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.     

Prostaglandins and thromboxanes in amniotic fluid during rivanol-induced abortion and labour

Abortion or delivery were induced by extra-amniotic instillation of Rivanol during the second trimester in twelve patients and during the third trimester in two patients with fetal death and one patient with fetal acrania. Serial sampling of amniotic fluid was performed through a transabdominal catheter and the levels of free arachidonic acid (AA), prostaglandin F₂α (PGF₂α), prostaglandin E₂ (PGE₂), 6-keto-prostaglandin F₁α (6-keto-PGF₁α) and thromboxane B₂ (TXB₂) were determined. The levels of AA, PGF₂α, PGE₂, 6-keto-PGF₁α and TXB₂ in amniotic fluid increased significantly during induction with the exception of AA in fetal death which was high and remained constant during induction. Furthermore, PGF₂α, 6-keto-PGF₁α and TXB₂ were all significantly correlated to AA.These observations suggested that free AA is released during Rivanol-induction of abortion and labour giving an increased synthesis of PGF₂α, PGE₂ prostacyclin and thromboxane A₂ in the fetal membranes and the decidua but not in the fetus. This increase might be relevant for the initiation and progress of abortion and labour in these patients.     

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.     

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.     

Prostaglandins and oxytocin: Their effects on uterine smooth muscle

A subcellular fraction was isolated from uteri of non-pregnant and pregnant cows. ATP-dependent calcium binding was shown to take place in this fraction. This calcium binding was inhibited in a dose related fashion when increasing amounts of prostaglandin (PG) E₂ or F_2α were added to the in vitro experimental medium. The physiologically inactive PGF_1β had no inhibitory effect. Oxytocin caused inhibition of calcium binding in preparations from both pregnant and non-pregnant cows. The response to PGE₂ and PGF_2α was somewhat greater in preparations from pregnant uteri than from non-pregnant uteri. The response to oxytocin was very much greater in pregnant uteri. Because of the high PG sensitivity of calcium binding in preparations from the non-pregnant uterus, it is concluded that the PGs may be the more suitable agent in the control of reproduction.     

Lipoxygenase- and cyclooxygenase-reaction products and incorporation into glycerolipids of radiolabeled arachidonic acid in the bovine retina

The metabolism of radiolabeled arachidonic acid (AA) by the intact bovine retina has been studied. Synthesis of prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs), and incorporation of AA into glycerolipids has been measured by reverse-phase and straight-phase high performance liquid chromatography with flow scintillation detection, and by thin-layer chromatography. AA was actively acylated into glycerolipids, particularly triglycerides, phosphatidylcholine and phosphatidylinositol. AA was also converted to the major PGs, PGF_2α, PGE₂, PGD₂, 6-keto-PGF_1α and TXB₂, and to the lipoxygenase reaction products, 12-HETE, 5-HETE, and other monohydroxy isomers. Approximately 6% of the radiolabeled AA was converted to eicosanoids. The synthesis of HETEs was inhibited in a concentration-dependent manner (IC₅₀ = 8.3 NM) by nordihydroguaiaretic acid (NDGA). PG synthesis was inhibited by aspirin (10 μM), indomethacin (1 μM) and NDGA (IC₅₀ = 380 nM). Metabolism of AA via lipoxygenase, cyclooxygenase and activation-acylation was inhibited by boiling retinal tissue prior to incubation. These studies demonstrate an active system for the uptake and utilization of AA in the bovine retina, and provide the first evidence of lipoxygenase-mediated metabolism of AA, resulting in the synthesis of mono-hydroxyeicosatetraenoic acids, in the retina.     

Effects of indomethacin, prostaglandin E2, prostaglandin F2α and 6-keto-prostaglandin F1α on hatching of mouse blastocysts

The present study has been performed to investigate how PGs would participate the hatching process. Effects of indomethacin, an antagonist to PGs biosynthesis, on the hatching of mouse blastocysts were examined in vitro. Furthermore, it was studied that prostaglandin E₂ (PGE₂), prostaglandin F_2α (PGF_2α) or 6-keto-prostaglandin F_1α (6-keto-PGF_1α) were added to the culture media with indomethacin. (1) The hatching was inhibited by indomethacin yet the inhibition was reversible. (2) In the groups with indomethacin and PGE₂, no improvement was seen in the inhibition of hatching and the inhibition was irreversible. (3) In the groups with indomethacin and PGF_2α, inhibition of hatching was improved in comparison with the group with indomethacin. (4) In the groups with indomethacin and 6-keto-PGF_1α, no improvement was seen. The above results indicated that PGF_2α possibly had an accelerating effect on hatching and a high concentration of PGE₂ would exert cytotoxic effect on blastocysts.     

Effects of 17 β estradiol on the metabolism of labelled arachidonic acid in uteri isolated from intact and ovariectomized rats. Influence of a restricted diet

The effects of restricted diet (50% of the normal intake during 25 days) on the metabolism of ¹⁴U arachidonic acid, were explored in uterine horn strips isolated from intact and ovariectomized rats, treated by 17 β-estradiol or controls. The metabolism of arachidonic acid into different eicosanoids, PGE₂, PGF_2α, 6-keto PGF_1α and TXB₂, showed that the restricted diet diminished PGE₂ and PGF_2α, in intact rats, significantly. In contrast, this kind of feeding did not produce any change in castrated rats.Tissue preparations from previously estrogenized intact and castrated normal-fed rats showed that the production of different metabolites decreased. A similar result was obtained in intact rats subjected to a restricted diet. Nevertheless, in castrated underfed rats, estrogens did not produce any effect on the various eicosanoids analysed.These results showed that in isolated uteri, the effects of 17 β-estradiol, on metabolite production from labelled arachidonic acid, are different from controls in ovariectomized diet-restricted rats.     

Changes in the levels of prostaglandins and thromboxane and their roles in the accumulation of exudate in rat carrageenin-induced pleurisy — a profile analysis using gas chromatography-mass spectrometry —

Injection of γ-carrageenin into t he pleural cavity of rats caused the accumulation of the pleural exudate. When levels of prostaglandins (PGs) and thromboxane (TX) B₂ were quantified by gas chromatography-mass spectrometry as their methyl ester (ME)-dimethyllisopropylsilyl (DMiPS) ether or ME-methoxine-DMiPS ether derivatives, 6-keto-PGF_1α reached the maximum at 1 hr after carrageenin, then PGE₂ and TXB₂ showed peaks at 3 hr and waned off before 9 hr. he PGF_2α level was kept low, but PGD₂, PGE₁ and PGF_1α were not detected. Aspirin (100 mg/kg, i.p.) significantly decreased the PG and TXB₂ levels and suppressed the rate of plasma exudation until 5 hr, but did not at 7 hr, when it was measured by the amount of exuded pontamine sky blue injected intravenously. OKY-025 (300 mg/kg, i.p.), a selective TXA synthetase inhibitor, and tranylcypromine (20 mg/kg, i.p.), a PGI synthetase inhibitor, could not extensively inhibit the accumulation of the exudate. These results suggest that the cyclooxygenase products of arachidonic acid, particularly PGE₂, definitely play an important role in the exudation during the first 5 hr.     

Prostaglandin and thromboxane production by human and guinea-pig macrophages and leucocytes

The ability of partially purified human and guinea-pig haematogenous cell populations, when cultured in vitro, to metabolise arachidonic acid (AA) has been studied. Supernatants from 24 hour cell culture have been subjected to analysis for products of AA metabolism by gas chromatography with electron-capture detection.The cell types studied were human peripheral blood monocytes (both glass adherent and non-adherent), neutrophils, eosinophils and leukemic leucocytes; thoracic duct lymphocytes and lung alveolar macrophages. From the guinea-pig, induced and non-induced macrophage or neutrophil enriched peritoneal exudate populations, lymph node cells, peritoneal eosinophils and peripheral blood platelets were examined. Supernatants were assayed for the presence of PGE₂, PGD₂, PGF_2α, TXB₂ and 6-keto-PGF_1α. In all types studied PGE₂ and TXB₂ were the major products formed. The identification of PGE₂ and TXB₂ was confirmed by GC/MS with multiple ion monitoring.The results have been compared with other reports and their possible significance discussed in relation to the proposed role of prostaglandins as mediators and modulators in immunopathology.     

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.     

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.     

Effects of experimental diabetes on spontaneous contractions, on the output of prostaglandins and on the metabolism of labelled arachidonic acid, in uteri isolated from ovariectomized rats. Influences of estradiol

Spontaneous changes in isometric developed tension (IDT) as a function of time after isolation (contractile constancy) in uteri from control-castrated and castrated chronic streptozotocin-diabetic rats, were explored. The effects of injecting 17-beta estradiol (Eo) were also studied. No differences in the minor changes of contractile constancy, between control and diabetic preparations, during a period of 60 min, were detected, whereas uteri from non-diabetic Eo injected animals (0.5 + 1.0 ug, prior to sacrifice), exhibited a profound reduction of IDT, significantly greater than in tissues obtained from Eo injected-diabetic rats. Moreover, basal generation and outputs into the suspending solution of prostaglandins (PGs) E1, E2 and F2 alpha, were explored in the same groups, at 60 min following tissue isolation. The basal outputs of these three PGs were similar in castrated control rats, but preparations from castrated-diabetics released significantly more PGE1. The administration of Eo to castrated-diabetics, failed to alter the releases of the three PGs explored. In addition, the metabolism of labelled arachidonic acid (AA) into different prostanoids (6-keto-PGF1, PGF2, PGE2 and thromboxane B2-TXB2), was also investigated. The non-diabetic spayed rat uterus converted AA into these four prostanoids, the transformation into 6-keto-PGF1 alpha (as an index of PGI2 formation) being the most prominent. In preparations from diabetic rats the formation) being the most prominent. In preparations from diabetic rats the formation of 6-keto-PGF1 alpha, PGF2 alpha and PGE2, was significantly smaller than in controls, whereas a greater % of TXB2 formation (as an index of TXA2), was detected. On the other hand uterine preparations from non-diabetic spayed rats injected with Eo formed less 6-keto-PGF1 alpha and PGE2 and similar amounts of PGF2 alpha or of TXB2 from AA, than Eo injected controls, whereas uteri from castrated diabetic animals injected with Eo, formed a similar % of 6-keto-PGF1 alpha, PGF2 alpha and PGE2 from AA, than tissue preparations from non-estrogenized controls. However, the enhanced transformation of the labelled fatty acid precursor (AA) into TXB2 in the diabetic group, was significantly reduced by the steroid. The role of the augmented generation and release of PGE1 in uteri from diabetic rats is discussed in terms of precedents indicating the relevance of PGs type E supporting rat uterine motility. In addition the influence of Eo is attractive, because its reducing effect on TX production, in diabetes, a disease known to be accompanied by enhanced synthesis of vasoconstrictor and platelet aggregation TXA2, and by frequent obstructive circulat     

Formation of prostanoids in human umbilical vessels perfused

Four major prostanoids (6-keto-PGF_1α, PGE₂, PGF_2α and TXB₂) were measured by specific radioimmunoassays in the outputs from human umbilical vessels perfussed . As evaluated by scanning electron microscopy (SEM) only few blood platelets were attached to the vessel wall. After an initial flush with decreasing concentrations of all four prostanoids, a stable stage was reached, lasting for 4–5 hours. During this stage the production could be inhibited by indomethacin and only slightly stimulated with arachidonic acid. The TXA₂ synthetase inhibitor UK 38485 depressed the TXB₂ production, while only slightly affecting the other three prostanoids at very high concentrations. The arteries produced relatively more 6-keto-PGF_1α than did the vein.     

Histamine alters prostaglandin output from diestrous rat uteri. Involvement of H2-receptors and 9-keto-reductase

The effects of exogenous histamine (H) on prostaglandin (PG) generation and release in uteri isolated from diestrous rats and the influences of H₂-receptors blockers (cimetidine and mitiamide) on the output of uterine PGs, were explored. Moreover, the action of H on the uterine 9-keto-reductase, was also studied. Histamine (10⁻⁴M) failed to alter the basal output of PGE₁ but reduced significantly the generation and release of PGE₂ and augmented the output of PGF_2α. On the other hand, cimetidine (10⁻⁵M) enhanced the basal release of PGE₂ but had no action on the outputs of PGs E₁ or F_2α. The enhancing effect of H on the production and release of PGF_2α was abolished in the presence of cimetidine. Also, the antagonist reversed the influence of H on the output of PGE₂. Metiamide, another H₂-receptor antagonist, did not alter the basal control generation and release of uterine PGs, but antagonized the augmenting influence of H on PGF_2α uterine output, as much as cimetidine did, and prevented the depressive action of H on the release of PGE₂ from uteri. Histamine (10⁻⁴M) significantly stimulated uterine formation of cyclic-adenosine monophosphate, an action which was antagonized by the presence of cimetidine (10⁻⁵M), a blocker of H₂ receptors. Also, histamine (10⁻⁵M) and dibutyril-cyclic-adenosine monophosphate (DB-cAMP) at 10⁻³M, enhanced significantly the formation ³H-PGF_2α from ³H-PGE₂. Results presented herein demonstrate that H is able to diminish the generation of PGE₂ in uteri from rats at diestrus augmenting the synthesis of PGF_2α, apparently via the activation of H₂-receptors, enhancing adenylate-cyclase. These effects appear to increase uterine 9-keto-reductase activity which transforms PGE₂ into PGF_2α. Relationships between the foregoing results and those evoked by estradiol, are also discussed.     

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.     

Prostaglandin synthesis by the cochlea

The exogenous and endogenous syntheses of prostaglandins (PG's) by the cochlea of adult mongolian gerbils were studied . After incubation of the whole membraneous cochlea with [³H]-arachidonic acid (AA), syntheses of PGF_2α, 6-keto PGF_1α, PGE₂, thromboxane (TX) B₂ and PGD₂ were evidenced in this order. The synthesis of radioactive PG's was almost completely inhibited by incubation with 10⁻⁵ M indomethacin. No significant amounts of those PG's were detected by radioimmunoassay (RIA) in the cochlea obtained from animals killed by microwave irradiation at 5.0 kw for 0.8 sec. However, when the homogenate of the whole membraneous cochlea obtained from animals without microwave irradiation was incubated at 37°C for 0–15 min, PGD₂, PGE₂, PGF₂α and 6-keto PGF₁α were found to be formed from endogenous AA in the cochlea by RIA. PG's were formed already at 0 time to considerable level (PGD₂, PGF₂α and 6-keto PGF₁α, 90–120 pg/cochlea; PGE₂, 370 pg/cochlea), reached to the maximum level (PGD₂, PGF₂α and 6-keto PGF₁α, 170–200 pg/cochlea; PGE₂, 500 pg/cochlea) at a 5-min incubation, and then gradually decreased. On the other hand, the amount of TXB₂ was lower than the detection limit by RIA (<50 pg/cochlea) even after the incubation. The cochlea was dissected into three parts: organ of Corti + modiolus (OC + M), lateral wall (LW), and cochlear nerve (CN), and then PG's formed by these tissues were determined after a 5-min incubation of the homogenates. In the CN and OC + M, PGE₂ was the major PG (100 and 160 pg/tissue, respectively), and the amounts of PGD₂, PGF₂α and 6-keto PGF₁α were about of those of PGE₂. In the LW, the amounts of PGD₂, PGE₂, PGF₂α and 6-keto PGF₁α were about the same level (70–100 pg/LW).     

Morphine diminishes the constancy of spontaneous uterine contractions, antagonizes the positive inotropic effects of prostaglandin E2, but not of prostaglandin F2α and inhibits prostaglandin E and F outputs from the uterus of ovariectomized rats

The effects of morphine on the constancy of spontaneous contractions (isometric developed TENSION = IDT and contractile FREQUENCY = CF), in uterine strips isolated from ovariectomized rats and the influence of naloxone, were explored. The inotropic responses to added prostaglandins (PGs) E₂ and F_2α and the influences of morphine and of morphine in the presence of naloxone on PG actions, were also determined. Moreover, the synthesis and outputs of PGs E and F from uteri and the effects of morphine alone and of morphine plus naloxone, were studied. Morphine (10⁻⁶ M) significantly depressed uterine constancy of IDT during the first hours following delivery, but its action on CF did not differ from controls. Naloxone, neither at 10⁻⁸ M nor at 10⁻⁶ M, altered the negative inotropoic influence of morphine on IDT. Exogenous PGs E₂ and F_2α, stimulated uterine inotropism in a concentration-dependent fashion. Morphine altered dose-response curves for exogenous PGE₂, evoking a parallel surmountable shift to the right, but did not affect the inotropic action of added PGF_2α. This antagonistic effect of the opioid was not altered by preincubation with naloxone. Basal synthesis and outputs of PGs E and F in uteri from ovariectomized rats were significantly depressed by morphine (10⁻⁶ M) but not altered by incubating tissues with morphine in presence of naloxone. Results are discussed in terms of a presumptive dual action of morphine on uterine motility, i.e., antagonizing PGE₂ receptors and inhibiting the synthesis of some PGs by the uterus. These influences of morphine do not appear to be subserved by the activation of μ opioid receptors. Moreover, the possibility that endogenous opioids could play a relevant role modulating uterine PG influences, is also discussed.     

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.     

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.