Bioconversion of arachidonic acid by human uterine cervical tissue and endocervix in late pregnancy

Prostaglandins (PGs) may play an important role on cervical ripening in late pregnancy, namely cervical dilatation and softening. To investigate this, arachidonic acid metabolites of cervical tissue and endocervix were studied. To separate and identify the metabolites, silicic acid chromatography, thin layer chromatography, reversed phase chromatography, gas-liquid chromatography and GC-MS were used. In cervical tissue, arachidonic acid was converted to 6-ketoPGF1 alpha, PGF2 alpha, PGE2, thromboxane B2, and 12-HETE. In endocervix, arachidonic acid was converted to PGF2 alpha, PGE2, thromboxane B2, 12-hydroxy-5, 8, 10-heptadecatrienoic acid, and 12-HETE. There was no relation between the arachidonic acid conversion rate and the Bishop score (points of cervical ripening).     

Prostaglandin and thromboxane production by rat decidual microsomes

The preparation of a microsomal fraction from the decidual tissue of pregnant rat uteri is described. Incubation of such microsomes with a mixture of radiolabelled and cold arachidonic acid (51 micrometer) plus cofactors resulted in a 30% substrate conversion. Products were resolved into four peaks (A, B, C and D) by thin-layer chromatography. Combined gas-liquid chromatography-mass spectrometry and further thin-layer chromatography identified the products as PGF2 alpha (A); thromboxane B2 (B); a mixture of 6-OXO PGF1 alpha and PGE2 (C); PGD2 and PGE2 (D). PGE2 was the major product.     

A radioimmunoassay for 6-keto-prostaglandin F1alpha

A radioimmunoassay for 6-keto-prostaglandin F1alpha has been developed. The assay is accurate and sensitive but since the antiserum cross-reacts 5-10% with prostaglandins (PGs) of the E and F series, solvent extraction and thin layer chromatography are required for absolute specificity. The assay has been validated by comparison with a radiochemical assay and by the use of an inhibitor of 6-keto PGF1alpha formation, 15-hydroperoxy arachidonic acid. 6-Keto PGF1alpha was found to have a low cross reaction with antisera directed against PGE2, PGF2alpha and thromboxane B2.     

Differential production of prostaglandins within the human uterus

The ability of broken cell preparations of human endometrium, myometrium and a mixture of endometrium and myometrium to convert 14C arachidonic acid to prostaglandins (PG's) was compared. Endometrium metabolished arachidonic acid predominantly to a mixture of PGF2 alpha and PGE2. A similar weight of myometrium showed relatively little activity, the major product identified was 6 oxo PGF1 alpha. However, a combination of endometrium and myometrium showed an enhanced conversion of arachidonic acid to 6 oxo PGF1 alpha associated with a decreased production of PGF2 alpha and PGE2. This suggests that human endometrium and myometrium differ in their ability to metabolize arachidonic acid and in their ability to convert the endoperoxides formed, to PG's.     

Eicosanoids production in endometriosis

In order to investigate the production of eicosanoids in human endometrium, myometrium, leiomyoma, adenomyosis, normal ovary, non-endometrial cyst and endometrial cyst, slices of each tissue were incubated. 6-Keto-prostaglandin (PG) F1 alpha, thromboxane (TX) B2, PGF2 alpha and PGE2 concentrations in the incubation medium were measured by direct RIA. 6-Keto-PGF1 alpha production of adenomyosis was significantly higher than that of endometrium, myometrium and leiomyoma, especially in the menstrual phase. The production of eicosanoids in endometrial cyst was significantly higher than that of non-endometrial cyst and normal ovary. These results suggest that endometriosis is associated with increased eicosanoid production in vivo.     

The effects of prostanoids on estrogen-dominated rat myometrial longitudinal muscle in vitro

The purpose of these experiments was to characterize the contractile response of longitudinal muscle from the estrogen-dominated rat uterus to natural and synthetic prostanoids. The biological significance is 1) to provide evidence for or against a physiological role for each natural prostanoid in the regulation of myometrial activity, 2) to determine if each prostanoid has pharmacological potential for the manipulation of myometrial activity, and 3) to understand the structural requirements for prostanoid action on the myometrium. All analogs tested produced excitation of the myometrium in vitro through what appeared to be a direct action on the muscle. The order of potency of the natural prostanoids was prostaglandin (PG) F2 alpha = PGD2 = PGE2 = PGE1 greater than PGA2 = PGB2 = 6-keto-PGF1 alpha. This order of potency was not consistent with any single currently recognized prostanoid receptor. Furthermore, PGF2 alpha had an EC50 (effective concentration that produces 50% of the maximal response) of 0.5 microM, which was low in comparison to other PGF2 alpha-sensitive tissues. There were large differences in the maximum tension developed in response to the prostanoids tested, only PGF2 alpha, PGE2 and 6-keto PGF1 alpha were full agonists. Although the simplest explanation of these data was that the rat uterus contains a single novel type of prostanoid receptor, the existence of multiple receptor subtypes could not be disproved. Evidence from the effect of synthetic analogs suggested that neither thromboxane A2 nor PGI2 are physiological regulators of activity in this tissue.     

Prostaglandin release from in vitro guinea-pig gallbladder

In order to study prostaglandin release from guinea pig gallbladder, full thickness tissue sections were incubated for one hour in Krebs solution. Extraction and two dimensional chromatography of incubation media obtained in the presence of radio-labelled arachidonic acid demonstrated the presence of PGE2, PGF2 alpha, 6-keto-PGF1 alpha and thromboxane B2. These results were supported by radioimmunoassay of incubations conducted in the absence of exogenous arachidonate and in the presence of varying concentrations of unlabelled exogenous arachidonate. The previously reported predominance of PGE2 was only seen at high concentrations of exogenous arachidonate.     

Effective use of rabbit gastric antral mucosal slices in prostaglandin synthesis and metabolism studies

Intact slice preparations of rabbit stomach (antral mucosa, corporal mucosa, antral muscle and corporal muscle) were incubated and the released prostaglandins (PGs) were measured by reverse-phase high-performance liquid chromatography using 9-anthryldiazomethane for derivatization. With respect to total PG production, the highest amounts were generated by antral mucosal slices. Antral mucosal slices produced PGE2, 6-keto PGF1 alpha, thromboxane B2, PGF2 alpha and PGD2 (in descending order of magnitude) and possessed a high capacity for producing 13,14-dihydro-15-keto derivatives of both PGE2 and PGF2 alpha. Studies utilizing aspirin, EGTA or Ca2+ revealed that PG release by antral mucosal slices in the present in vitro system reflects a composite of the activities of phospholipase A3, PG cyclooxygenase and PG-metabolizing enzymes. These results show that antral mucosal slices will be useful in physiological and pharmacological studies on PG synthesis and metabolism of the stomach.     

Sex differences in gallbladder prostaglandin synthesis mediated by acute inflammation

The influences of sex and acute inflammation on prostaglandin biosynthesis in rabbit gallbladder were examined by radiochromatography. Male rabbit gallbladder microsomes converted small amounts of labelled arachidonate to total prostaglandin synthesis with PGE2, 6-keto PGF1 alpha (stable metabolite of PGI2) and PGF2 alpha as the major products synthesized. Microsomes from the male rabbit gallbladder inflamed by bile duct ligation for 3 days increased total prostaglandin synthesis five-fold with 6-keto PGF1 alpha being the major prostaglandin produced. Female rabbit gallbladder microsomes converted three times more arachidonate to total prostaglandin synthesis than did microsomes from the male rabbit. Bile duct ligation did not alter total prostaglandin biosynthesis in the female rabbit gallbladder, but significantly decreased synthesis of PGE2, thromboxane B2 and PGF2 alpha and increased synthesis of 6-keto PGF1 alpha. These data suggest that although bile duct ligation had different effects on male and female gallbladder total prostaglandin synthesis, 6-keto PGF1 alpha is the major product induced by this stimulus for acute inflammation.     

Prostaglandin E2 and F2 alpha in mid-pregnant rat uterus and at parturition

Prostaglandin E2 (PGE2) and F2 alpha (PGF2 alpha) produced by 15 days pregnant rat myometrium, by parturient rat myometrium and myometrium plus endometrium were measured in vitro. The results showed that the PGs produced by parturient myometrium were higher than these obtained during mid-pregnancy. Myometrium with endometrium released more PGs than myometrium alone, and the addition of arachidonic acid (AA) at 10 DM did not show any significant effect. Exogenous progesterone or estradiol-17b at a concentration of 1 Dmol had no effect on parturient uterine PG secretions.     

The relation between thromboxane and prostaglandin synthesis in human decidua tissue: a comparison of eicosanoid synthesis in minced tissue with that in a cell-free preparation

Radiotracer studies and radioimmunoassay measurements demonstrate that minced tissues of human decidua produce chiefly thromboxane B2 (TxB2) (70% of total eicosanoids) and small amounts of prostaglandin F2 alpha (PGF2 alpha) (13%) PGD2 (8%), 6-keto-PGF1 alpha (5%) and PGE2 (4%). Inhibition of thromboxane synthesis with a specific inhibitor (OKY-1581: sodium (E)-3-[4(-3-pyridylmethyl)-phenyl]-2-methyl propenoate) increased prostaglandin formation in general, with the main product being PGF2 alpha (38%), a nonenzymic derivative of PGH2. Crude particulate fractions prepared from the same tissue synthesized two major products from [3H]arachidonate, TxB2 and 6-keto-PGF1 alpha (54 and 30%, respectively) and some PGF2 alpha and PGE2 (8-8%). However, in the presence of reduced glutathione (GSH), PGE2 became the main product (81%) (TxB2, 15%; PGF2 alpha, 2%; and 6-keto-PGF1 alpha, 2%). Half-maximal stimulation of PGE2 synthesis occurred at 46 microM GSH. The GSH concentration of tissue samples was found to be 110 +/- 30 microM. We conclude that human first trimester decidua cells possess the key enzymes of prostaglandin and thromboxane synthesis. Apparently, the production of these compounds is controlled by a specific mechanism in the tissue, which keeps PGE and prostacyclin synthesis in a reversibly suppressed state, whereas the formation of thromboxane is relatively stimulated.     

Prostaglandin and thromboxane synthesis by tissue slices from human aortic aneurysms

Sliced portions of the walls of human aortic aneurysms were incubated with extracts of human plasma and serum to determine the profile of prostanoid production. 6-Oxo-prostaglandin (PG) F1 alpha, PGE2, PGF2 alpha, and thromboxane (TX) B2 were measured by gas chromatography/electron capture mass spectrometry. 6-Oxo-PGF1 alpha, the stable hydrolysis product of PGI2, was the major cyclooxygenase product but substantial amounts of TXB2 (the hydrolysis product of TXA2), with smaller amounts of PGE2 and PGF2 alpha were also synthesised. These prostanoids could contribute to the response of the vascular wall to injury, thereby influencing the disease process. Serum extracts stimulated PGI2 and TXA2 synthesis, probably as a result of their Ca2+ content.     

Prostaglandin production by phagocytic cells of the mouse thymic reticulum in culture and its modulation by indomethacin and corticosteroids

The production of prostaglandins by phagocytic cells of the thymic reticulum in culture (P-TR) was studied by using high pressure liquid chromatography and radioimmunoassay. Radioimmunologic determinations showed that thromboxane B2 (TXB2), prostaglandin E2 (PGE2), and 6-keto-prostaglandin F1 alpha (6 keto-PGF1 alpha) were the major compounds released into the culture medium, whereas prostaglandin F2 alpha (PGF2 alpha) was only a minor component. Indomethacin and dexamethasone exerted a similar pattern of differential inhibition of the secretion of prostanoids. PGE2 and 6-keto PGF1 alpha productions were markedly decreased by these anti-inflammatory drugs, whereas those of TXB2 and PGF2 alpha were not or were only slightly affected. Experiments performed with an antiglucocorticoid compound (RU 38486) showed that the steroid-induced inhibition of prostanoid secretion is a classical receptor-mediated action. These results demonstrated that phagocytic cells of the thymic reticulum, which resemble the thymic interdigitating cells, produce several types of prostaglandins. Because it has been described that P-TR regulate thymocyte proliferation in vitro via the secretion of both interleukin 1 and PGE2, these results suggest that anti-inflammatory agents may be able to modulate the thymic microenvironment and, consequently, thymocyte proliferation.     

Effect of prostaglandins against alloxan-induced diabetes mellitus

Previously, we observed that alloxan-induced in vitro cytotoxicity and apoptosis in an insulin secreting rat insulinoma, RIN, cells was prevented by prior exposure to prostaglandin (PG) E(1), PGE(2), PGI(2), PGF(1)(alpha), and PGF(3)(alpha) (P<0.05 compared to alloxan), whereas thromboxane B(2) (TXB(2)) and 6-keto-PGF(1)(alpha) were ineffective. In an extension of these studies, we now report that prior intraperitoneal administration of PGE(1), PGE(2), PGF(1)(alpha), and PGF(3)(alpha) prevented alloxan-induced diabetes mellitus in male Wistar rats, whereas PGI(2), TXB(2), and 6-keto PGF(1)(alpha) were not that effective. PGE(1), PGE(2), PGF(1)(alpha), and PGF(3)(alpha) not only attenuated chemical-induced diabetes mellitus but also restored the antioxidant status to normal range in red blood cells and pancreas. These results suggest that PGE(1), PGE(2), PGF(1)(alpha), and PGF(3)(alpha) can abrogate chemically induced diabetes mellitus in experimental animals and attenuate the oxidant stress that occurs in diabetes mellitus.     

Effect of bacterial cell wall and lipopolysaccharide on arachidonic acid metabolism by caruncular and allantochorionic tissues from cows that calved normally and those that retained fetal membranes

Immunoactive eicosanoids may have a role in both placental separation and uterine involution in cattle. In the present study, we examined the effects of bacterial cell wall preparation and endotoxins on in vitro prostaglandin synthesis and arachidonic acid (AA) metabolism by caruncular and allantochorionic tissues. Placentomes were obtained about 6 h post partum from cows that delivered normally (n = 10) or those with retained fetal membranes (n = 4); the tissue explants were incubated for 6 h in the presence of labeled or nonlabeled AA. Prostaglandin F(2alpha) (PGF(2alpha)) and E(2) (PGE(2)) were measured by radioimmunoassay, and labeled AA metabolites were separated by reverse phase-high pressure-liquid chromatography. There was no effect of bacterial cell wall preparations or endotoxins on in vitro caruncular PGF(2alpha) secretion. However, bacterial products increased caruncular PGE(2) secretion in both cows that delivered normally and those with retained fetal membranes. For normal delivery cows caruncular tissue, bacterial product also increased leukotriene B(4) (LTB(4)) and decreased both thromboxane B(2) (TXB(2)) and hydroxy-eicosatetranoic acids (HETE) in vitro secretion. For the allantochorion, bacterial products increased in vitro PGF(2alpha) secretion only in cows that delivered normally and increased PGE(2) secretion essentially in cows with retained fetal membranes. In general, 6 keto PGF(1alpha) was the main metabolite secreted by both allantochorionic and carucular tissues. However, in cows with retained fetal membranes, PGE(2) became the most important metabolite secreted by allantochorion, especially in the presence of endotoxin. In conclusion, these results suggest that bacteria found in the early postpartum uterus or their endotoxin affect primarily caruncular and allantochorionic PGE(2) synthesis.     

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 microgram/g tissue) to both PGE2 (10.7 microgram/g tissue) and TXB2 (6.2 microgram/g tissue. Smaller amounts of PGF2alpha (0.9 microgram/g) and 6-oxoPGF1alpha 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, PGF2alpha or 6-oxoPGF1alpha. Homogenates from rabbit lungs converted arachidonic acid (100 microgram/g) mainly to PGE2, both before and after birth. The amount of PGE2 formed increased during gestation to a maximum of about 6 microgram/g tissue at 28 days of gestation. It then decreased to a minimum (1.5 microgram/g) which was observed 8 days after birth, followed by an increase to about 4 microgram/g in older rabbits.     

The urinary excretion of prostaglandins and thromboxane B2 in healthy volunteers: a study in males and females, and on the influence of seminal fluid contamination

Radioimmunoassay measurements of prostaglandins (PGs) E2, F2 alpha, 6-keto-PGF1 alpha and thromboxane (Tx) B2 in 24 h urine specimens from a male and a female healthy volunteer on several consecutive days revealed a dramatic increase of PGE2, PGF2 alpha, 6-keto-PGF1 alpha on days, upon which they had sexual intercourse; only TxB2 remained stable. Furthermore, the PGE2/PGF2 alpha ratio rose to values greater than 0.5 on days with sexual intercourse. This was found to be due to contamination of the urine samples by seminal fluid. Two 24 h urine samples from each of 26 healthy male and female volunteers (HV) revealed higher (p less than 0.01) mean PGE2 and PGF2 alpha values in males than in females. The results show that the interpretation of the urinary PG excretion as a measure of renal PG synthesis should be considered carefully, and that a PGE2/PGF2 alpha ratio greater than 0.5 indicates probable seminal contamination of urine.     

Arachidonic acid metabolites in pregnant rat uterus

Production of prostaglandin E2 (PGE2), F2 alpha (PGF2 alpha) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) by pregnant rat uterus were measured in vitro. At mid-pregnancy, myometrium incubated with decidua attached released more prostanoids into the culture medium than when incubated without. As pregnancy progressed to 21 days more prostanoids were detected in the culture medium. However, no significantly increased conversion of exogenous arachidonic acid (AA) by myometrium was found.     

Redirection of eicosanoid metabolism in mPGES-1-deficient macrophages

Microsomal prostaglandin E synthase (mPGES)-1 is one of several prostaglandin E synthases involved in prostaglandin H2 (PGH2) metabolism. In the present report, we characterize the contribution of mPGES-1 to cellular PGH2 metabolism in murine macrophages by studying the synthesis of eicosanoids and expression of eicosanoid metabolism enzymes in wild type and mPGES-1-deficient macrophages. Thioglycollate-elicited macrophages isolated from mPGES-1-/- animals and genetically matched wild type controls were stimulated with diverse pro-inflammatory stimuli. Prostaglandins were released in the following order of decreasing abundance from wild type macrophages stimulated with lipopolysaccharide: prostaglandin E2 (PGE2)>thromboxane B2 (TxB2)>6-keto prostaglandin F1alpha (PGF1alpha), prostaglandin F(2alpha) (PGF2alpha), and prostaglandin D2 (PGD2). In contrast, we detected in mPGES-1-/- macrophages a >95% reduction in PGE2 production resulting in the following altered prostaglandin profile: TxB2>6-keto PGF1alpha and PGF2alpha>PGE2, despite the comparable release of total prostaglandins. No significant change in expression pattern of key prostaglandin-synthesizing enzymes was detected between the genotypes. We then further profiled genotype-related differences in the eicosanoid profile using macrophages pre-stimulated with lipopolysaccharide followed by a 10-min incubation with 10 microm [3H]arachidonic acid. Eicosanoid products were subsequently identified by reverse phase high pressure liquid chromatography. The dramatic reduction in [3H]PGE2 formation from mPGES-1-/- macrophages compared with controls resulted in TxB2 and 6-keto PGF1alpha becoming the two most abundant prostaglandins in these samples. Our results also suggest a 5-fold increase in 12-[3H]hydroxyheptadecatrienoic acid release in mPGES-1-/- samples. Our data support the hypothesis that mPGES-1 induction in response to an inflammatory stimulus is essential for PGE2 synthesis. The redirection of prostaglandin production in mPGES-1-/- cells provides novel insights into how a cell processes the unstable endoperoxide PGH2 during the inactivation of a major metabolic outlet.     

High performance liquid chromatography and UV detection for the separation and quantitation of prostaglandins

A fast and reliable method for the separation and quantitation of arachidonic acid metabolites PGF1 alpha, PGF2 alpha, PGD2, PGE1, PGE2, PGB2, PGA2, 6-keto PGE1, 6-keto PGF1 alpha, TxB2 and 15-keto PGE2 by high-performance liquid chromatography has been developed. Utilizing a single reverse-phase column and a UV spectrophotometer, sensitivity as little as 30 nanograms of each of these prostaglandins can be separated and subsequently detected. Although this study was performed using standards, it is highly promising for future application to biological fluids.