The use of tritiated prostaglandins in metabolism studies. I: Evaluation of the kinetic isotope effect in the prostaglandin dehydrogenase reactions

Although numerous data exist concerning tritium kinetic isotope effect in enzymic reactions, little is related to the metabolism of tritiated prostaglandins. The present study reports an evaluation of the kinetic isotope effect which occurs during the oxidation of 15-hydroxyl group of tritium-labeled prostaglandins E2 and F2 alpha by the 15-hydroxyprostaglandin dehydrogenase and during the oxidation of 9-hydroxyl group of tritium-labeled prostaglandin F2 alpha by the 9-hydroxyprostaglandin dehydrogenase. The large kinetic isotope effect tends to limit the validity of the dehydrogenase assay using tritium-labeled prostaglandins as substrate. However these assays can be considered to be an indication of relative enzyme activity.     

The use of tritiated prostaglandins in metabolism studies. II: Kinetic isotope effect: an useful tool to investigate catabolizing sequence of prostaglandins

It is well established that prostaglandin catabolism involves sequential actions of a 15-hydroxyprostaglandin dehydrogenase, a 15-keto-prostaglandin delta 13-reductase and a 15-ketoprostaglandin reductase. This pathway must be confirmed in never investigated tissues before any enzyme assay is carried out. We have developed a new, simple, rapid and reliable method to investigate catabolizing sequence of prostaglandins based on the tritium kinetic isotope effect which occurs during the oxidation of the 15-hydroxyl group of the prostaglandin into a 15-keto group.     

Prostaglandin-sepharose: Application to the purification of bovine lung 15 (S) -hydroxyprostaglandin dehydrogenase

Prostaglandins E₁, F_1α and A₂ were covalently joined to the surface of Sepharose as carboxamide linkages. The insolubilized prostaglandins were shown to function effectively in the purification of 15(S) -hydroxyprostaglandin dehydrogenase.     

Detection of 15-hydroxyprostaglandin dehydrogenase in bovine mesenteric blood vessels

Two types of 15-hydroxyprostaglandin dehydrogenase (NAD⁺ and NADP⁺ dependent) were demonstrated in bovine mesentric arteries and veins. The 15-hydroxyprostaglandin dehydrogenase activity was found in the high-speed supernatant, suggesting that these enzymes are associated with the cytoplasmic fraction of the blood vessels. The levels of activities of both NAD⁺- and NADP⁺-dependent dehydrogenases were similar in mesentric blood vessels. Prostaglandin F_2α was preferred to the prostaglandin E₂ as subtrate by both NAD⁺ and NADP⁺ dependent enzymes. The presence of 15-hydroxyprostaglandin dehydrogenase in blood vessels may play a siginificant role in the regulation of intracellular levels of prostaglandins of the E and F series in blood vessels.     

An NADP-linked prostacyclin dehydrogenase in rabbit kidney

An NADP-linked 15-hydroxyprostaglandin dehydrogenase specific for prostacyclin was purified 1,300-fold from rabbit kidney. Prostaglandins E₂, F_2α, and 6-Keto PGF_1α and thromboxane B₂ were oxidized by the purified enzyme with rates of reaction less than 4% that of PGI₂. Unlike other rabbit kidney NADP-linked 15-hydroxyprostaglandin dehydrogenases, this enzyme catalyzes oxido reduction more rapidly at the 15- position than at the 9- position and does not utilise NAD as a cofactor. It has a molecular weight of 62,000 and migrates on polyacrylamide disc gel electrophoresis as a single diffuse band. The reaction product was identified by thin-layer chromatography as 6,15-diketo PGF_1α. Prostacyclin dhydrogenase is the first 15-hydroxyprostaglandin dehydrogenase described which is specific for the metabolism of prostacyclin.     

Comparison of substrate specificities of the human placental NAD- and NADP-linked 15-hydroxyprostaglandin dehydrogenases

A study of the relative activity of the purified placental NAD- and NADP-linked 15-hydroxyprostaglandin dehydrogenases with various prostaglandins and thromboxane B₂(TxB₂) suggests that most, if not all, oxidation in the placenta of the 15-hydroxyl group of prostaglandins of the A, E, and F series as well as PGI₂ (prostacyclin) and 6-keto PGF_1α is catalyzed by the NAD-linked enzyme. Prostaglandin B₁ is an excellent substrate for the NADP-linked enzyme. Despite the conformational similarities between PGB₁ and PGI₂, the latter molecule is a poor substrate for the NADP-linked enzyme. Thromboxane B₂ is not oxidized by the NAD-linked enzyme and is oxidized slowly by the NADP-linked enzyme.     

The influence of prostaglandins on sperm motility

Prostaglandin E₂ and F_2α were measured in ejaculates from 10 fertile and 55 infertile men. Prostaglandin F_2α was negatively correlated with motility (r=0.77; p<0.01) in normal men. In patients with disturbed fertility, prostaglandin F_2α was always higher than in the controls, while prostaglandin E₂ was elevated only in patients with persisting varicocele and in those with very low sperm counts and severely impaired motility. There was neither synthesis of prostaglandins in spermatozoa nor were binding sites for prostaglandin E₂ and F_2α detectable. Inactivation of seminal prostaglandins by incubation with prostaglandin 15-hydroxydehydrogenase resulted in a dramatic fall in motility. The results suggest that prostaglandin F_2α act on motility, but the action is not mediated by receptors.     

Partial purification of human placental 15-hydroxyprostaglandin dehydrogenase: Kinetic properties

The enzyme system, 15-hydroxyprostaglandin dehydrogenase, which catalyzes the first inactivation step in the catabolism of the prostaglandins has been isolated and purified 107-fold from human placenta. Kinetic studies reveal different Michaelis-Menten constants for most of the naturally occurring prostaglandins. The K_m for PGE₂ was found to be 10 μM, for PGE₁, 27 μM; for PGA₂, 32 μM; for PGA₁, 33 μM; and for PGF_2α 59 μM. The enzyme has a sharp pH-optimum between 7.5 and 8.8. Prostaglandin dehydrogenase appears to be isoenzymic as judged by separation on polyacrylamide disc gel electrophoresis. Inhibition studies with the partially purified enzyme indicate that progesterone and estrogen may influence the conversion of biologically active prostaglandins into the biologically inactive 15-ketoprostaglandins. These findings offer evidence for the control of prostaglandin metabolism in the human placenta.     

Basal level of human platelet prostaglandins: PGE1 is more elevated than PGE2

Radioimmunoassays of platelet prostaglandins E₁ and F_1α in platelet rich plasma or platelet suspension, demonstrate that both PGE₁ and PGF_1α are present at higher concentrations than prostaglandins E₂ and F_2α. Gas chromatography — mass spectrometry determinations of prostaglandins E₁ and E₂ in resting washed platelets confirm this difference. Lastly, there is a greater incorporation of [1-¹⁴C] acetate into prostaglandins E₁ and F_1α compared to that into prostaglandins E₂ and F_2α.     

Fetal-maternal secretion of prostaglandins in the cow

A study was conducted to measure the blood plasma concentrations of prostaglandin F₂α (PGF₂α), 13,14-dihydro-15-keto-prostaglandin F (PGFM), 6-keto-prostaglandin F₁α (6-keto), prostaglandin E₂ (PGE₂), and thromboxane B₂ (TBX₂) in the ovarian vein, uterine artery, uterine vein, umbilical artery and umbilical vein in 24 cows from days 80 to 260 of pregnancy. Blood was collected during surgery and all prostaglandins were measured using specific radioimmunoassay procedures. Results indicate that PGF₂α blood levels are higher in the umbilical vessels and uterine vein than in the ovarian vein and uterine artery. PGFM and PGE₂ showed a trend towards higher values in the umbilical than in the maternal vessels, but the levels of 6-keto and TBX₂ were not different among the vessels studied. No differences across time couls be observed in any of the prostaglandins measured, partly due to the great variability in blood levels among animals during the same stage of pregnancy.     

metabolism of prostaglandins of the A-type

, originally introduced as an inadvertent contaminant in solutions used for evaluating the stability of prostaglandins, proved to lead to the rapid disappearance of the cyclopentenone unit of PGA₂ (as monitored by circular dichroic spectroscopy). The cyclopentenone unit is converted, in various metabolites, to a 9-keto, 9α or 9β-hydroxy group lacking the ring unsaturation. The major EtoAc-soluble 9-hydroxy metabolite (Compound-I) was shown to be 9α, 15α-dihydroxy-2,3,4,5-tetranor-13- -prostenoic acid. Similar tetranor 9-hydroxy metabolites with one additional degree of unsaturation, and with a 9β-hydroxy group, also occur but these have not been fully characterized. Only two of the wide range of 9-keto metabolites are fully characterized by mass spectral (MS) data: 9,15-oxo-2,3,4,5-tetranorprostanoic acid and 9,15-oxo-2,3,4,5-tetranor-13- -prostenoic acid. The water soluble metabolites have not been characterized further.The fully characterized metabolites together with MS data from mixtures of minor metabolites indicate that can perform the following transformations: β-oxidation, dehydrogenation at C-15, reduction of the enone carbon-carbon double bonds (both Δ^10,11 and Δ^13,14), reduction of the 9-ketone, and possibly migration of the cyclopentyl double bond (Δ^10,11 → Δ^11,12). metabolizes 15-epimeric PGA₂ equally readily with the production of similar products. PGA₁ affords less 9-keto metabolites with compound I constituting 33% of the product by HPLC analysis. displays some enantioselectivity, PGA₂ and 15-epi-PGA₂ are each metabolized more rapidly than their enantiomers. Other prostaglandins appear to be less readily metabolized.     

Structure-activity relationships in a series of 11-deoxy prostaglandins

A series of 11-deoxy prostaglandin derivatives and some naturally occurring prostaglandins have been investigated in the anaesthetized artificially respired guinea-pig for their effect on blood pressure, bronchial resistance (overflow pressure at constant volume), tracheal segment pressure, and on intestinal and uterine smooth muscle. The compounds were administered intravenously. Prostaglandins E₁, E₂ and F_2α produced responses that were qualitatively similar to those in the literature. Prostaglandin A₂ (100 μg) was a bronchoconstrictor, although it decreased tracheal segment pressure and blood pressure. Prostaglandin B₂ (100 μg) caused double elevations in blood pressure, tracheal segment pressure and bronchial resistance. The intensity of bronchoconstriction produced by PGB₂ was of the same order as with PGF_2α. A number of structure-activity relationships were found. 11-Deoxygenation lowered the biological activity of the natural prostaglandins PGE₁ and PGF_1α. The vasodepressor and bronchodilator responses of 11-deoxy PGE₁ were converted to vasopressor and bronchoconstrictor by epimerisation at C-15. Introduction of a methyl group at C-15 of 11-deoxy PGF_1α both increased and prolonged vasopressor and bronchoconstrictor activity. At C-9 both the keto and β-hydroxy group imparted vasodepressor and bronchodilator activity, while the α-hydroxy led to vasopressor and bronchoconstrictor activity. Extension of the omega sidechain by two methylene groups radically reduced the activity of 11-deoxy PGF_1α and its derivatives.These experiments indicate that steric differences in the prostaglandin structures studied can result in diametrically opposed profiles of biological activity. Further, small variations in the prostaglandin molecule can lead to differences in potency and/or profile of activity in the guinea-pig.     

Characterization of two enzyme proteins catalyzing NADP+/NADPH-dependent oxidoreduction of prostaglandins at C-9 and C-15 from swine kidney

Two proteins (pI 4.8 and 5.8) capable of catalyzing NADP⁺/NADPH-dependent oxidoreduction of prostaglandins at C-9 and C-15 but not at C-11 have been purified to homogeneity from swine kidney. Both proteins exhibited identical molecular weight and subunit size. Similar amino acid composition, antigenic determinants, and coenzyme and substrate specificity were also found. The molecular weight of the enzyme as determined by gel filtration was 29,000. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gel gave a value of 29,500 indicating the presence of a single polypeptide chain. Either enzyme protein utilized a variety of prostaglandins (PGS) as substrates. PGA₁-glutathione conjugate and PGB₁ were found to be the best substrates for prostaglandin 9-ketoreductase and 15-hydroxyprostaglandin dehydrogenase activities, respectively. For prostaglandins having dual reactive groups in a single molecule, the rate of oxidation of PGF_2α at C-15 was comparable to that at C-9, whereas the rate of reduction of 15-keto-PGE₂ at C-15 was far greater than that at C-9.     

Analysis of metabolic profiles of prostaglandins in urine using a lipophilic anion exchanger

A method is described for the fractionation of prostaglandins and their metabolites in urine. Following acidification and extraction on Amberlite XAD-2, samples were separated by chromatography on the lipophilic anion exchanger diethyl-aminohydroxypropyl Sephadex LH-20 into fractions containing neutral compounds, monocarboxylic, dicarboxylic and polycarboxylic acids. The compounds in resulting fractions were further separated by reversed phase partition chromatography. As an application, the metabolic profiles in urine of [9β-^3H]-labeled prostaglandin F_2α¹ and prostaglandin analogs 15-methyl-PGF_2α and 16,16-dimethyl-PGF_2α were investigated in the cynomolgus monkey. It was demonstrated that the resolution of individual prostaglandin metabolites by reversed phase partition chromatography was considerably simplified by initial group separation on the anion exchanger, and several metabolites were much purified. A glucuronic acid conjugate of the main metabolite of 15-methyl-PGF_2α (dinor-15-methyl-PGF_2α) was tentatively identified using computerized gas chromatography - mass spectrometry.     

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.     

A Novel Transporter of SLC22 Family Specifically Transports Prostaglandins and Co-localizes with 15-Hydroxyprostaglandin Dehydrogenase in Renal Proximal Tubules

We identified a novel prostaglandin (PG)-specific organic anion transporter (OAT) in the OAT group of the SLC22 family. The transporter designated OAT-PG from mouse kidney exhibited Na⁺-independent and saturable transport of PGE₂ when expressed in a proximal tubule cell line (S₂). Unusual for OAT members, OAT-PG showed narrow substrate selectivity and high affinity for a specific subset of PGs, including PGE₂, PGF_2α, and PGD₂. Similar to PGE₂ receptor and PGT, a structurally distinct PG transporter, OAT-PG requires for its substrates an α-carboxyl group, with a double bond between C13 and C14 as well as a (S)-hydroxyl group at C15. Unlike the PGE₂ receptor, however, the hydroxyl group at C11 in a cyclopentane ring is not essential for OAT-PG substrates. Addition of a hydroxyl group at C19 or C20 impairs the interaction with OAT-PG, whereas an ethyl group at C20 enhances the interaction, suggesting the importance of hydrophobicity around the ω-tail tip forming a “hydrophobic core” accompanied by a negative charge, which is essential for substrates of OAT members. OAT-PG-mediated transport is concentrative in nature, although OAT-PG mediates both facilitative and exchange transport. OAT-PG is kidney-specific and localized on the basolateral membrane of proximal tubules where a PG-inactivating enzyme, 15-hydroxyprostaglandin dehydrogenase, is expressed. Because of the fact that 15-keto-PGE₂, the metabolite of PGE₂ produced by 15-hydroxyprostaglandin dehydrogenase, is not a substrate of OAT-PG, the transport-metabolism coupling would make unidirectional PGE₂ transport more efficient. By removing extracellular PGE₂, OAT-PG is proposed to be involved in the local PGE₂ clearance and metabolism for the inactivation of PG signals in the kidney cortex.     

Biosynthesis of prostaglandins in human ovarian tissues

Experiments in vitro demonstrate, that there are different patterns of PG-biosynthesis in the corpus luteum and in the follicles containing cortical substance of the human ovary. In the follicles 6-keto-F₁α. the transformation product of prostacyclin, is the main fraction; prostaglandins F₂α and E 2 being of inferior importance with regard to their amounts. The formation of the corpus luteum is in close correlation with a strongly increased prostaglandin E₂ and a diminished prostacyclin biosynthesis; prostaglandin F₂α hardly seems to be involved in this process.By means of indomethacin the formation of all three examined prostaglandins can be prevented almost completely, in the cortical substance as well as in the corpus luteum. LH (or HCG) at concentrations ranging from 2 ng to 20 μg per ml homogenate produce no stimulating effect of statistical significance on the rate of biosynthesis in both tissues.     

The effects of prostaglandins on the beating activity of cultured heart cells

The effects on the beating behavior of cultured rat heart cells of fourteen prostaglandins of the A, B, D, E, and F series were investigated, together with adrenaline and ouabain, at dose levels of 10⁻⁷ and 10⁻⁵M. Single heart cell beating activity was monitored photo-electrically and five parameters of beating behavior measured. Only PGF_2a markedly increased rate while PGF_2B reduced it. Maintenance of a stable rate (rate range) was minimally affected by prostaglandins with PGF_2β possibly reducing, and PGF_1α and 2-decarboxy E₁ possibly increasing, rate range. PGF_1α and F_2α both statistically reduced the percentage of cells beating while the other prostaglandins had no effect. Most of the prostaglandins either produced no change, or reduced, indices of contractile force (optical density changes with contractions and its first derivative (dOD/dt)). Only the negative chronotropic agent PGF_2β positive density effect. In conclusion, except for PGF_2α, prostaglandins generally have limited actions on the beating activity of cultured heart cells.     

Simple direct radioimmunoassay of the F prostaglandins

A simplified and accurate method of determining the F prostaglandins in 0.1 ml of serum without previous extraction is described. The procedure involves addition of anti-prostaglandin F_2α to serum followed by tritiated prostaglandin, equilibration for 4 hours, removal of unbound prostaglandin with dextran-coated charcoal and subsequent liquid scintillation counting of the supernatant. The mean ± S.D. concentration of prostaglandin F_2α in the serum of 15 healthy men was 90 ± 33 pg/ml and in 20 women 108 ± 43 pg/ml.     

Specific receptors for prostaglandins in airways

The relative bronchomotor activities of prostaglandins (PG) E₁, E₂, F_2α, F_2β and I₂ and of three synthetic E prostaglandin analogues (TR4161, TR4367 and TR4752) were determined on a large number of isolated preparations of guinea-pig trachea and human bronchial muscle. Each prostaglandin was capable of eliciting both contraction and relaxation, the relative incidence of these responses partly depending on concentration. TR4161 was a virtually pure relaxant; TR4367 was virtually devoid of bronchomotor activity; and TR4752 was a potent relaxant, devoid of contractant activity. The results also provided distinct rank orders of approximate potency for contraction and relaxation. Tachyphylaxis to the relaxant activities of PGE₁ and TR4752 confirmed the underlying contractant activity of the two natural E prostaglandins. Antagonism with a high dose of indomethacin of the contractant actions of PGE₁, PGE₂ and PGF_2α confirmed the presence of relaxant activities in each.Inhaled aerosols of the same natural and synthetic prostaglandins were evaluated for irritant activity on the airways, using the cough response of the restrained conscious cat. All of them, except TR4161, elicited severe coughing. The rank order of potencies for irritancy differed from those for tracheobronchial contractant and relaxant activities.These findings suggest that the three responses studied arise from the activation of three distinct PG receptors in the airways. We propose the terms χ (contractant), ψ (relaxant) and ω (irritant) for these putative receptors for prostaglandins or possibly other prostanoids.