Synthesis of tritium-labeled N-acetyl PGE2 carboxamide (CP-27,987)

The preparation of N-acetyl PGE₂ carboxamide (CP-27,987) regioselectively labeled at C-18 and C-19 with tritium is described. The overall radiochemical yield was 8.0% at a specific activity of 0.49 Ci/mmole. The synthesis employed is applicable to the preparation of tritium labeled natural prostaglandins and a variety of analogs.     

Prostaglandin E2 promotes proliferation of skeletal muscle myoblasts via EP4 receptor activation

We recently demonstrated that conditioned media (CM) from osteocytes enhances myogenic differentiation of myoblasts, suggesting that signaling from bone may be important for skeletal muscle myogenesis. The effect of CM was closely mimicked by prostaglandin E₂ (PGE₂), a bioactive lipid mediator in various physiological or pathological conditions. PGE₂ is secreted at high levels by osteocytes and such secretion is further enhanced under loading conditions. Although four types of receptors, EP1 to EP4, mediate PGE₂ signaling, it is unknown whether these receptors play a role in myogenesis. Therefore, in this study, the expression of EPs in mouse primary myoblasts was characterized, followed by examination of their roles in myoblast proliferation by treating myoblasts with PGE₂ or specific agonists. All four PGE₂ receptor mRNAs were detectable by quantitative real-time PCR (qPCR), but only PGE₂ and EP4 agonist CAY 10598 significantly enhance myoblast proliferation. EP1/EP3 agonist 17-phenyl trinor PGE₂ (17-PT PGE₂) and EP2 agonist butaprost did not have any significant effects. Moreover, treatment with EP4 antagonist L161,982 dose-dependently inhibited myoblast proliferation. These results were confirmed by cell cycle analysis and the gene expression of cell cycle regulators. Concomitant with the inhibition of myoblast proliferation, treatment with L161,982 significantly increased intracellular reactive oxygen species (ROS) levels. Cotreatment with antioxidant N-acetyl cysteine (NAC) or sodium ascorbate (SA) successfully reversed the inhibition of myoblast proliferation and ROS overproduction caused by L161,982. Therefore, PGE₂ signaling via the EP4 receptor regulates myogenesis by promoting myoblast proliferation and blocking this receptor results in increased ROS production in myoblasts.     

Utero-ovarian venous concentrations of prostaglandin E2 (PGE2 and prostaglandin F2α (PGF2α) following PGE2 intrauterine infusions

The uterine horns and utero-ovarian veins of nine crossbred mature gilts were bilaterally cannulated on day 9 of the estrous cycle (day 0 - first day of estrus). Each uterine horn in treated gilts (N=5) was infused with 150 μg PGE₂ in 3 ml of saline at 0900 h on day 12, 15 and 18 of the estrous cycle. Control gilts (N=4) received 3 ml saline intrauterine infusions on the corresponding day. Blood samples were collected from the utero-ovarian veins 15 min before each infusion and for the following 6 h with 15, 30 and 60 min intervals through the first, second and third two-hour periods, respectively. Venous concentrations of PGE₂ and PGF_2α were determined by radioimmunoassay procedures. Infusion of PGE₂ resulted in an immediate elevation in PGE₂ concentration in utero-ovarian venous drainage. Coincident elevations of PGF_2α utero-ovarian venous concentrations were observed after PGE₂ infusion. Plasma PGF_2α concentrations in the utero-ovarian veins were elevated (P<.01) in PGE₂ treated gilts for one hour post-treatment. The duration of PGE₂ and PGE₂α elevations as well as the peak values were influenced by day of the cycle.     

Phagocytes produce prostaglandin E2 in response to cytosolic Listeria monocytogenes

Listeria monocytogenes is an intracellular bacterium that elicits robust CD8⁺ T-cell responses. Despite the ongoing development of L. monocytogenes-based platforms as cancer vaccines, our understanding of how L. monocytogenes drives robust CD8⁺ T-cell responses remains incomplete. One overarching hypothesis is that activation of cytosolic innate pathways is critical for immunity, as strains of L. monocytogenes that are unable to access the cytosol fail to elicit robust CD8⁺ T-cell responses and in fact inhibit optimal T-cell priming. Counterintuitively, however, activation of known cytosolic pathways, such as the inflammasome and type I IFN, lead to impaired immunity. Conversely, production of prostaglandin E₂ (PGE₂) downstream of cyclooxygenase-2 (COX-2) is essential for optimal L. monocytogenes T-cell priming. Here, we demonstrate that vacuole-constrained L. monocytogenes elicit reduced PGE₂ production compared to wild-type strains in macrophages and dendritic cells ex vivo. In vivo, infection with wild-type L. monocytogenes leads to 10-fold increases in PGE₂ production early during infection whereas vacuole-constrained strains fail to induce PGE₂ over mock-immunized controls. Mice deficient in COX-2 specifically in Lyz2⁺ or CD11c⁺ cells produce less PGE₂, suggesting these cell subsets contribute to PGE₂ levels in vivo, while depletion of phagocytes with clodronate abolishes PGE₂ production completely. Taken together, this work demonstrates that optimal PGE₂ production by phagocytes depends on L. monocytogenes access to the cytosol, suggesting that one reason cytosolic access is required to prime CD8⁺ T-cell responses may be to facilitate production of PGE₂.     

The release of prostaglandins in human aqueous humour following intraocular surgery. Effect of indomethacin

Inflammatory reaction to ocular trauma is known to be related to prostaglandins. To further evaluate this phenomenon in human, PGE₁ and E₂ were measured by R.I.A. after silicic acid chromatography in aqueous humour of 26 patients before and 3 days after surgery for unilateral senile cataract (posterior chamber implant after extracapsular surgery). 13 out of these patients were treated with indomethacin by enteral route and 13 were not treated.PGE₂ levels increased in all non-treated patients from : m < 214 pg/ml before surgery, to 2666 ± 869 pg/ml (range : 257 - 8728) p < 0.001 after surgery. PGE₂ levels did not increase in indomethacin-treated patients. PGE₁ levels did not increase significantly in non-treated as in treated patients.1) Intra-ocular surgery is followed in human by a constant increase of the only PGE₂ in the aqueous humour. 2) Indomethacin inhibits this increase. 3) The post-surgical increase in the permeability of blood-aqueous barrier appears to be related to a release of PGE₂.     

Studies on the involvement of prostaglandins and their precursors in the rejection of Nippostrongylus brasiliensis from the rat

In vitro incubation of 6-day Nippo-strongylus brasiliensis in the presence of PGE₁ at 1000 ng/ml and PGE₂ at 500–10,000 ng/ml of medium did not affect worm motility nor in vivo survival of worms implanted into the small intestine of recipient rats. The intraduodenal injection of 250 and 500 μg PGE₁ or PGE₂ did not lead to expulsion of worms from infected rats. An in vitro exposure to precursor fatty acids of PGE₁ and PGE₂, dihomo-γ-linolenic acid and arachidonic acid, respectively, at concentrations of 1000–15,000 ng/ml of medium also failed to inhibit worm motility and in vivo worm survival. These results are at variance with some earlier reports and do not suggest that prostaglandins are directly involved in the immune rejection of N. brasiliensis. No prostaglandins could be demonstrated in worm homogenates.     

Microsomal prostaglandin E synthase-2 is not essential for in vivo prostaglandin E2 biosynthesis

Prostaglandin E₂ (PGE₂) plays an important role in the normal physiology of many organ systems. Increased levels of this lipid mediator are associated with many disease states, and it potently regulates inflammatory responses. Three enzymes capable of in vitro synthesis of PGE₂ from the cyclooxygenase metabolite PGH₂ have been described. Here, we examine the contribution of one of these enzymes to PGE₂ production, mPges-2, which encodes microsomal prostaglandin synthase-2 (mPGES-2), by generating mice homozygous for the null allele of this gene. Loss of mPges-2 expression did not result in a measurable decrease in PGE₂ levels in any tissue or cell type examined from healthy mice. Taken together, analysis of the mPGES-2 deficient mouse lines does not substantiate the contention that mPGES-2 is a PGE₂ synthase.     

Prostaglandin E1 (PGE1), but not prostaglandin E2 (PGE2), alters luteal and endometrial luteinizing hormone (LH) occupied and unoccupied LH receptors and mRNA for LH receptors in ovine luteal tissue to prevent luteolysis

Loss of luteal progesterone secretion at the end of the ovine estrous cycle is via uterine PGF₂α secretion. However, uterine PGF₂α secretion is not decreased during early pregnancy in ewes. Instead, the embryo imparts a resistance to PGF₂α. Prostaglandins E (PGE; PGE₁ + PGE₂) are increased in endometrium and uterine venous blood during early pregnancy in ewes to prevent luteolysis. Chronic intrauterine infusion of PGE₁ or PGE₂ prevents spontaneous or IUD, estradiol-17β, or PGF₂α-induced premature luteolysis in nonbred ewes. The objective was to determine whether chronic intrauterine infusion of PGE₁ or PGE₂ affected mRNA for LH receptors, occupied and unoccupied receptors for LH in luteal and caruncular endometrium, and luteal function. Ewes received Vehicle, PGE₁, or PGE₂ every 4 h from days 10 to 16 of the estrous cycle via a cathether installed in the uterine lumen ipsilateral to the luteal-containing ovary.Jugular venous blood was collected daily for analysis of progesterone and uterine venous blood was collected on day-16 for analysis of PGF₂α and PGE. Corpora lutea and caruncular endometrium were collected from day-10 preluteolytic control ewes and day-16 ewes treated with Vehicle, PGE₁ or PGE₂ for analysis of the mRNA for LH receptors and occupied and unoccupied receptors for LH. Luteal weights on day-16 in ewes treated with PGE₁ or PGE₂ and day-10 control ewes were similar (P ≥ 0.05), but were greater (P ≤ 0.05) than in day-16 Vehicle-treated ewes. Progesterone profiles on days 10–16 differed (P ≤ 0.05) among treatment groups: PGE₁ > PGE₂ > Vehicle-treated ewes. Concentrations of PGF₂α and PGE in uterine venous plasma on day-16 were similar (P ≥ 0.05) in the three treatment groups. Luteal mRNA for LH receptors and unoccupied and occupied LH receptors were similar (P ≥ 0.05) in day-10 control ewes and day-16 ewes treated with PGE₂ and were lower (P ≤ 0.05) in day-16 Vehicle-treated ewes. PGE₂ prevented loss (P ≤ 0.05) of day-16 luteal mRNA for LH receptors and occupied and unoccupied LH receptors. Luteal and caruncular tissue mRNA for LH receptors and occupied and unoccupied LH receptors were greater (P ≤ 0.05) on day-16 of PGE₁-treated ewes than any treatment group. mRNA for LH receptors and occupied and unoccupied receptors for LH in caruncules were greater (P ≤ 0.05) in day-16 Vehicle or PGE₂-treated ewes than in day-10 control ewes. It is concluded that PGE₁ and PGE₂ share some common mechanisms to prevent luteolysis; however, only PGE₁ increased luteal and endometrial mRNA for LH receptors and occupied and unoccupied LH receptors. PGE₂ prevents a decrease in luteal mRNA for LH receptors and occupied and unoccupied receptors for LH without altering endometrial mRNA for LH receptors or occupied and unoccupied receptors for LH.     

Direct Melanoma Cell Contact Induces Stromal Cell Autocrine Prostaglandin E2-EP4 Receptor Signaling That Drives Tumor Growth,Angiogenesis, and Metastasis

The stromal cells associated with tumors such as melanoma are significant determinants of tumor growth and metastasis. Using membrane-bound prostaglandin E synthase 1 (mPges1^−/−) mice, we show that prostaglandin E₂ (PGE₂) production by host tissues is critical for B16 melanoma growth, angiogenesis, and metastasis to both bone and soft tissues. Concomitant studies in vitro showed that PGE₂ production by fibroblasts is regulated by direct interaction with B16 cells. Autocrine activity of PGE₂ further regulates the production of angiogenic factors by fibroblasts, which are key to the vascularization of both primary and metastatic tumor growth. Similarly, cell-cell interactions between B16 cells and host osteoblasts modulate mPGES-1 activity and PGE₂ production by the osteoblasts. PGE₂, in turn, acts to stimulate receptor activator of NF-κB ligand expression, leading to osteoclast differentiation and bone erosion. Using eicosanoid receptor antagonists, we show that PGE₂ acts on osteoblasts and fibroblasts in the tumor microenvironment through the EP4 receptor. Metastatic tumor growth and vascularization in soft tissues was abrogated by an EP4 receptor antagonist. EP4-null Ptger4^−/− mice do not support B16 melanoma growth. In vitro, an EP4 receptor antagonist modulated PGE₂ effects on fibroblast production of angiogenic factors. Our data show that B16 melanoma cells directly influence host stromal cells to generate PGE₂ signals governing neoangiogenesis and metastatic growth in bone via osteoclast erosive activity as well as angiogenesis in soft tissue tumors.     

Metabolism of prostaglandins and xenobiotics by adrenal microsomal monooxygenase in the guinea pig

The possibility that prostaglandins could serve as substrates for the guinea pig adrenal microsomal monooxygenase was investigated. The binding of PGE₁ to adrenal microsomes was found to exhibit a reverse type I spectral change. Also PGE₁ diminished the magnitude of type I spectrum elicited by cortisol binding to adrenal microsomes. The incubation of [³H]PGE₁ or of [³H]PGE₂ with adrenal microsomes supplemented with NADPH yielded primarily the respective 19-hydroxy metabolite. The enzymatic activity catalyzing this hydroxylation appears to be a typical monooxygenase, requiring NADPH for activity and being strongly inhibited by metyrapone, SKF 525A, and cytochrome c. Carbon monoxide at a ratio of 9:1 to oxygen moderately inhibited the hydroxylation of PGE₁. Whereas the liver catalyzed the hydroxylation of PGE₁ and PGA₁ equally well, the adrenal microsomes preferentially catalyzed the hydroxylation of PGE₁. This finding and the observation that α-naphthoflavone is a weak inhibitor of the adrenal PGE₁ hydroxylation points to significant differences between the adrenal and liver prostaglandin hydroxylation activities. Cortisol, which is a substrate for adrenal monooxygenase, strongly inhibited PGE₁ and PGE₂ hydroxylation. By contrast, certain xenobiotics (ethylmorphine, hexobarbital, benzpyrene), which are also metabolized by adrenal microsomes, only slightly inhibited the hydroxylation of PGE₁. Similarly, PGE₁ only weakly inhibited ethylmorphine and benzphetamine demethylation and hexobarbital hydroxylation. These observations suggest that adrenal microsomes contain several monooxygenases with different affinities for prostaglandins and for the different xenobiotic substrates.     

Novel M4 positive allosteric modulators derived from questioning the role and impact of a presumed intramolecular hydrogen-bonding motif in β-amino carboxamide-harboring ligands

This letter describes a focused exercise to explore the role of the β-amino carboxamide moiety found in all of the first generation M₄ PAMs and question if the NH₂ group served solely to stabilize an intramolecular hydrogen bond (IMHB) and enforce planarity. To address this issue (and to potentially find a substitute for the β-amino carboxamide that engendered P-gp and contributed to solubility liabilities), we removed the NH₂, generating des-amino congeners and surveyed other functional groups in the β-position. These modifications led to weak M₄ PAMs with poor DMPK properties. Cyclization of the β-amino carboxamide moiety by virtue of a pyrazole ring re-enforced the IMHB, led to potent (and patented) M₄ PAMs, many as potent as the classical bicyclic β-amino carboxamide analogs, but with significant CYP1A2 inhibition. Overall, this exercise indicated that the β-amino carboxamide moiety most likely facilitates an IMHB, and is essential for M₄ PAM activity within classical bicyclic M₄ PAM scaffolds.     

Differences in PGE2 Production between Primary Human Monocytes and Differentiated Macrophages: Role of IL-1β and TRIF/IRF3

Prostaglandin E2 (PGE₂) is induced in vivo by bacterial products including TLR agonists. To determine whether PGE₂ is induced directly or via IL-1β, human monocytes and macrophages were cultured with LPS or with Pam3CSK4 in presence of caspase-1 inhibitor, ZVAD, or IL-1R antagonist, Kineret. TLR agonists induced PGE₂ in macrophages exclusively via IL-1β-independent mechanisms. In contrast, ZVAD and Kineret reduced PGE₂ production in LPS-treated (but not in Pam3CSK4-treated) monocytes, by 30–60%. Recombinant human IL-1β augmented COX-2 and mPGES-1 mRNA and PGE₂ production in LPS-pretreated monocytes but not in un-primed or Pam3CSK4-primed monocytes. This difference was explained by the finding that LPS but not Pam3CSK4 induced phosphorylation of IRF3 in monocytes suggesting activation of the TRIF signaling pathway. Knocking down TRIF, TRAM, or IRF3 genes by siRNA inhibited IL-1β-induced COX-2 and mPGES-1 mRNA. Blocking of TLR4 endocytosis during LPS priming prevented the increase in PGE₂ production by exogenous IL-1β. Our data showed that TLR2 agonists induce PGE₂ in monocytes independently from IL-1β. In the case of TLR4, IL-1β augments PGE₂ production in LPS-primed monocytes (but not in macrophages) through a mechanism that requires TLR4 internalization and activation of the TRIF/IRF3 pathway. These findings suggest a key role for blood monocytes in the rapid onset of fever in animals and humans exposed to bacterial products and some novel adjuvants.     

Quantitative determination of prostaglandins E1 and F1α by multiple ion analysis

Quantitative assays for prostaglandins (PG) E₁ and PGF_1α are described using [3,3,4,4,5,6-²H₆]labeled prostaglandins as carriers and methyl ester-O-methyloxime-acetate (PGE₁) and methyl ester-acetate (PGF_1α) derivatives for gas - liquid chromatography/mass spectrometric analysis. Thin-layer argentation chromatography was used to separate PGE₁ from PGE₂ and 13, 14-dihydro-PGE₂. These latter compounds, which do not separate from PGE₁ using conventional thin-layer chromatography or under the gas - liquid chromatographic conditions used, can significantly interfere with the quantitative analysis of PGE₁. The method described prevents this interference and is therefore suitable for the accurate analysis of PGE₁ in biological samples containing a high concentration of PGE₂ and/or 13, 14-dihydro-PGE₂.     

Effects of Bothrops asper snake venom on the expression of cyclooxygenases and production of prostaglandins by peritoneal leukocytes in vivo,and by isolated neutrophils and macrophages in vitro

In this study, the ability of Bothrops asper snake venom (BaV) to increase the production of prostaglandins PGE₂ and PGD₂ was assessed in a mouse model in vivo and in inflammatory cells in vitro. In addition, the expressions of COX-1 and COX-2 were assessed. BaV induced an increment in the in vivo synthesis of PGE₂ and PGD₂, together with an enhanced expression of COX-2, but not of COX-1. However, enzymatic activities of COX-1 and COX-2 were increased. Incubation of isolated macrophages and neutrophils with a sub-cytotoxic concentration of BaV in vitro resulted in increased release of PGE₂ and PGD₂ by macrophages and PGE₂ by neutrophils, concomitantly with an increment in the expression of COX-2, but not of COX-1 by both cell types. Our results demonstrate the ability of BaV to promote the expression of COX-2 and to induce the synthesis of proinflammatory prostaglandins. Macrophages and neutrophils may be important targets for this venom under in vivo situation.     

Synthesis of [3H]- and -[14C]-labelled sulprostone (CP-34,089/ZK-57,671)

The synthesis of N-methanesulfonyl 16-phenoxy-ω-tetranor PGE₂ carboxamide (sulprostone — CP-34,089/ZK-57,671) labeled with tritium and carbon-14 is described. Sulprostone labeled with tritium in the phenoxy moiety by means of catalytic hydrogenolysis was obtained in a 17% radiochemical yield with a specific activity of 1.0 Ci/mmol. The methanesulfonamide-¹⁴C derivative of sulprostone was prepared from methyl-¹⁴C iodide in an 11.8% radiochemical yield having a specific activity of 18.8 mCi/mmol.     

Inhibition of cytotoxic responses by prostaglandin E2 in the presence of interleukin 2

Experiments are described which provide direct evidence for a strong suppressive effect of prostaglandin E₂ (PGE₂) on the activation of cytotoxic T lymphocytes (CTL). The experiments also tested the hypothesis that PGE₂ inhibits cytotoxic responses exclusively by preventing the helper T cells from producing interleukin 2 (IL-2). In support of this hypothesis we found that indomethacin enhanced cytotoxic responses in the absence but not in the presence of concanavalin A-activated spleen cell supernatant (CSCS) indicating that the endogenously produced prostaglandin(s) inhibited primarily the production of IL-2. However, the addition of PGE₂ to microcultures was found to inhibit in a dose-dependent manner the activation of cytotoxic responses against I-region compatible stimulator cells even in the presence of CSCS. This inhibition was not observed in the presence of I-region incompatible stimulator cells, indicating that the inhibitory effect of PGE₂ did not result from a general toxic effect on the culture. The results argue against the notion that PGE₂ inhibits cytotoxic responses exclusively by preventing the production of IL-2.     

EP2 receptor activation by prostaglandin E2 leads to induction of HO-1 via PKA and PI3K pathways in C6 cells

Recently we proposed that COX-2 induction precedes expression of HO-1 in ischemic preconditioned rat brain. In the current study, we investigated the molecular mechanism by which prostaglandin E₂, one of COX-2 metabolites, induces HO-1 in rat C6 brain cells. We demonstrated that concentration of PGE₂ increased HO-1 expression in C6 cells in vitro. The effects of PGE₂ were mimicked by PGE₂ receptor EP₂ agonists, 11-deoxy PGE₂, and cAMP analog, dibutyl-cAMP. HO-1 expression by PGE₂ was inhibited by LY294002, PI3K inhibitor and H89, PKA inhibitor. The EP₂-specific antagonist, AH8006 also inhibited PGE₂-mediated HO-1 expression in a concentration-dependent manner. Finally, PGE₂ inhibited GOX-induced apoptosis as assayed by FACS analysis or DNA strand breaks assay, and this cell death was reversed by ZnPPIX, HO-1 inhibitor. In addition to HO-1 induction, PGE₂ also increased phosphorylation of Bad by PKA- and PI3K-depednent manner. Taken together, we conclude that PGE₂ induces HO-1 protein expression through PKA and PI3K signaling pathways via EP₂ receptor in C6 cells. The induction of HO-1 along with increase of p-Bad by PGE₂ is responsible for anti-apoptosis against oxidant stress.     

A defective prostaglandin E synthase could affect egg formation in the silkworm Bombyx mori

We had previously reported a prostaglandin E synthase (bmPGES) in the silkworm Bombyx mori that catalyzes the isomerization of PGH₂ to PGE₂. The present study aimed to provide a genome-editing characterization of bmPGES in B. mori. Results showed bmPGES gene disruption to result in a reduced content of PGE₂. The change affected the expression of chorion genes and egg formation in silkworms. Collectively, the results indicated that bmPGES could be involved in reproduction of B. mori. Therefore, this study provides insights into the physiological role of bmPGES and PGE₂ in silkworms.