Characterization of prostaglandin E2 generation through the cyclooxygenase (COX)-2 pathway in human neutrophils

In the present study, we characterized the generation of prostaglandin (PG)E2 in human neutrophils. We found that the Ca2+-dependent type IV cytosolic phospholipase A2 (cPLA2) was pivotally involved in the COX-2-mediated generation of PGE2 in response to a calcium ionophore, as determined by the use of selected PLA2 inhibitors. PGE2 biosynthesis elicited by bacterial-derived peptides or by phagocytic stimuli acting on cell surface receptors also showed to be dependent on cPLA2 activity. We then assessed metabolism of unesterified arachidonic acid (AA), and observed that PGE2 production becomes favored over that of LTB4 with higher AA concentrations. Withdrawal of calcium prevented the generation of PGE2 in response to a calcium ionophore but did not affect the up-regulation of COX-2 or its capacity to convert AA, thus limiting its implication at the level of cPLA2 activation. Of the main eicosanoids produced by neutrophils, only LTB4 was able to up-regulate COX-2 expression. Finally, the only PGE synthase isoform found in neutrophils is microsomal PGE synthase-1; it co-localized with COX-2 and its expression appeared mainly constitutive. These results highlight key differences in regulatory processes of the 5-LO and COX pathways, and enhance our knowledge at several levels in the PGE2 biosynthesis in neutrophils.     

Effect of prostaglandin E2, DL-cloprostenol,and prostaglandin E2 in combination with D-cloprostenol on uterine motility during diestrus in experimental cows

Prostaglandin F(2alpha) is used in dairy herd management because of its luteolytic properties and for its direct effect on the myometrium in cows diagnosed with endometritis. Prostaglandin E(2) has a contractile effect on the bovine uterus. In human medicine, prostaglandin E(2) is routinely used to maintain labor and to ripen the cervix. We hypothesized, that a combination of prostaglandin F(2alpha) and prostaglandin E(2) would provoke a long-lasting increase in intrauterine pressure (IUP) and uterine motility as compared to either prostaglandin group. Intrauterine pressure was recorded during the diestrus of eight lactating dairy cows using a transcervically placed intraluminal pressure microtransducer. After recording of physiologic uterine motility for 30min, prostaglandins (DL-cloprostenol, PGE(2), PGE(2) in combination with D-cloprostenol) or placebo were administered, followed by a 2h recording period. Significant differences were found for the area under the curve, the mean amplitude and the intrauterine pressure, whereas the number of pressure waves did not differ significantly among treatments. Peak values for area under the curve and mean amplitude were found during the first 15min for the combination of PGE(2) and D-cloprostenol. During the last 15min of the recording session, area under the curve and mean amplitude were increased only for the combination of PGE(2) and D-cloprostenol as compared to placebo. Although PGF(2alpha) and PGE(2) provoke an increase in intrauterine pressure, only their combination guarantees a significant effect over a 2h recording period.     

Cardiovascular effects of prostaglandin F(2 alpha) and prostaglandin E(2) in Atlantic cod (Gadus morhua)

Little is known of the cardiovascular functions of prostaglandins in non-mammalian vertebrates. There are indications that prostaglandins may have a function in haemostasis by constricting blood vessels in filament arteries in the fish gill after injury. Our aim was to examine the cardiovascular effect of the prostaglandins F(2 alpha) (PGF(2 alpha)) and E(2) (PGE(2)) with emphasis on branchial circulation. Intra-arterial injections of PGF(2 alpha) (10, 40, 160, 400 nmol kg(-1)) in cod caused a dose-dependent increase in ventral aortic blood pressure, a reduction in cardiac output, and an increase in gill vascular resistance. A contraction of filament arteries was observed with in vivo microscopy only seconds after injection. PGF(2 alpha) may therefore possibly be involved in a haemostatic vasoconstriction. In contrast, the most significant effects of PGE(2) appeared to be on the heart. PGE(2) also reduced dorsal aortic blood pressure.     

Ethylene evolution from various developing organs of olive (Olea europaea) after excision

Ethylene evolution from leaves, stems, inflorescences and fruits of the olive plant ( Olea europaea L.) cv. Manzanillo was studied at various stages of their development. Mature non-growing organs, particularly leaves, have a constant, low, and uniform rate of ethylene evolution. Ethylene evolution from detached mature olive leaves was constant during the first 12 h after excision. Leaves on shoots maintained in vitro kept a constant rate of ethylene evolution for at least the first 5–6 days. Leaf injury significantly increased ethylene evolution. Ethylene evolution from injured and non-injured control leaves could be markedly inhibited aminoethoxyvinylglycine (AVG) applied to the leaves or fed to the shoot. The use of excised olive shoots and leaves as an in vitro model system for studies of induced metabolic processes such as abscission and developing water stress was suggested.     

Association between prostaglandin E2 receptor gene and essential hypertension

BACKGROUND: Essential hypertension (EH) is a complex multifactorial polygenic disorder that is thought to result from an interaction between an individual's genetic makeup and various environmental factors. In the kidney, prostaglandins (PGs) are important mediators of vascular tone and salt and water homeostasis, and are involved in the mediation and/or modulation of hormonal action. In previous studies, mice deficient in the prostaglandin E2 (PGE(2)) EP2 receptor had resting systolic blood pressure (BP) that was significantly lower than that of wild-type controls. The BP of those mice increased when they were put on a high-salt diet, suggesting that the EP2 receptor is involved in sodium handling by the kidney. In the present study, we investigated the association between EH and nucleotide polymorphisms in the gene encoding the prostaglandin E2 receptor subtype EP2 (PTGER2). METHODS: We selected three single-nucleotide polymorphisms (SNP) in the human PTGER2 gene (rs1254601, rs2075797, and rs17197), and we performed a genetic association study of 266 EH patients and 253 age-matched normotensive (NT) controls. RESULTS: There was no significant difference in overall distribution of genotypes or alleles of any of the SNP between the EH and NT groups. However, among men, the A/A type of the SNP rs17197 (rs17197, A/G in 3'UTR) was significantly more frequent in EH subjects than in NT subjects (P=0.041). CONCLUSION: The present findings suggest that rs17197 is useful as a genetic marker of EH in men.     

Determination of prostaglandin E(2) by on-line solid-phase extraction-liquid chromatography with ultraviolet detection for microsomal prostaglandin E(2) synthase-1 inhibitor screening

A rapid, robust and selective on-line solid-phase extraction-liquid chromatographic method with ultra-violet detection (on-line SPE-LC-UV) for microsomal prostaglandin E(2) synthase-1 (mPGES-1) inhibitor screening was developed and validated. Disrupted A549 cells were used as mPGES-1 source and the formation of prostaglandin E(2) (PGE(2)) out of the substrate prostaglandin H(2) (PGH(2)) was determined at 195 nm. Direct on-line sample clean up was achieved by automated column switch (C18 trap column) prior isocratic separation using a C18 analytical column. The on-line SPE-LC-UV method was accurate, precise and reproducible in the range of 71-1763 ng/ml for PGE(2) and met the generally accepted criteria for bioanalytical methods. The method was successfully applied to determine the IC(50) value of the known mPGES-1 inhibitor NS-398.     

Stimulation of CFU-f formation by prostaglandin E(2) is mediated in part by its degradation product, prostaglandin A(2)

Prostaglandins (PG) of the E series are known to rapidly undergo non-enzymatic dehydration in culture medium containing serum albumin to produce the cyclopentenone PGs of the A series. We investigated the actions of PGA(1) and A(2) in the in vitro calcifying fibroblastic-colony forming unit assay which can partially mimic the in vivo anabolic effects of PGE(2). It was found that PGA(1) and A(2) both stimulated colony formation in a dose-dependent manner with a maximum at 10(-6) M and to a similar degree to PGE(2). In contrast to PGE(2), PGA(1) and PGA(2) both caused an inhibition of cAMP accumulation. Furthermore, the addition of protein kinase A inhibitors, H8 and H89, had no significant effect on the stimulation of colony number by PGE(2). These data suggest that (a) the bone anabolic effects of PGE(1) and E(2) are, in part at least, mediated by their dehydration products PGA(1) and A(2) and (b) that they are mediated via pathways not necessarily involving the cAMP/protein kinase A cascade.     

Reduced prostaglandin E2 (PGE2) receptors on atopic T lymphocytes

We have recently demonstrated that atopic T lymphocytes have decreased sensitivity to prostaglandin E2 (PGE2). In order to determine whether this decreased sensitivity was reflected at the receptor level, we have employed a radioligand binding assay utilizing [3H]PGE2. We have demonstrated a single specific reversible binding site for [3H]PGE2 on normal T cells (N = 10) with a mean KD (+/-SD) of 32.2 (+/-25.0) nM, a binding capacity of 20.2 (+/-13.0) pM, and a mean of 1004 (+/-118) receptors per cell. Atopic T cells (N = 10) were also found to have a single specific binding site for [3H]PGE2 with a mean KD of 24.9 (+/-17.8) nM, a binding capacity of 7.1 (+/-10.1) pM, and a mean of 372 (+/-61) receptors per cell. These radioligand binding studies were correlated with functional studies in the same subjects. Phytohemagglutinin-stimulated protein synthesis ([3H]leucine uptake) was suppressed in a dose-dependent fashion by PGE2 (10(-6)-10(-12) M). The maximal effect of PGE2 on normal T cells was 10(-6) M PGE2 with an IC50 of 10(-12) M. Atopic T cells responded quantitatively less than normal T cells to PGE2. Further, the maximum suppression of protein synthesis by PGE2 occurred at 10(-6) M with an IC50 of 10(-10) to 10(-11) M. These studies suggest that part of the decreased sensitivity of atopic T cells to PGE2 may result from a reduction in PGE2 binding sites.     

Mammalian oocytes are targets for prostaglandin E2 (PGE2) action

Background  

The ovulatory gonadotropin surge increases synthesis of prostaglandin E2 (PGE2) by the periovulatory follicle. PGE2 actions on granulosa cells are essential for successful ovulation. The aim of the present study is to determine if PGE2 also acts directly at the oocyte to regulate periovulatory events.     

Interferon-gamma and interleukin 4 inhibit interleukin 1beta-induced delayed prostaglandin E(2)generation through suppression of cyclooxygenase-2 expression in human fibroblasts

Interleukin (IL-)1 stimulates prostaglandin E(2)(PGE(2)) generation in fibroblasts, and preferential couplings between particular phospholipase A(2)(PLA(2)) and cyclooxygenase (COX) isozymes are implicated with IL-1-induced delayed PGE(2)generation. The regulatory effects of interferon (IFN)-gamma and IL-4 on IL-1beta-induced COX, PLA(2)isoforms expression and terminal delayed PGE(2)generation were examined in three types of human fibroblasts. These human fibroblasts constitutively expressed cytosolic PLA(2)(cPLA(2)) and COX-1 enzymes, and exhibited delayed PGE(2)generation in response to IL-1beta. IL-1beta also stimulated expression of cPLA(2)and COX-2 only, while constitutive and IL-1beta-induced type IIA and type V secretory PLA(2)s (sPLA(2)s) expression could not be detected. A COX-2 inhibitor and cPLA(2)inhibitor markedly suppressed the IL-1beta-induced delayed PGE(2)generation, while a type IIA sPLA(2)inhibitor failed to affect it. IFN-gamma and IL-4 dramatically inhibited the IL-1beta-induced delayed PGE(2)generation; these cytokines apparently suppressed IL-1beta-stimulated COX-2 expression and only weakly suppressed cPLA(2)expression in response to IL-1beta. These results indicate that IL-1beta-induced delayed PGE(2)generation in these human fibroblasts mainly depends on de novo induction of COX-2 and cPLA(2), irrespective of the constitutive presence of COX-1, and that IFN-gamma and IL-4 inhibit IL-1beta-induced delayed PGE(2)generation by suppressing, predominantly, COX-2 expression.     

Effect of different doses of prostaglandin E2 on intrauterine pressure and uterine motility during diestrus in experimental cows

Studies in human medicine proved the important role of prostaglandin E2, which stimulates uterine contractions in vivo and in vitro and has been extensively used to ripen the cervix around labor. We wanted to demonstrate that increasing the dosage of prostaglandin E2 (1.25 mg, 2.5 mg, 5 mg and 10 mg) provokes an increase in intrauterine pressure and uterine motility in cattle. Five healthy, lactating dairy cows were used as experimental animals for this study. Intrauterine pressure was recorded during the diestrus phase (1 recording per cow and diestrus phase) by means of a transcervically placed intraluminal pressure microtransducer. Physiologic uterine motility was recorded for 30 min, then placebo or one of the prostaglandin E2- dosages was administered through an indwelling catheter in the jugular vein, followed by a 2-h recording period (eight 15-min periods). Area under the curve (AUC), mean amplitude, frequency of pressure waves and intrauterine pressure were analyzed. Furthermore, we recorded protocols for monitoring heart and respiratory rates and side effects at 9 given examination times. Significant differences were found for the AUC, the mean amplitude and the intrauterine pressure (P < or = 0.05), whereas the number of pressure waves per 15 min did not differ significantly among treatments. Peak values for AUC, mean amplitude and intrauterine pressure were found during the first 15 min after administration of 10 mg of prostaglandin E2. Dose-effect curves showed that the 2.5 mg dosage provided the optimal ratio between myometrial stimulation and undesirable side-effects.     

Nitric oxide production in murine spleen cells: role of interferons and prostaglandin E(2) in the generation of cytotoxic activity

The production of nitric oxide (NO) was measured in cultures of spleen cells stimulated by lipopolysaccharide (LPS), IL-2 or LPS + IL-2. We observed that NO synthesis is increased by IFN-gamma but inhibited by IFN-alpha/beta. This is not the case when IL-2 is present in the cultures, since interferons play a minor role in the regulation of the NO production. When IL-2 and LPS were associated in the cultures, the IFN-alpha/beta role seems more important than that of IFN-gamma. PGE(2) inhibits NO production in LPS supplemented cultures but has a slight effect in the presence of IL-2 and no effect with IL-2 + LPS. 3-isoButyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterases, induces a decrease of IFN production. In the presence of H-7, an inhibitor of protein kinase C (PKC), NO production is reduced when the cultures are supplemented by LPS or IL-2 but not when IL-2 and LPS are both added. H-7 also reduced IFN production. In the presence of N(G)-monomethyl-L-arginine (N-MMA), an inhibitor of NO synthesis, IFN production was increased, with no change in the cytotoxic activity. Hence, interferons regulate NO production by mouse spleen cells and, in return, NO modulates the generation of IFN.     

Evidence for the presence of a prostaglandin E 2 -9-keto reductase in rat organs

Antibodies directed toward PGF_2α which cross react with PGE₂ only slightly were used to detect conversion of PGE₂ to PGF_2α by homogenates of several rat tissues. This conversion by rat heart homogenates was demonstrated to be reversible, lost after trypsin digestion, and inhibited by several sulfhydryl blocking agents. The activity of the rat heart homogenate was precipitable by ammonium sulphate, was not dialyzable, and was 50% destroyed when the homogenate was incubated at 50° for 5 min. In the rat, the heart had the highest activity, followed by the kidney, brain, and liver. Negligible activity was found in smooth muscle, skeletal muscle, and whole blood of rat.     

Expression of prostaglandin E(2) receptor subtypes in mouse hair follicles.

We investigated the mRNA distribution of the prostaglandin (PG) E(2) receptor subtypes and cyclooxygenases (COXs) in hair follicles of the mouse dorsal skin. In the 3-week hair follicles, which are in the anagen phase, EP3 and EP4 mRNA were expressed in the dermal papilla cells and the outer root sheath cells located in the hair bulb region, respectively. In the 8-week hair follicles, which are in the telogen phase, the signals for both EP3 and EP4 mRNAs had disappeared. To study the hair cycle-dependent expression of mRNAs for the EPs and COXs, an area of dorsal hair was depilated from 8-week-old mice. On days 8 and 12 after depilation, EP3 and EP4 mRNA were reexpressed in the dermal papilla cells and the outer root sheath cells, and the induction of COX-2 mRNA was also observed in the outer root sheath cells, the upper area of EP4 expression site. These results suggest that EP3 and EP4 receptors may involve in the development and regrowth of the hair follicles.     

Uterine transport of prostaglandin E(2)-releasing simulated embryonic vesicles in mares

Transrectal ultrasonography was used to test the hypothesis that prostaglandin E(2) (PGE(2)) would increase the uterine transport of simulated embryonic vesicles in mares. Uterine transport of PGE(2)-releasing (PGE) vesicles, vehicle-releasing (sham) vesicles, and equine embryos was contrasted on Day 12 or Day 13 post ovulation. In Experiment 1, there was no difference (P>0.10) in transport of PGE vesicles, sham vesicles, Day-12 embryos, and Day-12 embryos after cervical manipulation (n = 3 per group). In Experiments 2 and 3, respectively, transport of PGE and sham vesicles was contrasted with transport of Day-13 embryos after the vesicles (1 vesicle per mare) were placed into the uterine lumen with the embryo, (n = 7 per group). In Experiment 2, PGE vesicles were transported less often (P<0.05) from horn to body and from segment to segment than Day-13 embryos before vesicle insertion. In Experiment 3, sham vesicles were transported less often from horn to body (P<0.10) and from segment to segment (P<0.01) than Day-13 embryos before vesicle insertion. However, there was no difference (P>0.10) in the transport of PGE vesicles and embryos (Experiment 2) or sham vesicles and embryos (Experiment 3) together in the uterine lumen. In Experiment 4, transport of PGE and sham vesicles was contrasted by placing them together into the uterine lumen of nonpregnant mares on Day 13 (n = 7). There was no difference (P>0.10) in the transport of PGE and sham vesicles together in the uterine lumen. These results do not support the hypothesis that PGE(2) increases uterine transport of simulated embryonic vesicles. In addition, these results do not support the hypothesis that equine embryos stimulate uterine transport.     

Platelet-activating factor and prostaglandin E2 impair esophageal ACh release in experimental esophagitis

ACh is a neurotransmitter in cat esophageal circular muscle, as atropine nearly abolishes contraction of in vitro circular muscle strips in response to electric field stimulation (EFS) (5, 12). Experimental esophagitis reduced EFS- but not ACh-induced contraction of esophageal circular muscle, suggesting that esophagitis impairs neurotransmitter release. Because IL-1beta and IL-6 are produced in esophagitis and reproduce these changes in normal esophageal muscle (12), we examined the role of IL-1beta and IL-6 in this motor dysfunction. IL-1beta, IL-6 (12), H2O2, PGE2, and platelet-activating factor (PAF) were elevated in esophagitis specimens. Normal muscle incubated (2 h) in IL-1beta and IL-6 had increases in H2O2, PGE2, and PAF levels. H2O2 contributed to increased PGE2 and PAF, as the increase was partially (60-80%) reversed by the H2O2 scavenger catalase. EFS-induced [3H]ACh release from muscle strips significantly (42%) decreased in esophagitis and after 2 h incubation in PGE2 and in PAF C-16. Similarly, EFS-induced but not ACh-induced muscle contraction decreased in esophagitis and after incubation in PGE2 and PAF C-16. Finally, in normal muscle strips treated with IL-1beta electrical field stimulation (EFS)-induced contraction was partially restored by indomethacin or by the PAF antagonist CV3988 and was completely restored by the combination of CV3988 and indomethacin, whereas in strips treated with IL-6, EFS-induced contraction was partially restored by the PAF antagonist CV3988 and not affected by indomethacin. We conclude that IL-1beta-induced production of H2O2 causes formation of PGE2 and PAF that inhibit ACh release from esophageal cholinergic neurons without affecting ACh-induced contraction of esophageal circular muscle. IL-6 causes production of H2O2, PAF, and other unidentified inflammatory mediators.     

Helicobacter pylori-induced prostaglandin E(2) synthesis involves activation of cytosolic phospholipase A(2) in epithelial cells

Helicobacter pylori initiates an inflammatory response and gastric diseases, which are more common in patients infected with H. pylori strains carrying the pathogenicity island, by colonizing the gastric epithelium. In the present study we investigated the mechanism of prostaglandin E(2) (PGE(2)) synthesis in response to H. pylori infection. We demonstrate that H. pylori induces the synthesis of PGE(2) via release of arachidonic acid predominately from phosphatidylinositol. In contrast to H. pylori wild type, an isogenic H. pylori strain with a mutation in the pathogenicity island exerts only weak arachidonic acid and PGE(2) synthesis. The H. pylori-induced arachidonic acid release was abolished by phospholipase A(2) (PLA(2)) inhibitors and by pertussis toxin (affects the activity of G alpha(i)/G alpha(o)). The role of phospholipase C, diacylglycerol lipase, or phospholipase D was excluded by using specific inhibitors. An inhibitor of the stress-activated p38 kinase (SB202190), but neither inhibitors of protein kinase C nor an inhibitor of the extracellular-regulated kinase pathway (PD98059), decreased the H. pylori-induced arachidonic acid release. H. pylori-induced phosphorylation of p38 kinase and cytosolic PLA(2) was blocked by SB202190. These results indicate that H. pylori induces the release of PGE(2) from epithelial cells by cytosolic PLA(2) activation via G alpha(i)/G alpha(o) proteins and the p38 kinase pathway.