Differential effects of probenecid on the levels of endogenous PGF2α and TXB2 in brain cortex

Probenecid in single or repeated doses does not modify levels of PGF_2α and TXB₂ in rat brain cortex. After administration of subconvulsant dose of pentamethylene tetrazone (PMT) PGF_2α increases sharply and rapidly declines subsequently, whereas the elevation of TXB₂ is smaller but of longer duration. After probenecid pretreatment PGF_2α levels do not decline up to 30 minutes after the initial peak and are still elevated after 60 minutes. Levels of TXB₂ tend to be reduced after pretreatment. Differences in transport process or in biosynthetic compartments for these arachidonic acid (AA) metabolites may account for the observed data.     

Determination of 6-keto-PGF1α, 2,3-dinor-6-keto-PGF1α, thromboxane B2, 2,3-dinor-thromboxane B2, PGE2, PGD2 and PGF2α in human urine by gas chromatography—negative ion chemical ionization mass spectrometry

A method for quantification of 6-keto-PGF_1α, 2,3-dinor-6-keto-PGF_1α, TXB₂, 2,3-dinor TXB₂, PGE₂, PGD₂ and PGF_2α in human urine samples, using gas chromatography—negative ion chemical ionization mass spectrometry, is described. Deuterated analogues were used as internal standards. Methoximation was carried out in urine samples which were subsequently applied to phenylboronic acid cartridges, reversed-phase cartridges and thin-layer chromatography. The eluents were further derivatized to pentafluorobenzyl ester trimethylsilyl ethers for final quantification by gas chromatography—mass spectrometry. The overall recovery was 77% for tritiated 6-keto-PGF_1α and 55% for tritiated TXB₂. Urinary levels of prostanoids were determined in a group of six volunteers before and after intake of the thromboxane synthase inhibitor Ridogrel, and related to creatinine clearance.     

Hyperglycemia promotes elevated generation of TXA2 in isolated rat uteri

The relationship between high glucose concentrations and arachidonic acid metabolism in uterine tissue from control and diabetic ovariectomized rats was evaluated. Uterine tissue from diabetic rats produced amounts of PGE₂ and PGF_2α similar to controls, while a lower production of 6-keto-PGF_1α (indicating the production of prostacyclin) and a higher production of TXB₂ (indicating the generation of TXA₂) was found in the diabetic group. A group of diabetic rats was treated with phlorizin to diminish plasma glucose levels. Phlorizin treatment did not alter production of PGE₂, PGF_2α, and 6-keto-PGF_1α in the diabetic group. A diminished production of TXB₂ was found in the treated diabetic uteri when compared to the non-treated diabetic group. Moreover, a positive correlation between plasma glucose levels and uterine TXB₂ generation was observed. When control uterine tissue was exposed in vitro to high concentrations of glucose (22 mM) and compared to control tissue incubated in the presence of glucose 11 mM alterations in the generation of PGE₂, PGF_2α, and 6-keto-PGF_1α were not found, but a higher production of TXB₂ was observed and values were similar to those obtained in the diabetic tissue. Alteration in the production of the prostanoids evaluated were not observed when diabetic tissue was incubated in the presence of high concentrations of glucose. These results provide evidence of a direct relationship between plasma glucose levels and uterine production of TXA₂.     

Effects of nitrogen dioxide exposure on the contents of prostaglandins and thromboxane B2 in broncho alveolar lavage

Effects of 10 ppm nitrogen dioxide (NO₂) exposure on the contents of prostaglandins (PGs) and thromboxane (TX) B₂ in broncho-alveolar lavage (BAL) of rats were studied. In the BAL of normal rats, the amounts of PGs and TXB₂ in the whole lavage were 6-keto-PGF_1α (38.0 ± 6.4 ng) > TXB₂ (11.8 ± 4.0 ng) > PGF_2α (5.7 ± 1.6 ng) PGE (0.5 ± 0.3 ng). Rats were exposed to NO₂ for 1, 3, 5, 7 and 14 days. The NO₂ exposure decreased in the level of 6-keto-PGF_1α by about 35% throughout the exposure. The level of TXB₂ was higher in the day 5 exposure group (155%). The contents of PGF_2α and PGE first, decreased and then transiently increased on days 3 and 5. PG 15-hydroxy-dehydrogenase activity of lung homogenate decreased correspondingly on day 3 and 5. Then the contents PGF_2α and PGE decreased on day 7 and 14.6-keto-PGF_1α and TXB₂ are stable metabolites of PGI₂, a strong bronchorelaxant and TXA₂, a strong bronchoconstrictor respectively. Therefore the results suggested that the decrease in 6-keto-PGF_1α, a major prostanoid in the BAL and the increase in TXB₂ may correlate with broncho constriction by NO₂ exposure.     

Biosynthesis of prostaglandins and thromboxane B2 by fetal lung homogenates

The conversion of arachidonic acid to prostaglandins (PG's) and thromboxane B₂ (TXB₂) was investigated in homogenates from fetal and adult bovine and rabbit lungs. Adult bovine lungs were very active in converting arachidonic acid (100 μg/g tissue) to both PGE₂ (10.7 μg/g tissue) and TXB₂ (6.2 μ/g tissue). Smaller amounts of PGF_2α (0.9 μ/g) and 6-oxoPGF_1α were formed. Homogenates from fetal calf lungs during the third trimester of pregnancy were quite active in converting arachidonic acid to PGE₂, but formed very little TXB₂, PGF_2α or 6-oxoPGF_1α. Homogenates from rabbit lungs converted arachidonic acid (100 μg/g) mainly to PGE₂, both before and after birth. The amount of PGE₂ formed increased during gestation to a maximum of about 6 μg/g tissue at 28 days of gestation. It then decreased to a minimum (1.5 μg/g) which was observed 8 days after birth, followed by an increase to about 4 μg/g in older rabbits.     

Metabolism and effect of prostaglandin H2 in adipose tissue

Prostaglandin H₂ (PGH₂) inhibited noradrenaline induced cyclic AMP accumulation in isolated rat fat cells in a dose-dependent manner. IC₅₀ was 10 – 25 ng/ml both in the absence and in the presence of theophylline. The degree of inhibition produced by PGH₂ increased with time of incubation. A stable PGH₂ analog did not inhibit cyclic AMP accumulation. PGH₂ was rapidly converted by isolated fat cells to PGD₂, PGE₂ and PGH_2α, but no formation of thromboxane B₂ was found either or . PGE₂ was a more potent inhibitor than PGH₂ of noradrenaline induced cyclic AMP accumulation. PGD₂ enhanced cyclic AMP accumulation in a limited concentration interval, while PGF_2α was essentially uneffective.Our results suggest that PGH₂ is an inhibitor of cyclic AMP formation in isolated rat fat cells only after conversion to PGE₂. A physiological role for PGH₂ as a modulator of lipolysis is considered unlikely.     

Chemotactic activity of thromboxane B2, prostaglandins and their metabolites for polymorphonuclear leucocytes

(1) The chemotactic activities of thromboxane B₂ (TxB₂, PGE₂, PGF_2α, the 15-oxo, 15-oxo-13,14-dihydro and 13,14-dihydro metabolites of PGE₂, PGF_2α, and a metabolite of TxB₂ for polymorphonuclear leucocytes (PMN) have been investigated.(2) Thromboxane B₂ increased the directional migrationm of rat peritoneal PMN at a concentration of 2.0 μg/ml and of human peripheral neutrophils at a concentration of 0.5 μg/ml.(3) Neither PGE₂, PGF_2α nor their metabolites showed chemotactic activity for rat peritoneal PMN.(4) PGF_2α and 15-oxo-13,14-dihydro-thromboxane B₂ showed no chemotactic activity for human peripheral PMN.(5) The possible role of thromboxane B₂ in inflammation is discussed.     

Bio-synthesis of PGD2 and TXB2 by rabbit blood monocytes

A method for the preparation of a highly purified sample of rabbit blood monocytes is described. The metabolism of arachidonic acid (AA) in these cells was studied. Mononuclear cells were prepared by centrifugation on Ficoll-Paque gradients and the monocytes were obtained by further centrifugation and adherence onto plastic culture dishes. These procedures provided a preparation which contained 95% monocytes (non-specific esterase positive). Incubation of [1-¹⁴C]-AA with these cells produced four major metabolites which were separated by TLC; these corresponded to prostaglandin (PG) D₂, thromboxane (TX) B₂, 12-hydroxyheptadecatrienoic acid (HHT) and 12-/15- hydroxyeicosatetraenoic acid (HETE). A minor product which co-migrated with PGE₂ was also detected but neither 6-keto-PGF_1α nor PGF_2α were detected. Also, there was no evidence of the formation of 5-lipoxygenase products (5-HETE and LTB₄) by rabbit monocytes with or without calcium-ionophore A23187-stimulation. The production of PGD₂, TXB₂ and PGE₂ was further confirmed by analyzing [³H]-AA metabolites using high-performance liquid chromatography (HPLC) with tritiated standards as references. The biosynthesis of these compounds from endogenous substrate in A23187-stimulated monocytes was confirmed by specific radioimmunoassays with or without prior HPLC separation. The synthesis of immunoreactive LTB₄ and LTC₄ by A23187-stimulated cells was also monitored and found to be relatively low. The synthesis of PGD₂, TXB₂ and PGE₂ from both exogenous and endogenous substrate was suppressed by treatment of the monocytes with indomethacin (10⁻⁶ M).     

Regional distribution of arachidonic acid metabolites in rat brain following convulsive stimuli

Seizures were induced in female Wistar rats by electroconvulsive shock (ECS) or administration of pentetrazole (PTZ). Brain content of various prostanoids measured by radioimmunoassay showed time-dependent changes after the induction of convulsions; highest levels were found for PGD₂ followed by PGF_2α, PGE₂, TXB₂ and 6-keto-PGF_1α. Analysis of the various arachidonic acid metabolites in seven parts of the rat brain dissected according to the method of Glowinski and Iversen revealed the largest increases in hippocampus and cerebral cortex and smaller ones also in hypothalamus and corpus striatum both after ECS and PTZ. The ratios of the different cyclo-oxygenase products remained virtually the same in whole brain as well as in those regions where the formation of prostaglandins was markedly elevated. 15-keto-13,14-dihydro-PGF_2α also increased simultaneously in parallel to its parent compound, PGF_2α and was detected in significant amounts only in hippocampus and cerebral cortex. However, concentrations of 15-keto-13,14-dihydro-PGF_2α in these brain regions as well as in whole brain represented only 3–10% of the amounts found for PGF_2α. Thus, the metabolizing enzymes 15-hydroxy-PG-dehydrogenase and Δ13-PG-reductase seem to be of minor importance for the inactivation of prostanoids in brain tissue.     

Relation between prostaglandin E2, F2α, and cyclic nucleotides levels in rat brain and induction of convilsions

Fully convulsant doses of pentamethylenetetrazole cause marked increase in rat brain cortical PGF_2α, PGE₂, cGMP and cAMP during seizures, whereas subconvulsant doses cause an increase of rat brain cortical PGF_2α without affecting the other biochemical parameters considered. Rat cerebellar prostaglandins were not modified by the convulsant agent at either dosage.     

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

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

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

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

Effects of prostaglandin F2α on cerebral cortical evoked potentials

The cerebral cortical action of prostaglandin F_2α (PGF_2α) has been determined by recording the effects of intracarotid injections of PGF_2α on cerebral evoked potentials. PGF_2α differentially reduced cortical evoked potentials. The cortical action of PGF_2α appeared to be qualitatively identical with that of norepinephrine (NE) but weaker. A protection of the cortex from the inhibitory action of NE by a preceding dose of PGF_2α was demonstrated. The actions of both PGF_2α and NE appear to be on the same or related postsynaptic receptors. The actions described were at doses that did not reduce oxygen availability. PGF_2α may act as a modulator of adrenergic transmission in the cortex. The intracellular recording in the companion paper supplies the further critical evidence that PGF_2α has a synaptic inhibitory action.     

Preparation and quantitation of urinary metabolites of prostaglandin F2α by radioimmunoassay

Radioimmunoassay systems are described which have been developed to quantitate two principle urinary metabolites of PGF_2α; 9α,11α-dihydroxy-15-oxo-2,3,4,5-tetranorprostanoic acid (I) and 9α-11α-dihydroxy-15-oxo-2,3,4,5-tetranorprosta-1,20-dioic acid (II). Preparation of the required metabolites was achieved by total synthesis (I) or by bioconversion (isolation from urine of animals treated with 15-keto-PGF_2α^*, II). These metabolites were used to prepare conjugates for immunization. Labeled metabolites, suitable as binding markers, were prepared by metabolism of ³H-PGF_2α (I) or (II). Specificity of the resulting antibodies was compared to an antibody to PGF_2α and to 13,14-dihydro-15-keto PGF_2α. Antisera of II had little or no affinity for 20-carbon precursors (PGF_2α or 13,14-dihydro-15-keto PGF_2α), but had nearly equal affinity for metabolite I. Antisera of I, however, had little or no affinity for antigen of II. Therefore, analysis of samples by both assay systems enables quantitation of these excretion products of PGF_2α. Other assay parameters (binding, affinity, recovery, precision and the repeatability of the assays) were similar to those previously described for other RIA systems, and were considered satisfactory for quanitation of compounds in biological fluids.Quantitation of 24 hour urinary excretion of di-acid metabolite in humans was in close agreement with previously published values determined by physical-chemical means. Greater quantity of di-acid metabolite was excreted by human males (42.0 μg/24 hr) than by females sampled either during the follicular (20.0) or luteal phase (21.2) of the menstrual cycle. The total quantity of C-16 metabolites (as approximated by system II) excreted/kg body weight by the rhesus monkey was similar to that excreted by the human. However, the ratio of di-acid to mono-acid was much nearer unity in the monkey than the human.     

Transport of Eicosapentaenoic Acid-Derived PGE3, PGF3α, and TXB3 by ABCC4

Background

Eicosapentaenoic acid-derived prostaglandin (PG) E₃, PGF_3α, and thromboxane (TX) B₃ are bioactive lipid mediators which have anti-cancer and anti-inflammatory effects. To exert their effects, PGE₃, PGF_3α, and TXB₃ must be released to the extracellular space from cells, but the release mechanism has been unclear. We therefore investigated the contribution of ATP-binding cassette transporter C4 (ABCC4), which has been known as a prostanoids efflux transporter, to the release of PGE₃, PGF_3α, and TXB₃.

Materials and Methods

ATP-dependent transport of PGE₃, PGF_3α, and TXB₃ via ABCC4 was investigated by using inside-out membrane vesicles prepared from ABCC4-overexpressing HEK293 cells. To evaluate the contribution of ABCC4 to the release of PGE₃, PGF_3α, and TXB₃, we measured the extracellular and intracellular levels of PGE₃, PGF_3α, and TXB₃ in A549 cells when we used ABCC4 inhibitors (dipyridamole, MK571, and probenecid) or ABCC4 siRNAs. The quantification of PGE₃, PGF_3α, and TXB₃ was performed by using liquid chromatography-tandem mass spectrometry.

Results

The apparent K_m values for ABCC4-mediated transport were 2.9±0.1 µM for PGE₃, 12.1±1.3 µM for PGF_3α, and 11.9±1.4 µM for TXB₃ and the ATP-dependent accumulation of PGE₃, PGF_3α, and TXB₃ into vesicles was decreased by using typical substrates and inhibitors of ABCC4. ABCC4 inhibitors and ABCC4 knockdown showed the reduction of extracellular/intracellular ratio of PGE₃ (40–60% of control) and PGF_3α (60–80% of control) in A549 cells.

Conclusions

Our results suggest that PGE₃, PGF_3α, and TXB₃ are substrates of ABCC4 and ABCC4 partially contributes to the release of PGE₃ and PGF_3α.     

Prostaglandin E1 and F2α specific binding in bovine corpora lutea: Comparison with luteolytic effects

Preliminary characterization indicated the presence of separate prostaglandin (PG)E₁ and (PG)F_2α binding sites in membrane fractions prepared from bovine corpora lutea. These differ in the rate and temperature dependence of the specific binding. Equilibrium binding data indicate the apparent dissociation constants as 1.32 × 10⁻⁹M and 2.1 × 10⁻⁸M for PGE₁ and PGF_2α, respectively. Competition of several natural prostaglandins for the PGE₁ and PGF_2α bovine luteal specific binding sites indicates specificity for the 9-keto or 9α-hydroxyl moiety, respectively. Differences in relative ability to inhibit ³H-PG binding were found due to sensitivity to the absence or presence of the 5,6-cis-double bond as well.Bovine luteal function was affected following treatment of heifers with 25 mg PGF_2α as measured by reduced estrous cycle length, decreased corpus luteum size and significantly decreased plasma progesterone levels. In contrast, treatment with 25 mg PGE₁ resulted in cycle lengths comparable to those of non-treated herdmates with no apparent modification in corpus luteum size. However, plasma progesterone levels were increased significantly following PGE₁ treatment compared to pretreatment values. In so far as data obtained on PGF_2α relative binding affinity to the bovine CL can be compared to data obtained independently on PGF_2α induced luteolysis in the bovine, PGF_2α relative binding to the CL and luteolysis appeared to be associated. By similar reasoning, there was no apparent relationship between PGE₁ relative binding affinity in the luteal fractions and luteolysis in estrous cyclic cattle.     

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.     

Stereospecificity of enzymatic reduction of prostaglandin E2 to F2α

Antibodies directed toward PGF_2β were prepared in rabbits. The serologic specificity of the immune reaction was determined by inhibition of sodium borohydride-reduced (³H) PGE₂ anti-PGF_2β binding by several prostaglandins. The antibodies to PGF_2β recognize the β-hydroxyl configuration in the cyclopentane ring of PGF_2β. With the use of both anti-PGF_2α and anti-PGF_2β, the product of PGE₂ reduction by 9-ketoreductase purified from chicken heart was identified as PGF_2α. Guinea pig liver and kidney homogenates were examined for PGE 9-ketoreductase activity. Although enzyme activity was present, no evidence of PGF_2β production was found.     

Regulations of macrophage-mediated tumor cell killing by prostaglandins: Comparison of the effects of PGE2 and PGI2

Mouse resident peritoneal macrophages stimulated by purified bacterial lipopolysaccharide (LPS) produced both prostaglandin E₂ (PGE₂) and prostaglandin I₂ (PGI₂), the latter detected as its stable metabolite, 6-keto PGF_1α. Maximum production, induced in each case by 1 ng/ml purified LPS, was in the range of 10⁻⁷M for PGI₂ and 3 × 10⁻⁸M for PGE₂. A quantitatively similar increase in intracellular levels of macrophage cyclic AMP (measured on a whole cell basis), with a similar duration of effect, was stimulated by PGE₂ and PGI₂; however, only PGE₂ had a negative regulatory effect on macrophage activation for tumor cell killing. These data confirm that more than a whole cell increase in the concentration of cyclic AMP is needed to shut off nonspecific tumor cell killing mediated by LPS-activated resident peritoneal macrophages.     

Prostaglandin E1 and F2α specific binding in bovine corpora lutea: Comparison with luteolytic effects

Preliminary characterization indicated the presence of separate prostaglandin (PG)E₁ and (PG)F_2α binding sites in membrane fractions prepared from bovine corpora lutea. These differ in the rate and temperature dependence of the specific binding. Equilibrium binding data indicate the apparent dissociation constants as 1.32 × 10⁻⁹M and 2.1 × 10⁻⁸M for PGE₁ and PGF_2α, respectively. Competition of several natural prostaglandins for the PGE₁ and PGF_2α bovine luteal specific binding sites indicates specificity for the 9-keto or 9α-hydroxyl moiety, respectively. Differences in relative ability to inhibit ³H-PG binding were found due to sensitivity to the absence or presence of the 5,6-cis-double bond as well.Bovine luteal function was affected following treatment of heifers with 25 mg PGF_2α as measured by reduced estrous cycle length, decreased corpus luteum size and significantly decreased plasma progesterone levels. In contrast, treatment with 25 mg PGE₁ resulted in cycle lengths comparable to those of non-treated herdmates with no apparent modification in corpus luteum size. However, plasma progesterone levels were increased significantly following PGE₁ treatment compared to pretreatment values. In so far as data obtained in vitro on PGF_2α relative binding affinity to the bovine CL can be compared to data obtained independently in vitro on PGF_2α induced luteolysis in the bovine, PGF_2α relative binding to the CL and luteolysis appeared to be associated. By similar reasoning, there was no apparent relationship between PGE₁ relative binding affinity in the luteal fractions and luteolysis in estrous cyclic cattle.