Thromboxane and pulmonary hypertension following E. coli endotoxin infusion in sheep: Effect of an imidazole derivative

We assessed the effect of a specific thromboxane synthetase inhibitor (an imidazole derivative) on pulmonary hemodynamics and the concentrations of TxB₂ (TxA₂), 6-keto-PGF_1α (PGI₂), and PGF_2α in pulmonary lymph and transpulmonary blood samples following intravenous administration of E. coli endotoxin (1 μg/kg) in sheep. In control animals the rise in pulmonary artery pressure correlated with increases in plasma and lymph TxB₂ concentrations and large transpulmonary concentration gradients of this metabolite were measured. In imidazle treated animals both pulmonary hypertension as well as increases in plasma and lymph TxB₂ concentrations were substantially reduced. In contrast, peak concentrations of 6-keto-PGF_1α (PGI₂) and PGF_2α were severalfold higher than those measured in control animals. This suggests a shunting of endoperoxide metabolism towards prostacyclin and primary prostaglandins and documents the specificity of the thromboxane synthetase inhibitor. Out study provides evidence that endotoxin-induced pulmonary hypertension is mediated by pulmonary synthesis of TxA₂.     

Dose-dependent effects of a pyridoquinazoline thromboxane synthetase inhibitor on arachidonic acid metabolites and hemodynamics during E. coli endotoxemia in anesthetized sheep

We investigated the effects of a new pyridoquinazoline thromboxane synthetase inhibitor infused before administering Escherichia Coli endotoxin into 18 anesthetized sheep with lung lymph fistulas. In normal sheep increasing plasma Ro 23-3423 concentrations were associated with increased plasma levels of 6-keto-PGF1 alpha, a reduced systemic vascular resistance (SVR, r = -0.80) and systemic arterial pressure (SAP, r = -0.92), the mean SAP falling from 80 to 50 mm Hg at the 20 and 30 mg/kg doses. Endotoxin infused into normal sheep caused transient pulmonary vasoconstriction associated with increased TxB2 and 6-keto-PGF1 alpha levels while vasoconstriction and TxB2 increase were significantly inhibited by pretreatment with Ro 23-3423 in a dose-dependent manner. When compared to controls, plasma and lymph levels of 6-keto-PGF1 alpha, PGF2 alpha and PGE2 after endotoxin infusion were increased several-fold by administering Ro 23-3423 up to plasma levels of 10 micrograms/ml. Doses over 30 mg/kg with blood levels above 10 micrograms/ml reduced plasma and lymph levels of 6-keto-PGF1 alpha, PGF2 alpha and PGE2, suggesting cyclooxygenase blockade at this dose. The peak 6-keto-PGF1 alpha levels at 60 min after endotoxin infusion in sheep with Ro-23-3423 levels below 10 micrograms/ml were associated with the greatest systemic hypotension due to a reduced SVR (r = -0.86). After endotoxin infusion the leukotrienes B4, C4, D4 and E4 in lung lymph were assayed by radioimmunoassay and high pressure liquid chromatography and remained at baseline values.     

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.     

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.     

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.     

Sulindac does not preserve renal prostacyclin synthesis during endotoxemia

Previous reports have suggested that sulindac is a unique non- steroidal anti-inflammatory (NSAID) agent, because it does not inhibit renal prostaglandin synthesis in doses that inhibit platelet thromboxane B₂ synthesis when tested . NSAIDS are of potential therapeutic benefit in the treatment of septic or endotoxic shock. Therefore, this study was designed to investigate the proposed unique action of sulindac in experimental endotoxemia. In the current study, the effect of sulindac on aortic, portal and renal venous immunoreactive (i) 6-keto-PGF_1α levels, the stable metabolite of prostacyclin, was investigated during endotoxemia in the rat. In doses sufficient to reduce the elevation in aortic and portal venous plasma 16-keto- PGF_1α levels, sulindac also significantly (p < 0.05) attenuated the elevated renal venous plasma 6-keto-PGF_1α levels, compared to the vehicle group. Using lower doses, sulindac failed to reduce the endotoxin associate increased in either aortic or renal venous plasma 16-keto-PGF_1α levels. Thus, sulindac failed to demonstrate any selective sparing effect on renal prostacyclin generation during endotoxemia.     

The pericardium as a source of prostacyclin in the dog, ox and rat

Cyclo-oxygenase products of arachidonic acid metabolism formed by the pericardium and epicardial surface of dog heart were identified and quantitated by radioimmunoassay after separation by high-pressure liquid chromatography. Pieces of pariental pericardium, of dog, ox and rat, when incubated produced mainly 6-keto-PGF_1α, with lesser amounts of PGE₂, PGF_2α and thromboxane B₂. Biosynthesis of all prostanoids increased during incubation of the pariental pericardium of each species with arachidonic acid, but 6-keto-PGF_1α was still the major metabolite. When slices of dog heart were incubated with arachidonic acid (1 μg/ml) the rates of 6-keto-PGF_1α formation by the pariental pericardium was much greater than that of the myocardium and endocardium. Epicardial slices appeared to be intermediate in 6-keto-PGF_1α formation. The hearts of anesthetized dogs were also irrigated with Krebs' solution, and during the first 5 min of epicardial irrigation the pericardial fluid leaving the heart again contained high levels of 6-keto-PGF_1α, with lesser amounts of the other prostanoids. Addition of arachidonic acid (3 μg/ml) to the irrigating fluid caused an increase in all measured prostanoid levels, although 6-keto-PGF_1α remained the predominant metabolite. In contrast, intravenous infusion of isoproterenol selectively increased the release of 6-keto-PGF_1α from the irrigated heart. It is concluded that the pericardium and epicardium continuously release prostacyclin into the pericardial fluid, and that the increased release of this substance observed when cardiac workload increases derives mainly from these membranous sources. This raises the interesting possibility that pericardial prostacyclin might influence coronary vascular tone and chemoreflexes which arise from the epicardium during myocardial ischemia.     

Effects of hydrocortisone, oestradiol and progesterone on A23187-stimulated prostaglandin output from the guinea-pig uterus superfused in vitro

Hydrocortisone (10 μg/ml) had no effect on the basal outputs and A23187-stimulated outputs of PGF_2α, PGE₂ and 6-keto-PGF_1α from the Day 15 guinea-pig uterus superfused . These findings indicate that the high output of PGF_2α from the guinea-pig uterus during the last one-third of the oestrous cycle is not modulated by the adrenal glucocorticoid hormones. Progesterone (10 gmg/ml) had no effect on the A23187-induced increases in PG output from the Day 15 guinea-pig uterus. However, oestradiol (10 gmg/ml but not 1 μg/ml) significantly reduced the increases in outputs of PGF_2α, PGE₂ and 6-keto-PGF_1α induced by A23187 from the Day 15 guinea-pig uterus, without affecting basal PG outputs. The increase in uterine tone induced by A23187 in the Day 15 guinea-pig uterus was reduced by 20–50% by oestradiol (10 μg/ml). The addition of oestradiol (10 μg/ml) and progesterone together (10 gmg/ml) produced the same effects on the Day 15 guinea-pig uterus as oestradiol alone. Oestradiol (10 μg/ml) also reduced the A23187-induced increases in PG output from the Day 7 guinea-pig uterus, but did not reduce the increase in uterine tone. Oestradiol (10 gmg/ml) reduced the increases in outputs of PGF_2α, PGE₂ and 6-keto-PGF_1α induced by exogenous arachidonic acid from the Day 7 and Day 15 guinea-pig uterus. Previous studies have shown that oestradiol is not a cyclo-oxygenase inhibitor. The present findings suggest that oestradiol, at a relatively high concentration, may interfere with the access of arachidonic acid to the cyclo-oxygenase enzyme. This action of oestradiol may explain its anti-luteolytic action when administered to guinea-pigs in large doses after Day 9 of the cycle.     

Effect of in vivo prostaglandin treatment on H-PGF2α uptake in hamster corpora lutea

Pregnant hamsters were administered (SC) prostaglandin or vehicle on the morning of the 4th day of pregnancy. Serum progesterone was significantly depressed (p<.01) at 0.5, 2, and 6 hours after treatment with 100 μg PGF_2α. Serum progesterone levels were unchanged 2 hours and 6 hours after treatment with 100 μg PGF_2β and 2 hours after treatment with 1 mg PGF_2β. Progesterone levels were depressed to less than 1 ng/ml 6 hours after treatment with 1 mg PGF_2β. The specific uptake of ³H-PGF_2α in whole hamster corpora lutea was significantly depressed 2 hours and 6 hours following 100 μg PGF_2α treatment. A 15% depression in specific uptake occurred 0.5 hour post-treatment. Treatment with 100 μg PGF_2β resulted in no change. Administration of 1 mg PGF_2β resulted in depressed ³H-PGF_2α uptake at both 2 and 6 hours post-treatment.Prostacyclin (PGI₂) treatment resulted in no change in either ³H-PGF_2α specific uptake or serum progesterone 2 hours after 100 μg treatment SC. These parameters were both reduced approximately 30% 6 hours post-treatment. Treatment with 6-keto-PGF_1α resulted in a complete lack of measurable ³H-PGF_2α uptake and serum progesterone levels less than 1 ng/ml at both 2 and 6 hours after treatment with 1 mg SC.     

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.     

Dose-related action of estradiol on placental prostanoid prediction

Prostanoids play an important role throughout all of pregnancy and during the initiation and progress of labor. The human placenta at term produces large quantities of prostanoids, yet little is known of the factors that regulate their biosynthesis. Herein, we report the effect of estradiol or estradiol and progesterone on the basal release of placental prostanoids from fresh human term placental explants using a perifusion system.The basal release of prostaglandin E₂ (PGE₂, prostaglandin F_2α (PGF_2α), thromboxane (TxB₂) and 6-keto-prostaglandin F_1α (6-keto-PGF_1α) increased about 50% from the fifth to the ninth hour in culture, while the release of 13, 14-dihydro-15-keto-PGF (PGFM) remained constant and hCG release decreased. The dose-related effect of estradiol (20–2,000 ng/ml) in the perifusing medium starting at the fifth hour of perifusiOn (i.e., the zero treatment time) effected no change in the release of TxB₂, PGF_2α, PGFM or hCG. A biphasic action on the release of 6-keto-PGF,. was observed, i.e. it was significantly decreased when incubated with 20 ng/ml of estradiol, but effected an increase after exposure to 200 ng/ml. The concomitant addition of progesterone (2,000 ng/ml) with estradiol (200 ng/ml) significantly inhibited the stimulatory action of estradiol at this dose. The release of PGE₂ was inhibited in a dose-related fashion with increasing dose of estradiol. The addition of progesterone with estradiol (2,000 and 200 ng/ml, respectively) reversed the inhibition of PGE₂ by estradiol alone.These data demonstrate that physiologic levels of estradiol affect 6-keto-PGF_α and PGE₂ release from the human term placenta, but do not significantly alter production of TxB₂, PGFM or hCG under these conditions.     

14C-labeled arachidonic acid bioconversion in guinea pig placenta during the last third of gestation

In the presence study we investigated the arachidonic acid metabolism in guinea pig placenta during the last third of gestation. Homogenates were incubated with 14C-labeled subtrate, and eicosanoid formation was determined using rp HPLC. Arachidonic acid was substantially converted to cyclooxygenase products i.e.-keto-PGF_1α, TxB₂, PGF_2α, PGE₂, PGD₂ and 12-HHT. Lipoxygenase activity was also found but of a much lower degree and represented by the mono-hydroxy acid 12-HETE and 15-HETE. The total conversion of arachiodonic acid exhibited a progressive rise from day 50 to term, due principally to the increasing part of TxB₂, PGE₂ and 12-HHT throughout this gestational perid and in addition, near term, of 6-keto-PGF_1α and PGF_2α. The results suggest that there is an increasing concentration and/or activity of cyclooxygenase system enzymes with placenta development in guinea pig, which may contribute to the augmented intrauterine availability of prostanoids under parturition.Additional experiments were performed to compare the metabolism of exogenously added 14C-arachidonic acid and endogenously present 12C-arachidonic acid during placental homogenate incubation by means of isotopes dilution GC-MS. Although the 14C- and 12-C prostanoid patterns were comparable, the 14C/12C ratios of the prostanoids formed during incubation were significantly different. These data indicate that exogenous arachidonic acid and endogenous arachidonic acid in placental homogenate do not follow up extractly the same metabolic pathway so that assumption of biochemical identity between exogenous radio-tracer and studied endogenous substrate is not quite true.     

Some interrelationships between glucose levels thromboxane production and prostacyclin production in normal and diabetic mice

We investigated the relationship between glucose levels and platelet thromboxane production or aortic prostacyclin production, using radioimmunoassay (RIA) to measure thromboxane B₂ and 6-keto-PGF_2α. We found a direct relationship (p<.05) between plasma glucose levels and thromboxane A₂ production by arachidonate stimulated platelets in platelet rich plasma of normal mice. However, when mice were deprived of food overnight, the glucose level fell but the TxB₂ production rose significantly. Moreover, mice with streptozotocin diabetes had significantly elevated glucose levels. but normal TxB₂ production, which also rose significantly after fasting. Thus in our laboratory both fasting and diabetes nullify or reverse the direct relationship between glucose levels and TxB₂ production seen in normal fed mice. This makes it diffisult to ascribe the correlation between glucose and TxB₂ levels in normal fed animals to cause and effect. RIA revealed an inverse correlation between glucose levels and 6-keto-PGF_1α production which was highly significant in aortas taken from fasted mice and stimulated for 10 minutes with 0.1mM arachidonate. This inverse correlation was present with either normal or diabetic aortas. Moreover, fasting increased the production of 6-keto-PGF_1α. However there was a significant elevation of 6-keto production by aortas of mice with diabetes of 5–6 weeks duration, compared to aortas of normal mice. Therefore either diabetes in these mice reversed a normal inhibitory effect of glucose on 6-keto production, or else the inverse correlation between glucose levels and 6-keto production does not represent a cause and effect relationship between the two variables.     

Infuence of dietary fish on eicosanoid metabolism in man

Two groups of 40 volunteers were given a dietary supplement consisting of 135 g of mackerel or meat (control) paste per day for 6 weeks. Compliance was about 80% in both groups and the daily intake of 20:5(n−3) and 22:6(n−3) from the mackerel supplement was about 1.3 and 2.3 g, respectively. In collagen-activated platelet rich plasma, the potency of blood platelet to produced HHT from arachidonic acid (AA) clearly reduced in the mackerel group, whereas the formation of HHTE from timnodonic acid (TA) increased slightly. Changes in the formation of HHT and HHTE, measured by HPLC, correlated significantly with those of TxB₂ and TxB₃, respectively, measured by GC/MS. Changes in the formation of the lipoxygenase products HETE (ex AA) and HEPE (ex TA) were qualitatively similar to that seen for the cyco-oxygenase products, but quantitatively the responses were smaller. Formation of ir TxB₂ in clotting blood significantly reduced in the mackerel group. In collagen-activated, citrated whole blood, TxB₂ formation tended to be reduced in the mackerel-supplemented volunteers. Mackerel consumption was associated with the formation of considerable amounts of PGl₃, as judged from the appearance of 2,3-dinor-Δ 17-6-keto-PGF_1α in urine. The amount of the major metabolite of PGl₂, 2,3-dinor-6-keto-PGF_1α was not reduced, or even increased. The daily amount of tetranor prostaglandin metabolites in the urine did not change significantly, which indicates that mackerel supplementation did not alter the formation of prostaglandins E and F.     

Prostacyclin,thromboxane and prostaglandin F2αin suction blister fluid of human skin: Effect of systemic aspirin and local glucocorticosteroid treatment

Changes in prostaglandin (PG) metabolism are known to be involved in various skin diseases. To elucidate the behavior of hree vasoactive PGs in human skin, namely prostacyclin (PG1₂), thromboxane A₂ (TxA₂) and PGF_2α, their stable metabolites, 6-keto-PGF_1α. TxB₂, and 13, 14 dihydro-15-keto PGF_2α (MPGF_2α), respectively, were measured by radioimmunoassays in suction blister fluids of 29 healthy male subjects. Nine of them were treated with acetylsalicylic acid (0.5 g × 4/day for one day beforehand), eight with local glucocorticoid (clobetasol-17-propionate, Dermovat^R Cream, twice a day for seven days) and 12 served as controls. All three PGs were detected in blister fluid. In controls the mean (±SD) concentration of 6-keto-PGF_1α was 1160 ± 470 pg/ml (n=12) that of TxB₂ 1590 ± 610 pg/ml (n=12) and that of MPGF_2α 1800 ± 710 pg/ml (n=12), levels which are higher than the respective concentrations in human plasma. The preceding aspirin treatment decreased the 6-keto-PGF_1α levels by 40 % (P<0.005), the TxB₂ levels by 80 % (P<0.001) and MPGF_2α levels by 35 % (P<0.05), whereas the preceding local glucocorticoid caused no changes in these PG levels. The results show that 1) PG1₂, TxA₂, and PGF_2α are locally released in the suction blister fluid of healthy human skin, 2) systemic treatment with a PG synthesis inhibiting drug, acetysalicylic acid, reduces this release, and 3) locally applied clobetasol-17-propionate does not affect the levels of prostaglandins and thromboxane as measured by our methods.     

Effects of oestradiol, progesterone, hydrocortisone and oxytocin on prostaglandin output from the guinea-pig endometrium maintained in tissue culture

The effects of oestradiol, oxytocin, progesterone and hydrocortisone on prostaglandin (PG) output from guinea-pig endomerium, removed on days 7 and 15 of the oestrous cycle and maintained in tissue culture for 3 days, have been investigated. Oetradiol (3.7 to 3700nM) and oxytocin ( 2 to 200pM) did not stimulate endometrial PGF_2α output, thus not confirming the findings of a previous report (Leaver & Seawright, 1928), nor did they stimulate the outputs of PGE₂ and 6-keto-PGF_1α. In fact, oestradiol (3700nM) inhibited the outputs of PGF_2α, PGE₂ and, to a lesser extent, 6-keto-PGF_1α. Progesterone (3.2 to 3200nM) inhibited the outputsof PGF_2α and PGE₂; hydrocortisone (2.8 to 2800nM) had no effect on endometrial PG output. These findings indicate that the inhibitory effect of progesterone on endometrial PG synthesis and release in the guinea-pig is not due to progesterone having a glucocorticoid-like action. Furthermore, progesterone had no effect on 6-keto-PGF_1α output, suggesting that the mechanisms controlling endometrial PGI₂ synthesis (as reflected by measuring 6-keto-PGF_1α) are different from those controlling endometrial PGF_2α and PGE₂ synthesis.     

Metabolism of arachidonic acid in vitro by ovine conceptuses recovered during early pregnancy

Metabolism of [³H] arachidonic acid ([³H] AA) and synthesis of prostaglandins were examined with ovine conceptuses and endometrial slices collected on various days after mating. Tissues were incubated for 24 hr with or without 5 μCi of [³H] AA and with 200 μg radioinert AA. In experiment 1, results of chromatography indicated that conceptuses collected on days 14 and 16 after mating metabolized [³H] AA to PGE₂, PGF_2α, PGFM, 6-keto-PGF_1α, and to unidentified compounds in three chromatographic regions. One of these regions (region 1) contained triglycerides. Endometrial slices metabolized only small amounts of the [³H] AA to prostaglandins. In experiment 2, results of radioimmunoassays indicated that day 14 conceptuses released somewhat similar amounts (ng/mg tissue) of PGF_2α (32.1 ± 17.9), PGFM (8.4 ± 6.2), PGE₂ (12.3 ± 7.5) and 6-keto-PGF_1α (41.4± 4.8), whereas day 16 conceptuses released more (P<.05) PGF_2α (9.0 ± 4.1) and 6-keto-PGF_1α (15.9 ± 2.7) than PGE₂ (0.9 ± 0.2) or PGFM (0.5 ± 0.08). Day 14 and 16 endometrial slices released (ng/mg tissue) more (P<.05) PGFM (3.0 ± 0.2) and 6-keto-PGF_1α (4.0 ± 0.4) than PGF_2α (0.5 ± 0.08) or PGE₂ (0.05 ± 0.02). In experiment 3, conceptuses were recovered on days 16, 20 and 24 of pregnancy and incubated with [³H] AA to determine the effects of indomethacin on [³H] AA metabolism. In general, indomethacin (Id; 4 × 10⁻⁴ M) reduced (P<.05) the percentage of total dpm recovered as prostaglandins, but Id increased the release of chromatographic region I. Experiment 4 was conducted with day 16, 20 and 24 conceptuses to evaluate the time course of metabolism of [³H] AA, and the appearance of region I and of prostaglandins. In general, the percentage of total dpm in region I increased as the percentage of dpm as [³H] AA decreased. The percentage of dpm as prostaglandins increased as the percentage of dpm in region I decreased. Prostaglandins, probably essential for embroynal survival and development, were synthesized in vitro by ovine conceptuses.     

Excretion of primary prostanoids and their metabolites during acute volume expansion

Simultaneous determination of urinary excretion rates of primary unmetabolized prostanoids and their enzymatic metabolites were performed by gas chromatography-mass spectrometry (GC/MS) or tandem mass spectrometry (GC/MS/MS). Changes in kidney function were induced by acute (4 h) volume expansion. Despite marked changes in urine flow, GFR, urinary pH, osmolality, sodium and potassium excretion, only a insignificant or transient rise in the enzymatic prostanoid metabolites (2,3-dinor-6-keto-PGF_1α, PGE-M, 2,3-dinor-TxB₂ and 11-dehydro-TxB₂) was observed. The excretion rates of the primary prostanoids were elevated in parallel with the rise in urine flow: PGE₂ rose (p < 0.05) from 14.2 ± 4.0 to 86.2 ± 20.7, PGF_2α from 60.0 ± 4.9 to 119.8 ± 24.0, 6-keto-PGF_2α from 7.2 ± 1.3 to 51.5 ± 17.0, and txB₂ from 11.2 ± 3.3 to 13.6 ± 3.6 ng/h/1.73 m² ( ) at the maximal urine flow. Except for 6-keto-PGF_1α and TxB₂, this rise in urinary prostanoid levels was only transient despite a sustained fourfold elevated urine flow. We conclude that urine flow rate acutely affect urine prostanoid excretion rates, however, over a prolonged peroid of time these effects are not maintained. The present data support the concept that urinary levels of primary prostanoids mainly reflect renal concentrations whereas those of enzymatic metabolites reflect systemic prostanoid activity. From the excretion pattern of TxB₂ one can assume that this prostanoid represents renal as well as systemic TxA₂ activity.     

Ascorbic acid promotes prostanoid release in human lung parenchyma

Ascorbic acid reduces airway reactivity to inhaled bronchoconstrictor agents in man and guinea pigs. The precise mechanism(s) responsible for this effect are unknown, but in both species an acute indomethacin treatment reverses the action of the ascorbic acid. To determine if ascorbic acid promotes prostanoid synthesis and/or inhibits degradation, human lung parenchymal slices (100–200mg) were incubated for 60 minutes in oxygenated Tyrode's solution alone or with sodium ascorbate (0.001M–1M) and/or methacholine (1μM–100μM) and/or indomethacin (0.17μM–17μM). Aliquots of the incubation medium were assayed by radioimmunoassay for PGE₂, PGF_2α, thromboxane B₂ and 6-keto-PGF_1α. Ascorbic acid increased the accumulation of all four prostanoids in the incubation medium, especially thromboxane B₂ and 6-keto-PGF_1α. This stimulatory effect of ascorbic acid was concentration-dependent and was inhibited by indomethacin. We conclude that ascorbic acid can alter prostanoid generation by human lung tissue and this effect may, in part, explain its antibronchoconstrictor activity in man.     

The effect of physiological levels of non-esterified fatty acids on the radioimmunoassay of prostaglandins

Non-esterified fatty acids (NEFA) can significantly interfere with the radioimmunoassay of PGE and PGF_2α using commercially available anti-sera. PGB₁ antigen-antibody binding is 50% inhibited by 110 pg of PGB₁, 48 ng of PGE₁, 3.5 μg of PGF_2α, or 9.0 μg linoleic, 14 μg arachidonic, 22 μg δ-linoleic, 40 μg palmitoleic or 45 μg oleic acids. PGF_2α antigen-antibody binding is 50% inhibited by 270 pg of PGF_2α, 70 ng of PGE₁, or 4.2 μg arachidonic, 14 μg δ-linolenic, 22 μg linoleic, 70 μg palmitoleic or 110 μg oleic acids. Physiological levels of NEFA, such as the quantities found in small volumes of plasma, are sufficient to prohibit accurate prostaglandin measurements. Chromatography on small columns of silicic acid proved to be an effective technique for separation of NEFA and prostaglandin from lipid extracts, however, the results of this study suggest that the interference produced by the presence of NEFA in the measurement of prostaglandin from certain physiological fluids may be avoided if the prostaglandins are not extracted prior to radioimmunoassay.