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.     

Simultaneous quantification of prostaglandins in human synovial cell-cultured medium using liquid chromatography/tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric (LC/MS-MS) method was developed for the simultaneous quantification of prostaglandin (PG) E(2), PGF(2alpha), 6-keto-PGF(lalpha) and thromboxane (TX) B(2). These eicosanoids and their deuterium derivatives, using as internal standards, were extracted by solid-phase extraction and analyzed using LC/MS-MS in the selected reaction-monitoring (SRM) mode. A good linear response over the range of 10 pg to 10 ng for each eicosanoid was demonstrated. The accuracy of added eicosanoids ranged from 94.1 to 106.6% and coefficients of variation ranged from 0.62 to 7.8%. Furthermore, we applied this method for the determination of eicosanoids in the human synovial cell-cultured medium, stimulated by lipopolysaccharide (LPS). LPS produced each eicosanoid and they increased in a time-dependent manner. The production levels after 24 h stimulation were 6-keto-PGF(1alpha) > PGE(2) > TXB(2) > PGF(2alpha). This simultaneous quantification method is so useful to clarify the function of synovial cells in rheumatoid arthritis (RA).     

Effects of exogenous prostanoids on the proliferation of osteoblast-like cells in vitro

The effects of several prostaglandins on the proliferation of secondary cultures of osteoblast-like cells, as measured by the incorporation of [3H]-thymidine into DNA and total DNA content of the cultures, were studied. PGE2 in the concentration range of 10(-8) to 10(-5) M caused a direct, dose-related stimulation of proliferation, while PGF2 alpha and PGD2 were less effective. PGA2 and 6-keto-PGF1 alpha were inactive in the osteoblasts in concentrations of 10(-7) to 10(-6) M. A similar stimulation profile was observed for the induction of ornithine decarboxylase (ODC, L-ornithine decarboxy-lyase, EC 4.1.1.17): the order of potency of the different prostaglandins in the induction of the ODC activity was PGE2 greater than PGF2 alpha = PGD2; again, PGA2 and 6-keto-PGF1 alpha were without effect in concentrations up to 10(-6) M. These results show that the primary prostaglandins, in order of potency PGE2 greater than PGF2 alpha = PGD2, can have a direct, stimulatory effect on the proliferation of osteoblasts, which is closely related to the induction of ODC activity.     

Simultaneous analysis of prostaglandin glyceryl esters and prostaglandins by electrospray tandem mass spectrometry

A high-performance liquid chromatography (HPLC) positive-ion electrospray ionization tandem mass spectrometry method for the quantification of prostaglandin glyceryl esters (PG-Gs), a newly discovered class of eicosanoids, is described. All four PG-Gs (PGE(2)-G, PGD(2)-G, PGF(2alpha)-G, and 6-keto-PGF(1alpha)-G) and the prostaglandins (PGs) that are formed by their hydrolysis are simultaneously quantified. Analytes were purified via reverse-phase solid-phase extraction, separated by reverse-phase HPLC, and quantified on a triple-quadrupole mass spectrometer using selected reaction monitoring. Quantification was achieved by stable isotope dilution employing penta-deuterated (PG-Gs) or tetra-deuterated (PGs) analogs. Analyte recovery from cell culture medium was >43% for all analytes at four different concentration levels. The limit of quantification is in the range of 25fmol on-column for each analyte and the analytes exhibit a linear response over approximately a 500-fold range. This method allows simultaneous profiling of several PG-Gs and PGs without multistep sample purification or derivatization.     

Analysis of leukotrienes, prostaglandins, and other oxygenated metabolites of arachidonic acid by high-performance liquid chromatography

High-performance liquid chromatography procedures were developed which separate leukotrienes (LTs), hydroxy-fatty acids (HETEs), prostaglandins (PGs), the stable metabolite of prostacyclin (6-keto-PGF1 alpha), the stable metabolite of thromboxane A2 (TXB2), 12-hydroxyheptadecatrienoic acid (HHT), and arachidonic acid (AA). Two methods employing reverse-phase columns are described. One method uses a radial compression system, the other a conventional steel column. Both systems employ methanol and buffered water as solvents. The radial compression system requires 60 min for separation of the AA metabolites, while the conventional system requires 100 min. Both methods provide good separation and recovery of 6-keto-PGF1 alpha, TXB2, PGE2, PGF2 alpha, PGD2, LTC4, LTB4, LTD4, LTE4, HHT, 15-, 12-, and 5-HETE; and AA. The 5S,12S-dihydroxy-6-trans, 8-cis, 10-trans, 14-cis-eicosatetraenoic acid (5S,12S-diHETE), a stereoisomer of LTB4, coelutes with LTB4. To determine the applicability of the methods to biologic systems, AA metabolism was studied in two models, guinea pig lung microsomes and rat alveolar macrophages. Both HPLC systems demonstrated good recovery and resolution of eicosanoids from the two biological systems. A simple evaporation technique for HPLC sample preparation, which avoids the use of chromatographic and other time-consuming methodology, is also described.     

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 microCi of [3H] AA and with 200 micrograms radioinert AA. In experiment 1, results of chromatography indicated that conceptuses collected on days 14 and 16 after mating metabolized [3H] AA to PGE2, PGF2 alpha, PGFM, 6-keto-PGF1 alpha, and to unidentified compounds in three chromatographic regions. One of these regions (region I) 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 PGF2 alpha (32.1 +/- 17.9), PGFM (8.4 +/- 6.2), PGE2 (12.3 +/- 7.5) and 6-keto-PGF1 alpha (41.4 +/- 4.8), whereas day 16 conceptuses released more (P less than .05) PGF2 alpha (9.0 +/- 4.1) and 6-keto-PGF1 alpha (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 less than .05) PGFM (3.0 +/- 0.2) and 6-keto-PGF1 alpha (4.0 +/- 0.4) than PGF2 alpha (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 X 10(-4) M) reduced (P less than .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 embryonal survival and development, were synthesized in vitro by ovine conceptuses.     

Liquid chromatographic-mass spectrometric determination of cyclooxygenase metabolites of arachidonic acid in cultured cells

A liquid chromatographic-electrospray ionization-mass spectrometric (LC-ESI-MS) technique was developed to simultaneously determine the cyclooxygenase metabolites of arachidonic acid (6-keto-PGF(1alpha), PGD(2), PGE(2), PGF(2alpha), and PGJ(2)) produced by cultured cells. Samples were separated on a C(18) column with water-acetonitrile mobile phase, ionized by electrospray, and detected in the positive mode. Selected ion monitoring (SIM) of m/z 353, 335, 335, 319, and 317 were used for quantifying 6-keto-PGF(1alpha), PGD(2), PGE(2), PGF(2alpha), and PGJ(2), respectively. Prostaglandins were detected at concentrations as low as 1 pg (S/N=3) on the column. The method was used to determine the production of PGs from bovine coronary artery endothelial cells (ECs) and human prostate cancer cells (PC-3) with different degree of invasiveness. Bradykinin (10(-6) M) stimulated a marked increase in the production of 6-keto-PGF(1alpha), PGE(2), and PGF(2alpha) and a small increase of PGD(2) by ECs. 6-Keto-PGF(1alpha) was the major metabolite in these cells. The production of PGE(2) was threefold higher and PGD(2) was twofold higher in PC-3-S (invasive) cells than in PC-3-U (non-invasive) cells.     

Selective inhibition of arachidonic acid metabolite release from human lung tissue by antiinflammatory steroids

The effects of antiinflammatory steroids on arachidonic acid metabolite release from human lung fragments were analyzed. Incubation of lung fragments for 24 hr with 10(-6) M dexamethasone inhibited the net release of the prostacyclin metabolite 6-keto-PGF1 alpha, PGE2, and PGF2 alpha from lung fragments stimulated with anti-IgE but failed to inhibit the anti-IgE-induced release of PGD2, TXB2, and iLTC4. The IC50 of dexamethasone for inhibition of both spontaneous and anti-IgE-induced 6-keto-PGF1 alpha release was approximately 2 X 10(-8) M, and a 6-hr preincubation with the drug was required for 50% inhibition of prostaglandin release. Other agents were tested for activity in stimulating arachidonic acid metabolite release from human lung fragments. FMLP (fmet-leu-phe) stimulated the release of all metabolites tested (6-keto-PGF1 alpha, PGD2, PGE2, PGF2 alpha, TXB2, iLTC4); platelet-activating factor (PAF), but not lysoPAF, stimulated the release of PGD2, TXB2, and iLTC4. In contrast to the case with anti-IgE, where dexamethasone failed to inhibit net PGD2 and TXB2 release, the steroid inhibited the release of these metabolites stimulated by both FMLP and PAF. The steroid inhibited iLTC4 release induced by the highest concentration of PAF (10(-6)M) but did not inhibit iLTC4 release stimulated by either 10(-7) M PAF, FMLP, or anti-IgE. Because neither FMLP nor PAF caused the release of PGD2 or TXB2 from purified human lung mast cells, and because they also failed to induce histamine release from lung fragments, it is suggested that these stimuli produce PGD2 and TXB2 release in lung fragments through an action on a cell distinct from the mast cell. This suggestion is supported by the selective inhibition of the release of these arachidonic acid metabolites by dexamethasone. We suggest that the inhibitory action of steroids on arachidonic acid metabolite in human lung fragments contributes to their therapeutic efficacy in pulmonary diseases.     

Measurement of icosanoids. Report of the Group for Standardization of Methods in Icosanoid Research

This statement from laboratories highly qualified in icosanoid analysis identifies the urgent need for the availability of the following compounds in labeled (deuterium and tritium) and unlabeled form: PGE2 PGF2 alpha PGD2 6-keto-PGF1 alpha Thromboxane B2 9 alpha,20-dihydroxy-11,15-dioxo-2,3- dinorprost -5-enoic acid 9 alpha-hydroxy-11,15-dioxo-2,3,18,19- tetranorprost -5-ene-1,20-dioic acid 15-keto-13,14-dihydro-PGE2 15-keto-13,14-dihydro-PGF2 alpha 5 alpha-7 alpha-dihydroxy-11- ketotetranorprosta -1,16-dioic acid 7 alpha-hydroxy-5,11-diketo- tetranorprosta -1,16-dioic acid 2,3 dinor-thromboxane B2 2,3 dinor-6-keto-PGF1 alpha 2,3 dinor-6,15-diketo 13,14 dihydro-20-carboxyl-PGF1 alpha 2,3 dinor-13,14-dihydro-6,15-diketo-PGF1 alpha LTB4 LTC4 LTD4 LTE4 LTF4 20-OH-LTB4 20-COOH-LTB4 5-HETE 12-HETE 15-HETE omega-OH-12-HETE 5S, 12S-di HETE 5S, 15S-di HETE HHT other hydroxylated polyunsaturated fatty acids and their epoxides.     

Determination of urinary 8-epi-prostaglandin F(2alpha) using liquid chromatography-tandem mass spectrometry: increased excretion in diabetics

Liquid chromatography-tandem mass spectrometry (LC/MS-MS) was applied to the quantitative analysis of urinary 8-epi-prostaglandin F(2alpha) (8-epi-PGF(2alpha)) level. 8-Epi-PGF(2alpha) and its internal standard, [(2)H(4)]-8-epi-PGF(2alpha), were extracted from urine by using a solid phase extraction cartridge and loaded to LC/MS-MS in selected reaction monitoring (SRM) mode. The standard curve showed good linearity in the range of 40 pg to 10 ng (r = 0. 997). The accuracy of the added 8-epi-PGF(2alpha) ranged from 96.8 to 104.9% with a mean +/- SD of 99.5+/-2.5%. The average level +/- SD of urinary 8-epi-PGF(2alpha) in 13 healthy volunteers (five women and eight men, 31+/-7.4 years old) was 429.4+/-149.6 pg/mg creatinine. The level of seven patients with noninsulin dependent diabetes mellitus (two women and five men, 40+/-13.6 years old), 630.9+/-275.6 pg/mg creatinine, was statistically higher than that of healthy volunteers (P<0.05). This finding suggested that diabetics are in a highly oxidative condition. This simple and rapid LC/MS-MS method can be used to elucidate the pathophysiological feature of diabetes or for monitoring the curative effect.     

Dissociation of the contractile and hypertrophic effects of vasoconstrictor prostanoids in vascular smooth muscle.

To more clearly define the physiologic roles of thromboxane (TX)A2 and primary prostaglandins (PG) in vascular tissue we examined vascular contractility, cell signaling, and growth responses. The growth-promoting effects of (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619; TXA2 agonist), PGF2 alpha, and PGE2 consisted of protein synthesis and proto-oncogene expression, but not DNA synthesis or cell proliferation. U46619 contracted rat aortas and increased cultured rat aortic vascular smooth muscle cell intracellular free calcium concentration [Ca2+]i, [3H]inositol monophosphate (IP) accumulation, myosin light chain phosphorylation, and protein synthesis ([3H]leucine incorporation) with EC50 values ranging from 10 to 50 nM. Each of these responses was inhibitable with the TXA2 receptor antagonist [1S]1 alpha,2 beta(5Z),3 beta,4 alpha-7-(3-[2- [(phenylamino)carbonyl]hydrazino]methyl)-7-oxabicyclo[2.2.1]hept-2- yl-5-heptenoic acid (SQ29548). In contrast, PGF2 alpha increased [Ca2+]i, [3H]IP, and protein synthesis with EC50 values of 30-230 nM but contracted rat aortas with an EC50 of 4800 nM. PGE2 increased [Ca2+]i, [3H]IP accumulation, protein synthesis, and contracted rat aortas with EC50 values of 2.5-3.5 microM. TXA2 receptor blockade prevented PGF2 alpha- and PGE2-induced aortic contraction and cell myosin light chain phosphorylation, but not cell signaling or protein synthesis. Binding studies to vascular smooth muscle TXA2 receptors using 1S-[1 alpha,2 beta(5Z),3 alpha(1E,3S),4 alpha]-7-(3-[3-hydroxy-4-(p- [125I]iodophenoxy)-1-butenyl]7-oxabicyclo[2.2.1]hept-2-yl)-5-hepte noic acid ([125I]BOP) showed U46619, SQ29548, PGF2 alpha, and PGE2 competition for TXA2 receptor binding at concentrations similar to their EC50 values for aortic contraction, while binding competition with [3H]PGF2 alpha and [3H]PGE2 demonstrated the specificity of [125I]BOP and SQ29548 for TXA2 receptors. The results suggest that 1) PGF2 alpha- and E2-stimulated vessel contraction is due to cross-agonism at vascular TXA2 receptors; 2) PGF2 alpha stimulates TXA2 receptor-independent vascular smooth muscle protein synthesis at nanomolar concentrations, consistent with an interaction at its primary receptor; and 3) TXA2 is a potent stimulus for vascular smooth muscle contraction and protein synthesis. We suggest that the main physiologic effect of PGF2 alpha may be as a stimulus for vascular smooth muscle cell hypertrophy, not as a contractile agonist.     

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.     

Prostaglandin synthesis by the cochlea

The exogenous and endogenous syntheses of prostaglandins (PG's) by the cochlea of adult mongolian gerbils were studied in vitro. After incubation of the whole membraneous cochlea with [3H]-arachidonic acid (AA), syntheses of PGF2 alpha, 6-keto PGF1 alpha, PGE2, thromboxane (TX) P2 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 degrees C for 0-15 min, PGD2, PGE2, PGF2 alpha and 6-keto PGF1 alpha 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, PGF2 alpha and 6-keto PGF1 alpha, 90-120 pg/cochlea; PGE2, 370 pg/cochlea), reached to the maximum level (PGD2, PGF2 alpha and 6-keto PGF1 alpha, 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 (less than 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, PGF2 alpha and 6-keto PGF1 alpha were about 1/3 of those of PGE2. In the LW, the amounts of PGD2, PGE2, PGF2 alpha and 6-keto PGF1 alpha were about the same level (70-100 pg/LW).     

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-PGF2 alpha; 13,14-diOH-15-keto-PGF2 alpha; 6-keto-PGF1 alpha 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 PGF2 alpha was shifted to the right of that for PGF2 alpha itself; the curve for 6-keto-PGF1 alpha 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 PGF2 alpha and its metabolites was long lasting (more than 3 hours) and so it was the activity evoked by PGI2;6-keto-PGF1 alpha and BaCl2, but not the contractions following 6-keto-PGE1, which disappeared much earlier. The contractile tension after PGF2 alpha; 15-keto-PGF2 alpha; 13,14-diOH-15-keto-PGF2 alpha and PGI2, increased as time progressed whilst that evoked by 6-keto-PGF1 alpha 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-PGF1 alpha, or another not tested) or as a consequence of a factor which might protects prostacyclin from inactivation.     

Uteroplacental production of eicosanoids in ovine pregnancy

Dramatic cardiovascular alterations occur during normal ovine pregnancy which may be associated with increased prostaglandin production, especially of uteroplacental origin. To study this, we examined (Exp 1) the relationships between cardiovascular alterations, e.g., the rise in uterine blood flow and fall in systemic vascular resistance, and arterial concentrations of prostaglandin metabolites (PGEM, PGFM and 6-keto-PGF1 alpha) in nonpregnant (n = 4) and pregnant (n = 8) ewes. To determine the potential utero-placental contribution of these eicosanoids in pregnancy, we also studied (Exp 2) the relationship between uterine blood flow and the uterine venous-arterial concentration differences of PGE2, PGF2 alpha, PGFM, 6-keto-PGF1 alpha, and TxB2 in twelve additional late pregnant ewes. Pregnancy was associated with a 37-fold increase in uterine blood flow and a proportionate (27-fold) fall in uterine vascular resistance (p less than 0.01). Arterial concentrations of PGEM were similar in nonpregnant and pregnant ewes (316 +/- 19 and 245 +/- 38 pg/ml), while levels of PGFM and PGI2 metabolite 6-keto-PGF1 alpha were elevated 23-fold (31 +/- 14 to 708 +/- 244 pg/ml) and 14-fold (12 +/- 4 to 163 +/- 78 pg/ml), respectively (p less than 0.01). Higher uterine venous versus uterine arterial concentrations were observed for PGE2 (397 +/- 36 and 293 +/- 22 pg/ml) and 6-keto-PGF1 alpha (269 +/- 32 and 204 +/- 32 pg/ml), p less than 0.05, but not PGF2 alpha or TxB2. Although PGFM concentrations appeared to be greater in uterine venous (1197 +/- 225 pg/ml) as compared to uterine arterial (738 +/- 150 pg/ml) plasma, this did not reach significance (0.05 less than p less than 0.1). In normal ovine pregnancy arterial levels of PGI2 are increased, which may in part reflect increased uteroplacental production. Moreover the gravid ovine uterus also appears to produce PGE2 and metabolize PGF2 alpha.     

Age-associated decreases in prostaglandin contents in human gastric mucosa.

This study was designed to clarify effects of ageing on human gastric mucosal prostaglandin (PG) contents. Forty examinees were divided into 5 age groups of 8 persons each, as follows: age under 40, age 40-49, age 50-59, age 60-69, and age over 70. PG contents in human gastric mucosa were measured by microcolumn high performance liquid chromatography (HPLC) with helium/cadmium laser induced fluorescence detection using biopsy samples obtained by endoscopy. The contents of 6-keto-PGF1 alpha, PGF2 alpha, PGE2, and PGD2 in the under 40 group were 638 +/- 39, 97 +/- 16, 468 +/- 68, 497 +/- 86 (pg/mg tissue), respectively. No significant differences in PG contents among groups aged under 70 were observed. In contrast, significantly low PG contents in the over 70 group were observed, i.e., the contents of 6-keto-PGF1 alpha, PGF2 alpha, PGE2, and PGD2 were 311 +/- 58, 36 +/- 8, 196 +/- 48, 171 +/- 40, respectively, and their contents were significantly lower than those in other age groups. In conclusion, gastric mucosal PG contents decrease significantly in over 70 years-old and this might be a contributing factor in the pathogenesis of gastric ulcers in elderly people.     

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 PGF2 alpha and TXA2, as monitored from the formation of its stable degradation product TXB2 (PGF2 alpha greater than TXB2 greater than 6-keto-PGF1 alpha, the stable degradation product of PGI2 = PGD2 = PGE2 = 13,14-dihydro-15-keto-PGF2 alpha). 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 much greater than PGD2 greater than 13,14-dihydro-15-keto-PGE2 greater than PGF2 alpha = TXB2 = 6-keto-PGF1 alpha greater than 13,14-dihydro-15-keto-PGF2 alpha). 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 PGF2 alpha. 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.     

Transcardiac 8-iso-prostaglandin F(2 alpha)generation from acute myocardial infarction heart: insight into abrupt reperfusion and oxidant stress

8-iso-prostaglandin F(2 alpha)(8-iso-PGF(2 alpha)), a representative isoprostane, has been reported to be a reliable marker for oxidant stress in vivo. To examine if 8-iso-PGF(2 alpha)is generated in patients with acute myocardial infarction (AMI), we measured the level of immunoreactive 8-iso PGF(2 alpha)in the great cardiac vein as well as classical eicosanoids, 6-keto-prostaglandin F(1 alpha)(6-keto-PGF(1 alpha)) and thromboxane B(2)(TXB(2)) in the process of urgent coronary balloon angioplasty. Fourteen patients with anterior AMI were divided into two groups: the totally occluded (n=7) and the already perfused groups (n=7). In the former, transient elevation of 8-iso-PGF(2 alpha)was observed immediately after the angioplasty, i.e. the ratio of post-angioplasty level to pre-level was approximately 2.4 for 8-iso-PGF(2 alpha), 14 for 6-keto-PGF(1 alpha), and 5 for TXB(2). In the already perfused group, the levels of these eicosanoids were unchanged. In the totally occluded group, peak creatine phosphokinase in a peripheral vein was correlated with the level of 8-iso-PGF(2 alpha)(r(2)=0.841, P<0.01), but not with those of the other two eicosanoids. In conclusion, transcardiac 8-iso-PGF(2 alpha)generation is a reliable marker for the size of myocardium exposed to oxidant stress.     

Prostaglandin moieties that determine receptor binding specificity in the bovine corpus luteum.

This study provided a pharmacological evaluation of prostaglandin binding to bovine luteal plasma membrane. It was found that [3H]PGF2 alpha' [3H]PGE2' [3H]PGE1 and [3H]PGD2 all bound with high affinity to luteal plasma membrane but had different specificities. Binding of [3H]PGF2 alpha and [3H]PGD2 was inhibited by non-radioactive PGF2 alpha (IC50 values of 21 and 9 nmol l-1, respectively), PGD2 (35 and 21 nmol l-1), and PGE2 (223 and 81 nmol l-1), but not by PGE1 (> 10,000 and 5616 nmol l-1). In contrast, [3H]PGE1 was inhibited by non-radioactive PGE1 (14 nmol l-1) and PGE2 (7 nmol l-1), but minimally by PGD2 (2316 nmol l-1) and PGF2 alpha (595 nmol l-1). Binding of [3H]PGE2 was inhibited by all four prostaglandins, but slopes of the dissociation curves indicated two binding sites. Binding of [3H]PGE1 was inhibited, resulting in low IC50 values, by pharmacological agonists that are specific for EP3 receptor and possibly EP2 receptor. High affinity binding of [3H]PGF2 alpha required a C15 hydroxyl group and a C1 carboxylic acid that are present on all physiological prostaglandins. Specificity of binding for the FP receptor depended on the C9 hydroxyl group and the C5/C6 double bond. Alteration of the C11 position had little effect on affinity for the FP receptor. In conclusion, there is a luteal EP receptor with high affinity for PGE1' PGE2' agonists of EP3 receptors, and some agonists of EP2 receptors. The luteal FP receptor binds PGF2 alpha' PGD2 (high affinity), and PGE2 (moderate affinity) but not PGE1 due to affinity determination by the C9 and C5/C6 moieties, but not the C11 moiety.     

Salivary isoprostanes indicate increased oxidation injury in periodontitis with additional tobacco abuse

Isoprostanes (IPs) are indicators of in-vivo oxidative stress, and have been successfully used as markers for chronic inflammatory processes. The presence of chronic periodontal disease and cigarette smoking has been individually linked to the development of atherosclerosis, yet data regarding oxidative stress in this context are not available yet. The aim of this study was to evaluate levels of the salivary prostaglandins (PGs) 8-epi-PGF(2alpha), 6-oxo-PGF(1alpha), thromboxane B(2) (TXB(2)) and PGF(2alpha) in association with periodontal disease status with and without additional cigarette smoking. We analyzed saliva samples from 121 adults, (aged 21-73 years, 90 non-smokers, 31 smokers) for levels of 8-epi-PGF(2alpha), 6-oxo-PGF(1alpha), TXB(2) and PGF(2alpha). On the basis of periodontal disease indices the periodontal status of each subject was assessed and outcomes were then correlated with smoking status and laboratory findings. Salivary 8-epi-PGF(2alpha) levels increased with deteriorating plaque index, and were significantly higher (115.5 +/- 23.5 pg/ml) in smoking individuals, when compared to non-smokers (70.2 +/- 20.4 pg/ml, p<0.0001). In addition, smokers showed higher TXB(2) and PGF(2alphas) and lower 6-oxo-PGF(1alpha) levels p<0.0001). Oxidative stress, as reflected by elevated salivary 8-epi-PGF(2alpha) levels, is associated with the extent of periodontal disease and is significantly aggravated by concomitant tobacco abuse. Chronic inflammation and smoking have been individually associated with the development of atherosclerosis. The results of this study indicate that: 1) salivary IPs can reliably assess the degree of oxidative stress, and: 2) smoking and periodontal disease are two modifiable cardiovascular risk factors, able to potentiate each other.