Effect of prostaglandin I2 and related compounds on vascular permeability response in granuloma tissues

The effects of prostaglandin I₂, 6-ketoprostaglandin F_1α, prostaglandin E₁ and thromboxane B₂ on the vascular permeability response in rat carrageenin granuloma were studied with the aid of ¹³¹I- and ¹²⁵I-human serum albumin as indicators for the measurement of local vascular permeability.A single injection of 5 μg of prostaglandin I₂ methyl ester or I₂ sodium salt into the locus of the granulomatous inflammation elevated local vascular permeability 2.0–2.5 times over the control within 30 min. The potency was equal to that of the positive control prostaglandin E₁ which has been known to be the most potent mediator in this index among several candidate prostaglandins for chemical mediator of inflammation. The other prostaglandin and thromboxane B₂ tested were essentially inactive.     

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.     

Improved sensitivity of iodinated histamine-prostaglandin radioimmunoassay by prostaglandin methyl esters

Use of (¹²⁵I)-labeled histamine-prostaglandin tracer increases the sensitivity of the radioimmunoassays of prostaglandin derivatives. Six different antisera were produced for prostaglandins and their derivatives (prostaglandins E₁, E₂, F_1α, F_2α, 13,14-dihydro-15-ketoprostaglandin E₂, and 13,14-dihydro-15-ketoprostaglandin F_2α) and were investigated with the corresponding tritiated and lodinated tracers. Displacement of iodinated tracers by the methyl esters of the prostaglandin compounds resulted, in most cases, in a three- to fivefold increase in sensitivity compared to unesterified inhibitors. Esterification also caused some alteration in the specificities observed. Our results suggest that conformational changes in the esterified prostaglandins (tracer and inhibitor) could explain these charges.     

Differential separation of thromboxanes from prostaglandins by one and two-dimensional thin layer chromatography

[¹⁴C]-labelled thromboxane B₂ and hydroxy fatty acids were isolated using thin layer and gas chromatographic procedures from human platelets incubated with [1-¹⁴C]-arachidonic acid. A number of TLC solvent systems were evaluated for differential separation of thromboxanes and hydroxy fatty acids from prostaglandins E₂, A₂, D₂ and F_2α. Chromatographic properties in nine different solvent systems are tabulated. Two dimensional TLC procedures suitable for complete resolution of mixtures of these compounds on a single plate were developed. The systems were used to demonstrate conversion of [1-¹⁴C]-arachidonic acid to thromboxane B₂ and prostaglandin E₂ by human lung fibroblasts in tissue culture.     

β2-Adrenergic regulation of prostaglandin D2 receptor in rabbit platelets

[³H]Prostaglandin D₂ binding to rabbit platelets was increased by about 150% in the presence of β-adrenoceptor agonist, isoproterenol. The isoproterenol-induced potentiation of the [³H]prostaglandin D₂ binding gave a bell-shaped dose-response relationship (maximum response at 3·10⁻⁸ M) in a stereospecific manner. Similar and moderate potentiation was obtained with terbutaline. On the other hand, β-adrenoceptor antagonists such as alprenolol, propranolol and butoxamine (β₂-specific) had no potentiating effect on [³H]prostaglandin D₂ binding; rather, they abolished the isoproterenol-induced increase of [³H]prostaglandin D₂ binding. The β₁-specific antagonist, metoprolol, did not have any effect. Rabbit platelets were found to possess one [³H]prostaglandin D₂ binding site (K_d = 6·10⁻⁷ M, B_max = 787 fmol/mg protein). In the presence of isoproterenol at 3·10⁻⁸ M, B_max was increased with unaltering K_d value. Isoproterenol did not increase [³H]prostaglandin E₁, [³H]prostaglandin E₂ and [³H]prostaglandin F_2α bindings to platelets. The potential effect of isoproterenol was mimicked by forskolin, theophylline, dibutyryl cyclic AMP, prostaglandin E₁ and prostaglandin I₂, but it was abolished by 2′, 5′-dideoxyadenosine, an inhibitor of adenylate cyclase, indicating that elevated level of cyclic AMP may be available for the induction of the increase of [³H]prostaglandin D₂ binding. Prostaglandin D₂-induced cyclic AMP synthesis and antiaggregation activity were also augmented in the presence of isoproterenol. These results suggest a β₂-adrenoceptor-mediated cyclic AMP-dependent mechanism for the regulation of prostaglandin D₂ receptor binding in rabbit platelets.     

Substrate specificity of three prostaglandin dehydrogenases

Studies on the substrate specificity, kcat/Km, and effect of inhibitors on the human placental NADP-linked 15-hydroxyprostaglandin dehydrogenase (9-ketoprostaglandin reductase) indicate that it is very similar to a human brain carbonyl reductase which also possesses 9-ketoprostaglandin reductase activity. These observations led to a comparison of three apparently homogeneous 15-hydroxyprostaglandin dehydrogenases with varying amounts of 9-ketoprostaglandin reductase activity: an NAD- and an NADP-linked enzyme from human placenta and an NADP-linked enzyme from rabbit kidney. All three enzymes are carbonyl reductases for certain non-prostaglandin compounds. The placental NAD-linked enzyme, which has no 9-ketoprostaglandin reductase activity, is the most specific of the three. Although it has carbonyl reductase activity, a comparison of the Km and kcat/Km for prostaglandin and non-prostaglandin substrates of this enzyme suggests that its most likely function is as a 15-hydroxyprostaglandin dehydrogenase. The results of similar comparisons imply that the other two enzymes may function as less specific carbonyl reductases.     

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.     

Purification and partial characterization of an NADH-linked delta13-15-ketoprostaglandin reductase from human placenta.

A Δ¹³-15-ketoprostaglandin reductase has been isolated from human placenta and purified 800-fold. The enzyme utilizes NADH as a cofactor but not NADPH. It reduces the 13,14 double bond in 15-ketoprostaglandin E₁, E₂ and F_2α. The K_M apparent for NADH is 54.8 μM and the K_M apparent for 15-ketoprostaglanding E₂ is 7.0 μM. The partially purified enzyme contains no 15-hydroxyprostaglandin dehydrogenase activity.     

Identification of an unusual cyclooxygenase metabolite of arachidonic acid in rabbit renal medulla

A renal medulla 100,000g pellet metabolized arachidonic acid, C_20:4, to the previously described prostaglandins prostaglandin E₂, 6-ketoprostaglandin F_1α, thromboxane B₂, 12-hydroxyheptadecatrienoic acid, and 11-hydroxyeicosatetraenoic acid. In addition, under conditions of low enzyme to substrate ratios, the renal medulla also produced an unusual metabolite from arachidonic acid. This metabolite was inhibited by indomethacin, and thus suggested that it was a product of the cyclooxygenase. Addition of GSH to the incubation inhibited its formation, while p-hydroxymercuri-benzoate enhanced its formation. This compound was identified by HPLC purification, uv absorption, and gas chromatography-mass spectroscopy. The compound was 9,15 dioxo,11-hydroxyprosta-5,13-dienoic acid.     

Effects of prostaglandin D2 on membrane potential in neuroblastoma X glioma hybrid cells as determined with a cyanine dye

A cyanine dye, diS-C₃-(5) was used to determine the effects of prostaglandins on the membrane potential in neuroblastoma X glioma cells (NG 108-15). The largest depolarization was seen with prostaglandin D₂ (ED₅₀ = 1.5 μM), and relative potencies of various prostaglandins (3 μM) were: D₂, 100; I₂, 41; E₁, 17; E₂, 7; and F_2α, 7. 5-Hydroxytryptamine in a dose over 100 μM also depolarized the membrane. The effect of prostaglandin D₂ was observed in a Na⁺-free medium or when Ca²⁺ was replaced by Sr²⁺. The addition of 3 mM ethylene-glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid or 5 mM Co²⁺ partially inhibited the effects. These observations suggest that the depolarization of membrane by prostaglandin D₂ may primarily be related to alteration of Ca²⁺ permeability in the cell membrane.     

Tissue specific regulation of prostaglandin production stimulation of 6-ketoprostaglandin F1α biosynthesis by rat kidney cytosol

Boiled cytosol of various rat tissues each affected prostaglandin biosynthesis by bovine seminal vesicle microsomes in a specific way. Kidney cytosol enhanced 6-ketoprostaglandin F_1α production in a dose-dependent manner. This stimulatory effect was lost after dialysis. Liver, spleen and carrageenin granuloma cytosol inhibited 6-ketoprostaglandin F_1α production but enhanced prostaglandin E₂ production.     

The effect of acute hypoxia on prostaglandin release in perfused human fetal placenta

The release of prostaglandin E₂ and F_2α, thromboxane B₂ and 6-keto-prostaglandin F_1α was measured in isolated human placental cotyledons perfused under high- and low-oxygen conditions. Also the effect of reoxygenation on prostaglandin production was studied. During the high-oxygen period, prostaglandin E₂ accounted for 44 % and 6-keto-prostaglandin F_1α for 28 % of all prostaglandin release, and the rank order of prostaglandin release was E₂ > 6-keto-prostaglandin F_1α > thromboxane B₂ > prostaglandin F_2α. Hypoxia had no significant effect on quantitative prostaglandin release, but the ration of prostaglandin E₂ to prostaglandin F_2α was significantly increased. After the hypoxic period during reoxygenation the release of 6-keto-prostaglandin F_1α was significantly decreased, as was the ratio of 6-keto-prostaglandin F_1α to thromboxane B₂. Also the ratio of the vasodilating prostaglandins (E₂, 6-keto-prostaglandin F_1α) to the vasocontricting prostaglandins (thromboxane B₂, prostaglandin F_2α) was decreased during reoxygenation period. With the constant flow rate, the perfusion pressure increased during hypoxia in six and was unchanged in three preparation. The results indicate that changes in the tissue oxygenation in the placenta affect prostaglandin release in the fetal placental circulation. This may also have circulatory consequences.     

Fluorescent derivatives of prostaglandins and thromboxanes for liquid chromatography

Fluorescent esters of the prostablandins D₂, E₂, F₂α, and 6-keto-F₁α and of thromboxane B₂ have been prepared using the reagent 4-bromomethyl-7-methoxycoumarin. All of these derivatives can be separated in a single run either by thin-layer or high-performance liquid chromatography (TLC or HPLC). As little as 20 ng of PGE₂ can be detected after derivatization and HPLC analysis. Identification of thromboxane B₂ produced by human platelets and of 6-keto-PG F₁α produced by bovine aortic microsomes has been achieved with this method.     

Prostaglandin D2 in rat brain,spinal cord and pituitary: Basal level and regional distribution

A highly sensitive and specific radioimmunoassay for prostaglandin D₂ has been developed and used to determine the basal level and regional distribution of this prostaglandin in rat brain, spinal cord and pituitary. The assay can detect as little as 20 pg of prostaglandin D₂, and the antiserum used shows 20% cross-reactivity to prostaglandin D₁, 0.1% cross-reactivity to 13,14-dihydro-15-ketoprostaglandin D₂ and even lower cross-reactivity to other prostaglandins. Prostaglandin D₂-like immunoreactivity was extracted with ethanol from the rat tissues. The immunoreactivity comigrated with authentic prostaglandin D₂ on silica gel thin layer chromatography, showed the dilution curve parallel to that of the authentic compound, and decreased in amounts by the pretreatment of animals with indomethacin, suggesting that it was prostaglandin D₂ itself. To avoid a postmortem formation of prostaglandins, we sacrificed animals by microwave irradiation at 4.5 kW for 1.2 sec under which conditions both prostaglandin D synthetase and prostaglandin D dehydrogenase were completely inactivated. The amount of prostaglandin D₂ in whole brain measured under these conditions was 3.42±0.59 ng (mean+S.E.M.), and those of prostaglandin E₂ and F_2α measured by the respective radioimmunoassays were 1.32±0.24 and 0.96±0.20 ng, respectively. Prostaglandin D₂ was widely distributed in rat brain, spinal cord and pituitary. The highest concentrations were seen in pineal gland and neurointermediate pituitary followed by anterior pituitary. Lower but significant concentrations were observed in other parts of brain, among which hypothalamus and septum showed the relatively high concentrations.     

In vitro prostaglandin synthesis by various rat renal preparations

Prostaglandin synthesis by eight different structures from the rat kidney (whole cortex, cortical tubules, glomeruli, outer medulla, papilla, glomerular cultured epithelial and mesangial cells, cultured interstitial medullary cells) was measured in vitro after incubation with [¹⁴C]arachidonic acid using high-performance liquid chromatography followed by RIA with four specific anti-prostaglandin antibodies (prostaglandin E₂, prostaglandin F_2α, 6 keto-prostaglandin F_1α, thromboxane B₂). Prostaglandin production by the whole cortex and cortical tubules was very low. The order of abundance for isolated glomeruli was thromboxane B₂ > prostaglandin E₂ > prostaglandin F_2α > 6 keto-prostaglandin F_1α. Mesangial cells synthesized prostaglandin E₂ at a markedly high rate, and in decreasing order: prostaglandin F_2α, thromboxane B₂ and 6 keto-prostaglandin F_1α. The same order of abundance was observed for epithelial cells. The papilla synthesized essentially prostaglandin E₂ and prostaglandin F_2α, whereas the main product for the outer medulla was 6 keto-prostaglandin F_α. Cultured interstitial cells synthesized mainly prostaglandin E₂ and to a lesser extent prostaglandin F_2α. Unidentified peaks eluting between 6 keto-prostaglandin F_α and thromboxane B₂ were also observed chiefly with glomeruli but they were absent with the medullary preparations. They disappeared after incubation with indomethacin or aspirin and represented for glomeruli the greatest percentage of conversion of [¹⁴C]arachidonic acid. These results show that the prostanoid profile varies markedly with the different regions and cells of the rat kidney.     

Stimulative effect of prostaglandins on production of hexosamine-containing substances by cultured fibroblasts (2) early effect of various prostaglandins at various doses

Early effects of various prostaglandins on the production of hexosamine-containing substances by cultured fibroblasts, which were derived from a rat carrageenin granuloma, were studied. At the stationary phase, the cells were exposed for 6 h to one of the prostaglandin A₁ (PGA₁), A₂, B₁, B₂, D₂, F_1α, F_2α, E₁, E₂ or arachidonic acid in various concentrations ranging from 0.01 to 10 μg/ml for all the stimuli and from 10 pg to 10 μg/ml for PGF_2α. The activity of the cells in incorporating ³H-glucosamine into hexosamine-containing substances (acidic) glycosaminoglycans and glycoproteins) during this period was compared with that of control cells. All the stimuli tested showed more or less stimulative effect on the synthesis of hexosamine-containing substances at their specific concentrations. PGF_2α was found to be the most potent stimulant and its stimulative effect was found significant even at the low concentration of 100 pg/ml. PGD₂, F_1α and E₂ were the next potent stimuli. Their optimum dose were around 1 μg/ml but they still had significant stimulation at the concentration of 0.01 μg/ml. Effect of PGE₂ was rather mild. Stimulation by PGA₁, A₂, B₁ and B₂ or arachidonic acid was seen at high dose, and its seemed to be non-specific. The results suggested that these prostaglandins such as PGF_2α, D₂, F_1α and E₂ play some important role on regulating the production of intercellular ground substances.     

Endogenous formation of the prostaglandin endoperoxine metabolite,thromboxane B2, by brain tissue

Thromboxane B₂ was formed from endogenous precursors during short incubations of guinea pig and rat cerebral cortex. The amount formed by guinea pig brain tissue was 5–6 times the formation of prostaglandin F_2α and E₂. Noradrenalin stimulated and indomethacin and mercaptoethanol inhibited thromboxane B₂ formation. The mass spectrum of the brain compound was identical to thromboxane B₂ formed from arachidonic acid by guinea pig lung and human platelets.     

Characterization of imidazo[1,5-a]pyridine-5-hexanoic acid (CGS 13080) as a selective thromboxane synthetase inhibitor using in vitro and in vivo biochemical models

CGS 13080 inhibited cell-free thromboxane synthetase with an IC₅₀ of 3 nM. It was at least five orders of magnitude less potent toward other key enzymes involved in arachidonic acid metabolism. Submicromolar concentrations inhibited calcium ionophore-induced formation of thromboxane B₂ by intact human platelets with concomitant accumulation of prostaglandin E₂. Oral doses lower than 1 mg/kg in rats suppressed the elevations of plasma thromboxane B₂ induced by calcium ionophore. This was attended by shunting of endoperoxide substrate to 6-keto-prostaglandin F₁α and prostaglandin E₂. CGS 13080 is one of the most potent and selective thromboxane synthetase inhibitors yet identified.     

Comparison of the effects of prostaglandin I2 and prostaglandin E2 stimulation of the rat kidney adenylate cyclase-cyclic AMP systems

Prostacyclin (Prostaglandin I₂) effects on the rat kidney adenylate cyclase-cyclic AMP system were examined. Prostaglandin I₂ and prostaglandin E₂, from 8 · 10^?4 to 8 · ^?7 M stimulated adenylate cyclase to a similar extent in cortex and outer medulla. In inner medulla, prostaglandin I₂ was more effective than prostaglandin E₂ at all concentrations tested. Both prostaglandin I₂ and prostaglandin E₂ were additive with antidiuretic hormone in outer and inner medulla. Prostaglandin I₂ and prostaglandin E₂ were not additive in any area of the kidney, indicating both were working by similar mechanisms. Prostaglandin I₂ stimulation of adenylate cyclase correlated with its ability to increase renal slice cyclic AMP content. Prostaglandin I₂ and prostaglandin E₂ (1.5 · 10^?4 M) elevated cyclic AMP content in cortex and outer medulla slices. In inner medulla, with Santoquin® (0.1 mM) present to suppress endogenous prostaglandin synthesis, prostaglandin I₂ and prostaglandin E₂ increased cyclic AMP content. 6-Ketoprostaglandin F_1α, the stable metabolite of prostaglandin I₂, did not increase adenylate cyclase activity or tissue cyclic AMP content. Thus, prostaglandin I₂ activates renal adenylate cyclase. This suggests that the physiological actions of prostaglandin I₂ may be mediated through the adenylate cyclase-cyclic AMP system.     

Partial purification and some properties of human erythrocyte prostaglandin 9-ketoreductase and 15-hydroxyprostaglandin dehydrogenase.

Human erythrocytes were found to contain two prostaglandin metabolizing enzymes: a prostaglandin E 9-ketoreductase catalyzing the reduction of prostaglandin E₂ to form prostaglandin F_2α and a 15-hydroxyprostaglandin dehydrogenase that catalyzes the oxidation of prostaglandin F_2α to form 15-ketoprostaglandin F_2α. Both enzymes are found in the cytoplasmic fraction of erythrocytes and both enzymes use the triphosphopyridine nucleotides as cofactors more effectively than the diphosphopyridine nucleotides. These two enzymes were partially purified from erythrocyte homogenates and some of their properties were studied.