Receptor sites on human prostate tissue for prostaglandin F2 alpha.

The binding of high specific activity ³H-prostaglandin F₂ alpha to membranes derived from human prostate tissue is described. Binding is specific for PGF₂ alpha and is readily displaced by cold PGF₂ alpha. The influence of testosterone and lactogen on the binding of the prostaglandin is investigated. Testosterone appears to enhance binding while the binding observed in the presence of lactogen is difficult to interpret.     

A novel effect of indomethacin on prostaglandin F2 alpha synthesis and metabolism by the human prostate

Human prostate tissue has been found to be an excellent organ for synthesizing and metabolizing prostaglandin F₂ alpha. Employing ³H-arachidonic acid as substrate, tritium incorporation into PGF₂ alpha and three different metabolites, namely, 15-keto-PGF₂ alpha, 13, 14-dihydro-PGFP₂ alpha, and 13, 14-dihydro-15-keto-PGF₂ alpha, has been shown. The identity of these substances was corroborated by gas chromatography and chemical ionization mass spectrometry. Indomethacin has been widely reported as an inhibitor of PGF₂ alpha synthesis. In our experiments, upon adding indomethacin, slight to no inhibition of PGF₂ alpha synthesis was seen. Instead, we found a highly significant increase in the incorporation of the tritium of arachidonic acid into 15-keto-PGF₂ alpha. We interpret these findings as evidence that the indomethacin may block not PGF₂ alpha synthetase, but the Δ13-reductase, thus causing the accumulation of this enzyme's substrate, 15-keto-PGF₂ alpha.     

Measurement of prostaglandin F2α metabolites by radioimmunoassay

Antibodies against 15 keto PGF_2α and 13,14 dihydro 15 keto PGF_2α were produced in goats and rabbits using the appropriate prostaglandin protein conjugate. Tritium labeled 15-keto, and 13,14 dihydro 15-keto PGF_2α were prepared from ³H-PGF_2α. These antibodies and ³H-labeled compounds were used to develop radioimmunoassays for the respective F_2α metabolites. The antibodies had relatively little cross-reactivity (≤0.1%) with the parent F_2α molecule. Infusion of PGF_2α in monkeys increased 15-keto-h₂ levels 10–20 fold higher than PGF_2α in peripheral plasma. The levels of this metabolite were not altered detectably during clotting, indicating relatively slow rates of PGF_2α metabolism in vitro. These assays should be useful to follow release rates of exogenous prostaglandins from various formulations and delivery systems, and in vivo tissue synthesis of PGF_2α, where low levels preclude measuring the parent compound.     

Catabolism of prostaglandin F2α in rat ovary : Differences between ovarian and uterine tissues

The catabolism of prostaglandin F_2α(PGF_2α) in rat ovarian homogenate was studied comparing with uterine homogenate. Two kinds of metabolite were recognized by incubation of PGF_2α with ovarian or uterine homogenate; 13,14-dihydro-15-keto-PGF_2α(15KD-PGF_2α) and 13,14-dihydro-PGF_2α(13,14H₂-PGF_2α) in ovarian homogenate and 15-keto-PGF_2α(15K-PGF_2α) and 15KD-PGF_2α in uterine homogenate. Incubation of 15KD-PGF_2α with ovarian homogenate resulted in the formation of 13,14H₂-PGF_2α but incubation with uterine homogenate did not produce 13,14H₂-PGF_2α. 13,14H₂-PGF_2α was in accord with Rf value of a compound formed by reduction of 15KD-PGF_2α with sodium borohydride.     

Determination of cyclooxygenase products and prostaglandin metabolites using high-pressure liquid chromatography and radioimmunoassay.

A method is described for measurement of the cyclooxygenase products, thromboxane,prostacyclin, and prostaglandins (PG), and several prostaglandin metabolites. The procedure involves separation of the compounds by high-pressure liquid chromatography combined with identification and estimation by serologic analysis. These combined procedures have been used to identify and estimate five such products, PGE₂, PGE₁ PGF2α, PGF_1α, and 6-keto-PGF_1α, in the culture fluids of dog kidney cells stimulated by a tumor-promoting phorbol diester. The prostaglandin metabolites, 13,14-dihydro-15-keto-PGE₂, 13,14-dihydro-15-keto-PF2_2α, 13,14-dihydro-PGE₂, and 13,14-dihydro-PGF_2α, were not found in these culture fluids.     

Bronchopulmonary effects of prostaglandin F2α and three of its metabolites in the dog

The airway and lung dynamics of prostaglandin F_2α (PGF_2α) and three of its metabolites were examined in the spontaneously-ventilated, pentobarbital anesthetized dog. Changes in expiratory flow rate, tidal volume, respiration rate, lung resistance and dynamic lung compliance were evaluated and compared quantitatively. In a dose range of 0.3–3.0 μg/kg i.v., PGF_2α and its 13,14-dihydro metabolite were found to be exceptionally potent agents. This metabolite was approximately twice as potent as PGF_2α on most parameters studied. Two other metabolites, 15-keto-PGF_2α and 15-keto-13,14-dihydro-PGF_2α, were only slightly effective, even in a dose range of 1.0–30.0 μg/kg i.v. These latter two metabolites produced dose-response curves with significantly shallower slopes than PGF_2α and were shown to be at least thirty-five times less potent than the parent compound. Therefore, oxidation of PGF_2α at the carbon-15 position by 15-hydroxy prostaglandin dehydrogenase appears to produce compounds with minimal bronchopulmonary activity.     

Metabolism of arachidonic acid by caruncular and allantochorionic tissues in cows with retained fetal membranes (RFM)

The metabolism of arachidonic acid (AA) by caruncular and allantochorionic tissues and its regulation was studied in normal cows (n=13) and those with retained fetal membranes (RFM; n=9). Tissues were taken via the vagina about 6 hours postpartum and incubated for 6 hours in minimum essential medium containing tritiated AA alone or in the presence of oxytocin, platelet activating factor (PAF), epidermal growth factor (EGF) or ionophore calcium (A23187). The metabolites of AA were separated by reverse phase-high pressure-liquid chromatography. Tissue concentrations of prostaglandin F_2α (PGF_2α) and prostaglandin E₂ (PGE₂) and plasma 13,14-dihydro-15-keto-PGF_2α (PGFM) concentration were also measured by radioimmunoassay. For caruncular tissue, less thromboxane B₂ (TXB₂) and more 6-keto prostaglandin F_1α (PGIM) was synthesized in tissue from the animals with RFM than in the controls. Oxytocin, PAF, EGF and A23187 increased only PGIM production in the control animals; A23187 also decreased TBX₂ synthesis. For the allantochorion, more PGE₂, leukotriene B₄ (LTB₄) and PGIM and less TXB₂, PGF_2α and hydroxyecosatetranoic acids (HETE) was synthesized in tissue from cows with RFM than from animals that delivered normally. All of the substances used in this study increased PGIM, PGF_2α and LTB₄ and decreased TXB₂ production by the allantochorionic tissue in control animals. The metabolism of AA by the allantochorionic tissue seems quantitatively under hormonal control. The metabolism of AA at the level of both maternal and fetal components of the placenta in cows with RFM differed from that seen in animals that expelled the membranes normally.     

Transient cerebral ischemia and brain prostaglandins.

Prostaglandin levels were measured in gerbil brain during cerebral ischemia for up to 2 hours, and during reperfusion after various periods of transient ischemia. The levels of PGF_2α and its metabolite (13,14-dihydro-15-keto-PGF_2α) were low and did not change during ischemia. However, PGF_2α, 13,14-dihydro-15-keto-PGF_2α, PGE₂, and thromboxane B₂ increased during reperfusion after brief episodes of ischemia. Indomethacin or aspirin inhibited this increase. Animals pretreated with indomethacin recovered more rapidly and were more active during reperfusion than those without treatment.     

PRESENCE OF 15-HYDROXY PROSTAGLANDIN DEHYDROGENASE, PROSTAGLANDIN-Δ13-REDUCTASE AND PROSTAGLANDIN E-9-KETO(α)-REDUCTASE IN THE FROG SPINAL CORD

Metabolism of prostaglandin E₁ (PGE₁) and F_1α (PGF_1α) was studied in the frog spinal cord, using a hemisected preparation in vitro and tissue homogenates (whole honiogenate and tissue fractions). In the intact tissue, PGE, was converted to three Metabolites, 1 to 111, whereas only Metabolites 11 and 111 werc detected in experiments with PGF_1α. Work with tissue homogenatcs confirmed that PG transformation is enzymatic, and endproducts were identified as PGF_1α (Metabolite 1), 15-kcto metabolite (Metabolite 11) and 15-keto-13,14-dihydro metabolite (Metabolite 111). The 15-keto-13,14-dihydro metabolite was formed via the 15-keto metabolite which is consistent with findings elsewhere. These results establish the presence in the frog spinal cord of two pathways for PG metabolism, consisting one of the 15-hydroxy prostaglandin dehydrogenase (15-PGDH) and the prostaglandin-A13- reductase (13-PGR), the other of the prostaglandin E 9-keto(α)-reductase (9K-PGR). 9K-PGR is regarded as an inactivating enzyme because amphibian spinal neurons are less responsive to PGF, than to PGE₁. In the intact or in the homogenized tissue, PGE, is metabolized more efficiently by the 15-PGDH/13-PGR than by the 9K-PGR route. The 15-PGDH metabolizes PGE, more readily than PGF_1α. The present findings, together with the previous demonstration of active PG synthesis in the tissue and the potent actions of exogenous PGs, strongly suggest that the PGs play an important role in the function of neurons in the frog spinal cord.     

Measurement of 11-ketotetranor PGF metabolites: An approach for monitoring prostaglandin F2α release in the mare

The appearance and disappearance of 15-keto-13,14-dihydro-PGF_2α and 11-ketotetranor PGF metabolites have been measured in the blood and urine of the mare following i.v. injection of prostaglandin F_2α (PGF_2α). The basal plasma concentration of the 11-ketotetranor PGF metabolites was 10-fold greater than 15-keto-13,14-dihydro PGF_2α; after injection of PGF_2α, however, 15-keto-13,14-dihydro PGF_2α increased rapidly to concentrations exceeding those of the 11-ketotetranor PGF metabolites, which also increased but to a much lesser extent. The half-life for the disappearance of 15-keto-13,14-dihydro PGF_2α was about 30-fold shorter than that of the 11-ketotetranor PGF metabolites. Similar profiles were seen in the urine, except that the concentration of the 11-ketotetranor PGF metabolites was always greater than that of the 15-keto-13,14-dihydro PGF_2α. In the mare, the main plasma metabolite of PGF_2α appears to be 15-keto-13,14-dihydro PGF_2α, whereas the 11-ketotetranor PGF metabolites predominate in the urine.Similar patterns were seen for both types of metabolite in the plasma during luteolysis and early pregnancy. Because of the differences in rates of appearance and disappearance of these metabolites, measurement of both allows the detection of peaks of PGF_2α release in samples taken less frequently than is necessary when either type is measured alone.     

A radioimmunoassay for 13,14-dihydro-15-keto prostaglandin F2α with chromatography and internal recovery standard

Antibodies to the 13,14-dihydro-15-keto metabolite of prostaglandin F_2α(PGF_2αM) were raised in sheep using a bovine thyroglobulin conjugate of PGF_2αM. Labeled 13,14-dihydro-15-keto prostaglandin A₂ (PGA₂M), 13,14-dihydro-15-keto prostaglandin E₂ (PGE₂M) and PGF_2αM were prepared from their corresponding high specific activity parent prostaglandins with swine kidney homogenate and purified using reverse phase liquid-liquid partition chromatography. A rapid method of column chromatography for use prior to radioimmunnoassay was developed.Mathematical corrections for the effect of recovery tracer on the logit/log transformation are presented with a new approach to expression of radioimmunoassay cross reactions allowing continuous expression of the variation of these cross reactions with displacement. These mathematical approaches are widely applicable to other radioimmunoassay systems and transformations.The assay was used for measurement in groups of human volunteers: males, females, women at delivery and paired venous umbilical cord bloods. A correlation between venous cord and maternal peripheral PGF_2αM levels is demonstrated with a gradient from the cord plasma to maternal plasma.     

Responsiveness of the lamb ductus arteriosus to prostaglandins and their metabolites

The relative potencies of the prostaglandins A₁, A₂, E₁, E₂, F_2α and their 15-keto-, 15-keto-13,14-dihydro-, and 13,14-dihydro-metabolites were investigated on isolated lamb ductus arteriosus preparations contracted by exposure to elevated PO₂. All the prostaglandins (except PGF_2α and its 15-keto-metabolites) relaxed the tissue. However, only PGE₁, E₂, and their 13,14-dihydro-metabolites, were effective at concentrations below 10⁻⁸ M. Therefore, events that alter metabolism of circulating PGs in the perinatal period may have significant effects on the relative patency or closure of the ductus arteriosus.     

Differential release of prostaglandins by organ cultures of human fetal trachea and lung

Summary Human fetal lung at 16–19 weeks gestation has a partially differentiated epithelium, and in organ culture, distal airsacs dilate and the epithelium autodifferentiates to type I and II pneumatocytes, processes regulated by endogenous prostaglandin PGE₂. Human fetal trachea, at the same gestation, has a terminally differentiated mucociliary epithelium but after 4–6 d in organ culture, develops squamous metaplasia. Tracheal cultures restricted to 3 d have normal phase-contrast and light microscopy appearances and immunohistochemical reactivities (epithelium: cytokeratin 7,8,18; glutathione S-transferase pi-isozyme; epithelial membrane antigen and mesenchyme; desmin; vimentin). In human fetal trachea organ cultures, the predominant prostaglandins released are 6-keto-PGF_1α, PGF_2α, and PGE₂, a pattern similar to that previously described for human adult trachea and lung. In fetal lung cultures, 13,14-dihydro-15-keto-PGF_2α is the major prostaglandin released with lesser amounts of 13,14-dihydro-15-keto-PFG_2α, PGF_2α, PGE₂, and 6-keto-PGF_1α. Human fetal lungin vitro has the competence to self-differentiate, as early as 12 weeks gestation and presence of high levels in fetal lung of the inactive metabolite 13,14-dihydro-15-keto-PGE₂ relative to PGE₂ suggests that active prostaglandin catabolism may be one of the mechanisms to retard this stage of maturationin vivo by limiting PGE₂ availability. Surprisingly, the profile of prostaglandins released from fetal lung organ culture does not change to that of a mature lung with terminal differentiation of the epithelium, and this may indicate differences in the expression of key prostaglandin-metabolizing enzymes in developing human fetal lung in culture and within utero ontogeny.     

Patterns of prostaglandin synthesis and degradation in isolated superficial and proliferative colonic epithelial cells compared to residual colon

Prostaglandin (PG) synthesis and degradation were examined in different regions (epithelial versus non-epithelial structures) of the rat distal colon by both HPLC analysis of [¹⁴C] arachidonate (AA) metabolites and by specific radioimmunoassays. Intact isolated colonic epithelial cells synthesized mainly PGF₂α and TXA₂, as monitored from the formation of its stable degradation product TXB₂ (PGF_2α > TXB₂ > 6-keto-PGF_1α, the stable degradation product of PGI₂=PGD₂=PGE₂=13,14-dihydro-15-keto-PGF_2α). The profile of PG products of isolated surface epithelial cells was identical to that of proliferative epithelial cells. However, generation of PGs by surface epithelium was 2 to 3-fold higher than by proliferative cells both basally and in the presence of a maximal stimulating concentration (0.1 mM) of AA. The latter implied a greater synthetic capacity of surface epithelium, rather than differences due to endogenous AA availability. The major sites of PG synthesis in colon clearly resided in submucosal structures; the residual colon devoid of epithelial cells accounted for at least 99% of the total PGs produced by intact distal colon. The profile of AA metabolites formed by submucosal structures also differed markedly from that of the epithelium. The dominant submucosal product was PGE₂. PGE₂ and its degradation product 13,14-dihydro-15-keto-PGE₂ accounted for 63% of the PG products formed by submucosal structures (PGE₂ PGD₂ > 13,14-dihydro-15-keto-PGE₂ > PGF_2α=TXB₂=6-keto-PGF_1α > 13,14-dihydro- 15-keto-PGF_2α). By contrast, epithelial cells, and particularly surface epithelium, contributed disproportionately to the PG degradative capacity of colon, as assessed from the metabolism of either PGE₂ or PGF_2α. When expressed as a percentage, epithelial cells accounted for 71% of total colonic PGE₂ degradative capacity but only 23% of total colonic protein. Approximately 15% of [³H] PGE₂ added to the serosal side of everted colonic loops crossed to the mucosal side intact. Thus, at least a portion of the PGE₂ formed in the submucosa reaches, and thereby can potentially influence functions of the epithelium.     

Measurement of Prostaglandins in the Cerebrospinal Fluid in Cat, Dog, and Man

Prostaglandins are involved in the modulation of various central functions (neurotransmitters and hypothalamic hormone release, thermoregulation, cerebro-vascular tone) and their levels increase in pathological situations [subarachnoid hemorrhage (SAH), stroke, convulsive disorders, etc.]. This study, using sensitive and specific antibodies, examined levels of four eicosanoids, Prostaglandins E₂ and F_2α(PGE₂, PGF_2α); and the metabolites of PGI₂, 6-keto-prostaglandin F_1α (6-keto-PGF1_α) and of thromboxane A₂, thromboxane B₂ (TxB₂), in the cerebrospinal fluid (CSF) obtained atraumatically from three species (human, canine, and feline). An assessment of the methodologic procedures (extraction and radioimmunoassay) was carried out. Human lumbar cerebrospinal fluid was shown to contain PGF_2α (15–44 pg/ml), 6-keto-PGF_1α (undetectable to 39 pg/ml), and TxB₂ (un-detectable to 28 pg/ml), whereas PGE₂ was undetectable (>18 pg) in all cases. In both animals species the eico-sanoid concentrations were 3-to 30-fold higher than humans for every prostaglandin examined. Although the prostaglandin profile for a given species remained constant (cat, PGE₂:6-keto-PGF_1α:TxB₂:PGF_2α; dog, TxB₂:PGE₂:6-keto-PGF_1α:PGF_2α), the absolute levels were found to be lower in the pentobarbital-anesthetized animals than in conscious cats. The correspondence of the prostaglandin profiles found in cerebrospinal fluid with the profiles reported in the literature in brain homogenates for the same species supports the hypothesis that cerebrospinal fluid levels of prostaglandins reflect the relative rates of synthesis in neural tissue.     

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.     

Metabolism of prostaglandin D2 in isolated rat lung: the stereospecific formation of 9α, 11β-prostaglandin F2 from prostaglandin D2

The metabolic transformation of exogenous prostaglandin D₂ was investigated in isolated perfused rat lung. Dose-dependent formation (2–150 ng) of 9α,11β-prostaglandin F₂, corresponding to about 0.1% of the perfused dose of prostaglandinD₂, was observed by specific radioimmunoassay both in the perfusate and in lung tissue after a 5-min perfusion. To investigate the reason for this low conversion ratio, we analyzed the metabolites of tritium-labeled 9α,11β-prostaglandin F₂ and prostaglandin D₂ by boric acid-impregnated TLC and HPLC. By 5 min after the start of perfusion, 9α,11β-prostaglandin F₂ disappeared completely from the perfusate and the major product formed remained unchanged during the remainder of the 30-min perfusion. The major product was separated by TLC and identified as 13,14-dihydro-15-keto-9α,11β-prostaglandin F₂ by GC/MS. In contrast, pulmonary breakdown of prostaglandin D₂ was slow and two major metabolites in the perfusate increased with time, each representing 56% and 11% of the total radioactivity at the end of the perfusion. The major product (56%) was identified as 13,14-dihydro-15-ketoprostaglandin D₂ and the minor one (11%) was tentatively identified as 13,14-dihydro-15-keto-9α,11β-prostaglandin F₂ based on the results from radioimmunoassays, TLC, HPLC, and the time course of pulmonary breakdown. These results demonstrate that the metabolism of prostaglandin D₂ in rat lung involves at least two pathways, one by 15-hydroxyprostaglandin dehydrogenase and the other by 11-ketoreductase, and that the 9α,11β-prostaglandin F₂ formed is rapidly metabolized to 13,14-dihydro-15-keto-9α,11β-prostaglandin F₂.     

Synthesis and metabolism of prostaglandins E2, F2α and D2 by the rat gastrointestinal tract. Stimulation by a hypertonic environment in vitro

Whole cell preparations of rat stomach corpus, jejunum, and colon were incubated and the released prostaglandin E₂ (PGE₂), PGF_2α, PGD₂, 15 keto-13,14 dihydro PGE₂, and 15 keto-13,14 dihydro PGF_2α were measured by combined gas chromatography-mass spectrometry. All regions made PGD₂ and possessed a high capacity for producing 15 keto-13,14 dihydro derivatives of both PGE₂ and PGF_2α. Hypertonic sucrose solutions resulted in concentration-dependent increases in prostaglandin release, particularly of PGE₂ and its metabolite. It is suggested that PG's may play a role in the local effects of luminal hyperosomolarity on digestive tract functions.     

Maternal and fetal prostaglandin concentrations during late gestation in dairy cattle

A study was conducted to measure the blood plasma concentrations of prostaglandin F_2α (PGF_2α), 13,14-dihydro-15-keto-prostaglandin F_2α (PGFM), prostaglandin E₂ (PGE₂) and 13,14-dihydro-15-keto-prostaglandin E₂ (PGEM) in the jugular vein, umbilical vein and artery and uterine vein of 18 Holstein Friesian cows during late gestation. A caesarean section was performed on all cows before term in order to obtain blood samples from the different sources. Plasma PG concentrations in the uterine or fetal circulation were significantly higher than in jugular vein plasma. Correlations between peripheral PG metabolite concentrations and primary PG concentrations in the various sources of the uterus or fetus were not significant (r = .17 − .47) and demonstrated that prostaglandin values based upon peripheral blood alone are of limited value.     

Preparation of two dinor-PGI2 metabolites from 6-keto-PGF1α by mycobacterium rhodochrous

The transformation of 6-keto-PGF_1α to two prostacyclin metabolites, 2,3-dinor-6-keto-PGF_1α (I) and 2,3-dinor-6,15-diketo-13,14-dihydro-PGF_1α (II) by $\text{Mycobacterium rhodochrous}$ UC-6176 is described. The finding that the bacterium oxidized 6-keto-PGF_1α to the 6,15-diketo metabolite II shows that it contains 15-hydroxy prostaglandin dehydrogenase and Δ¹³ reductase enzyme systems.