be distinguished from prostaglandins of the A, B, D, E and F series. It is reduced on reaction with borohydride, but reduction does not yield PGF_1α. NaOH has no effect on the metabolite, a finding that rules out the presence of a β-hydroxy-ketone configuration of the ring structure. The chromatographic behavior of the metabolite and its reaction with borohydride are those expected of 9,11-hydroxy-15-ketoprostanoic acid.