The regulatory influence of prostaglandins on protein synthesis in the human non pregnant cervix

Cervical connective tissue was obtained from non pregnant fertile women undergoing hysterectomy. Tissue specimens were mechanically chopped into 1 mm thick slices which were preincubated in Krebs-Ringer bicarbonate buffer containing PGE₂ or PGF_2α (300 ng/ml). After 60 min the slices were transferred into fresh buffer with the corresponding PG-concentration and ³H-labelled proline or hydroxyproline. Following incubation (60 min) the protein bound radioactivity was determined and related either to the dry weight or to the protein content of each slice. The two methods used did not show any qualitative differences. The experiments showed that PG:s had a marked effect on protein synthesis in the cervical tissue. During the follicular phase incubation with both types of PG:s was followed by a decreased incorporation indicating decreased net synthesis of collagen while there was an increased incorporation and hence increased synthesis in the luteal phase. There was no significant influence on the distribution of the model amino acid ¹⁴C-AIB in the presence of PG:s neither in the follicular nor in the luteal phase. The present data point to an acute effect of PGE₂ and PGF_2α on cervical collagen metabolism and indicate furthermore that the process is steroid hormone dependent. It is concluded that these substances may exert their effect by modulating the incorporation of amino acids into protein.     

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.     

Facile method for preparation of 2,3-dinor-6-keto PGF1α, the major urinary metabolite of prostacyclin

We report a convenient and efficient method for the preparation of prostaglandin 2,3-dinor-6-keto-F_1α by incubating prostaglandin 6-keto-PGF_1α (6-keto-PGF_1α) with dispersed rat hepatocytes. Chromatographic separation revealed a single product from the hepatocyte metabolism of 6-keto-PGF_1α whose structure was positively confirmed by mass spectrometry as 2,3 dinor-6-keto-PGF_1α. This methods allowed for the preparation of high specific activity radioactive 2,3-dinor-6-keto-PGF_1α which can be utilized to determine the recovery of urinary dinor-6-keto-PGF_1α during extraction and separation of the compound for radioimmunoassay measurements, as well as deuterated 2,3-dinor-6-keto-PGF_1α which can be used as an internal standard in the gas chromatography-mass spectrometric assay of this compound.     

Defibrotide, by enhancing prostacyclin generation, prevents endothelin-1 induced contraction in human saphenous veins

Spirals of human saphenous veins (HSV), mounted in a 5 ml organ bath containing Krebs-Henseleit solution (37°C), when kept in contact with defibrotide (100–200 ug/ml) for 15 min, enhance (2 and 3 fold) their own basal release of 6-keto-PGF_1α (61 ± 1.3 pg/mg w.t. n = 12). The phenomenon was long lasting upon repeated washing and sensitive to indomethacin (1 ug/ml). Endothelin-1 (ET-1, 20–40 ng) induced a sustained contraction of HSV and concomitantly released from the venous tissue a proportional amount of 6-keto-PGF_1α.Indomethacin (1 ug/ml), by inhibiting cyclo-oxygenase enzyme, potentiated the contractile activity of ET-1 in HSV whereas exogenous PGE₂ (20 ng/ml) considerably reduced the tension developed by the peptide on this venous tissue.Defibrotide (200 ug/ml), by releasing 6-keto-PGF_1α, and other vasoactive prostaglandins, antagonized the contractile effect ET-1 (20 ng) in HSV. This data indicates that the eicosanoid metabolism is involved in the modulation of the potent vasoconstrictor effect of ET-1 in HSV and that PGI₂-releaser, such as defibrptide, may have therapeutical value against immoderate changes of venous tone.     

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.     

Effects of oestradiol, progesterone, hydrocortisone and oxytocin on prostaglandin output from the guinea-pig endometrium maintained in tissue culture

The effects of oestradiol, oxytocin, progesterone and hydrocortisone on prostaglandin (PG) output from guinea-pig endomerium, removed on days 7 and 15 of the oestrous cycle and maintained in tissue culture for 3 days, have been investigated. Oetradiol (3.7 to 3700nM) and oxytocin ( 2 to 200pM) did not stimulate endometrial PGF_2α output, thus not confirming the findings of a previous report (Leaver & Seawright, 1928), nor did they stimulate the outputs of PGE₂ and 6-keto-PGF_1α. In fact, oestradiol (3700nM) inhibited the outputs of PGF_2α, PGE₂ and, to a lesser extent, 6-keto-PGF_1α. Progesterone (3.2 to 3200nM) inhibited the outputsof PGF_2α and PGE₂; hydrocortisone (2.8 to 2800nM) had no effect on endometrial PG output. These findings indicate that the inhibitory effect of progesterone on endometrial PG synthesis and release in the guinea-pig is not due to progesterone having a glucocorticoid-like action. Furthermore, progesterone had no effect on 6-keto-PGF_1α output, suggesting that the mechanisms controlling endometrial PGI₂ synthesis (as reflected by measuring 6-keto-PGF_1α) are different from those controlling endometrial PGF_2α and PGE₂ synthesis.     

Assay of prostacyclin synthesis in intact aorta by aqueous sampling

Conversion of 1-¹⁴C-arachidonic acid (AA) to 6-keto-PGF_1α, the stable metabolite of prostacyclin (PGI₂) was assayed kinetically by employing an aqueous sampling technique. In this way, one can arrive at a kinetic view of PGI₂ synthesis from AA in intact tissue. The assay appears to be particularly suitable to tissues such as the aorta where PGI₂ constitutes the major metabolite of AA. The assay avoids the need for organic solvent extraction and relies on the essential absence of tissue binding of 6-keto-PGF_1α. The disappearance of AA can also be followed in this system but quantitation is complicated by avid tissue binding of the fatty acid. The assay, as described should be applicable to other vascular tissues and should greatly simplify kinetic analyses of prostacyclin synthesis.     

Differential production of PGF and 6-keto-PGF1α by the rat endometrium and myometrium in response to oxytocin, catecholamines and calcium ionophore

Uterine horns from castrated, estrogen-treated rats were superfused for 6 hours in 95% O₂/5% CO₂ at 37°C. The method of superfusion in which medium flows separately over the inner and outer surfaces of the horn allows prostaglandin synthesis in the myometrium and endometrium to be measured independently while their anatomical relationship is undisturbed. Prostaglandins were measured by radioimmunoassay. The myometrium formed more 6-keto-PGF_1α than PGF whereas the opposite was true of the endometrium. Production rates of TxB₂ in both tissues were relatively low. The addition of ionophore A-23187, oxytocin or phenylephrine to the superfusion medium not only increased the myometrial production rates of both 6-keto-PGF_1α and PGF but also increased the ratio 6-keto-PGF_1α:PGF. Neither ionophore nor phenylephrine affected the rate of prostaglandin synthesis in the endometrium whereas oxytocin caused a significant increase in the production rate of PGF. We conclude that the large amounts of 6-keto-PGF_1α in the myometrial superfusate probably originate in both the smooth-muscle cells of the myometrium and the endothelium of the myometrial blood vessels. The differential responses to ionophore A-23187, phenylephrine and oxytocin suggest differences in the mode of their regulation of prostaglandin synthesis.     

Effects of superovulation, arachidonic acid or endometrium on release of prostaglandins and synthesis of proteins by bovine trophoblast

This study was conducted in vitro to examine factors that may regulate prostaglandin release by bovine trophoblast and endometrial slices. Trophoblastic tissues and endometrial slices were recovered from superovulating and normally-ovulating cattle on day 16 or 20 of pregnancy and incubated for 24 h. Release of PGF_2^α and 13,14-dihydro-15-keto-PGF_2^α (PGMF), and incorporation of [¹⁴C]-leucine into proteins were quantified and expressed per μg DNA, which gives a measure of cellular activity. Activity of trophoblastic tissue for synthesizing protein was decreased (P<.05) and for releasing PGMF was increased (P<.05) on day 20 compared to day 16 of pregnancy. Neither supercovulation nor day of pregnancy altered trophoblastic activity for releasing PGF_2^α. Supercovulation increased (P<.05) endometrial release of PGF_2^α. Endometrial release of PGF_2^α was less (P<.05) on day 20 than on day 16 of pregnancy. When arachidonic acid (0, 100, 200 or 400 μg) was added at the start of incubation, trophoblastic release of PGF_2^α changed (P<.05) quadratically with dose of arachidonic acid. When arachidonic acid was added 8 h after the start of incubation, triphoblastic release of PGF_2^α increased linearly (P<.01) with dose of arachidonic acid. Adding arachidonic acid to incubation medium did not affect trophoblastic or endometrial protein synthesis. Endometrial slices suppressed (P<.05) trophoblastic protein synthesis and release of PGF_2^α. Apparently, endometrium can modulate trophoblastic release of prostaglandins and synthesis of proteins in vitro, and trophoblastic tissue from supercovulated cattle 16 or 20 days pregnant can be used to study trophoblastic synthesis of prostaglandins and proteins.     

Prostaglandins and the rat seminal vesicle: Effects of mating and adrenergic stimulation

The concentrations of PGE, PGF, and 6-keto-PGF_1α were increased in rat seminal vesicle tissue following mating activity. Likewise, synthesis of PGE and PGF was stimulated by epinephrine (3 × 10⁻⁷to 3 × 10⁻⁶ M) in tissues and media from incubations of intact rat seminal vesicles. The stimulation was inhibited by phentolamine, an α-adrenoreceptor blocking agent. Carbamylcholine (2 × 10⁻⁶ M) and bradykinin (1 × 10⁻⁶ M) had no effect on PGE or PGF synthesis, even though both compounds stimulated contractility of the rat seminal vesicle at these concentrations. These data suggest that mating and adrenergic stimulation increase prostaglandin synthesis in] the rat seminal vesicle, probably through an α-adrenergically mediated mechanism.     

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.     

Ascorbic acid promotes prostanoid release in human lung parenchyma

Ascorbic acid reduces airway reactivity to inhaled bronchoconstrictor agents in man and guinea pigs. The precise mechanism(s) responsible for this effect are unknown, but in both species an acute indomethacin treatment reverses the action of the ascorbic acid. To determine if ascorbic acid promotes prostanoid synthesis and/or inhibits degradation, human lung parenchymal slices (100–200mg) were incubated for 60 minutes in oxygenated Tyrode's solution alone or with sodium ascorbate (0.001M–1M) and/or methacholine (1μM–100μM) and/or indomethacin (0.17μM–17μM). Aliquots of the incubation medium were assayed by radioimmunoassay for PGE₂, PGF_2α, thromboxane B₂ and 6-keto-PGF_1α. Ascorbic acid increased the accumulation of all four prostanoids in the incubation medium, especially thromboxane B₂ and 6-keto-PGF_1α. This stimulatory effect of ascorbic acid was concentration-dependent and was inhibited by indomethacin. We conclude that ascorbic acid can alter prostanoid generation by human lung tissue and this effect may, in part, explain its antibronchoconstrictor activity in man.     

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.     

Studies of the mechanisms involved in the fate of prostacyclin (PGI2) and 06-keto-PGF1α in the pulmonary circulation

We have investigated the metabolism of [3]H-prostaglandin (PG)I₂ and its non-enzymatic breakdown product [3]H-6-keto-PGF_1α by rat pulmonary tissue and their possible uptake and metabolism upon passage through the isolated perfused rat lung. When incubated with rat lung homogenate in the presence of β-NAD, [3]H-PGI₂ was extensively degraded into at least one metabolite, while [3]H-6-keto-PGF_1α was only minimally metabolized. However, on passage through isolated perfused rat lungs, neither [3]H-PGI₂ nor [3]H-6-keto-PGF_1α were removed from the circulation into the lung or degraded. This demonstration that PGI₂ is not a substrate for the transport system for the removal of PGs from the circulation into the lung further illustrates that this system is a critical determinant for the pulmonary inactivation of circulating prostaglandins. The experimental findings are discussed in reference to the structure-activity requirements necessary for pulmonary transport and subsequent metabolism.     

Prostacyclin induces a surprising long-lasting motility in quiescent uterine strips (indomethacin-treated) isolated from ovariectomized rats

Dose-response curves for several prostaglandins (PGI₂; PGD₂; PGF₂ and PGE₂); BaCl₂ or prostaglandin metabolites (15-keto-PGF_2α; 13, 14-diOH-15-keto-PGF_2α; 6-keto-PGF_1α and 6-keto-PGE₁ in quiescent (indomethacin-treated) uterine strips from ovariectomized rats, were constructed. All PGs tested as well as BaCl₂, triggered at different concentrations, evident phasic contractions. Within the range of concentrations tested the portion of the curves for the metabolites of PGF_2α was shifted to the right of that for PGF_2α itself; the curve for 6-keto-PGF_1α was displaced to the right of the curve for PGI₂ and that for 6-keto-PGE₁ to the left.It was also demonstrated that the uterine motility elicited by 10⁻⁵ M PGF_2α and its metabolites was long lasting (more than 3 hours) and so it was the activity evoked by PGI₂; 6-keto-PGF_1α and BaCl₂, but not the contractions following 6-keto-PGE₁, which disappeared much earlier. The contractile tension after PGF_2α; 15-keto-PGF_2α; 13, 14-diOH-15-keto-PGF_2α and PGI₂, increased as time progressed whilst that evoked by 6-keto-PGF_2α or BaCl₂ fluctuated during the same period around more constant levels.The surprising sustained and gradually increasing contractile activity after a single dose of an unstable prostaglandin such as PGI₂, on the isolated rat uterus rendered quiescent by indomethacin, is discussed in terms of an effect associated to its transformation into more stable metabolites (6-keto-PGF_1α, or another not tested) or as a consequence of a factor which might protects prostacyclin from inactivation.     

Prostaglandin profiles in nervous tissue and blood vessels of the brain of various animals

The endogenous formation of prostaglandin (PG) D₂, E₂, F_2α, and 6-keto-PGF_1α was determined in homogenates of mouse, rat, and rabbit brain, and of rat cerebral blood vessels, using gas chromatography mass spectrometry. In all species tested, 6-keto-PGF_1α could be identified in the brain homogenates, but was a minor component in relation to other PGs. In contrast 6-keto-PGF_1α was the most abundant PG in the blood vessels, being present in about 40-fold higher levels than in the brain tissue. PGD₂ was the most abundant PG in rat and mouse brains, but was below detection limits in the analyzed blood vessels. These studies indicating differential metabolism of PG endoperoxides in nervous and vascular tissue, provide a biochemical basis for further studies on the role of the PGs in brain circulation and neuronal activity.     

Formation of 6-keto-PGF1α by collecting tubule cells isolated from rabbit renal papillae

Homogeneous populations of collecting tubule epithelial cells have been isolated from rabbit renal papillae by a sequence of procedures involving: (a) dissociation of the tissue by mincing and treatment with trypsin; (b) destruction of contaminating non-collecting tubule cells by differential lysis in hypotonic media and (c) collection and washing by repeated centrifugation. The isolated cells have been characterized as being derived from the collecting tubules on the basis of anatomical source, size and histological staining for both NADH diaphorase activity and cyclooxygenase antigenicity. The cells are judged to be viable by several criteria including their ability to exclude both trypsin and vital dyes, their capacity to metabolize glucose and leucine and their ability to retain distinctive morphology following 10–14 days in culture media. Homogenates of freshly isolated collecting tubule cells when incubated with [³H]-arachidonic acid yielded radioactive products identified by thin-layer chromatographic behavior in multiple solvent systems as 6-keto-PGF_1α, PGF_2α, PGE₂, PGD₂ and a monohydroxy acid, probably HHT. No lipoxygenase-like activity was detected. At arachidonate concentrations of 2 or less, the major product was 6-keto-PGF_1α; while at substrate concentrations of greater than 10 , PGE₂ was the major radioactive prostaglandin formed. Similar distributions of products were observed when homogenates of dissociated renal papillae enriched in medullary interstitial cells were incubated with arachidonic acid. Our results indicate that collecting tubule cells do contain significant prostacyclin synthetase activity and suggest that PGI₂ plays a role in the function of mammalian collecting tubules.     

Estrogen increases male rat aortic endothelial cell (RAEC) PGI2 release

Estrogen has been proposed as a negative risk factor for development of peripheral vascular disease yet mechanisms of this protection are not known. This study examines the hypothesis that estrogen stimulates rat aortic endothelial cell (RAEC) release of PGI₂. Male Sprague-Dawley rat abdominal aortic 1-mm rings were placed on 35 mm matrigel plates, and incubated for 1 week. The cells were transferred to a Primaria 60-mm dish and maintained from passage 3 in RAEC complete media and experiments performed between passages 4–10. Cells were incubated with Krebs-Henseleit buffer (pH 7.4) containing carrier or increasing concentrations of β-estradiol or testosterone for 60 min. The effluent was analyzed for eicosanoid release of 6-keto-PGF_1α (6-keto, PGI₂ metabolite), PGE₂ and thromboxane B₂ (TXB₂) by EIA (hormone stimulated — basal). Cells were analyzed for total protein by the Bradford method and for cyclooxygenase-1 (COX-1) and prostacyclin synthase (PS) content by Western blot analysis and densitometry. Testosterone did not alter RAEC 6-keto-PGF_1α release, whereas estrogen increased RAEC 6-keto-PGF_1α release in a dose-related manner. Estrogen preincubation (10 ng/ml) decreased COX-1 and PS content by 40% suggesting that the estrogen-induced increase in male RAEC PGI₂ release was not due to increased synthesis of COX-1 or PS. These data support the hypothesis that estrogen stimulation can increase endogenous male RAEC release of PGI₂.     

Prostaglandin synthesis and metabolism in isolated pancreatic islets of the rat

Isolated pancreatic islets of Langerhans of the rat which were sonicated and incubated with radiolabeled arachidonic acid for 1 hr synthesized several species of prostaglandins (PGs). Both thin-layer and high-performance liquid (HPLC) chromatographic techniques demonstrated the synthesis by islet sonicates of PGF_2α and PGE₂ equivalents, in addition to the 15-keto-13, 14-dihydro metabolites of these primary PGs. In addition, HPLC allowed the identification of 6-keto-PGF_1α (the metabolite of prostacyclin) as a major PG synthesized from arachidonate by this tissue. Islet vascular elements, as well as endocrine cells, may contribute to the synthesis of the latter compound. Lesser amounts of arachidonate were incorporated into PG-like compounds eluting as thromboxane. The synthesis of PGs was sensitive to the protein concentration of islet sonicate, and a five-fold dilution of protein resulted in a comparable reduction in arachidonate incorporation into PGs. Labeled arachidonate was also incorporated into compounds which elute as hydroxy or hydroperoxy-eicosatetrainoic acids on HPLC. Thus, isolated pancreatic islets synthesize a variety of PGs which may have a physiological role in hormone secretion form this endocrine organ.     

Metabolism of arachidonic acid in vitro by ovine conceptuses recovered during early pregnancy

Metabolism of [³H] arachidonic acid ([³H] AA) and synthesis of prostaglandins were examined with ovine conceptuses and endometrial slices collected on various days after mating. Tissues were incubated for 24 hr with or without 5 μCi of [³H] AA and with 200 μg radioinert AA. In experiment 1, results of chromatography indicated that conceptuses collected on days 14 and 16 after mating metabolized [³H] AA to PGE₂, PGF_2α, PGFM, 6-keto-PGF_1α, and to unidentified compounds in three chromatographic regions. One of these regions (region 1) contained triglycerides. Endometrial slices metabolized only small amounts of the [³H] AA to prostaglandins. In experiment 2, results of radioimmunoassays indicated that day 14 conceptuses released somewhat similar amounts (ng/mg tissue) of PGF_2α (32.1 ± 17.9), PGFM (8.4 ± 6.2), PGE₂ (12.3 ± 7.5) and 6-keto-PGF_1α (41.4± 4.8), whereas day 16 conceptuses released more (P<.05) PGF_2α (9.0 ± 4.1) and 6-keto-PGF_1α (15.9 ± 2.7) than PGE₂ (0.9 ± 0.2) or PGFM (0.5 ± 0.08). Day 14 and 16 endometrial slices released (ng/mg tissue) more (P<.05) PGFM (3.0 ± 0.2) and 6-keto-PGF_1α (4.0 ± 0.4) than PGF_2α (0.5 ± 0.08) or PGE₂ (0.05 ± 0.02). In experiment 3, conceptuses were recovered on days 16, 20 and 24 of pregnancy and incubated with [³H] AA to determine the effects of indomethacin on [³H] AA metabolism. In general, indomethacin (Id; 4 × 10⁻⁴ M) reduced (P<.05) the percentage of total dpm recovered as prostaglandins, but Id increased the release of chromatographic region I. Experiment 4 was conducted with day 16, 20 and 24 conceptuses to evaluate the time course of metabolism of [³H] AA, and the appearance of region I and of prostaglandins. In general, the percentage of total dpm in region I increased as the percentage of dpm as [³H] AA decreased. The percentage of dpm as prostaglandins increased as the percentage of dpm in region I decreased. Prostaglandins, probably essential for embroynal survival and development, were synthesized in vitro by ovine conceptuses.